The Immune Response to Infection

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The Immune Response to Infection

This page intentionally left blank

The Immune Response to Infection edited

by

Stefan H. E.Kaufmann

Max Planck Institute for Infection Biology Berlin, Germany

Barry T. Rouse

College of Veterinary Medicine, University of Tennessee Knoxville, Tennessee

David L. Sacks

Laboratory of Parasitic Diseases, National Institutes of Health Bethesda, Maryland

W A S H I N G T O N ,

D C

Front cover: Macrophage in the process of engulfing Mycobacterium tuberculosis organisms. Back cover: Left (red): Helicobacter pylori; right (blue): Neisseria gonorrhoeae attached to epithelial cells. All three photos courtesy of Volker Brinkmann and Stefan H.E. Kaufmann (Max Planck Institute for Infection Biology, Berlin, Germany).

Copyright © 2011

ASM Press American Society for Microbiology 1752 N Street, N.W. Washington, DC 20036-2904

Library of Congress Cataloging-in-Publication Data The immune response to infection / edited by Stefan H.E. Kaufmann, Barry T. Rouse, David L. Sacks. p. ; cm. Includes bibliographical references and index. ISBN 978-1-55581-514-1 (alk. paper) 1. Immune response. I. Kaufmann, S. H. E. (Stefan H. E.) II. Rouse, Barry T. III. Sacks, David Lawrence. [DNLM: 1. Adaptive Immunity—immunology. 2. Immunity, Innate—immunology. 3. Host-Pathogen Interactions—immunology. 4. Infection—immunology. QW 541 I326 2011] QR186.I445 2011 616.07'9—dc22 2010019964 All Rights Reserved Printed in the United States of America 10

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Address editorial correspondence to: ASM Press, 1752 N St., N.W., Washington, DC 20036-2904, U.S.A. Send orders to: ASM Press, P.O. Box 605, Herndon, VA 20172, U.S.A. Phone: 800-546-2416; 703-661-1593 Fax: 703-661-1501 Email: [email protected] Online: estore.asm.org

Contents

8 Regulation of Antimicrobial Immunity / 109

Contributors / ix Preface / xv

yASMINE BELKAID, SHARVAN SEHRAWAT, AND BARRy T. ROUSE

The Immune Response to Infection: Introduction / 1

STEFAN H. E. KAUFMANN, BARRy ROUSE, AND DAVID SACKS

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SECTIon I

SECTIon II

HoST DEFEnSE: GEnERAL / 5 1

Memory and Infection / 121

DAVID MASOPUST AND MARK K. SLIFKA

THE PATHoGEnS / 131

Invertebrate Innate Immune Defenses / 7

LAURE EL CHAMy, CHARLES HETRU, AND JULES HOFFMANN

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overview of Viral Pathogens / 133

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overview of Parasitic Pathogens / 143

12

overview of Bacterial Pathogens / 155

13

overview of Fungal Pathogens / 165

14

Prionoses and the Immune System / 173

JONATHAN W. yEWDELL AND JACK R. BENNINK

2 The ontogeny of the Cells of the Innate and the Adaptive Immune System / 21

RICK L. TARLETON AND EDWARD J. PEARCE

FRITz MELCHERS

3 The Evolutionary origins of the Adaptive Immune System of Jawed Vertebrates / 41

PHILIPPE J. SANSONETTI AND ANDREA PUHAR

JIM KAUFMAN

AxEL A. BRAKHAGE AND PETER F. zIPFEL

4 Host Defense (Antimicrobial) Peptides and Proteins / 57 LAURENCE MADERA, SHUHUA MA, AND ROBERT E. W. HANCOCK

JüRGEN A. RICHT AND ALAN yOUNG

5 Reactive oxygen and Reactive nitrogen Intermediates in the Immune System / 69

SECTIon III

InnATE IMMUnITY To MICRoBIAL InFECTIonS / 183

CHRISTIAN BOGDAN

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Complement in Infections / 85

15

WILHELM J. SCHWAEBLE, yOUSSIF MOHAMMED ALI, NICHOLAS J. LyNCH, AND RUSSELL WALLIS

7

Innate Immunity to Viruses / 185

AKIKO IWASAKI

16 natural Killer Cell Response against Viruses / 197

Immune Defense at Mucosal Surfaces / 97

MARIAN R. NEUTRA AND JEAN-PIERRE KRAEHENBUHL

JOSEPH C. SUN AND LEWIS L. LANIER v

vi

17

Contents

Innate Immunity against Bacteria / 209

THOMAS ARESCHOUG, ANNETTE PLüDDEMANN, AND SIAMON GORDON

18 Innate Immunity to Parasitic Infections / 225

CHRISTOPHER A. HUNTER AND ALAN SHER

SECTIon IV

ACQUIRED IMMUnITY To MICRoBIAL InFECTIonS / 237 19 Acquired Immunity against Virus Infections / 239

EVA SzOMOLANyI-TSUDA, MICHAEL A. BREHM, AND RAyMOND M. WELSH

20 Immune Responses to Persistent Viruses / 255

E. JOHN WHERRy AND PAUL KLENERMAN

21 Acquired Immunity: Acute Bacterial Infections / 269 DENNIS W. METzGER

22 Acquired Immunity: Chronic Bacterial Infections / 279

ANDREA M. COOPER AND RICHARD ROBINSON

23 Acquired Immunity: Fungal Infections / 289 LUIGINA ROMANI

24 Acquired Immunity to Intracellular Protozoa / 301 PHILLIP SCOTT AND ELEANOR M. RILEy

25 Acquired Immunity to Helminths / 313

29 Pathology and Pathogenesis of Malaria / 361

CHANAKI AMARATUNGA, TATIANA M. LOPERA-MESA, JEANETTE G. TSE, NEIDA K. MITA-MENDOzA, AND RICK M. FAIRHURST

30 Pathology and Pathogenesis of Virus Infections / 383 CARMEN BACA JONES AND MATTHIAS VON HERRATH

SECTIon VI

EVASIon AnD SUPPRESSIon oF THE AnTIMICRoBIAL HoST RESPonSE / 391 31 Viral Immune Evasion / 393

LILA FARRINGTON, GABRIELA O’NEILL, AND ANN B. HILL

32 Growing old and Immunity to Viruses / 403 JANKO NIKOLICH-ŽUGICH AND MARCIA A. BLACKMAN

33 Growing old and Immunity to Bacteria / 413 JOANNE TURNER

34 Bacterial Strategies for Survival in the Host / 425

ANNA D. TISCHLER AND JOHN D. McKINNEy

35 Suppression of Immune Responses to Protozoan Parasites / 441 DAVID L. SACKS

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Immune Evasion by Parasites / 453

JOHN M. MANSFIELD AND MARTIN OLIVIER

DAVID ARTIS AND RICK M. MAIzELS

SECTIon VII SECTIon V

PATHoLoGY AnD PATHoGEnESIS / 325

GEnETICS oF THE AnTIMICRoBIAL HoST RESPonSE / 471

26 Pathology and Pathogenesis of Bacterial Infections / 327

37 Genetics of Antibacterial Host Defenses / 473

WARWICK J. BRITTON AND BERNADETTE M. SAUNDERS

27 Helicobacter pylori: the Role of the Immune Response in Pathogenesis / 337

STEVEN M. HOLLAND

38 Immunogenetics of Host Response to Parasites in Humans / 483 JENEFER M. BLACKWELL

KAREN ROBINSON AND JOHN C. ATHERTON

28 Pathogenesis of Helminth Infections / 347

THOMAS A. WyNN AND JUDITH E. ALLEN

39 Immunogenetics of Virus Pathogenesis / 491

SEAN WILTSHIRE, DAVID I. WATKINS, EMIL SKAMENE, AND SILVIA M. VIDAL

Contents

SECTIon VIII

AUToIMMUnITY AnD CAnCER / 509 40 Viruses, Autoimmunity, and Cancer / 511 MEGHANN TEAGUE GETTS, LIES BOGAERT, W. MARTIN KAST, AND STEPHEN D. MILLER

41 The Role of Bacterial and Parasitic Infections in Chronic Inflammatory Disorders and Autoimmunity / 521 STEFAN EHLERS AND GRAHAM A. W. ROOK

42 Theileria-Induced Leukocyte Transformation: an Example of oncogene Addiction? / 537 MARIE CHAUSSEPIED AND GORDON LANGSLEy

SECTIon IX

IMMUnE InTERVEnTIon / 547 43 Systems Vaccinology: Using Functional Signatures To Design Successful Vaccines / 549 TROy D. QUEREC AND BALI PULENDRAN

44 Meeting the Challenge of Vaccine Design To Control HIV and other Difficult Viruses / 559 BARNEy S. GRAHAM AND CHRISTOPHER WALKER

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45 Immune Intervention Strategies against Tuberculosis / 571

PETER ANDERSEN AND STEFAN H. E. KAUFMANN

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Immune Intervention in Malaria / 587

CAROLE A. LONG AND FIDEL P. zAVALA

47 Targeting Components in Vector Saliva / 599

MARy ANN McDOWELL AND SHADEN KAMHAWI

SECTIon X

THE MAJoR KILLERS (CLInICS, EPIDEMIoLoGY, AnD IMMUnE PARAMETERS) / 609 48 AIDS Vaccines: the Unfolding Story / 611 STEPHEN NORLEy

49 Tuberculosis / 623

GERHARD WALzL, PAUL VAN HELDEN, AND PHILIP R. BOTHA

50 Malaria: Clinical and Epidemiological Aspects / 633 ANDREA A. BERRy, MyAING M. NyUNT, AND CHRISTOPHER V. PLOWE

51 The Epidemiology and Immunology of Influenza Viruses / 643 RAFAEL A. MEDINA, IRENE RAMOS, AND ANA FERNANDEz-SESMA

Index / 653


Contributors

yOUSSIF MOHAMMED ALI

ANDREA A. BERRy

Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom

Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201

MARCIA A. BLACKMAN

The Trudeau Institute, Saranac Lake, Ny 12983

JUDITH E. ALLEN

Institutes of Evolution, Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3JT, UK

JENEFER M. BLACKWELL

Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Subiaco, Western Australia, Australia

CHANAKI AMARATUNGA

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852

LIES BOGAERT

Departments of Molecular Microbiology & Immunology and Obstetrics & Gynecology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033. Department of Surgery and Anesthesiology of Domestic Animals, Faculty of Veterinary Medicine, University of Ghent, Merelbeke, B-9820, Belgium

PETER ANDERSEN

Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark

THOMAS ARESCHOUG

Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sölvegatan 23, 22362 Lund, Sweden

CHRISTIAN BOGDAN

Microbiology Institute Clinical Microbiology, Immunology and Hygiene, Friedrich Alexander University Erlangen, Nuremberg, and University Clinic of Erlangen, Wasserturmstraße 3/5, D-91054 Erlangen, Germany

DAVID ARTIS

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-4539

PHILIP R. BOTHA

Division of Infectious Diseases, Department of Medicine Tygerberg Academic Hospital, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa

JOHN C. ATHERTON

Nottingham Digestive Diseases Centre Biomedical Research Unit, University Hospital, Nottingham, NG7 2UH, United Kingdom

AxEL A. BRAKHAGE

CARMEN BACA JONES

Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Friedrich Schiller University Jena, Department Molecular and Applied Microbiology and Department of Infection Biology, Beutenbergstrasse 11a, 07745 Jena, Germany

yASMINE BELKAID

MICHAEL A. BREHM

Center for Type 1 Diabetes Research, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 Mucosal Immunology Unit, Laboratory of Parasitic Diseases, Division of Intramural Research, National Institute of Health, 4 Center Drive B1-28, Bethesda, MD 20892

Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655

WARWICK J. BRITTON

JACK R. BENNINK

Centenary Institute, Locked Bag No 6, Newtown, 2042; and Discipline of Medicine, Sydney Medical School, University of Sydney (D06), Sydney, 2006, NSW, Australia

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-3209

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contributors

MARIE CHAUSSEPIED

Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. Inserm, 1016, Paris, France. Laboratory of Comparative Cell Biology of Apicomplexa, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France

ANDREA M. COOPER

Trudeau Institute, Inc., 154 Algonquin Ave., Saranac Lake, Ny

STEFAN EHLERS

Microbial Inflammation Research, Research Center Borstel, Parkallee 1, D-23845 Borstel, Germany, and Molecular Inflammation Medicine, Institute of Experimental Medicine, Christian-Albrechts-University, Arnold-Heller-Str. 3, D-24105 Kiel, Germany

LAURE EL CHAMy

Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France

RICK M. FAIRHURST


LILA FARRINGTON

Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239

ANA FERNANDEz-SESMA

Department of Microbiology and the Emerging Pathogens Institute, Mount Sinai School of Medicine, New york, Ny 10029

MEGHANN TEAGUE GETTS

Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611

SIAMON GORDON

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, Ox1 3RE, United Kingdom

BARNEy S. GRAHAM

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892

ROBERT E. W. HANCOCK

Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada

CHARLES HETRU


ANN B. HILL

Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239

JULES HOFFMANN

CHRISTOPHER A. HUNTER

Department of Pathobiology School of Veterinary Medicine, University of Pennsylvania, Rm 313, Hill Pavilion, 380 South University Avenue, Philadelphia, PA 19104-4539

AKIKO IWASAKI

Department of Immunobiology, yale University School of Medicine, New Haven, CT 06520

SHADEN KAMHAWI

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, 12735 Twinbrook Parkway, Rockville, MD 20852

W. MARTIN KAST

Departments of Molecular Microbiology & Immunology and Obstetrics & Gynecology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033. Cancer Research Center of Hawaii, University of Hawaii, Honolulu, HI 96822

JIM KAUFMAN

University of Cambridge Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, Department of Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, United Kingdom

STEFAN H. E. KAUFMANN

Department of Immunology, Max Planck Institute for Infection Biology, Chapritéplatz 1, D 10117 Berlin, Germany

PAUL KLENERMAN

Nuffield Dept of Medicine and NIHR Biomedical Research Centre Programme, Peter Medawar Building, University of Oxford, Oxford Ox1 3Sy, UK

JEAN-PIERRE KRAEHENBUHL

Health Sciences eTraining (HSeT) Foundation, CH 1066 Epalinges, Switzerland

GORDON LANGSLEy

Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. Inserm, 1016, Paris, France. Laboratory of Comparative Cell Biology of Apicomplexa, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France

LEWIS L. LANIER

Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, CA 94143

CAROLE A. LONG

Malaria Immunology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852

TATIANA M. LOPERA-MESA


NICHOLAS J. LyNCH



STEVEN M. HOLLAND

SHUHUA MA

Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1684

Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada

contributors

LAURENCE MADERA

MARTIN OLIVIER

RICK M. MAIzELS

GABRIELA O’NEILL

Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada Centre for Immunity, Infection and Evolution, and Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3JT, United Kingdom

JOHN M. MANSFIELD

Department of Microbiology and Immunology, Duff Medical Building, 3775 University, McGill University, Montréal, Québec, Canada. Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239

EDWARD J. PEARCE

Department of Bacteriology, Microbial Sciences Building, 1550 Linden Drive, University of Wisconsin-Madison, Madison, WI 53716

Trudeau Institute Inc., 154 Algonquin Avenue, Saranac Lake, Ny 12983

DAVID MASOPUST

Center for Vaccine Development, Howard Hughes Medical Institute and University of Maryland School of Medicine, Baltimore, MD 21201

Department of Microbiology, Center for Immunology, University of Minnesota, 2-182 Medical Biosciences Building, 2101 6th St. SE, Minneapolis, MN 55455

MARy ANN McDOWELL

The Eck Institute for Global Health and Infectious Diseases, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556

JOHN D. McKINNEy

Global Heath Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland

RAFAEL A. MEDINA


FRITz MELCHERS

Max Planck Institute for Infection Biology, Senior Research Group on Lymphocyte Development, Charitéplatz 1, D 10117, Berlin, Germany

DENNIS W. METzGER

Center for Immunology and Microbial Disease, Albany Medical College, Albany, New york 12208

STEPHEN D. MILLER

Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611

NEIDA K. MITA-MENDOzA


MARIAN R. NEUTRA

Department of Pediatrics, Harvard Medical School, and GI Cell Biology Laboratory, Children’s Hospital, Boston, MA 02115

JANKO NIKOLICH-ŽUGICH

Department of Immunobiology and the Arizona Center on Aging, University of Arizona College of Medicine, Tucson, Az 85718

STEPHEN NORLEy

Robert Koch Institute, 13353 Berlin, Germany

MyAING M. NyUNT

Department of International Health, Global Disease Epidemiology and Control Program, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205

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CHRISTOPHER V. PLOWE

ANNETTE PLüDDEMANN

Department of Primary Health Care, University of Oxford Old Road Campus, Oxford, Ox3 7LF, UK

ANDREA PUHAR

Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, 75724 Paris Cedex 15, France

BALI PULENDRAN

Emory Vaccine Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329

TROy D. QUEREC

Emory Vaccine Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329

IRENE RAMOS


JüRGEN A. RICHT

Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, K224B Mosier Hall, Manhattan, KS 66506-5601

ELEANOR M. RILEy

Department of Infectious and Tropical Diseases, London School of Tropical Medicine and Hygiene, London, UK

RICHARD ROBINSON

Trudeau Institute, Inc., 154 Algonquin Ave., Saranac Lake, Ny

KAREN ROBINSON

Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, United Kingdom

LUIGINA ROMANI

Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy

GRAHAM A. W. ROOK

Centre for International and Medical Microbiology (CIMM), Windeyer Institute for Medical Sciences, University College London (UCL), 46 Cleveland Street, GB - London W1T 4JF, United Kingdom

xii

contributors

BARRy T. ROUSE

JEANETTE G. TSE

DAVID L. SACKS

JOANNE TURNER

Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996 Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 126, 4 Center Dr., MSC 0425, Bethesda, MD 20892-0425.

PHILIPPE J. SANSONETTI

Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, 75724 Paris Cedex 15, France

BERNADETTE M. SAUNDERS

Centenary Institute, Locked Bag No 6, Newtown, 2042; and Discipline of Medicine, Sydney Medical School, University of Sydney (D06), Sydney, 2006, NSW, Australia

WILHELM J. SCHWAEBLE


PHILLIP SCOTT

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104

SHARVAN SEHRAWAT

Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996

ALAN SHER

Laboratory of Parasitic Diseases NIAID, NIH Building 50, Room 6140, 50 South Drive, MSC-8003, Bethesda, MD 20892-8003

EMIL SKAMENE

Department of Human Genetics, The McGill Life Sciences Complex, Bellini Pavilion, Room 356, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1, Canada

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852 Center for Microbial Interface Biology, and Department of Internal Medicine, Division of Infectious Diseases, The Ohio State University, 460 West 12th Ave., Columbus, OH 43210

PAUL VAN HELDEN

DST/NRF Centre of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa

SILVIA VIDAL


MATTHIAS VON HERRATH

Center for Type 1 Diabetes Research, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037

CHRISTOPHER WALKER

Nationwide Children’s Hospital and the Departments of Pediatrics, Pathology, and Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43205

RUSSELL WALLIS


GERHARD WALzL

MARK K. SLIFKA

Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th Ave., Beaverton, OR 97006

DST/NRF Centre of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa

JOSEPH C. SUN

DAVID I. WATKINS

Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, CA 94143

Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, 6152 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706

EVA SzOMOLANyI-TSUDA

RAyMOND M. WELSH



RICK L. TARLETON

E. JOHN WHERRy

Center for Tropical & Emerging Global Diseases, Coverdell Center for Biomedical Research (Rm 310B), 500 D.W. Brooks Drive, University of Georgia, Athens, GA 30602

Department of Microbiology and Institute for Immunology, University of Pennsylvania, School of Medicine, 421 Corie Blvd., Room 312, Philadelphia, PA 19104

ANNA D. TISCHLER


Global Heath Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland

SEAN WILTSHIRE

contributors

THOMAS A. WyNN

Immunopathogenesis Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892-8003

JONATHAN W. yEWDELL

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-3209

ALAN yOUNG

Department of Veterinary Science, South Dakota State University, Box 2175, ARW168F, Brookings, SD 57007

xiii

FIDEL P. zAVALA

Department of Molecular Microbiology and Immunity, Bloomberg School of Public Health, Malaria Research Institute, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205

PETER F. zIPFEL

Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Friedrich Schiller University Jena, Department Molecular and Applied Microbiology and Department of Infection Biology, Beutenbergstrasse 11a, 07745 Jena, Germany


Preface

“Excellent textbooks and review volumes on immunology, virology, parasitology, medical microbiology, and infectious diseases abound. So what gap is this book aimed to fill?” This question was posed in the preface of the ASM Press book Immunology of Infectious Diseases (edited by Stefan H. E. Kaufmann, Alan Sher, and Rafi Ahmed), published in 2002. The explanation provided then still holds true today. Microbiology and immunology, despite their common roots, have matured as distinct disciplines, and infectious diseases are too often viewed from the perspective of either the microbe or the host. A more holistic approach was provided by that book and a second one published by ASM Press in 2004, The Innate Immune Response to Infection (edited by Stefan H. E. Kaufmann, Ruslan Medzhitov, and Siamon Gordon). These books now urgently need updating because the knowledge base in immunology, as well as with all types of infectious agents, has expanded dramatically. The present volume, The Immune Response to Infection, covers all aspects of innate and adaptive immune mechanisms and describes how they interact with pathogens of different types, resulting in either success or failure to control infection and clinical disease. This volume also emphasizes how our understanding of mechanistic events is leading the design and production of more effective prophylactic and therapeutic control measures for infectious agents. Most of the chapters here consider host-pathogen interactions in the context of the broad divisions of the microbial world—either viruses, bacteria, or parasites—

and do not confine their discussion to any individual pathogen. The exceptions are for the agents of the “big three” infectious diseases—HIV/AIDS, tuberculosis, and malaria—which account for almost one-third of human deaths from infections, as well as influenza, which is the focus of much media and public attention. We have also included chapters that consider the detrimental sequelae of infection that are an indirect result of the infectious process, such as chronic inflammation, cancer, and autoimmunity. Finally, all of the chapters emphasize the special attributes that make pathogens difficult to control, and they appraise the prospects of current and future prophylactic and therapeutic vaccines. We hope that this book, which comprises the rich variety of aspects of infection and immunity, helps to further promote the important relationship between immunology and medical microbiology. We express our deep appreciation to the editorial staff of ASM Press, in particular, Greg Payne and Ellie Tupper. We also want to thank our associates Mary Louise Grossman and Lisa Washington for their secretarial help and for their wonderful dedication. Most of all, we thank our colleagues for sacrificing so much of their valuable time to generously share their outstanding expertise with us and with the readers of this book. STEFAN H. E. KAUFMANN, BARRy T. ROUSE, AND DAVID L. SACKS

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The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC

The Immune Response to Infection: Introduction STEfAN H. E. KAUfMANN, BARRy RoUSE, AND DAvID SACKS

HISTORICAL PERSPECTIVE

immune system. from a coevolutionary perspective, these different stratagems in the struggle for life between microbes and host might seem to offer neither any clear-cut advantage. yet humans have one major advantage: our brain that can envision, design, and develop new countermeasures. In the case of infectious disease these are anti-infectives, mostly antibiotics and vaccines. from the beginning, plagues have written history (fig. 1) (Brock, 1999). However, it took until the late 19th century until we understood what causes plagues and how our body fights against them. Investigations of plagues started almost 500 years bce in ancient Greece, when Thucydides (460–396 bc) assumed contagiousness of the plague in Athens because he realized that those who recovered from disease would not fall ill again. Even though Hippocrates (460–375 bc) and his school recognized that some disease can be transmitted, their main concept viewed disease as an imbalance among the four humors: phlegm, black bile, yellow bile, and blood. Hence their contribution to our understanding of contagious diseases was minimal. Some time later, Diodorus Siculus (1st century bc) impressively described plagues at the times of Pericles and considered that gas arising from the muddy ground was responsible. Bad air is termed miasma in Greek, and a long line of investigators believed in miasma as a cause of infectious diseases. Marcus Terentius varro (116–27 bc) argued against miasma in his treatise on agriculture, where he assumed that minute animals invisible to the naked eye cause severe diseases when they are inhaled through the nose and mouth. This knowledge would have been lost during the Middle Ages were it not continued by the Islamic world. Notably, the Persian Rhazes (864–830 ad), in his treatise about smallpox and measles, precisely described differences and similarities of these two plagues. A major breakthrough, however, came with the work of Girolamo fracastoro (1478–1553 ad), from verona, Italy. In his work About the Contagious Diseases and Their Treatment, he foresaw the major characteristic features of infectious diseases, speculating that small organisms were responsible for transmission. The German Jesuit Athanasius Kircher (1602–1680 ad) was a universally educated man who was also curious about the causes of plague, and was the first to claim to have seen microbes, which cause infectious diseases. This, however, was impossible because the magnification glasses he used were insufficient. What he probably

Microbes were the first living organisms to colonize the world some 3 billion years ago. When our human ancestors entered the scene some millions of years ago, microbes had already occupied every niche on the globe. The human body was no exception to this occupation. Microbes colonized and readily exploited the outer and inner surfaces of our ancestors’ bodies; every now and then they entered into deeper tissue sites where they caused harm—something we now call infectious disease. As long as humans were nomadic and did not settle in larger communities, infectious diseases were restricted to small groups of individuals and could not spread. once humans switched to stable settlements and started to grow crops and tend animals, microbes could better spread from contaminated water, soil, and vegetable sources to animal and human, from animal to human, and from human to human. farming and animal husbandry practices began 10,000 to 20,000 years ago and provided fertile ground for insects, which served as vectors for disease transmission. In fact, we can trace several major infectious diseases of today back to these times. Currently, we witness a resurgence of plagues with the emergence of new diseases such as acquired immunodeficiency syndrome (AIDS) and new strains of influenza virus as well as the reemergence of old diseases such as tuberculosis (TB). In the struggle between microbe and human, the central strategy microbes trust on is rapid replication time paired with frequent mutation so that a few microbes surviving under catastrophic conditions can emerge and conquer a new and previously hostile niche. In contrast, humans developed along a different path based on specialization and complexity. We possess specialized organs that serve as pumps for blood circulation, the heart; as site of metabolite production and detoxification, the liver; or as site for perception and thought, the brain. The combat of infectious agents is the responsibility of a specialized organ, the Stefan H. E. Kaufmann, Max Planck Institute for Infection Biology, Department of Immunology, Charitéplatz 1, 10117 Berlin, Germany. Barry Rouse, Department of Pathobiology, 1414 W. Cumberland Ave, WLS Rm B408, University of Tennessee, Knoxville, TN 37996-0845. David Sacks, Intracellular Parasite Biology Section, Laboratory of Parasitic Diseases, NIAID, Bldg 4, Rm 126, 4 Center Dr., MSC 0425, Bethesda, MD 20892-0425.

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INTRODUCTION

FIGURE 1 Timeline of infectious disease research. The timeline illustrates major breakthroughs in the area of infection and immunity.

observed were some blood cells rather than microbes. The first step from concept to experiment was carried out by the textile merchant Antonius van Leeuwenhoek (1632–1723 ad) from Delft, The Netherlands, who, in his free time, crafted small lenses that allowed him to see real microbes for the first time (which he called “Animalcules”). Thus Leeuwenhoek could be named the father of experimental microbiology, although he had no particular interest in infectious diseases. The next issue to clarify was whether life was generated spontaneously or was derived from living organisms. This question was resolved by the Italian Lazzaro Spallanzani (1729–1799 ad) who showed that after heating, a closed flask containing broth remained free of living organisms, whereas broth in an open flask was soon recolonized by microbes, even after being heated. Louis Pasteur (1822–1895 ad) refined this

experiment by using a flask with an s-shaped neck, which prevented entry of microbes when stored, but allowed exchange of air. Even when left open, the heated broth in these flasks was not colonized. Pasteur also showed that fermentation and putrefaction were not just chemical processes but the function of microorganisms. Pasteur went one step further and opened the way for infectious disease research by showing that putrefaction was the underlying mechanism of diseases that were accompanied by pus formation. Moreover, he laid the foundation for rational vaccine design by attenuating microbes and using them for prevention or therapy of infectious diseases. He is best known for the development of two vaccines, one to prevent anthrax and one for treating rabies. The AustroHungarian Ignaz Semmelweis (1818–1865 ad) emphasized the importance of careful hand washing before diagnosing

The Immune Response to Infection: Introduction

healthy pregnant women. In hospital stations where doctors who had been working in pathology prior to diagnosing pregnant women there was a far higher mortality rate of women and infants at delivery. Semmelweis’s advice for disinfection prior to contact with the patient during delivery was not appreciated by the medical community at the time. The Scottish surgeon Joseph Lister (1827–1912 ad) was more successful in convincing his colleagues to recognize the value of wound disinfection, and, together with Semmelweis, established hygiene as a principle for the prevention of the spread of infectious diseases. The Englishman John Snow (1813–1858 ad) furthered the principles of public health for control of plagues and established the discipline of epidemiology. Robert Koch (1843–1910 ad), by revealing the etiology of TB, anthrax, and cholera, founded the golden age of medical microbiology in Berlin, Germany (Kaufmann & Winau, 2005). Within the next decades, dozens of pathogens were discovered and the etiologies of equally numerous infectious diseases were established. Robert Koch’s school also paved the way for immunology, which was established by his scholars, Emil von Behring (1854–1917 ad) and Paul Ehrlich (1854–1915 ad) (fig. 2). Behring developed the concept of passive vaccination, and Ehrlich built the concepts of antibody specificity and specific immunity in general. Pasteur’s Russian colleague Elie Metchnikoff (1845–1916 ad) described phagocytosis and phagocytes as cellular mediators of nonspecific immunity. While the immunologic concepts introduced by Ehrlich and Metchnikoff provided the scientific basis for an understanding of the mechanisms underlying

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vaccination, it was Edward Jenner (1749–1823 ad), an English country physician, who had developed the first active vaccine a century before. Jenner used smallpox with low virulence for humans to protect against a highly virulent related human pox. Medical microbiology paved the way for the emergence of immunology as a new discipline. At the same time, it hampered its development because of its focus on exogenous invaders as being exclusively responsible for pathogenesis. This was a necessary step to challenge the prevailing dogma, which considered disease exclusively as result of malfunctioning host cells. Ehrlich and Metchnikoff balanced this out. The host is not a helpless prey for foreign invaders. A highly sophisticated host defense system—the immune system—exists to combat predators. At the same time, not only microbes, but also the host contributes to pathogenesis. The concept of infectious disease as the result of a cross talk between pathogen and host had developed. Even though the Paris and Berlin schools of immunology hotly disputed their opposing theories, we now know how much they complement each other. Both host cells as first described by Metchnikoff and soluble mediators as described by Ehrlich comprise the two pillars of the immune system: Metchnikoff’s nonspecific innate immune system and Ehrlich’s specific, acquired immune system (Kaufmann, 2008). first signs for a synergy between specific and nonspecific immunity emerged when researchers, notably Hans Buchner (1850–1902 ad), Jules Bordet (1870–1961 ad), as well as Ehrlich himself described complement as a soluble factor of innate immunity, which was activated by antibodies.

FIGURE 2 The merger of innate and acquired immunity. The figure depicts the major events that led to the merger of innate and acquired immunity to a global view of the immune response.

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INTRODUCTION

Complementarities were further emphasized by the Englishman Almroth Wright (1861–1947 ad), who demonstrated the cooperation between antibodies and phagocytes in a process termed opsonization, which allowed engulfment of microbes that resist phagocytosis by means of a capsule (fig. 2). Thus the specific antibodies of Behring and Ehrlich and the nonspecific phagocytes of Metchnikoff could be fused in a more general concept of immunology. It took another half century, however, before the specific mediators of acquired cellular immunity, T lymphocytes, were discovered. This was achieved in the 1960s when J. f. A. P. Miller described the thymusderived lymphocytes. Through the work of Jim Gowans, it was already known that such lymphocytes recirculated through the body and initiated acquired immune responses. The interaction between T lymphocytes and macrophages in defense against intracellular bacteria was described by George B. Mackaness (1924–2007 ad). Communication between T cells and macrophages was facilitated by soluble mediators now called cytokines, as revealed (independently) by Barry R. Bloom and John R. David. Toward the end of the 20th century, the central importance of the innate immune system became clear when it was shown to instruct the acquired immune system and hence play a much more decisive role than originally assumed. Even though numerous researchers contributed to these advances, one of the first who foresaw the intensive cross talk between innate and acquired immunity was Charles A. Janeway (1943–2003 ad). Today, most immunologists agree that cooperation between innate and adaptive immune mechanisms is critical to the success of the immune response. Even though at its birth, immunology was viewed as our defense mechanism against pathogens, it subsequently became the target of more academic research lines focusing on the molecular mechanisms underlying antigen specificity, cell–cell communication, and intracellular signaling processes. The ease with which lymphocytes can be obtained from peripheral blood of humans or lymphoid organs of experimental animals made them preferred targets for many investigators interested in more general biologic issues. In the mid-20th century, diagnosis of infectious diseases had become routine and major intervention measures— notably vaccination to prevent and antibiotics to treat infectious disease—had become broadly available. It is no wonder that the perceived threat of infectious disease had subsided substantially (Kaufmann, 2009). How wrong we were. In fact, globalization has increased the risk of emerging and reemerging plagues, and recent years witness heightened interest in immunity against microbial pathogens. It

soon became clear that the canonical approach to vaccine development had been exhausted. Thus far, vaccines had been developed mostly by trial and error with only minor impact from immunologic knowledge. Principally, these vaccines stimulate antibodies, which fight against pathogens or neutralize their toxic products. This approach, however, is insufficient for design of novel vaccines against agents that are not controlled by antibodies alone. These include the three major killers, malaria, HIv/AIDS, and TB, as well as vaccines against pandemic flu. Here, knowledge in immunology is required for rational vaccine design, since not only antibodies but also multiple subsets of T cells need to be stimulated. Broader immune responses against a variety of antigens are required, which are also directed not only against dominant epitopes, but also against subdominant ones. This needs novel adjuvants designed on the basis of our knowledge about pathogen recognition by the innate immune system, about antigen presentation by antigen-presenting cells through the different presentation pathways, and about stimulatory and inhibitory signalling pathways that control activation and suppression of T lymphocyte populations of distinct functions. It is against this background that we have decided to publish a book on the immunology of infectious diseases, which comprises the whole spectrum of immunology as it relates to infection. It is our goal to describe the cross talk between the different kinds of infectious agents with the different host factors involved in immunity. focus is not only laid on underlying mechanisms, but also at the different sequelae of host–pathogen interactions ranging from sterile eradication of the invader to controlled chronic infection to pathologic corollas of the host–pathogen crosstalk. We also consider the pathogenesis of certain autoimmune disorders and cancers that are induced by infectious agents but then apparently become independent of the infectious process.

REFERENCES Brock, T. D. 1999. Milestones in microbiology: 1546 to 1940. ASM Press, Washington, DC. Kaufmann, S. H. 2008. Immunology’s foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat. Immunol. 9:705–712. Kaufmann, S. H. E. 2009. The new plagues: pandemics and poverty in a globalized world. Haus Publishing, London. Kaufmann, S. H. E., and F. Winau. 2005. from bacteriology to immunology: the dualism of specificity. Nat. Immunol. 6:1063–1066.

host defense: general

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1 Invertebrate Innate Immune defenses LAURE EL CHAMY, CHARLES HETRU, AND JULES HOFFMANN

IntrodUCtIon

an attempt to trace the origin of essential elements of the innate immune system.

All metazoans are able to mount an innate immune response. Whereas invertebrate species rely solely on this type of defense, gnathostome vertebrates have developed, in addition, a sophisticated adaptive immune system centered on lymphocytes that are absent from invertebrates. Importantly, these adaptive responses require strong stimulatory signals from cells of the innate immune system, and during infections in vertebrates, both arms of defenses cross talk in their fight against invading microorganisms. Current estimates are that 95% of all metazoan species are invertebrates. Consequently, they lack adaptive immune defenses, which are believed to have appeared in ancestral cartilaginous fish some 450 million years ago and are restricted to vertebrates. In this review, we will focus on invertebrate immune defenses and address the question of when and how they evolved in sponges and in recent forms of arthropods. For lack of space, we will restrict our analysis to invertebrate protostomes and will not consider here immune defenses of deuterostomes such as sea urchins and sea squirts. Recent reviews on these groups are found in Rast and Messier-Solek (2008) and Khalturin et al. (2004). Beyond the long-standing concepts that innate defenses depend on the presence of barrier epithelia, phagocytosis by blood cells, and a variety of circulating proteins, our understanding of the mechanisms of invertebrate immune defenses largely depends on studies performed on the model organism Drosophila melanogaster. Flies are easily induced by septic injuries to mount an immune response and are amenable to large scale genetic analysis to decipher the gene cascades required to mount this response. The overall picture that has evolved on the Drosophila host defense is still unique among invertebrates and we will first present its general outlines. With this platform of information in mind, we will then turn our attention to other arthropod groups and to other invertebrate phyla and provide short overviews of recent advances in the studies of their immune defenses. We will extend our review to currently available data from the simplest metazoan species, the Porifera and Cnidaria, in

a PrototYPICal Innate IMMUne resPonse: the host defense of DROSOPHILA

The Drosophila host defense comprises both humoral and cellular reactions. The hallmark of the humoral response is the challenge-induced synthesis, mainly by the fat-body cells (equivalent of the mammalian liver), of potent antimicrobial peptides (AMPs) and their secretion into the hemolymph (blood), (which is the systemic immune response). AMPs are also produced by epithelial barriers where they contribute to repel the infections, (which is the local immune response). When these barriers are breached, an additional layer of defense is provided by rapidly activated proteolytic cascades leading to coagulation and melanization (i.e., synthesis and deposition of melanin) at the injury site and around the invading microorganisms. The cellular reactions essentially correspond to the phagocytosis of invading microorganisms by professional phagocytes (the so-called plasmatocytes). Encapsulation of parasites by dedicated blood cells (the lamellocytes) can also contribute to the cellular host defense of larvae. Each of these aspects of the Drosophila host defense is presented in the following sections.

the systemic Immune response

Septic injury of Drosophila larvae and adults induces the appearance of an antimicrobial activity in the hemolymph. Seven families of antimicrobial peptides have been identified to date in Drosophila. Their immune-inducibility relies on the activation of transcription factors of the NF-kB family by two signaling cascades, referred to as the Toll and IMD (immune deficiency) pathways. Interestingly, the molecular characterization of these pathways has pointed to significant similarities with NF-kB activating pathways controlling mammalian innate defenses, namely the interleukin 1 receptor/Toll-like receptor (IL-1R/TLR) and tumor necrosis factor-receptor (TNFR) pathways (reviewed in Hoffmann, 2003). The Toll pathway is predominantly activated upon fungal and gram-positive bacterial infections, whereas the IMD pathway is mainly induced by gram-negative bacterial

Laure El Chamy, Charles Hetru, and Jules Hoffman, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France.

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challenge, although there are some exceptions to this rule. The JNK (Jun N-terminal kinase) and JAK (Janus kinase)/ STAT (signal transducers and activator of transcription) pathways are also involved in some aspects of the Drosophila immune response, as described below.

Immune effectors

Antimicrobial peptides are the best characterized Drosophila immune effectors. The Drosophila genome encodes at least 25 immune-inducible AMPs that belong to seven families (reviewed in Imler & Bulet, 2005). Although structurally diverse, these peptides are mostly small in size, cationic, and predominantly membrane-active. Whereas Drosomycins and Metchnikowins target mainly fungi, Defensin is active against gram-positive bacteria, and Attacins, Cecropins, Diptericins, and Drosocin predominantly affect gram-negative bacteria. Note that the activity spectra can overlap (e.g., Cecropins have been reported to act against fungi). The combined concentrations of the AMPs can reach up to 400 μM in the hemolymph of infected flies. Some AMPs are very stable and are still detected in the hemolymph several weeks after challenge (e.g., Drosomycins) whereas others (e.g., Cecropins) are rapidly degraded by proteolysis. In addition to the genes encoding AMPs, a large set of genes is up-regulated following microbial infection (reviewed in Lemaitre & Hoffmann, 2007). Many of these can be assigned immune functions such as: regulation of AMP gene expression, phagocytosis, melanization, and production of reactive oxygen species (ROS). Other induced genes are related to iron sequestration. A large fraction of the induced genes have as of yet no known functions. Among these is the Turandot family consisting of eight proteins secreted by the fat body under stress conditions including bacterial infections, heat-shock, or exposure to ultraviolet light. Another group of induced genes encodes the DIMs (Drosophila immune-induced molecules) family, with at least 17 members of generally small sized cystein-containing peptides that do not exhibit antimicrobial activities in conventional in vitro assays.

sensing and signaling

The recent years have seen significant progress in the understanding of the mechanisms underlying microbial detection and the selective activation of the Toll and IMD signaling pathways by various classes of invaders. Our current view is that bacteria are detected through the sensing of distinct forms of peptidoglycan (PGN), whereas fungi are detected through the sensing of b-glucans. Genetic studies have revealed that these tasks are assigned to two families of germ-line encoded receptors, referred to as pattern recognition receptors PRRs: the peptidoglycan recognition proteins (PGRPs) and the glucan binding proteins (GNBPs) (reviewed in Ferrandon et al., 2007). PGRPs are innate immune proteins highly conserved from insects to mammals. These proteins are characterized by a PGRP domain that is evolutionarily related to bacteriophage type II amidases and is implicated in the immune response by either interacting with or degrading microbial PGN. The Drosophila genome encodes 13 PGRP members. Some of these PGRPs have retained the amidase activity while other members have lost crucial amino acid residues that are essential for catalysis and serve as recognition proteins (reviewed in Charroux et al., 2009). A remarkable feature of recognition PGRPs is their ability to discriminate between gram-positive and gram-negative bacteria based on single amino acid substitution in the composition of their respective PGNs and to subsequently initiate distinct signaling pathways. PGN consists of long glycan chains of alternating N-acetylglucosamine and N-acetylmuramic acid residues that are cross-linked to each other

by peptide bridges. The majority of gram-positive bacteria are characterized by a lysine residue at the third position of the stem peptide that is replaced by a meso-diaminopimelic acid (DAP) in the PGN of gram-negative bacteria (and the gram-positive Bacillus species) (Fig. 1). To date, four members of the PGRP family have been assigned PRR functions, namely PGRP-SA and PGRP-SD which mediate Toll pathway activation upon gram-positive bacterial infections, and PGRP-LC and PGRP-LE which trigger IMD signaling upon infections with gram-negative bacteria (reviewed in Charroux et al., 2009). GNBPs, also known as b-GRPs (b-glucan recognition proteins) are related to a group of b-glucanases that are found in bacteria, fungi, plants, and invertebrates. Two domains characterize insect GNBPs: a N-terminal glucan binding domain that binds to b-(1,3)-glucans, and a C-terminal glucanase-like domain (reviewed in Royet et al., 2005). The Drosophila genome encodes three GNBP members, of which GNBP3 was shown to mediate fungal detection upstream of Toll. Unexpectedly, GNBP1 was shown to be required for the sensing of gram-positive bacteria in complex with PGRP-SA (reviewed in Ferrandon et al., 2007).

The Toll Pathway

The Toll pathway was initially discovered for its role in the control of dorsoventral patterning in the Drosophila embryo. A genetic analysis revealed more recently that many components of this pathway are also used for the control of AMP gene expression following fungal and gram-positive bacterial infections (reviewed in Hoffmann, 2003) . Toll is the eponymous member of the conserved family of Tolls and Toll-like receptors (TLRs). Its extracellular domain is characterized by multiple leucine rich repeats (LRRs) and its intracytoplasmic domain shares significant sequence similarities with the corresponding domain of the IL-1R and is referred to as TIR (Toll/IL-1R) domain (reviewed in Leulier & Lemaitre, 2008). Toll signaling in Drosophila drives the expression of immune responsive genes through the activation of two closely related NF-kB transcription factors, DIF in adults, and DIF and/or dorsal in larvae (reviewed in Hoffmann, 2003; Lemaitre & Hoffmann, 2007). Unlike mammalian TLRs, which directly bind microbial elicitors, Drosophila Toll does not act as a prototypical PRR but is activated by an endogenous ligand, the cytokine-like spaetzle. Spaetzle is a cystine-knot polypeptide with structural and functional similarities to mammalian neutrophins (Zhu et al., 2008 and references therein). In the absence of stimuli, spaetzle is present in the hemolymph as an inactive dimeric precursor. Pattern recognition involves circulating PRRs, acting upstream of Toll, which activate a proteolytic cascade leading to the processing of spaetzle to its active Toll ligand form (Fig. 2). The data available to date indicate that recognition of gram-positive bacteria occurs in a protein complex of PGRP-SA and GNBP1; the exact role of the latter is still under investigation. Interestingly, PGRP-SD is required as a third partner in this PRR complex for sensing of some gram-positive bacterial strains. As regards fungal recognition, GNBP3 is the sole PRR that has been identified to date (reviewed in Ferrandon et al., 2007). Binding of microbial ligands to the receptors described above leads, through as yet unknown mechanisms, to the activation of a proteolytic cascade in which four zymogens, ModSP (modular serine protease), grass, spirit, and SPE (spaetzle processing enzyme), have been identified (Buchon et al., 2009 and references therein). ModSP is a modular serine protease characterized by four low-density lipoprotein-receptor class A (LDLa) domains and one complement control protein (CCP) domain at its N-terminus. Genetic analysis has revealed a role for ModSP in integrating signals from the circulating PRRs

1. Invertebrate Innate Immune defenses

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FIGURE 1 Peptidoglycans as inducers of the Drosophila Toll and IMD signaling pathways. Peptidoglycan (PGN) is a glucopeptide polymer consisting of long chains of alternating N-acetylglucosamine and N-acetylmuramic acid residues connected to each other by short peptide bridges. The nature of the peptide bridge varies depending on the bacterial strains. Most gram-positive bacterial PGN carries a lysine residue at the third amino acid position in the peptide stems (Lys-type PGN), that is replaced by a diaminopimelic acid residue in most gram-negative bacteria (DAP-type PGN). In Drosophila, sensing of Lys-type PGN and DAP-type PGN is mediated by dedicated recognition PGRPs that activate the Toll and IMD pathways, respectively. Catalytic PGRPs have a zinc-dependant amidase activity (scissors). They reduce the immune stimulatory potency of PGN by removing the peptide bridges from the sugar backbone.

and connecting them to the downstream elements of the proteolytic cascade. Grass, spirit, and SPE are characterized by an inhibitory domain, called clip-domain that is only found in arthropods. SPE is the ultimate protease in this cascade and directly cleaves spaetzle to its Toll ligand form. In addition to the detection of microbial moieties a Toll-dependent immune response is also driven by the sensing of microbial virulence factors, notably, by secreted proteases. This reaction is centered on the clip-domain serine protease persephone, which was shown to be activated by secreted fungal subtilisins (Gottar, 2006). Persephone mediates activation of SPE and subsequently that of Toll. This reaction can be mimicked by the injection of purified bacterial proteases, hence the proposal that persephone is a sensor of danger signals (El Chamy, 2008). In agreement with this hypothesis, a recent analysis has revealed that, in the absence of any infection, tracheal melanization triggers the systemic activation of the Toll pathway in a persephone-dependent manner. The current model proposes that a host factor deriv-

ing from the melanization reaction diffuses from the tracheal system into the hemolymph where it triggers persephone activation, further driving the systemic Toll response in the fat body (Tang et al., 2008). Activation of the Toll receptor triggers an intracellular signaling cascade, which has partial similarities with the TLR and IL-1R signaling pathways (Color plate 1 a and b) (reviewed in Hoffmann, 2003; Ferrandon et al., 2007). This process involves the assembly of a multivalent complex, including death-domain-containing proteins around the intracellular TIR domain. In Drosophila, this complex comprises two adaptor molecules: a homologue of myeloid differentiation factor 88 (MyD88), which, in addition to the death domain, has a TIR domain similar to that of Toll and tube, which has a bivalent death domain. Pelle, the third partner, is a member of the IL-1R associated kinase (IRAK) family of serine-threonine kinases. The end result of Toll signaling is the dissociation of the NF-kB transcription factors from the ankyrin repeat inhibitor protein cactus, a homologue of mammalian inhibitor

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FIGURE 2 Activation of the Drosophila Toll pathway during fungal and gram-positive bacterial infections. The Toll receptor is activated by a proteolytically processed form of the cytokine-like polypeptide spaetzle (present as a dimer in the hemolymph). Microbial cell wall components (right panel) interact with circulating receptors (fungal b-glucans with GNBP3; gram-positive bacterial peptidoglycan (PGN) with a PGRP-SA/GNBP1 complex). This recognition activates, via a still unidentified mechanism, a proteolytic cascade including the zymogen modular serine protease (ModSP) and the serine protease grass, leading to the activation of the spaetzle processing enzyme (SPE), which cleaves pro-spaetzle into its active Toll-ligand form. Alternatively, microbial secreted proteases (left panel) can activate the circulating zymogen persephone, which, directly or indirectly (still unknown), activates SPE to cleave pro-spaetzle.

NF-kB (IkB). This process involves signal-dependent phosphorylation of cactus and its subsequent degradation. By analogy with mammals, it was suggested that kinases related to mitogen activated protein kinase kinase kinase (MAPKKK) and to IkB kinase (IKK) families might be involved in this process. However, such molecules have not been assigned a function in the Toll pathway in Drosophila and the identity of the cactus kinase has remained elusive so far. Liberated DIF and/or dorsal translocate to the nucleus and trigger the expression of hundreds of genes including those encoding some of the AMPs, notably the antifungal drosomycin, which is often used as a read-out of Toll pathway activation. Dorsal and DIF seem to require additional modifications for full biological activity. A protein cassette, which includes dTRAF2 (Drosophila homolog of TNFR-associated factor 2), aPKC (atypical protein

kinase C), and the scaffolding protein Ref2(P) (homologue of mammalian p62), has been suggested to participate in this process. Toll signaling shows slow kinetics, as revealed by transcriptional profiling of AMP gene expression, with maximum peaks at 24 to 48 hours following gram-positive bacterial and fungal infections, respectively. Negative regulators of this pathway have not been investigated in detail. However, following septic injury, the expression of the gene encoding cactus was shown to be up-regulated in a Toll-dependent manner, thus pointing to a negative feedback loop on Toll signaling. In addition to Toll, the Drosophila genome encodes 8 Toll receptors (18 wheeler, Toll 3 to 9). An immune function has so far only been assigned to Toll. Toll 5 and Toll 9 were also proposed to induce AMP gene expression based on


transfection assays in hemocyte-derived Drosophila cell lines but the in vivo significance of these results is not clear at this stage. Conversely, all Drosophila Toll members seem to be required for developmental processes as suggested by their expression patterns during embryogenesis and metamorphosis. These observations were confirmed by genetic analysis for Toll 2 and Toll 8 mutants (reviewed in Leulier & Lemaitre, 2008). This contrasts with the situation in mammals where all TLRs are devoted to immune detection (reviewed in Kawai & Akira, 2009). Further, Drosophila Tolls share sequence characteristics both in the LRR ectodomain and the intracellular TIR domain that distinguish them from mammalian TLRs. Whereas mammalian TLRs (and all identified vertebrate TLRs) contain a single cystein cluster flanking the C-terminal end of the extracellular LRR, Drosophila Tolls contain multiple cystein clusters including two or more of the characteristic C-terminal cluster in addition to a N-terminal cluster. The only exception is Toll 9, which has a single cystein cluster and is more closely related to mammalian TLRs (Fig. 3).

The IMD Pathway

The IMD pathway controls the activation of the third Drosophila NF-kB transcription factor, relish (reviewed in Lemaitre & Hoffmann, 2007). The IMD gene encodes an intracellular death-domain-containing protein with significant similarities to mammalian RIP1 (receptor interacting protein 1), which is associated with the tumor necrosis factor-receptor 1 (TNF1R). As indicated above, the IMD pathway shows striking similarities with the intracellular signaling cascade activated downstream of the TNFR in mammals (reviewed in Hoffmann, 2003; Ferrandon et al., 2007) (Color plate 2 a and b).

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The IMD pathway is activated through the direct interaction of microbial ligands with the transmembrane receptor PGRP-LC (reviewed in Ferrandon et al., 2007; Charroux et al., 2009). The PGRP-LC receptor displays three distinct isoforms, PGRP-LCa, PGRP-LCx, and PGRP-LCy, resulting from alternative splicing of its transcript. These receptors share identical intracellular domains but present different PGRP exodomains. Our current understanding is that IMD pathway activation is driven by the association of at least two PGRP molecules: PGRP-LCx homodimers mediate the recognition of polymeric DAP-type PGN and PGRP-LCa/ LCx heterodimers sense monomeric PGN. In addition, a secreted form of the PGRP-LE protein, consisting of its PGRP domain, is proposed to enhance the PGRP-LC receptor activity by binding to PGN in circulation and bringing it into close proximity of the cell surface. In the malpighian tubules, the full-length, non-secreted form of PGRP-LE can function as an intracellular receptor and trigger the IMD pathway in a PGRP-LC independent way (reviewed in Charroux et al., 2009; Ferrandon et al., 2007). Upon stimulation of PGRP-LC, the IMD protein is recruited to the intracellular domain of this receptor with which it interacts. The N-terminal domain of PGRP-LC (and that of PGRP-LE) contains a core motif named RHIM domain (for its homology to the RIP homotypic interaction motif) which is crucial for signaling, but not for PGRP-LC and IMD interaction. Thus, signaling via the RHIM domain seems to require a so far unidentified third partner of the PGRP-LC receptor and the IMD adaptor protein. A signaling complex is further established around IMD. FADD, a homologue of mammalian Fas-associated death domain, interacts with IMD via its death domain

FIGURE 3 Characteristic structural domains of Drosophila Toll and Toll9 and of mammalian TLR2.

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and with the caspase-8 DREDD via its death effector domain (DED). This complex triggers the activation of the MAP3K, TAK1 (transforming growth factor [TGF]-b activated kinase 1), which in turn activates the IKK signalosome. The latter protein complex includes kenny, a regulatory subunit homologous to IKKg/nemo and IRD5, a catalytic subunit homologous of IKKb, which phosphorylates relish on specific serine residues. The precise molecular mechanisms leading to the activation of TAK1 and of the IKK signaling complex remain unclear. However, convincing evidence points to a K63-linked polyubiquitination process involving the ubiquitin conjugating enzyme complex (including the Drosophila homologues of Uev1a and Ubc13), the RING-finger domain protein DIAP2 (Drosophila inhibitor of apoptosis protein 2) and the adaptor protein TAB2 (TAK1 binding protein 2). In contrast to dorsal and DIF, relish carries its own IkB-like inhibitory sequences in the form of several ankyrin repeats located at its C-terminal end which is cleaved, following relish phosphorylation, most probably by the caspase-8 DREDD. The N-terminal fragment bearing the Rel-homology domain translocates to the nucleus and drives the expression of hundreds of genes including diptericin. Although the IKK complex is required for relish cleavage, this process does not depend on its phosphorylation. Instead, phosphorylation is critical for the adequate transcriptional activation of relish target genes (Erturk-Hasdemir, 2009). In addition, a newly identified nuclear protein, akirin is required for the control of relish transcriptional activity. Homology search has led to unravel two akirin homologs in mammals. One of these exerts similar functions in NF-kB -dependent gene expression downstream of TNFR, TLR, and IL-1R (Goto, 2008). It is currently understood that the differential expression profiles of IMD- and Toll-immune induced genes are related to distinct sequence codes of kB-response elements in the promoter regions of the target genes responsive to the various NF-kB family members of Drosophila (for references see recent reviews by Lemaitre & Hoffmann, 2007 and Ferrandon et al., 2007). In contrast to Toll, IMD pathway-dependent signaling is rapid and short-lived. Maximum levels of AMP gene expression are detected around six hours following infection. Negative regulation of the IMD pathway occurs at different levels (reviewed in Aggarwal & Silverman, 2008). First, secreted catalytic members of the PGRP family, namely PGRP-SC and PGRP-LB degrade DAP-type PGN, thus reducing its immune stimulatory potency. In addition, the transmembrane PGRP-LF prevents the inappropriate activation of the IMD pathway by sequestering circulating PGN (reviewed in Charroux et al., 2009). Further, a homologue of the Fas-associating factor 1, caspar, interferes with the DREDD-dependent cleavage of relish. Finally, an intracellular protein, pirk (also designated pims and rudra), is induced in a relish-dependent manner following gram-negative bacterial infections and interferes with the activation of the IMD pathway by interacting with PGRP-LC and IMD (Aggarwal et al., 2008; Lhocine, 2008; Kleino et al., 2008).

The JNK Pathway

The JNK signaling pathway is involved in early developmental processes of the Drosophila embryo where it controls dorsal closure. It is also required for wound healing in adults. Microarray analysis of Drosophila hemocyte-derived cell lines and adult flies indicated an immune induction of the JNK pathway through the upstream components of the IMD signaling cascade (reviewed in Lemaitre & Hoffmann, 2007). Thus, similar to TNFR signaling in mammals, the IMD pathway in Drosophila bifurcates downstream of TAK1,

activating both IKK and JNK signaling. The precise role of the JNK pathway in the Drosophila host defense is not yet firmly established, as genetic analyses are hampered by the early embryonic lethality of JNK pathway null mutants. The transcriptional program triggered by this MAPK signaling cascade is enriched for genes related to cytoskeleton remodeling. These findings are consistent with the role of the JNK pathway in controlling epithelial sheet movement and tissue repair following septic injury and may also be linked to hemocyte activation (all references are included in a recent review by Lemaitre & Hoffmann, 2007). JNK signaling also promotes pro-apoptotic gene expression, an observation that correlates with early lethality of transgenic flies overexpressing IMD and developmental defects associated with the reduction of PGRP-LF expression levels (Georgel, 2001; Maillet et al., 2008). JNK signaling mediates immediate but very short-lived responses (up to 3 hours) following septic injury. Some observations point to negative feedbacks between the IMD/relish and IMD/JNK branches (Aggarwal & Silverman, 2008).

The JAK/STAT Pathway

The JAK/STAT pathway was originally identified for its role in embryonic segmentation and was later shown to control other developmental processes including larval hematopoiesis (reviewed in Agaisse & Perrimon, 2004). The four main components of this pathway in Drosophila are: the cytokine-like ligand Unpaired (Upd), the receptor Domeless (a member of the type-I cytokine receptor family), the Janus Kinase Hopscotch, and the STAT92E transcription factor. Septic injury triggers a transient activation of the JAK/STAT pathway with maximum levels reached 6 hours following infection. The induced genes encode, among others, secreted proteins such as TEP (thiol ester-containing proteins) and Turandot (Tot) family members. TEPs show significant similarities to members of the complement C3/a2macroglobulin family and could function as opsonins as was shown for Anopheles gambiae TEP1 (reviewed in Blandin et al., 2008). The functional relevance of JAK/STAT pathway activation upon septic injury remains unclear, as JAK/STAT-deficient flies are resistant to bacterial infections. An interesting new finding has linked JAK/STAT pathway activation to the resistance to viral infections in Drosophila (reviewed in Kemp & Imler, 2009). Genome-wide expression profile analysis of flies infected by DCV (Drosophila C Virus) has revealed the activation of a dedicated transcriptional program, which is different from that induced following bacterial and fungal infections. Part of the induced genes, such as vir-1, were shown to be JAK/STAT dependent. In addition, Hopscotch mutant flies exhibited accelerated lethality following viral infection, which correlated with an increase in viral loads. These results suggest that the antiviral immune response in Drosophila triggers the production of a cytokine that activates the receptor Domeless and JAK/STAT-dependent induction of a set of genes to control the viral load in infected flies. In mammals, the innate antiviral defense is characterized by the production of interferons (IFN absent from the Drosophila genome), which trigger JAK/STAT pathway transcriptional activation of IFN-stimulated genes (ISGs). Thus, these observations in Drosophila point to an evolutionarily conserved role of the JAK/STAT pathway in the control of viral infections from insects to mammals. It is important to note here that, in addition to the inducible antiviral response, an intrinsic response based on RNA interference (RNAi) also plays a crucial role in the control of viral infection in Drosophila (reviewed in Kemp & Imler, 2009) (Fig. 4). This is reminiscent of the findings in plants and other invertebrates (Caenorhabditis elegans), suggesting that RNAi represents an ancestral response to


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FIGURE 4 The Antiviral Response in Drosophila. RNA interference and inducible gene reprograming are two major arms of the Drosophila antiviral defense. Viral nucleic acids are recognized by the RNase III enzyme dicer 2, which activates the small interfering RNA (siRNA) pathway leading to the degradation of viral RNA. In addition, dicer 2 can trigger the inducible expression of a variety of genes via a yet unidentified signaling pathway. Among the induced genes is that encoding the vago cystein-rich polypeptide, which is involved in the control of the viral load. Further, a cytokine-mediated response activated upon viral infection leads to the expression of genes via the activation of the JAK/STAT pathway in neighboring cells. The mechanism controlling this antiviral-inducible response is so far unknown.

viral infection. Viral double-stranded RNA is recognized by the RNAse III enzyme dicer 2, which drives the degradation of viral RNA through the small interfering RNA (siRNA) pathway. Intriguingly, dicer 2 (but no other mediators of the siRNA pathway) is also required for the expression of some DCV-inducible genes, such as vago, in the infected cells. Dicer 2 contains an amino-terminal DExD/H box helicase domain, which is important for the inducible vago expression. This domain is similar and is phylogenetically related to the helicase domain of mammalian RIG-I-like receptors, which mediate the sensing of viral nucleic acids in the cytosol and the induction of interferon gene expression (reviewed in Kawai & Akira, 2009). Hence, although Drosophila lacks interferon homologues and the mechanisms of antiviral responses are different between Drosophila and mammals, many similarities can be drawn on sensing (requirement of intracellular DExD/H box helicases) and signaling (cytokine production and JAK/STAT signaling) during viral infection in these evolutionarily distant organisms.

epithelial defense reactions

Epithelial barriers constitute critical interfaces between the host and microorganisms of the environment. Antimicrobial defense reactions deployed at the level of these epithelial layers provide a first tier of defense counteracting microbial invaders. In Drosophila, various spectra of AMPs have been reported in the epidermis, the reproductive system, the respiratory, and the digestive tracts. Whereas some AMPs are constitutively expressed (such as Drosomycin in the salivary glands and Cecropin in the ejaculatory duct), others are induced upon infection, and by contrast to the

systemic response, their regulation is solely dependent on the IMD pathway (including that of Drosomycin in the trachea and of Diptericin in the gut). The development of appropriate food-borne infection models in Drosophila has now uncovered the central role of locally synthesized AMPs in the gut for the survival of pathogenic infections such as Serratia marcescens or Pseudomonas entomophila. Recognition of ingested DAP-type PGN is mediated by the PGRP-LC receptor, triggering the local activation of the IMD pathway, and immune reactivity is attenuated by secreted catalytic PGRPs in the gut lumen, which degrade bacterial PGN into nonstimulatory molecules. It is proposed that this modulation of the IMD pathway protects the gut flora from excessive AMP production. Furthermore, in the absence of gut infection, a selective repression of NF-kB-dependent AMPs is provided by the homeobox gene caudal (Ryu, 2008). In addition to AMPs, ROS are rapidly produced following microbial ingestion and critically contribute to the bactericidal reactions in the gut. ROS production is mediated by dual oxidase (DUOX), a member of the conserved family of NADPH oxidases. DUOX enzymatic activity is Ca2+-dependent and is regulated by the Gaq-Phospholipase C-b (PLC-b) pathway mediating the mobilization of intracellular Ca2+. The identity of the receptor acting upstream of this pathway and the mechanism triggering this oxidative reaction remain unsolved (Ha, 2009). Excessive ROS produced following microbial infection are eliminated by a secreted antioxidant enzyme, the immune regulated catalase (IRC), thereby protecting the host from the deleterious effects of the oxidative burst. (All references for epithelial defense reactions in Drosophila are

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found in recent reviews by Lemaitre & Hoffmann, 2007 and Ferrandon et al., 2007.)

Melanization

Melanization is a particular feature of invertebrate immune reactions that is immediately activated at the site of cuticular wounding and around invading microorganisms. It relies on the activation of phenoloxidase (PO), a key enzyme that catalyzes the conversion of phenols to quinones, which polymerize nonenzymatically to form melanin. Melanization is generally assumed to contribute to the arthropod defense by facilitating wound healing, sequestrating microorganisms and preventing or retarding their growth, and by the production of toxic intermediates that can kill microbial intruders (for reference see Cerenius & Soderhall, 2004).

Cellular responses Drosophila Blood Cell lineages and functions

Compared to the complexity of blood cell lineages in mammals, the picture is relatively simple in Drosophila as there are no equivalents to lymphoid cells and as hemocyte lineages are restricted to three cell types: plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes represent the predominant population of embryonic and larval hemocytes (95%) and are the unique blood cells found in adults. They are professional phagocytes, which are in charge of the uptake and clearance of both apoptotic cells and microorganisms. Plasmatocytes also contribute to the humoral response by secreting AMPs and, as previously mentioned, they are required for the activation of the systemic response of adults exposed to stress conditions (i.e., JAK/STAT pathway activation). Crystal cells and lamellocytes have no mammalian counterparts and are both involved in defense reactions that are restricted to invertebrates (i.e., melanization for crystal cells and encapsulation for lamellocytes) (reviewed in Meister, 2004).

Phagocytosis

Several studies have revealed the importance of the cellular arm of defense in Drosophila. To give but one example: mutant larvae devoid of hemocytes are filled with microorganisms and cannot reach the adult stage unless reared on germ-free media. Further, adult flies deprived of hemocytes by targeted cell death of hemocyte lineages, are highly susceptible to some bacterial and fungal infections, and this lethality correlates with higher bacterial loads (Charroux & Royet, 2009; Defaye et al., 2009). Phagocytosis is a central theme in innate immunity and is conserved in all metazoans. High-throughput RNAi screens in the embryonic hemocyte-derived S2 cells have deciphered the phagocytic machinery and have identified multiple components of the basic mechanisms of phagocytosis, namely regulators of vesicle transport and actin cytoskeleton remodeling, which are preserved in other animal species. A recent proteomic analysis identified approximately 600 proteins potentially associated with the Drosophila S2 cell phagosome. Of the identified proteins, many are evolutionarily conserved and 70% have mammalian orthologues. By combining several approaches, this study generated a detailed model of the phagosome, which is also valid in mammalian cells. Finally, studies performed in S2 cells and other in vivo and ex vivo analysis using different microorganisms identified diverse phagocytic receptors, some of which have mammalian homologues required for the phagocytic process. (All references are found in a recent review on phagocytosis by Stuart & Ezekowitz, 2008.) These receptors belong to three families

of evolutionarily conserved proteins: scavenger receptors, EGF-like repeat containing receptors, and immunoglobulin repeat containing proteins. Other molecules, including complement-like opsonins are proposed to promote the phagocytic uptake of microorganisms.

Scavenger Receptors

Scavenger receptors represent a large family of structurally heterogeneous receptors, which bind to and internalize a broad array of endogenous molecules and microbial ligands with polyanionic character. Three members of the Drosophila scavenger receptor family were shown to be involved in phagocytosis: croquemort and peste are homologous to mammalian scavenger receptors, and dSR-CI belongs to an insect-specific family of scavenger receptors. Croquemort is required for the phagocytosis of apoptotic cells (in the Drosophila embryo) and is also involved in the phagocytosis of bacteria. In cell culture experiments, peste was shown to be required for the entry of intracellular Mycobacteria and Listeria. Finally, dSR-CI contributes to the phagocytic uptake of both gram-positive and gram-negative bacteria in Drosophila S2 cells.

Receptors Containing Multiple EGF-like Repeats

Receptors containing multiple EGF-like repeats are found in diverse species, including humans, and are emerging as a new family of phagocytic receptors. The best characterized member in Drosophila is eater, a type I transmembrane receptor with 32 typical EGF-like repeats in its extracellular domain. This receptor has a broad reactivity, like scavenger receptors, and directly binds to microbial surfaces. Eater seems to be the major phagocytic receptor in Drosophila, as silencing of its transcript dramatically decreases the phagocytic index of Drosophila S2 cells. Furthermore, eater mutant flies are highly susceptible to infections with both gram-positive and gram-negative bacteria. Another member of this family of EGF-like repeat receptors, nimrod C1, is involved in the uptake of Staphylococcus aureus and may participate to the phagocytosis of Escherichia coli. Finally, draper contains 16 EGF-like repeats and is required for the phagocytosis of apoptotic cells. Interestingly, this receptor contains an ITAM (immunoreceptor tyrosine-based activation motif) in its intracytoplasmic domain.

Immunoglobulin-like Repeat Proteins

Another member of Drosophila phagocytic receptors, Dscam (Down syndrome cell adhesion molecule), belongs to the immunoglobulin superfamily. Dscam is a single pass transmembrane receptor characterized by 10 immunoglobulinlike domains and 6 fibronectin type III domains in its extracellular part. This receptor was identified for its essential functions in neuron wiring and is also expressed in hemocytes. The dscam gene comprises a cluster of variable exons flanked by constant exons which can theoretically generate, by alternative splicing, a large number of distinct protein products. Silencing of the dscam transcript in hemocytes decreases the phagocytic uptake of E. coli by these cells. In addition, some recombinant isoforms of Dscam directly bind to E. coli. Secreted isoforms of Dscam are detected in Drosophila hemolymph and a role of these isoforms in opsonization of pathogens has also been proposed.

Complement-like Opsonins

Other molecules proposed to mediate phagocytosis in Drosophila comprise the secreted family of TEPs. Based on their homology to the complement C3/a2-macroglobulin superfamily, these molecules were anticipated to function either as opsonins to promote phagocytosis and/or as


protease inhibitors. Some data so far support the opsonin function, namely from the studies performed on Anopheles gambiae TEP1 (reviewed in Blandin et al., 2008). The Drosophila genome encodes four expressed TEPs (TEP I to IV) and a closely related gene, Mcr (macroglobulin complement related), which encodes a protein lacking the characteristic thiol ester motif of TEPs. Based on RNAi-mediated gene silencing in Drosophila S2 cells, TEP II and TEP III are proposed to mediate phagocytosis of E. coli and S. aureus respectively, whereas Mcr is proposed to mediate binding and internalization of the yeast Candida albicans. Confirmation of TEP functions during phagocytosis awaits further in vivo studies.

CoMParatIVe analYsIs of the Innate IMMUne sYsteM Insects other than drosophila

Most of the data obtained from the analysis of gene families related to the immune system in other insect species are compatible with the picture that we have described for Drosophila melanogaster. This is namely the case for the two dipteran species, Anopheles gambiae and Aedes aegypti, the hymenopteran Apis mellifera, the lepidopteran, Bombyx mori, and the coleopteran, Tribolium castaneum (Christophides et al., 2004; Waterhouse, 2007; Evans et al., 2006; Tanaka, 2008; Zou et al., 2007). Phylogenetic analysis of the insect immune repertoires (including those of twelve additional Drosophila species [Sackton et al., 2007]) has revealed dynamic changes of genes related to recognition and effector functions, probably as a result of the selective pressure imposed by distinct pathogens encountered by each insect species. These include PRRs (PGRPs and GNBPs) as well as phagocytic receptors (i.e., scavenger receptors) and opsonins (TEPs). With regard to the effector molecules, novelties are noted among insect orders mainly with the emergence of lineage-specific AMPs such as Drosomycin in Drosophila, Gambicin in mosquitoes, and Apisimin and others in bees (Christophides, 2002; Evans et al., 2006; Sackton et al., 2007). In contrast, intracellular signaling mediators are highly conserved in all studied insects. Notably, a nearly complete tool kit of both Toll and IMD pathways has been identified in all analyzed genomes. Some experimental data support the functional conservation of these pathways for immune defenses in the respective insect species. Mosquitoes lack the NF-kB transcription factor dif and rely on a dorsal orthologue, REL1, and on a relish orthologue, REL2. These mosquito REL factors have been reported to control the induced expression of AMPs and the resistance to bacterial infections (Shin et al., 2005; Bian, 2005; Antonova et al., 2009; Meister, 2005). In Anopheles gambiae, REL1 and REL2 are also involved in the resistance to the rodent malaria parasite Plasmodium berghei by controlling the basal expression of the major antiparasitic factors (Blandin et al., 2008). Of the five Toll receptors and the three spaetzle ligands encoded in the genome of the yellow fever mosquito, Aedes aegypti, a role for Toll5A and its putative ligand spaetzle1C is documented in the defense against the entomopathogenic fungus Beauveria bassiana. This receptor ligand complex triggers the activation of the REL1 transcription factor regulating the antifungal immune response (Shin et al., 2006; Shin et al., 2005; Bian, 2005). The Toll pathway, together with the JAK/STAT pathway, has also been linked to the transcriptional program triggered by Dengue virus infection in Aedes aegypti and a contribution of the Toll pathway in the defense against Dengue virus in this species has also been proposed (Xi et al., 2008).

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In the silkworm Bombyx mori and the beetle Tenebrio molitor, the expression of some AMPs is up-regulated following the injection of a maturated form of spaetzle orthologues (Wang, 2007; Roh et al., 2009). Due to the lack of appropriate genetic tools, functional data for immune-related genes are restrained in the lepidopteran and coleopteran species, as compared to Drosophila. However, these large insects have provided valuable biochemical tools for the investigation of pathogen recognition processes and the characterization of the proteolytic cascades subsequently activated downstream of PRRs (i.e., melanization and the processing of spaetzle orthologues) (reviewed in Iwanaga & Lee, 2005; Roh et al., 2009). Recently, the components of the extracellular pathway leading to the processing of spaetzle were purified from the hemolymph of the beetle Tenebrio molitor. Biochemical assays have allowed the in vitro reconstruction of the proteolytic cascade activated downstream of TmGNBP3, TmPGRP-SA, and TmGNBP1 (Roh et al., 2009). This work led to the identification of the apical protease, DmModSP, acting downstream of PRRs for the activation of the Toll pathway in Drosophila (Buchon et al., 2009).

horseshoe Crab

The horseshoe crab is often referred to as a “living fossil” and first appeared more than 500 million years ago. Its host defense largely relies on granular hemocytes, which constitute 99% of the circulating blood cells. These hemocytes contain secretory granules that store the major elements of the horseshoe crab’s defense arsenal. They include AMPs, serine protease zymogens, the clotting protein coagulogen and several lectins (reviewed in Iwanaga & Lee, 2005). The innate immune system of this chelicerate arthropod recognizes invading pathogens through a combinatorial system involving several lectins with distinct affinities for carbohydrates exposed on the surface of pathogens. Another recognition molecule playing a key role at the frontline of the horseshoe crab antimicrobial defense is the serine protease zymogen Factor C. This factor is mainly found in the hemocyte granules, but is also detected at the hemocyte surface. Factor C is highly sensitive to gram-negative bacterial lipopolysaccharide (LPS) and is autocatalytically activated upon stimulation by this microbial elicitor. This reaction leads to the secretion of the defense molecules from granular hemocytes by exocytosis (reviewed in Kurata et al., 2006). Of the released molecules, Factor C and another serine protease zymogen, factor G, which is responsive to b-(1-3)-glucan, trigger a two-step proteolytic cascade leading to hemolymph coagulation. This reaction consists in the conversion of coagulogen into an insoluble protein, coagulin. Hemolymph clotting is believed to contribute to the animal’s defense by preventing blood leakage and by trapping the pathogen. The immobilized microorganisms can subsequently be recognized by the large number of plasma and hemocyte-delivered lectins and killed by AMPs. Phagocytosis also contributes to the horseshoe crab defense reactions. Homologs of vertebrate complement C3 and C2/ Bf were reported from the horseshoe crab Carcinoscorpius rotundicauda and complement mediated phagocytosis of bacteria by hemocytes was observed (Zhu et al., 2005). These findings imply an early origin of core elements of the complement system. Further evidence for an archaic role of NF-kB signaling cascades in the antimicrobial defense is also provided from the study of this living fossil. Two NF-kB transcription factors, CrNF-kB and CrRelish, and an IkB homologue, CrIkB, have been cloned in C. rotundicauda. CrNF-kB belongs to the dorsal-like group of transcription factors and interacts with CrIkB, which inhibits its nuclear translocation. Bacterial

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infection causes rapid degradation of CrIkB and subsequent release of CrNF-kB, which activates the expression of immune-related genes such as Factor C (Wang, 2006). Experimental data support the conservation of the CrRelish function and point to its up-regulation following bacterial infection, suggesting an involvement of CrRelish in antibacterial defense (Fan, 2008). A TRAF homolog has also been reported from C. rotundicauda (Wang, 2006). How these signaling cascades are activated is not yet established. A Toll receptor has been identified in the Japanese horseshoe crab Tachypleus tridentatus, called tToll, and is closely related to Drosophila Toll. Its biological function has not been investigated so far and its ligand has not been defined. The expression pattern of tToll does not suggest an immune function (reviewed in Inamori et al., 2004).

Mollusks and annelids

Investigations of the immune reactions of several Annelid and Mollusk species have revealed a potent cellular arm of defense, involving phagocytosis, encapsulation, and production of lytic activities often referred to as natural killer-like (NK) activities. These cellular defenses are combined with humoral reactions involving lysozymes and AMPs (reviewed in Salzet et al., 2006; Cheng-Hua et al., 2009). These reactions are mostly inducible. At present, the mechanisms controlling these responses are still poorly defined. In Mollusks, recognition of nonself relies in particular on lectins. Of special interest is the family of fibrinogenrelated proteins (FREPs) identified from the gastropod Biomphalaria glabrata, an intermediate host of the human parasite Schistosoma mansoni. FREPs were first described as plasma proteins that are induced following trematode infection. They bind to trematode sporocysts and precipitate glucan-bearing molecules released by the parasite. FREPs consist of one or two amino-terminal immunoglobulin superfamily (IgSF) domains and a carboxyterminal fibrinogen domain. Interestingly, for one subfamily, FREP3, it was observed that the IgSF1 is diversified at the genomic level at higher rates than those recorded for control genes. All sequence variants were found to derive from a small set of source sequences by point mutation and recombinatorial processes. Importantly, diverse FREP3 transcripts were also produced. Loker and colleagues, who discovered this phenomenon, have hypothesized that snails have evolved a mechanism capable of diversifying molecules involved in recognition during innate defenses (Zhang et al., 2004). Further, members of the PGRP and GNBP families have also been identified in snails and are proposed to contribute to the microbial detection in these species (reviewed in Bayne, 2009). In the context of immune signaling in Mollusks, genome-based approaches identified several components of NF-kB signaling pathways in various species. In the squid Euprymna scolop, several ESTs (expressed sequence tags) encoding homologues of proteins associated with putative Toll and IMD pathways were identified. These include a Toll receptor; four PGRPs; orthologues of several intracellular signaling mediators including IRAK, TRAF, and the IKK complex; an IkB-like protein; and two NF-kB-like transcription factors (Goodson, 2005). Evidence for the presence of NF-kB signaling pathways have also been reported from other Mollusk species, including an NF-kB transcription factor from the marine gastropod Haliotis diversicolor supertexta, a Toll receptor and a MyD88 homologue in the marine bivalve Chlamys farreri, and orthologues of NF-kB and IKKb from the pacific oyster Crassostrea gigas (Jiang & Wu, 2007; Qiu et al., 2007; Escoubas, 1999; Qiu, 2007; Montagnani et al., 2004).

In Annelids, the molecular mechanisms underlying the antimicrobial defenses are, as yet, poorly understood. The analysis of genome sequence reads of the leech Helobdella robusta and the polychaete Capitella capitata have identified homologues of Toll receptors in both species (Davidson et al., 2008). Surprisingly, the family of TLRs is largely expanded in Capitella with at least 105 members inferred from this analysis. The majority of these receptors are structurally related to vertebrate TLRs (i.e., having a membrane proximal single cystein cluster in their LRR ectodomain). In contrast, of the 16 Toll homologues identified in Helobdella, most show multiple cystein-rich clusters in their LRR ectodomain, as is the case for most insect Toll receptors. Mediators of the NF-kB signaling cascades have also been identified in both species, notably, two dorsal-like transcription factors and most mediators of the Toll signaling (but not yet of the IMD) pathway.

orIgIn of the Innate IMMUne sYsteM: the Innate IMMUne rePertoIre of CnIdarIa and PorIfera

The phylum Cnidaria includes aquatic species such as sea anemones, corals, and jellyfish. These animals provide crucial insights into the early origin of metazoans. The genome sequence is now available for two Cnidarian species: the sea anemone Nematostella vectensis and the fresh water polyp Hydra magnipapillata. Amazingly, analysis of these databases uncovered a complete Toll/TLR pathway in the genome of N. vectensis, indicating that this pathway predates the divergence between Cnidarians and Bilaterians some 600 million years ago (reviewed in Hemmrich et al., 2007). The Nematostella Toll receptor is characterized by multiple cystein clusters in its LRR extracellular domain, suggesting that this form likely represents the ancestral structural organization of Toll receptors. A single NF-kB transcription factor and its IkB inhibitory protein have been identified in the genome of N. vectensis together with many intracellular signaling mediators of the NF-kB signaling pathways, including MyD88, IRAK/Pelle, TRAF, TAK1, and elements of a IKK signalosome. In contrast, and quite intriguingly, the Hydra genome apparently lacks a conventional NF-kB transcription factor and a canonical Toll receptor, although it features two genes encoding transmembrane TIR-domain containing proteins with a short ectodomain deprived of LRR. Hydra mounts an inducible expression of AMPs upon infection and it has been suggested that the TIR-domain transmembrane molecules might function in an atypical signaling complex with separate LRR-containing proteins to control these Cnidarian immune defenses (Bosch, 2009). Other potential immune molecules that have been identified in the genome of the sea anemone are absent from Hydra. These include homologs of C3, Bf, and MASP elements involved in the complement cascades. These observations suggest that the multicomponent complement system was already established in the genome of the common ancestor of Cnidaria and Bilateria (Kimura et al., 2009). The absence of these elements from Hydra likely reflects secondary gene losses during evolution. Evidence for MAPK signaling pathways (including JNK, p38, and AP1) has also been obtained from the analysis of the genomes of both Cnidarian species. Sponges, which belong to the phylum Porifera, represent an even earlier branching lineage than Cnidaria, occupying a basal position near the root of the metazoan tree of life. Investigations of cDNA libraries from the sponge Suberites domuncula and a thorough analysis of the genome of Amphimedon queenslandica have revealed the presence of an impressive armamentarium of genes encoding potential


recognition proteins, including scavenger receptors and several members of NF-kB signaling pathways (reviewed in Hemmrich et al., 2007). Of note, in contrast to the sea anemone NF-kB, the sponge NF-kB is structurally related to Drosophila relish, in that it carries its own inhibitory sequences. The expression pattern of AmqNF-kB during embryonic and larval stages suggests a developmental role, although these observations do not exclude a potential defense function of AmqNF-kB (Gauthier & Degnan, 2008).

ConClUsIons

As illustrated in this presentation, we now have at our disposal a wealth of information on innate defenses in invertebrates ranging from relatively simple organisms to highly evolved animals. Obviously, for many species, we still lack crucial functional data and the cellular and molecular mechanisms underlying the immune reactions still await further analysis in several large invertebrate phyla. In all frankness, we must admit that even our view of the much studied Drosophila immunity is still fragmentary and that it requires intense further investigations. With these caveats in mind, the essential message that we want to convey here is that innate immunity, as we understand it today, has appeared very early in life’s history, probably with the first multicellular organisms. There is clearly a common thread through all invertebrate phyla regarding recognition of microorganisms, activation of intracytoplasmic signaling cascades, and induction of gene programs that culminate in the synthesis of potent defense molecules, among which the antimicrobial peptides feature prominently. Recognition of microorganisms by the invertebrate immune system is a field that has significantly advanced in recent years. A variety of phagocytic receptors have been described, most of which have mammalian counterparts. They interact with an intracellular machinery of vesicular trafficking and cytoskeleton rearrangements, ultimately leading to large phagosomal structures where microorganisms can be disposed of. Again, from our recent information, it appears that most of the players in a few well-studied cases are highly similar to the molecules that govern these processes in mammals. This hardly comes as a surprise as it is largely surmised that phagocytosis evolved very early in evolution for purposes of nutrition, development, and defenses. Intriguingly, some putative phagocytic receptors, such as Dscam in Drosophila and the circulating opsonin-like FREPs in Mollusks, which feature IgSF domains, are encoded by genes that have the potential to generate multiple receptor isoforms. It will be interesting to follow future studies on the functional relevance of these arrangements. Parallels with the now well-established variable lymphocytes receptor (VLR) system of jawless fish (Agnathans) recently described by Cooper and colleagues (Pancer, 2004) appear premature at this stage. Every subfield has its own potential pitfalls. This is also the case in the Toll/TLR context. In the best documented cases, that is Drosophila and mammals, a very important difference is that the Toll receptor becomes activated by a cleaved form of the cytokine spaetzle, whereas mammalian TLRs interact directly with microbial ligands. The expression “pattern recognition receptors,” coined by Janeway (1989) thus perfectly applies to mammalian TLRs but not to that of the Drosophila Toll. This remark underlines that, pending functional studies, we are uncertain how to interpret results on dozens or more Toll/TLRs in various invertebrates. Therefore, it is certainly premature to propose that the increase in number may reflect environmental pressures by microorganisms.

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One of the beauties of innate immunity, as illustrated by the case of Drosophila, is that the number of microbial ligands that actually activate gene reprogramming is extremely limited: Firm evidence exists to date only for PGNs and b-glucans. More recently, viral RNAs have been added to the list. The receptors, as we see them today, have essentially derived from ancestral enzymes, namely amidases, glucanases, and helicases. In Drosophila at least, the number of documented receptors is less than a dozen proteins to date. Would a larger repertoire be beneficial for this organism? We believe that a larger repertoire of receptors has to be viewed in the context of lymphocyte development, clonal selection, and expansion and memory. In the absence of these features, increasing the repertoire of innate immune receptors might be of little improvement for the defense, but we are ready to reevaluate our view if clear and reproducible data will instruct us otherwise in the future. In the early days of our research on the signaling cascades that led to the expression of immune genes in Drosophila, we were extremely interested by the parallels between these cascades and those of mammalian immune defenses. This is now commonplace, but the data reviewed here strongly suggest that these cascades were in place already at the very earliest stages of evolution. Again, we lack functional data for most of the species in which NF-kB is present, but we anticipate that future studies will show that from sponges to insects, and further to mammals, similar molecular modules direct essentially similar cascades to transcribe gene programs that ultimately are beneficial to the host and deleterious to the invader. Further, as illustrated above, many of the effector molecules appear to be similar in the various phyla and are present in vertebrates as well. An interesting question pertains to the parallel evolutions of developmental regulations and immune defenses. How do we explain that Drosophila has nine Toll receptors and six spaetzle paralogs, three of which have been documented to act as neurotrophins (Zhu et al., 2008), all of which are involved in development and that just one pair, Toll-1 spaetzle, were selected to direct antifungal and some antibacterial defenses? We hope that functional studies in other phyla will help clarify our view on these dual roles in immune and developmental regulation during evolution. Finally, a word of apology to our friends in the growing field of Caenorhabditis elegans defense reactions. C. elegans lacks so many of the features described in this review (namely NF-kB) that we have decided to put this organism apart and refer to excellent recent reviews covering this special field (Schulenburg et al., 2004; Kim & Ausubel, 2005; Shivers et al., 2008). The common view in the community today is that C. elegans has lost most of the conventional traits of the innate immune system of invertebrates as they were described here. In fact, a similar situation might also prevail for the Cnidarian Hydra. Although innate immunity was discovered in invertebrates by Metchnikov nearly 130 years ago, it has long remained a relatively neglected area of research. In the 1990s, molecular genetics have given a potent stimulus to the field, initially in Drosophila and now in many phylogenetically diverse groups. The progress has been astounding over the last 15 years and hundreds of laboratories worldwide are now engaged in research on innate immunity. Moreover, in some precise areas of high significance (e.g., the discovery of antimicrobial peptides, the role of Toll receptors in innate immunity, and the role of peptidoglycan recognition proteins) invertebrate immunity has led the way. We hope that this thread will continue and that this review article has stimulated some interest among potential future students of the field.

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The authors apologize for their inability to quote many additional contributions due to space limitations. Work in the laboratory of the authors has been generously supported by the French National Research Agency (CNRS), the National Institute of Health (NIH), the European Union, and the University of Strasbourg. We thank our past and present associates in the laboratory for their contributions and for continuous helpful discussions.

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2 The Ontogeny of the Cells of the Innate and the Adaptive Immune System FRITZ MELCHERS

INTRODUCTION

Recognition of specific antigen by binding at sufficiently high avidity induces clonal lymphocyte proliferation and maturation to effector functions—in the case of T cells to helper, cytotoxic, or regulatory cells; in the case of B cells to antigen receptor-hypermutated, Ig class-switched cells and Ig-secreting plasma cells, which generate and maintain the Ig levels in the extracellular spaces, notably the blood, or, after transport through epithelial layers, in the lumen of the gut.

A single pluripotent hematopoietic stem cell (pHSC) can be the progenitor of all the erythrocytes, megakaryocytes, and blood platelets, of all the cells of the myeloid cell lineages, of all the natural killer (NK) cells, of all the different functional types of T lymphocytes, and of all the sublineages of B lymphocytes. Hence, the ontogeny of these different types of blood cells, the cells of the innate and the adaptive immune system, is very complex. Some of the major molecular steps and cellular stages, including a few of the interactions of the developing hematopoietic cells with the endogenous environment of cooperating cells, as well as with the exogenous environment of antigens and polyclonal activators will be described, mainly with the mouse but also with references to the development in humans. Development begins, shortly after the fertilization of the egg, with the development of embryonic stem (ES) cells; from there, the progenitors of the vascular and hematopoietic cells (called hemangioblasts) are developed; and thereafter is the generation of a pluripotent hematopoietic stem cell. From that point, it continues first in embryonic development and, thereafter, throughout life through the different stages of hematopoietic, myeloid, and lymphoid progenitors, and finally to mature cells of the myeloid cells and of NK cells (i.e., of the innate immune system) and to the development of antigen receptor-expressing lymphocytes of the adaptive immune system and their deposition and use in different parts of the immune system.

The Clonal Selection Hypothesis

The clonal selection hypothesis (Jerne, 1955; Burnet, 1957; Talmage, 1959; Nossal & Lederberg, 1958) proposes that one lymphocyte makes only one structure of an antigenbinding receptor, that this one structure on the surface of one lymphocyte binds one antigen of the wide world of structures (antigens) surrounding these lymphocytes, and that many different antigens bind to many different antigen receptors, but always one antigen to one of the wide world of lymphocytes in the system. To deal with the tolerance of the immune system to its own antigens, Lederberg (1959) extended this hypothesis by proposing that the emerging repertoires of antigen receptors on lymphocytes would first enter a phase—today recognized as immature lymphocytes—in which binding of the body’s own antigens, the autoantigens, would select their specific cells and eliminate them. The surviving lymphocytes would then further differentiate with the next stage of cellular development—now known as mature lymphocytes—in which binding of a foreign antigen would initiate a positive immune response (i.e., the proliferation and maturation of cells to effector functions that the system can use to eliminate the invading antigen). One prediction of this hypothesis was that the second allele of an antigen receptor-encoding gene would not be activated for expression in a single lymphocyte, termed allelic exclusion of the receptor loci. Another expectation was that a single lymphocyte would only rarely find a second, structurally nonidentical antigen to which it could bind to elicit a response. Furthermore, autoantigens would only negatively select and foreign antigens would only positively select cells of the system for a response.

The Adaptive Immune System of Lymphocytes

The major hallmark and specific feature of T and B lymphocytes is their capacity to produce a wide variety of antigenbinding receptors, in T cells, the a/b- or g/dT-cell receptors (TcR), and in B cells, immunoglobulin (Ig) heavy/light (H/L) chain-containing B-cell receptors (BcR), which they deposit for antigen recognition on their surface.

Fritz Melchers, Max Planck Institute for Infection Biology, Senior Research Group on Lymphocyte Development, Charitéplatz 1, D 10117, Berlin, Germany.

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Our current knowledge of the generation and selection of lymphocyte receptor repertoires shows that the clonal selection hypothesis, in its initial definition, is often no longer tenable. Nevertheless, the adaptive immune system manages to engage only a small part of its final, available repertoire of antigen receptors on lymphocytes for the recognition of, and response to, an antigen.

DEVELOPMENT OF HEMATOPOIETIC CELLS AND LYMPHOCYTES Embryonic and Adult Development In Vivo

Three waves of hematopoietic cell developments colonize the mouse embryo (Ling & Dzierzak, 2002; Godin & Cumano, 2002; Cumano et al., 2001). The first begins in extra embryonic tissue (i.e., in the yolk sac at day 7 to 7.5 of development). This so-called primitive hematopoiesis (which shows similarities to hematopoiesis in lower vertebrates) generates primitive erythrocytes (synthesizing “fetal” hemoglobin), megakaryocytes (with lower ploidy) and platelets, and myeloid cells (such as macrophages with a special set of enzymes) (Shivdasani et al., 1995). Lymphocytes are not generated. Although these primitive blood cell lineages are developed at extraembryonic, ventral sides of the embryo, they migrate to the embryonic, dorsal side as soon as blood circulation is established at day 8 to 9 of development with the development of vascular endothelium. At day 8.5 to 9 of mouse embryonic development, the second wave of hematopoiesis is started within the intraembryonic, anterior portion of the aorta-gonad-mesonephros region (Medvinsky & Dzierzak, 1996; Cumano et al., 1996). Undifferentiated, apparently pluripotent stem cells (Ohmura et al., 2001) then migrate through the blood and colonize the rudiments of the thymus and fetal liver. At these sites, the second, so-called definitive wave of hematopoiesis is initiated. It now generates erythrocytes producing adulttype hemoglobin, definitive megakaryocytes and platelets, and adult-type myeloid cells and lymphocytes. In the thymus, several waves of thymopoiesis are initiated. The T cells that are generated in these first, fetal waves of development from day 16 of mouse embryonic development onwards are first g/d and, thereafter, a/b TcRexpressing cells, all with little or no N-region insertions at their V(D)J junctions because of the lack of expression of the enzyme TdT, and they display a restricted repertoire of v-region combinations. They migrate to intraepithelial sites of the body where they might perform first line of defense functions. In the fetal liver of the mouse, between day 13 of embryonic development until birth at day 19, a first, apparently synchronous wave of B-lymphocytes is generated (Strasser et al., 1989). Two other sites of embryonic B-cell development, generating B cells with properties similar to those of the fetal liver, are the embryonic side of the placenta and the omentum (Kincade, 1981; Owen et al., 1976; Melchers, 1979; Gekas et al., 2005, Ottersbach & Dzierzak, 2005; Solvason & Kearney, 1992). Since most of these B cells do not express the enzyme TdT, they rearrange the V(D) J segment of the IgH and IgL chain loci without N-region diversity. The early waves of embryonic B cells preferentially home to the gut-associated lymphoid tissues, belong, in large part, to the BIa-types of B cells, and form a first line of defense and cohabitation against and with the developing intestinal bacterial flora. The third wave of hematopoiesis (i.e., the second definitive hematopoiesis) starts in bone marrow between days 17

and 19 (i.e., at around the birth of a mouse) (Kincade et al., 2002). It initiates a continuous process of hematopoietic cell generation throughout life. B cells develop to form at least three distinct lineages: B1a, B1b, and B2 cells (Melchers, 2009; Tung & Herzenberg, 2007). They are then distributed into at least two major subcompartments of mature, BcR-expressing B-lymphocytes of the mature, peripheral system. Approximately half of them are found in the follicular regions of the spleen and lymph nodes and in the recirculating blood and lymph, a majority of which are the B2-type B cells. The other half is mainly found in the gut-associated lymphoid tissues (e.g., in Peyer’s patches, either in follicular structures near the flat M-cell regions of the gut epithelia, or as single intraepithelial lymphocytes in the lamina propria of the gut). At least a majority of these B cells appear to belong to these so-called BI subpopulations. Most B1a cells develop during the prenatal period in the fetal liver from precursors that lack lg rearrangements and are distinct from those that give rise to B1b and B2 cells (Tung & Herzenberg, 2007). B1a cells persist for some time after birth even though they are derived from precursors that disappear during the neonatal period. B1a cells contribute a large part to the natural lgM antibodies in serum, respond to T cell-independent antigens, express unmutated V regions, contain very few (if any) N-region sequences in their complementarity determining region 3 (CDR3) sequences, and need the spleen to develop (Wardemann et al., 2002). T cells develop in the thymus. Progenitors from bone marrow with myeloid and lymphoid, or only lymphoid potentials migrate to the thymus to continuously generate, until sexual maturity, the different subpopulations of T cells. After sexual maturity, the thymus begins to involute and decreases T-cell production progressively. Even after adult thymectomy the mature T-cell compartments are maintained in size and function, indicating that T-cell maintenance in the system becomes extrathymic. The early stages of thymocytes are CD4 through CD8 and are either CD441CD252 (DN1), CD441CD251 (DN2), CD442CD251 (DN3) or CD442CD252 (DN4) cells in that developmental sequence. At the transition of DN2 to DN3 cells the expression of TcRs decides whether they develop either to a/bTcR- or to g/dTcR-expressing cells. The a/bTcR1 cells become CD41CD81 (DP) cells that are then selected to either the CD41 (SP) or the CD81 (SP) lineages of cells. After exit from the thymus, the a/bT cells mature to either regulatory, helper, or cytotoxic functions. The CD41 a/b T cells further differentiate into helper type 1 (Th1), helper type 2 (Th2), follicular B helper T, helper T-17 (Th17), or regulatory CD41CD251 T cells. The CD81 a/b T cells differentiate into type 1 or type 2 cytotoxic T cells (Tc1,Tc2), or NK T cells (NKT). g/dT cells also differentiate to helper or cytotoxic cells. It will be described below that the specificity of the TcRs determines lineage choice (Kyewski & Klein, 2006; Haks et al., 2005; Hayes et al., 2005; Kreslavsky et al., 2009). In human embryonic development, mature T and B lymphocytes are already detectable at 10 to 12 weeks, hence, well before birth. Therefore, human embryos have mature, antigen-reactive lymphocytes several months before birth, ready to react with a positive memory response to a foreign antigen if challenged by one. However, a more detailed description of prenatal human hematopoietic development is missing, as is a detailed assessment of the postnatal changes in these developments. It is safe to expect that immune systems of mice, as well as of humans, will be different at different times of an aging individual.

2. the ontogeny of the Cells of the Innate and the adaptive Immune system

Development of Hematopoietic Cells from Embryonic Stem (ES) Cells In Vitro

From the 64cell blastocyst stage of the mouse embryo at day 3.5 of development—and from the comparable stage of human embryonic development—ES cell lines have been established in tissue culture that will proliferate for many generations without losing their ability to participate in the in vivo development of all cell lineages when reinjected into a recipient blastocyst and retransplanted into a foster mother, leading to a fully chimeric mouse (Martin, 1981; Evans & Kaufman, 1981). Furthermore, mouse ES cells are capable of homologous recombination (i.e., of introduction of genes) without the help of retroviral transfection at their sites in the chromosomes, thereby allowing proper transgenic changes and repair of selected gene loci (Matise et al., 2000). Furthermore, ES cells can be induced in vitro to differentiate to various cell lineages, notably also to hematopoietic cells and, more specifically, to cells of the innate and the adaptive immune system. These ES cell-differentiated hematopoietic cells, depending on their state of differentiation, can be transplanted in recipients to populate some or all of the hematopoietic compartments. It is surprising that these tissue culture conditions can be chosen in such a way that they will follow the embryonic time program in a wave of development, such that mesodermal cells appear at day 5 (equivalent to 8.5 of embryonic development), pHSC at day 8 to 9 (equivalent to 11.5 to 12.5), and T- and B-lymphocyte progenitors and precursors after day 12 (equivalent to day 15.5) (Nakano et al., 1994; Tsuneto, in preparation). Since it is now also possible to generate ES cell-like iPS (induced pluripotent stem) cells from peripheral, differentiated cells such as epithelial cells or lymphocytes, (Okita et al., 2007; Wernig et al., 2007; Hanna, 2008) and from them transplantation-tolerant hematopoietic cells, all in tissue culture, the study and use of any defined or mutable gene in its function in the development of the innate and the adaptive immune system can be studied. Such studies will help to develop protocols for genetic therapies of defective, disease-inducing gene loci in histocompatible ES-like iPS cells and their subsequent in vitro development to transplantable pHSC, with the hope to cure immunodeficiencies and, hopefully, to interfere with autoimmune diseases.

CELLULAR STAGES OF HEMATOPOIETIC DEVELOPMENT Pluripotent Hematopoietic Stem Cells

Pluripotent hematopoietic stem cells (pHSCs) generate the cells of the innate and the adaptive immune system first as the embryo develops, and thereafter continuously throughout life, not only to build up, but to maintain the numbers of cells (estimated as more than 2 3 109 in an adult mouse, and more than 2 3 1012 in an adult human, of which a large part turns over with half-lives of less than a week. These pHSCs derive from embryonal mesoderm, which also generates the environments of the hematopoietic system. These environments are the vascular endothelial cells that form blood and lymph vessels through which the blood cells travel and the microenvironmental stromal cells, forming hematopoietic “niches” in bone marrow and thymus for the developing progenitors (Jordan & Lemischka, 1990; Spangrude et al., 1991; Smith et al., 1991; Morrison et al., 1995; Osawa et al., 1996a, 1996b). The cooperating stromal cells that help to induce these different hematopoietic lineage differentiations belong, in part, to mesenchymal stem-cell-derived osteoblasts, and, in other parts, to epithelial cells. Osteoblasts have been found

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to be stimulatory for some early hematopoietic progenitor functions, while differentiated adipocytes are either no longer stimulatory, or even inhibitory (unpublished observations). In part, this may be due to the spectrum of cytokines produced by these different cellular stages of mesenchymal cell differentiation. Thus, in contrast to a series of stromal cell lines derived from wild-type mouse bone marrow the OP9 stromal cell line derived from M-CSF-deficient mouse bone marrow stroma (Nakano et al., 1994) cooperates in early hematopoiesis, even from embryonic stem cells. Five properties characterize pHSCs: 1. Their capacity to renew themselves upon cell division, 2. Their pluripotency to develop into the various differentiation lineages of blood cells, 3. Their chemotactic attraction to home back to specific niches in bone marrow upon transplantation, 4. Their long-term reconstituting ability to again serve as a stem cell when used from the transplanted host in secondary transplantations, and 5. Their potency to protect a transplanted host from death as consequence of lethal irradiation after (socalled bone marrow) transplantation.

self-renewal

pHSCs have the capacity to renew themselves upon cell division. In a steady state of the hematopoietic system selfrenewal will be by asymmetric divisions, generating one further differentiated cell while preserving the other as pHSC when the cell divides.

Intracellular Control Mechanisms

Several factors control the self-renewing proliferation of pHSCs, most notably the Polycomb gene family member bmi-I (Iwama et al., 2004; Park et al., 2003) and the transcription factor HOX B4, (Sauvageau et al., 1995; Helgason et al., 1996; Thorsteinsdottir et al., 1999; Kyba et al., 2002; Antonchuk et al., 2002; Buske et al., 2002; Krosl et al., 2003), both of which are controlled in their gene expression by the nuclear factor NF-Ya (Zhu et al., 2003). The Wntsignaling pathway, including b-catenin (Baba et al., 2005; Duncan et al., 2005; Reya et al., 2003) has been found to be involved in the control of pHSC proliferation, as it does in other stem cell programs. Thus, transgenic expression of NF-Ya, bmi-I, HOX B4, or b-catenin favor long-term proliferation of pHSC. In tissue culture, pHSs can now be expanded and cloned in the presence of appropriate cytokines (e.g., TPO [thrombopoietin], IL3, IL6, and stem cell factor [SCF, the ligand for the receptor tyrosine kinase c-kit]), all on a supportive layer of mesenchymally derived stromal cells. Prolonged, transgenic expression of these genes, however, may inhibit efficient hematopoietic differentiation into the different blood cell lineages (Schiedlmeier et al., 2003).

Extracellular Controls

Mesenchyme-derived as well as epithelial stromal cells provide several pHSC-cooperative, stimulatory interactions, among them SCF, interacting with c-kit on pHSCs (Gilliland & Griffin, 2002); angiopoietin, interacting with Tie-2 on pHSC (Arai et al., 2004; Zhang et al., 2006); and bone morphogenic protein 4 (BMP-4), interacting with its receptor on pHSC (Maeno et al., 1996; Hogan, 1996; Graff, 1997). Also, they chemotactically attract pHSC by the production of CXCL-12 (SDF-1), interacting with its receptor CXCR4 on pHSC (Nagasawa et al., 1996; Egawa et al., 2001; Ma et al., 1999; Ma et al., 1998; Kawabata et al., 1999; Melchers et al., 1999) and Ca-2, which is the Ca11-receptor

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protein expressed on pHSC, probably sensing Ca11 near the marrow of bones (Adams et al., 2006). Activation of these stromal cells, especially the osteoblasts among them, in hematopoietic cell niches (Wilson & Trumpp, 2006) result in subsequent activation or inhibition of pHSC activities. Furthermore, nor-adrenaline produced by sympathetic nerves in collaboration with actions of the stem-cell-mobilizing cytokine G-CSF induce apoptosis of stromal cells, thereby mobilizing pHSC to leave the marrow and enter blood circulation (Osmond et al., 1985; Rico-Vargas et al., 1995; Medina et al., 1993; Medina et al., 2001; Katayama et al., 2006). Since early progenitors of B lymphocyte development in mice and humans have been shown to decrease in absolute numbers in bone marrow during life (Rolink et al., 1993), it is reasonable to expect that absolute numbers of earlier progenitors and of pHSC also decrease with age, even though they never disappear completely.

Pluripotency

A single pHSC can repopulate all cell lineages of blood and fill their compartments in the body with normal numbers of cells (Jordan & Lemischka, 1990 Spangrude et al., 1991; Smith et al., 1991; Morrison et al., 1995; Osawa et al., 1996a, 1996b). pHSCs give rise to erythrocytes, megakaryocytes, and platelets that are not considered to be parts of the immune system. pHSCs also generate the cells of the innate immune system (i.e., myeloid cells such as monocytes, macrophages, various forms of dendritic cells, granulocytes such as basophils, neutrophils and eosinophils, and osteoclasts). Finally, pHSCs are the origin of lymphoid cells (i.e., the various forms of NK cells and cells of the adaptive immune system, T and B lymphocytes). Pluripotency is extracellularly controlled by the environment of cell contacts, chemokines and cytokines provided by stromal cells, and is intracellularly regulated by hematopoietic cell–autonomous genetic programs (i.e., receptor-mediated signal transductions and regulations of gene expressions).

MYELOID-LYMPHOID PROGENITORS Generation by Environmental Influences

When pHSCs are exposed to the action of FLT3L, the ligand of the membrane-bound tyrosine kinase receptor flt3 they lose their long-term repopulating capacity (Adolfsson et al., 2001). FLT3L is provided by “niches” of stromal cells in the bone marrow. FLT3L induces an apparently irreversible change to short-term repopulating stem cells (ST-pHSC), also called multipotent progenitors (MPP). In tissue cultures on OP9 stromal cells these MPPs can be grown in the presence of FLT3L and SCF. When regrown in tissue cultures with TPO, IL3, IL6, and SCF, they do not regain pHSC potential. Upon transplantation, these cells give rise to one wave of myeloid and lymphoid cell differentiation which is first seen in bone marrow (for myeloid, progenitor lymphoid and B-lymphoid progenitors) and in the thymus (for T-lymphoid progenitors) at 3 to 4 weeks, then as mature myeloid, and T- and B-lymphoid cells in the periphery, such as the spleen, 6 to 8 weeks after transplantation. While myeloid cells disappear again due to their short half-life, long-lived T and B cells remain for more than 4 months in the periphery (e.g., spleen, peritoneum, blood) of the transplanted host.

Induction by Intracellular Controls

GATA-1 controls erythroid development (Pan et al., 2005) while Icaros and PU-I induce myeloid-lymphoid cell development (Klemsz et al., 1990; Hromas et al., 1993; Singh & Pongubala, 2006; Dakic et al., 2005; Nutt et al., 2005).

THE CHOICE BETWEEN MYELOID AND LYMPHOID DEVELOPMENT Environmental Influences

While stromal cell lines derived from wild-type mouse bone marrow most often do not support the development of lymphoid, but do support myeloid cells, it was found that the OP9 stromal cell line derived from M-CSF-deficient mouse bone marrow stroma (Nakano et al., 1994) cooperates in early hematopoiesis, even from embryonic stem cells, to proliferate pHSC with TPO, IL3, IL6 and SCF, and MPP with FLT3L and SCF. When M-CSF is added to such proliferating MPPs, they develop to myeloid cells that cease to proliferate after 1 week. On the other hand, when IL7 is added to the proliferating MPPs they become lymphoid progenitors, with the properties of common lymphoid progenitors (CLP) that can be induced to become either B lymphoid, T lymphoid, or NK cells. Hence, progenitors with the dual capacity to develop to myeloid and to lymphoid cells are induced on OP9 stromal cells, by M-CSF to myeloid, or by IL7 to lymphoid development. It appears that the constitutive production of M-CSF by wild-type stromal cells favors the development of myeloid progenitors, thereby disfavoring lymphoid progenitors. Once MPPs have chosen one of the two ways, the “other” cytokine no longer influences these cells in their continued differentiation (e.g., M-CSF is no longer active on lymphoid, and IL7 no longer on myeloid development). When MPPs or the IL7-induced CLPs are cultured on transgenic NOTCH-ligand DELTA-I expressing OP9 stromal cells, in vitro differentiation and proliferation of thymocytes to the CD4/CD8-double negative state is induced (Schmitt et al., 2004). Finally, osteoclast development is induced on stromal cells expressing transgenic TRANCE (Tsuneto et al., 2005).

Intracellular Controls

High levels of PU.1 induce myeloid and low levels lymphoid cell development (Nutt et al., 2005). Mef2 is required for proper expression of key lymphoid regulators, among them the IL7 receptor. It is in agreement with the expectation that external recognition of IL7 induces lymphoid lineage choice (Stehling-Sun et al., 2009), while at the same time, it represses myeloid development through the downregulation of C/EBPa. It appears to act in concert with PU.1 to modulate the intracellular decisions between myeloid and lymphoid lineage development, maybe differently at different concentrations of complexes containing PU.1 and Mef2c.

EXCLUSIVE B- VERSUS T-LYMPHOID LINEAGE DECISIONS

At least a part of the CD19-negative, CD42/CD8-double negative (DN), IL7-receptor-expressing, receptor-tyrosine kinases c-kit and flk-2-double positive CLP progenitor cells in bone marrow have yet to make the decision to enter either the T- or B-lymphoid lineage pathway of differentiation. Some of these early lymphoid progenitors in bone marrow express both the T-lymphoid lineage specific preTa gene as well as the surrogate L chain-encoding VpreB and 5 genes. Similarly, cellular functions control the proliferative expansion of DN2 to DN3 and 4 and large DP (CD41, CD81) thymocytes once the precursor have migrated to the thymus, and SL chain controls proliferative expansion of pre-BI to large pre-BII cells in the bone marrow. Hence, while these common lymphoid progenitors already express


both surrogate chains as well as the rearrangement machinery (i.e., RAG1, RAG2 and TdT), they still have to commit themselves to the expression of only one of the two—either pre-Ta, express CD3 and enter D- to J-rearrangements at the TcRd or b loci, or SL chain, Iga and b and enter D- to J-rearrangements at the IgH locus.

B-Lineage Commitment environmental Influences

We do not know which microenvironmental influences positively stimulate the development of sIgM1 immature B cells from B-lineage-committed progenitors and precursors, in contrast to the other lineages described above in bone marrow. B-progenitors of wild-type mice differentiate spontaneously—without addition of cytokines or exposure to stromal cells—to sIgM1 B-lymphocytes (Rolink et al., 1991). Contrary to the lineage-inducing capacities of other cytokines, IL7 locks DHJH-rearranged pre-BI cells into their early stage of B-lineage differentiation. In the added presence of stromal cells, which provide, for example, stem cell factors (SCF) for interaction with the receptor tyrosine kinase c-kit, these pre-BI cells proliferate but, again, do not differentiate to later stages of B-cell development (Rolink et al., 1995). Removal of IL7, rather than the addition of a different set of cytokines, induces the development of IgM1 B cells.

transcription Controls

Three transcription factors—E2A, EBF, and PAX5—control the initiation and maintenance of B-lymphoid development (Bain et al., 1994; Zhuang et al., 1994; Lin et al., 1995; Sigvardsson et al., 1997; Kee & Murre, 1998; Roessler & Grosschedl, 2006; Busslinger, 2004). Among the three, E2A dominates this control by not only initiating, but also maintaining the expression of EBF1, PAX5, and, thus, the B-cell program. E2A or EBF-deficient mice are arrested in B-cell development, just as PAX5-deficient mice are. However, E2Adeficient progenitor B cells carry no DHJH-rearrangements (Ikawa et al., 2004). By contrast, PAX5-deficiency still allows DH to JH-rearrangements on the IgH chain loci to the same extent (i.e., on both alleles) as PAX5-proficient, wild-type progenitor B cells (Rolink et al., 1999b). Transgenic expression of E2A (Singh & Pongubala, 2006), but not of EBF or PAX-5, or expression of important lineagespecific genes controlled by these early regulators (e.g., the IL7-receptor) (Choi et al., 1996) induce autonomous, environment-independent, B-lineage specific differentiation. Transfection, and subsequent transgenic expression of the E47 component of E2A in nonlymphoid fibroblasts, activates the terminal deoxynucleotidyl transferase (TdT) gene and the IgH chain locus (Choi et al., 1996). When transgenic expression of the rearrangement machinery (i.e., RAG1 and RAG2) is also provided in an embryonic kidney cell line, DH to JH rearrangements are induced at the endogenous IgH chain loci (Romanow et al., 2000). Endogenous rearrangements at the L chain loci in the same nonlymphoid cells require more selective actions of transcriptions factors; in the RAG1/RAG2-transgenic cells, E2A allows for endogenous VK to JK rearrangements, and EBF for V to J rearrangements (Romanow et al., 2000). Finally, myeloid cell differentiation can be autonomously induced by the transgenic expression of high levels of PU-I, or of the gene encoding the receptor for GM-CSF (Singh, 1996) as lymphoid differentiation can be directed by the transgenic expression of the receptor for IL7 (Singh & Pongubala, 2006). Both E2A-deficient, as well as PAX5-deficient progenitor B cells can be grown—like wild-type pre-BI cells—for

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long periods and cloned on stromal cells in the presence of IL7. In the case of PAX5-deficient pre-B cells, they are cell clones that are genetically identifiable by individual sets of DHJH rearrangements at both IgH chain alleles, just as wildtype, DHJH-DHJH rearranged pre-BI cells are (Rolink et al., 1991; Schaniel et al., 2002a, 2002b). The same is true for RAG-deficient as well as SL-chain-deficient, CD191 pro/ pre-B cells, the former without any DHJH rearranged IgH chain loci, the latter with normally DHJH/DHJH rearranged loci. Hence, the cellular differentiation program of the expression of the tyrosine kinases flk2 and ckit, and of CD19, accompanied by reactivity to IL7 and to stromal cell interactions that provide the ligands for flk2 and ckit and the cytokine IL7, can become disconnected from the DH to JH rearrangement program in progenitor B cells. The PAX5-deficient progenitors can be induced in vitro to (DHJH/DHJH rearranged) macrophages by M-CSF, to (DHJH/DHJH rearranged) dendritic cells by GM-CSF and M-CSF, to (DHJH/DHJH rearranged) granulocytes by GMCSF and G-CSF, to (DHJH/DHJH rearranged) NK cells by IL-2, to (DHJH/DHJH rearranged) osteoclasts by TRANCE expressed on mesenchyme-derived stromal cells, and to (DHJH/DHJH rearranged) thymocytes by NOTCH-ligand DELTA I, again expressed on stromal cells (Rolink et al., 2002a; Radtke et al., 2004; Höflinger et al., 2004). Transplantation of pre-BI cells, grown on stromal cells in the presence of IL7 from day 18 fetal liver, populate exclusively the B1a compartment in spleen, peritoneum, and blood, and do not populate the bone marrow, and only rarely and transiently populate the thymus. In contrast, pre-BI cells derived from bone marrow in SCF/FLT3L/IL7-stimulated cultures on stromal cells populate transiently (i.e., 2 to 4 weeks bone marrow with B-cell progenitors and thymus with T-cell progenitors and, thereafter, spleen and blood with T and B cells, and peritoneum with B cells resembling B1b cells (Vegh et al., 2010). On the other hand, transplantation of PAX5-deficient pre-BI-like cells, grown from bone marrow on stromal cells in the presence of IL7, populate all the myeloid, NK cell, and T cell compartments (with the exception of B-cell compartments in bone marrow), the thymus, and the peripheral lymphoid organs in a long-term repopulating fashion. Hence, such PAX5-deficient pre-BI-like cells can be isolated, regrown, and retransplanted several times into secondary, tertiary, and onward recipients, always repopulating all the different myeloid and lymphoid compartments except the B-lineage compartments (Schaniel et al, 2002).

T-Lineage Commitment Commitment by Internal Controls

Expression of NOTCH-1 accompanies development to the T lineage (Rolink et al., 2002a). Ectopic expression of “active” NOTCH-1 allows early thymocyte development in the absence of a thymus and, at the same time, inhibits B-cell development (Pui et al., 1999). Ectopic expression of Id2 induces differentiation to the NK cell lineage (Yokota et al., 1999; Ikawa et al., 2001; Engel & Murre, 2001) and, again, inhibits B-cell development, possibly by forming heterodimeric complexes with E2A (Engel & Murre, 2001). On the other hand, “inactive” NOTCH-1 inhibits T-cell development at the earliest, DNI stage of T-cell development, but promotes B-cell development even in the thymus (Radtke et al., 1999; Wilson et al., 2001). NOTCH-1 can also be inactivated by Lunatic Fringe (Koch et al., 2001) and by Deltex (Izon, 2002). Again, such activations lead to arrest of T-cell development and stimulation of B-cell development.

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Whenever activated, NOTCH-1 releases its intracellular domain, which acts on the transcription factor RBPJ to convert it from a repressor to an activator (Tanigaki & Honjo, 2007). Direct targets of the activator are Ptcra, CD25, and Hes-1.Thereafter and throughout a/b TcR T-cell differentiation from DN1 to CD41CD81DP thymocytes, activities of the transcription factors RUNX-1-CFBb, MYB, GATA-3, Icaros, PU.1, ETS-1, ETS-2, SPIB, E2A, HEB, and LEF1 all participate in controls at different developmental points, as described in detail in Rothenberg et al. (2008).

Commitment by external Controls

When CLPs are cultured in vitro not on OP9 stromal cells, but on OP9 stromal cells that express the ligand for NOTCH1 and DELTEX 1 constitutively after transgenic transfection, they develop in the presence of FLT3L and IL7 to the four stages of CD4/CD8-double negative (i.e., DN1, DN2, DN3, and DN4 cells, as well as to CD4/CD8double positive thymocytes) (Schmitt & Zuniga-Pflücket, 2002). In prolonged cultures, cells with a DN2/DN3 phenotype expand. The chemokine CCL25 attracts progenitors expressing its receptor, CCR9, within the thymus.

REVERSIBLE DECISIONS OF EXCLUSIVE B-CELL DIFFERENTIATIONS – THE FLEXIBILITY OF PROGENITOR CELLS

The hierarchical models of hematopoietic cell differentiation propose that commitment to a given cell lineage, with concomitant loss of the respective pluripotency or multipotency, is achieved in irreversible steps. This appears to be the case when FLT3L commits pHSCs to myeloid or lymphoid (i.e., differentiation to MPPs) and also when M-CSF commits MPPs to myeloid (i.e., to CML), and IL7 to lymphoid (i.e., CLP differentiation). By contrast, this appears not to be the case with PAX5deficient pre-BI-like cells. While they are blocked in further B-cell development, they differentiate to all the possible hematopoietic cell lineages (i.e., erythroid, myeloid, NK, and T-lymphoid cells), depending on the external environment of cytokines and niche cell contacts. However, they do so with rates that are too slow to allow protection of the transplanted host from irradiation-induced death. Hence, while they appear to be pluripotent, they are not pHSCs, although they home back to their original sites in bone marrow, retaining the original phenotype of ckit1flk-21CD192 IL7Ra1sca-11 cells (Rolink et al., 1999a; Schaniel et al., 2002b). The same multipotency has also been seen with E2A-deficient pro-B cells (Ikawa et al., 2004). By contrast, wild-type pre-BI cells do not home back to the bone marrow but rather, upon transplantation, give rise to a single wave of B-cell development, mainly to BI type (Rolink et al., 1991). B2-type B cells are developed when CD41 helper T cells, together with CD41CD251 regulatory T cells, are cotransplanted. Hence, PAX5-deficient progenitors are fully functional, pluripotent, long-term reconstituting, and flexible stem cells, but they are not pHSCs since they do not differentiate to all the hematopoietic cell lineages at appropriate rates to allow protection of the host from death by lethal irradiation. The expression of PAX5 can be seen as the final, decisive step to commit hematopoietic progenitors to the B-lineage pathway of development. The importance of PAX5 in this commitment process is further documented by experiments, in which lox-flanked PAX5 has been “knocked into” the PAX5 locus, rendering the locus conditionally inactive. When pre-BI cells of such conditionally PAX5-deficient pre-BI cells are treated with CRE-recombinase, thus deleting the PAX5-locus, these wild-type pre-BI cells revert

back to the PAX5-deficient pro/pre-B-cell phenotype. They become inducible to enter erythroid, myeloid, NK-, and Tlymphoid development (Mikkola et al., 2002). T-lymphoid cells in the thymus derived from the PAX5-inactivated and transplanted pre-BI B cells retain the DHJH/DHJH rearrangements of the original pre-B cells. When mature, sIgM1 B cells of conditionally PAX5deficient mice are rendered PAX-5 deficient by treatment with CRE-recombinase, the cells revert back to a pre-B-like stage (Schebesta et al., 2002). In vitro, such reverted cells regain their IL7-receptor expression and regain the capacity to respond to IL7 by proliferation. Since these cells have already expressed a set of productively VHDHJH/VLJL rearranged IgH/IgL-chain loci (i.e., sIgMs with μH- and kLor L-chains), and since PAX5 deficiency leads to lack of SLP65 expression (Schebesta et al., 2002), the resulting pre-B cells express a polyclonal spectrum of VH-domains on μH chains, and reexpress the SL chain (i.e., reexpress pre-BcRs but cannot signal downregulation of SL chain expression). Hence, they continue to proliferate, stimulated by IL7. Interestingly, the induced PAX5-deficiency turns off the expression of the productively VLJL rearranged Lchain loci (i.e., of L chains), suggesting a tight regulation of L chain expression by PAX5. In vivo transplantation of the CRE-induced, PAX5-deficient pre-B cells leads to repopulation by polyclonal μH chain-expressing thymocytes in the thymus, again without expression of the VLJL rearranged Lchain loci. It remains to be determined whether the CREinduced PAX5 inactivation dedifferentiates these cells to a large pre-BII cell-like stage or to a pro/pre-BI-like stage of B-cell differentiation.

THE GENERATION OF THE ANTIGEN RECEPTOR-EXPRESSING LYMPHOCYTE REPERTOIRES

In order to generate lymphocytes expressing antigen receptors, CLPs have to enter the appropriate primary lymphoid organs and begin to differentiate and rearrange antigen receptor loci. From the moment a lymphocyte precursor expresses the first chain of its antigen-specific receptor, that receptor becomes the dominating control element of all further steps of proliferation or death, and survival and location in the immune system. T- and B-cell antigen receptors (i.e., TCR, BCR) are generated by joining of V, D, and J segments at the TcR and Ig loci in a process ordered in time and cell differentiation stages. First, D to J rearrangements at the TcRb, TcRd, and IgH loci are initiated. For B-lineage cells this happens first during embryonic development in omentum and fetal liver, and later in adult life, in bone marrow, while for the T-lineage this is initiated in the fetal, and then in the adult thymus. Fetal cells appear to originate from progenitors that are distinct from those used after birth and all throughout life. In the B-lineage, D to J-rearrangements are begun in CD19-FLT31B2201c-kit1 pro/pre-B cells and are complete in CD191FLT3-B2201c-kit1pre-BI cells. T-lineage cells begin their D to J rearrangements at the transition from DN1 to DN2 cells, and have completed them in DN2 cells.

V to DJ Rearrangements and Synthesis, and Use of the First Chain of the Heterodimeric Antigen Receptors as Pre-Lymphocyte Receptors

The first functional Ig TcR chains are made when V to DJ rearrangements are initiated that can generate productively rearranged IgH, respectively, TcRb or TcRd loci. In B-lineage cells, V to DJ rearrangements occur in the


CD191B2201c-kit-CD251 large pre-BII stage, in the a/b TcR T-lineage at the DN3 stage (Kyewski & Klein, 2006; Melchers, 2005). All T-cell lineages with the possible exception of fetal TcRg/d expressing T cells, are derived from the same intrathymic precursors. All B cells, with the exception of the fetal wave of development in omentum and fetal liver generating B1a cells are derived from the same precursor in bone marrow. Productive TcRg and TcRd rearrangements result in expression of a g/d TcR, which commit cells mostly to the gd lineage (Kyewski & Klein, 2006). Since they do not form a TcRd chain-containing pre-lymphocyte receptor, these T-lineage thymocytes do not expand by proliferation in the thymus as TcRb chain-containing T-lineage cells do. Hence, they make up only 5% of all thymocytes and, consequently, generate correspondingly lower numbers of T cells for the peripheral immune system, at least in the spleen, lymph nodes, and blood. a/b TcR T-cell development and B-cell development show striking similarities. Productive IgH gene and TcRb gene rearrangements generate Igμ-H chains and TcRbchains which are assembled with SL chains—composed of the Vpre-B and 5 proteins, respectively—with pre-Ta chains to form pre-lymphocyte receptors (pre-BcR, pre-TcR) that are deposited on the cell surface of large pre-BII cells, such as DN3 thymocytes. In this way, the Vpre-B part of the SL chain in pre-BcRs (but not pre-TcRs, which lack such Vprobing Vpre-T parts) probe the newly generated IgH chains for their ability to form structurally intact heterodimers, first with the surrogate light chain, and later, in development with IgL chains (Melchers, 2005). Between 50% and 80% of the newly formed IgH chains do not pair well enough to form a pre-BcR on the cell surface, and hence, are excluded from the positive selection for further use in the B-lineage that follows. Initially, all TcRb chains may form a pre-TcR. The newly formed pre-BCRs and TcRb-pre-TcRs induce proliferation of large pre-BII cells, respectively, DN3 thymocytes. In the case of pre-BcRs, the number of induced cell cycles (between 2 and 7) depend on the fitness of the IgH chain/SL chain pairing, while fitness between TcRb chains and pre-Ta chains appear not to influence a larger number of induced cell cycles (7 or more). Mutations that abolish the proper structure of the preBcR (i.e., B-lineage progenitors deficient in VpreB1 and VpreB2 or 5, or VpreB1, VpreB2, and 5), or that interfere with pre-BcR signaling (Melchers, 2005; Melchers et al., 2000), abolish the proliferative expansion of large pre-BII cells, as do mutations that interfere with or abolish pre-TcR functions. Pre-BcRs and pre-TcRs may not even use the complementarity determining regions (CDRs) of the V-domains to bind ligands that could induce proliferation. The newly generated VH-domain repertoire of pre-BcRs is screened for fitness to pair (i.e., to be prepared for the eventual combination with conventional L chains). Thereby unfit H chains are excluded that may have other unwanted properties, such as the formation of self-aggregating immune complexes that might bear the danger of glomerulonephritis and vasculitis (Melchers, 2005). It appears that the non-Ig-portion of the 5-subunit of an SL chain is mandatory for the capacity of pre-BcRs to stimulate pre-BII cell proliferation (Ohnishi & Melchers, 2003). Crosslinking of pre-BCRs via positively charged arginine residues in the non-Ig portion of 5, possibly mediated by repetitive negative charges on molecules such as negatively charged nucleic acids and other molecules, either on the same pre-BII cells or on environmental stromal cells (Ohnishi & Melchers, 2003; Bradl et al., 2003), initiates pre-BCR signaling. Some CDR3 regions of newly generated

27

VH-domains contain argine residences that are apparently capable of mediated pre-BcR signaling functions even in the absence of an SL chain (Doyle et al., 2006). The pre-BCR also induces the downregulation of SL, thereby limiting proliferative expansion of IgH chain-expressing cells through exhaustion of pre-BcR formation. Furthermore, the pre-BcR down regulates recombinase activating gene (RAG1 and 2) expression, thereby preventing VDJ rearrangements on the second allele, possibly because the locus is rendered inaccessible for the rearrangement machinery. This should secure allelic exclusion at the IgH chain locus, so that one B cell produces only one type of H chain. Membrane-bound IgH chains signal the cell to turn off the rearrangement machinery and make the second, DHJH rearranged IgH chain allele inaccessible for further rearrangements. However, it appears that the classical pre-BcR with SL chain is not involved in mediating this allelic exclusion, since SL-triple-deficient (Vpre-B12/2 /vpreB22/252/2) B cells still are allelically excluded (Shimizu et al., 2002; Melchers, 2005). In pre-BII cells where pre-BCRs cannot be formed, these nonproliferating cells nevertheless proceed in differentiation (Grawunder et al., 1993; Rolink et al., 1996; Grawunder et al., 1995). However, their contribution to the developing B-cell compartment is, at best, 20- to 40-fold lower than that of their pre-BCR-expressing counterparts (Rolink et al., 1993b). Deletion of the gene encoding the adaptor protein SLP-65 directly by targeted mutagenesis (Flemming et al., 2003; Jumaa et al., 2003) or indirectly by targeted inactivation of PAX5 controlling SLP-65 expression (Koch et al., 2001) abolishes the capacity of large pre-BII cells to downregulate surrogate L-chain expression. This leads to continued pre-BcR formation in dividing large pre-BII cells so that, in fact, SLP-65-defective mice have a hyperplastic large pre-BII cell compartments. This chronic pre-BII cell proliferation is a breeding ground for secondary mutation so that, in a period of weeks, pre-B cell lymphomas (lymphocytic leukemias) develop in these mice. Do some autoantigens, nevertheless, influence—either positively or negatively—the pre-BcR repertoire development? It has been observed that a subset of autoreactive and polyreactive pre-BCRs expressed in pre-BII-like cells induced a proliferative boost in tissue culture cells, suggesting a positive rather than a negative selection by polyreactive pre-BCRs (Köhler et al., 2008) while other experiments indicated that autoantigens suppress pre-BcR-dependent pre-B-cell development (Keenan et al., 2008). Still other experiments indicate that pre-BCR cross-linking in fetal liver organ culture did not boost pre-B-cell proliferation, nor did it inhibit it (Ceredig et al., 1998). Pre-BCR formation leads to a shift in the originally generated heavy chain repertoire (ten Boekel et. al., 1997) and this shift is delayed in SL chain-deficient bone marrow. It facilitates a loss of positively charged and aromatic amino acid residue-containing polyreactive μH-chains, and thus may represent negative selection of μH-chain repertoires by the pre-BCR (Keenan et al., 2008). It is not clear how much of this negative selection of pre-BCRs is mediated by autoantigens binding to CDRs of μH-chain chains and how much is due to lack of pairing with SL. Productive TcRb rearrangements generate pre-TcRs, that induce proliferation of DN3 thymocytes in a cell autonomous fashion without any apparent requirement for a ligand. As for the B lineage in pre-BII cells, formation of a pre-TcR on the surface of DN3 thymocytes leads to the termination of V to DJ rearrangements on the second TcRb allele. More than 99% of thymocytes and T cells are allelically excluded at their TcRb locus. It is unknown whether

28


TcRb loci can replace their Vb -regions in secondary replacements. Polar residues in the extracellular domain of the pre-TcRa chain, as well as its cytoplasmic tail are likely to contribute to this autonomous signalling (Yamasaki et al, 2006; Aifantis et al., 2002). However, the only conserved portion of the pre-TCRa chain is the transmembrane region and not its polar residues, so that other structural features might contribute to this autonomous signaling. Pre-TcRs terminate further rearrangements at the TcRb locus, but, unlike the pre-BcRs, do not select cells expressing particular TcRb chains by pairing, because pre-Ta does not have a Vpre-B-like structure that would allow such pairing with V regions of TcRb chains. Hence, the distribution of TcRb V regions in unselected and pre-TCR-selected thymocytes is the same (von Boehmer et al., 2003). All pre-TcR-expressing cells commit to the a/b TcR T-lineage with the help of Notch signaling, and they subsequently all undergo TcRa rearrangements.

V to J Rearrangements and Synthesis and Use of the Second Chains of the Heterodimeric Antigen Receptors as TcRs and BcRs in Immature T- and B-Cell Compartments

V to J rearrangements at the TcRa loci occur last in DN4 and CD41/CD81 DP cells, as do rearrangements at the Igk and Igl loci in CD191 B2201c-kit-CD251small preBII and in IgM1 immature B cells. Furthermore, except for most of the embryonically generated V(D)J-joints, N-region sequences of differing lengths are inserted at most joining sites, although this is more evident in human, and less so in murine B-cell development. Most cells with productive TcRd and TcRg rearrangements develop to the gd lineage without allelic exclusion of either TcRd or TcRg chains (Rothenberg et al., 2008). It is in fact not the isotype of the TcR but the signaling strength (Schmitt & Zuniga-Pflücker, 2002; Mikkola et al., 2002) that commits cells to either the d/g or a/b lineage. Some 20% g/d TcR-expressing cells that undergo weak signaling can still enter the a/b lineage by silencing TcRg expression and deleting the TcRd locus by TcRa rearrangement (Nossal & Lederberg, 1958). Little is known about tolerance mechanisms in g/d T cells except that TcR ligands appear to have a role in committing these cells to the g/d lineage (Schmitt & Zuniga-Pflücker, 2002; Mikkola et al., 2002) rather than in causing apoptosis. In fact, TcR ligation results in the generation of a subset of g/d T cells that assume the characteristics of innate lymphocytes in that they express the PLZF transcription factor, which is also is expressed in NKT cells (Schebesta et al., 2002).

Secondary Rearrangements at the IgL and TcRa Loci

A first rearrangement on the Ig L-chain loci can be followed by replacements by secondary VL to JL rearrangements on an already VLJL rearranged L-chain locus (Yamagami et al., 1999a, 1999b). TcRa can enter secondary Va to Ja rearrangements, and they can be extensive since more than 50 Ja segments are available in the mouse for such secondary, tertiary, and other rearrangements (Nossal & Lederberg, 1958). Secondary rearrangements can be made in immature lymphocytes that lack IgL chain, respectively, TcRa chain protein expression as heterodimeric antigen receptors on the cell surface, either as a result of a nonproductive V to J rearrangement, or because the chains do not fit their corresponding partner chain in the heterodimer. They can also be the result of an inappropriately affine binding of a BcR to an autoantigen, in the case of an a/b TcR to MHC-self-peptide. Changing the Ig L chain, respectively TcR a chain in this way has been termed editing. Continued rearrangements at

the IgL chain loci of autoreactive BcR-expressing, immature B cells increase the chance save this cell from apoptosis by a change of the specificity away from autoantigen recognition. By contrast, continued rearrangements at the TcRa locus increase the chance to salvage an immature thymocyte by changing the TcR specificity towards self-MHC/self-peptide recognition. During the process of L-chain editing, evolutionarily selected editor Ig light chains that have low isoelectric points (due, for example, to the presence of aspartate residues) appear to neutralize the DNA binding properties of certain IgH chains; this may facilitate avoidance of diseases mediated by antibody DNA complexes such as SLE (Li et al., 2001). Editing can also lead to the expression of two or even more different L chains (and possibly also two different H chains) in a single immature B cell (Gerdes & Wabl, 2004; Khan et al., 2008; Doyle et al., 2006). If editing results in the expression of one autoreactive and one nonautoreactive BCR, immature B cells can become unreactive to autoantigen due to dilution of the autoreactive with the nonautoreactive BCR. These dual-expressor B cells can enter the mature B cell pool while remaining potentially autoreactive. L chain editing can also lead to polyreactivity. Interestingly, some human B cells coexpress V pre-B and conventional L chains together with H-chains containing CDR3 regions enriched in positively charged and/or aromatic amino acids. Two-thirds of these cells are autoreactive, hence, they appear to have escaped central tolerance. Although human Vpre-B, which has an isoelectric point of 5.67, may act as an “editor” by neutralizing positively charged CDR3 regions, mouse Vpre-B proteins have an isoelectric point of 9.37 and thus may not be able to perform this function.

THE SELECTION OF IMMATURE LYMPHOCYTES BY THEIR ANTIGEN SPECIFICITIES Selection For and Against Autoantigen Reactivities

From the random generation of the antigen-binding regions, especially of the third complementarity region (CDR3) of the V regions of TcRs and BcRs, it is predictable that some of these V regions expressed on BcRs and TcRs of immature T and B cells should be fully autoreactive. In fact, it has been estimated that more than half of all newly generated BCRs are capable of binding autoantigens (Nemazee, 1995). The reactivity or responsiveness of B cells to antigens and autoantigens remains imprecisely defined; however, as the correlation between the binding affinity of an antigen to a BCR and the functional response of the B cell expressing this BCR cannot be given as a quantitative term. Furthermore, such antigen-sensing mechanisms are not necessarily the same in developmentally distinct subsets of lymphocytes. The same limitations in the judgement of quantitations of avidities exist for the interactions of TcRs with MHC-self-peptide or MHC-foreign peptide interactions. There is no reason to expect that the diversities of the originally generated repertoires of TcRb and TcRd chains and, thereafter, of the TcRa and TcRg chains are different from those of the IgH and L chain, since all loci are rearranged by the same rearrangement machinery, including the insertion of N regions by TdT. The originally generated repertoires of TcRs have not been tested for binding to the same collection of autoantigens and foreign antigens as BcRs have. It is, therefore, reasonable to expect that these original TcR repertoires are similarily polyreactive and autoreactive, and many of them should be heterodimer-unfit


to pair. The more extensive pre-TcR-driven expansion of TcRb chains may just simply increase the chances to find a pairing TcRa chain later. However, neither polyreactive nor autoreactive a/b TcR matter, because all of them, in contrast to BcRs, are selected to fit only one selected set of autoreactivities, namely recognizing MHCI or MHCII-self-peptide complexes (see below). Only those will be allowed to mature to single-positive (SP) CD41 or CD81 thymocytes, and to leave the thymus and mature in the periphery to effector cytotoxic or helper T cells. It is surprising—and also not in agreement with the hypothesis of clonal selection—that a large part of the originally generated repertoire of IgM1 B cells are most likely are polyreactive (i.e., bind a large collection of structurally seemingly unrelated antigens).

Autoantigen-Presenting Cells in Primary Lymphoid Organs for t Cells

To begin T-cell development 10 to 100 CLP migrate through the blood from the adult bone marrow into the cortical regions of the thymus (Kyewski & Klein, 2006). After commitment to the T lineage they proliferate as CD4-CD8-DN1 and -DN2 thymocytes and, thereby, expand to 5 3 107 cells in 15 to 20 divisions within nearly 2 weeks. In these proliferating cells, D to J rearrangements are initiated and completed. V to DJ rearrangements follow in DN3 and 4 thymocytes. The pre-TcR of the a/b T lineage allows another expansion by more than seven divisions, so that the ratio of a/b versus g/d TcR thymocytes after V to J rearrangements settles at 95% to 5%. However, most of these newly generated thymocytes never leave the thymus; only 1 to 2 3 10106 cells do. Productive TcRa rearrangements in TcRb-expressing cells lead to expression of an a/b TcR on the cell surface if the two chains can form a proper heterodimer. In marked contrast, TcRa rearrangements are only halted when the TcR can bind to MHC I or II self-peptide complexes with sufficient avidities (Borgulya et al., 1992; Casanova et al., 1991). In fact, all mature a/b TcR T cells contain both TcRa alleles in VJ-rearranged forms, and 30% of these thymocytes and T cells express two productive TcRa rearrangements (Padovan et al., 1993). Therefore, the TcRa loci appear to be kept open for secondary rearrangements as long as no MHC-self-peptide complex fits, thereby increasing the chances to positively edit the a/b TcR specificity for self-MHC/self-peptide recognition. However, not all the 30% of T cells express two TcRs on the cell surface (Saito et al., 1989) because pairing preferences exist for some TcRb chains in pairing with certain TcRa chains (Heath et al., 1995). Hence, only 10% of T cells express two different TcRa chains on the cell surface. In summary, a/b TcR T cells are frequently not allelically excluded. The specificity of the a/b TcRs determines whether cells are neglected and die, or are positively or negatively selected (von Boehmer, 2004). a/b TcRs on CD41CD81 thymocytes that recognize MHC class I self-peptide complexes on antigen-presenting cells in the thymus bring these MHCI molecules on the antigen-presenting cells in contact with CD8, which induces their differentiation to naïve CD81 cytotoxic T cells (Tc), on which CD4 has been downregulated. Conversely, other a/b TcRs on other thymocytes that recognize MHC class II self on antigen-presenting cells allow the contact of MHCII with CD4 on thymocytes and induce the differentiation to CD41CD8− helper T cells (Th), or to CD41 CD251 regulatory T cells (Treg) (von Boehmer, 2008). The induction to cytotoxic and helper

29

T-cells differentiation—also called positive selection—is dependent on the avidity of interaction between MHC selfpeptide complexes and a/b TcRs. If these avidities are too high, the thymocytes are driven into programmed cell death (apoptosis), also called negative selection. High avidity interactions can, however, also result in a diversion of developing abTCR1 cells into unconventional lineages involved in innate immunity, such as NK T cells. For the selection of such NK T cells, the selecting ligand is expressed on lymphoid cells (Bendelac et al., 2007).

Generation and Selection of Treg Cells in the Thymus

High affinity TcR ligands can also induce the generation of Treg cells that express the transcription factor Foxp3 and have an essential role in preventing autoimmune disease (Yamagata et al., 2004; Guy-Grand & Vassalli, 2004). Lineage fate of such cells is instructed by TcR binding to agonist ligands expressed on stromal cells within the thymus (Guy-Grand & Vassalli, 2004; Jordan et al., 2001). The Treg cell lineage differentiation process might begin either with CD41CD81 double positive, FOXP3-expressing cells in the thymic cortex (Apostolou et al., 2002), or with CD418− single positive cells in the medulla. It is evident that in developing T cells, the TcR determines lineage fate. The developing thymocytes are confronted with different types of autoantigen-presenting cells: (i) epithelial cells in the subcapsular regions, cortical epithelial cells, and epithelial cells in the medulla (mTEC [medullary thymic epithelial cells]); (ii) hematopoietically derived dendritic cells (DC); and (iii) hematopoietically derived B lymphocytes. All of them can take up, process, and present autoantigens (proteins) as self-peptides in MHC I and II molecules. mTECs and DCs express the highest levels of MHC. Hence, these three types of cells are prime sites for thymocytes probing the specificities of their a/b TcRs on MHC-self-peptide complexes (Kyewski & Klein, 2006). High-avidity interactions lead to negative selection by apoptosis, or to anergy, and short time survival. The ignorance of TcRs with no avidity for MHC-self-peptide complexes does not allow such thymocytes to further differentiate to a state where they could leave the thymus. These thymocytes remain short-lived and die by neglect. Low-avidity interactions lead to positive selection to more mature, CD41 or CD81 cells that are allowed to leave the thymus. Hence, the emerging T-cell repertoire in the peripheral immune system is restricted in the recognition of peptides of foreign antigens by the MHC of the antigenpresenting cells that they have encountered in the thymus. mTECs have the most unusual capacity to transcribe and translate a large variety of autoantigen-encoding gene loci, a capacity that appears to be in part controlled by the AIRE gene product (Peterson et al., 2008). Some thymic DCs also express AIRE and may, therefore, have the same expanded autoantigen-presenting capacity. Therefore, positive and negative selection of helper CD41, cytotoxic CD81, and regulatory CD41CD251 thymocytes does not only include the autoantigens expressed in the normal cells of the thymus; thereby, central tolerance can be generated for all autoantigens.

for B Cells

In immature B cells, surface membrane expression of IgM appears stop further Ig rearrangements. At least two-thirds of such immature B cells are capable of doing so, because two-thirds of the immature, as well as the peripheral, splenic mature B1 and B2 compartments have one of the two IgL chain alleles in germline configuration. They are allelically excluded, since

30


less than 0.5% of all immature or mature B cell express more than one IgH chain allele as BcR on the surface. Quite comparably to T cells, the specificity of the Ig receptor influences the selection into the different B cell lineages, shown in experiments where the transgenic change of a B2-derived Ig receptor to a B1a-derived one changes the selection of the Ig-transgenic cells from B2 to B1a. It is remarkable that almost nothing is known of the autoantigen-presenting modes that select B-cell repertoires and establish central B-cell tolerance in the different B-cell compartments. Genetic deficiencies in the complement components C1q, C4, serum amyloid protein, complement receptor 2, or secreted natural serum IgM lead to systemic autoimmune disease characterized by production of autoantibodies against DNA, and other nuclear antigens suggest that these gene loci may contribute to central B-cell tolerance (Melchers & Rolink, 2006). Two models have been proposed to explain the emergence of autoantigen-reactive B-cell repertoires in these knockout animals. In one model (Carroll, 2004), macrophages expressing the appropriate complement receptors (e.g., C1qR, CR1) efficiently remove apoptotic cells that are bound by natural IgM, C1q, and C4b, thereby preventing accumulation of these cells and subsequent activation of mature B cells. The other model (Melchers & Rolink, 2006), suggests that autoantigens, including those released from apoptotic cells are presented to immature B cells by immune complexes containing C1q, C4b, and IgM on as yet unidentified cells expressing the appropriate receptors (e.g., C1qR, CR1, and possibly FcRμ). In this model, the result of the autoantigen presentation depends on the avidity of interaction of the immune complex-bound autoantigens with the BcR expressed on an immature B cell. If the avidity is very high, it can result in negative selection (i.e., induction of apoptosis and/or anergy in the immature B cell). If the avidity is low, the corresponding immature B cell can be positively selected, maybe into the B1 compartments, if this low autoantigen recognition is maintained in the B1 populated sites. If the BcR on an immature B cell does not bind any autoantigen with even a low avidity this cell is ignored and is allowed to enter (maybe into the B2) conventional B cell compartments. It is notable that, in this view, ignored B cells are allowed to enter the mature, peripheral compartments while T cells must be MHC-self-peptide-selected. A better understanding of the modes of central B-cell tolerance induced by apoptosis, editing, anergy, and ignorance requires identification of the relevant antigen-presenting cell populations. At this first checkpoint of B-cell repertoire development, the contribution of polyreactive BcRs to the total repertoire drops from 60% to 6%. In contrast, in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients with these autoreactive and polyreactive BcRs are not lost at this checkpoint (Carroll, 2004), providing strong evidence of the role of this checkpoint in preventing autoimmune disease.

POSITIVE SELECTION OF B CELLS

Autoreactive, in fact, often polyreactive B cells (Leslie et al., 2001) can be positively selected and appear to accumulate in the BI compartments of the gut-associated lymphoid tissues (i.e., in the follicles below the M cells of the flat epithelium of the gut). Some of them are also found as single, intraepithelial cells. Many positively selected B cells accumulate in the gutassociated lymphoid tissues (GALT), including the lamina propria. They produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epi-

thelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side it binds with low affinity and high crossreactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. B cells within the marginal zone of the spleen also appear to be positively selected. However, it remains to be seen whether this major traffic intersection, in fact, is a selection site for newly generated (as well as possibly of recirculating) B cells, potentially, these B cells could be in contact with cellular debris of dying blood cells and of killed bacteria, which are found at the same sites and which are about to be removed from the circulation. Many of them have a BI phenotype (Hayakawa et al., 1999; Stall et al., 1992). BcRs of BI B cells are often found to cross-react with a variety of auto-antigens and bacterial antigens at low avidities. B1 cells appear mildly activated (i.e., “tickled”) do not proliferate in their GALTs but appear to be long lived. Transplantation of BI cells into secondary hosts, in contrast to the transplantation of conventional B cells is easy and repeatable in subsequent hosts. Even low numbers of BI cells will fully replenish the GALTs. This suggests that the low avidity–autoreactivity, combined with a polyreactivity to several antigens, could be the stimulatory influence for BI cells to replenish their lymphoid sites after transplantation and to attain longevity (Craig & Cebra, 1971; Förster & Rajewsky, 1990). Many positively selected B cells appear to be BI cells, and many are accumulated in the gut. B cells in the GALT, including the lamina propria, produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epithelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side, it binds with low affinity and high cross-reactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. At least in parts this IgA production by gut-associated B cells appears to be quite distinct from the Ig-production elicited in a helper T-cell-dependent, inflammatory response of B cells in germinal centers of peripheral lymph nodes and the spleen. First, while the follicular B cell response beneath the M cells may be helper T-cell-dependent; hence, germinal center-derived, the submucosal, intraepithelial response in the lamina propria can be helper T-cell-independent. Onethird of the IgA might derive from such T-cell-independent B cell responses in these T-cell-independent responses (Rolink et al., 1991). The expression of the Ig class-switchinducing AID (activation-induced cytidine deaminase) gene (Muramatsu et al., 2000; Revy et al., 2000; Shinkura et al., 2004) is normally induced by a CD40 ligand-CD40 interaction. However, the cooperating cell providing the CD40 ligand in the GALT, especially in the intraepithelial spaces is not known, and must not be a helper T cell. Furthermore, these B cell responses could be costimulated by other cell–cell contacts and cytokines that might not be typical for a T-cell-dependent, germinal center response of B cells. Hypermutation of the V regions of the IgM, and later IgA, expressed and secreted by the lamina propria of the intestine BI cells could well occur in the AID-expressing, IgA class switching cells. However, the lack of the selection


of better fitting v region mutants by bacterial antigens and the lack of germinal center formation (i.e., exclusive B-cell proliferation) may decrease the probability to generate and select better fitting antibacterial antibodies—in as much as that also should decrease the probability of generating high affinity autoreactive antibodies.

IGNORANCE OF B CELLS

Emerging B cells with no apparent autoreactivity pass the two checkpoints in bone marrow and the spleen as long as they express a surface membrane-deposited BcR. A variety of Ig-transgenic mouse models support this view (Fields & Erikson, 2003). The ignored B cells enter the spleen via the terminal branches of the central arterioles and populate the follicular regions as conventional, B2-type B cells. Initially they are short-lived, with half-lives of 2 to 4 days. They then mature to longer-lived B cells, with half-lives longer than 6 weeks. Many positively selected B cells appear to be BI cells, and many are accumulated in the gut. B cells in the GALT, including the lamina propria, produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epithelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side it binds with low affinity and high cross-reactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. Conventional, B2-type B cells are most often triggered into responses by helper T-cell-dependent antigens. These foreign antigens also stimulate helper T cells to cooperate with the follicular B cells in a response, which takes place mainly in germinal centers within the follicular regions of secondary lymphoid organs. This induces B cells in a CD40 (B)-CD40 ligand (T) cell cytokine (e.g., IL4- or TGF-b-)dependent fashion to switch to IgG, IgE and IgA, and to preferentially hypermutate the V regions of the rearranged IgH- and L-chain genes, leading to affinity maturation of B cells. The switched, hypermutated BcR-expressing B cells have the choice either to mature to Ig-secreting plasma cells or BcR-expressing memory cells. Both of them gain longevity in such a T-cell-dependent response with half-lives changed from a few days to weeks and months of survival in the immune system. The memory B cells and the long-lived plasma cells appear to leave the germinal centers to lodge in special niches in the bone marrow until they are recalled by a secondary challenge of the same antigen. While BI B cells are apparently unable to be stimulated to longevity by BAFF, a member of the TNF family of cytokines, conventional B cells express BAFF-receptor, a member of the TNF-receptor family, and respond to BAFF by polyclonal maturation to long-lived B cells (Rolink & Melchers, 2002). The TNF family ligands BAFF and APRIL and their receptors BCMA, TACI, and BAFF-R control the selection of short-lived immature B cells with no apparent positively (or negatively) selecting specificities for autoantigens to long-lived mature B cells. Experiments of the in vivo administration of soluble BAFF-R ligands and of soluble decoy receptors, the analysis of BAFF-transgenic, BAFF-deficient and BAFF-receptor (BAFF-R)-deficient mice, as well as the in vitro responses of immature and mature B cells of BAFF (all reviewed in Rolink & Melchers, 2002) have shown that immature B cells from bone marrow and the spleen (initially

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immature as well as transitional B cells) and mature B cells respond to BAFF by polyclonal maturation to long-lived B cells without proliferation. BAFF and BAFF-R deficiencies arrest B-cell development at the transition from immature to transitional B cells. Although the action of BAFF in vitro is polyclonal and independent of BCR occupancy, it remains to be seen whether ligand selection through BCR occupancy plays a role in this selection of the conventional “virgin” antigen-reactive mature B cells. B2 cells can also respond, as B1 cells do, to T-cellindependent antigens (i.e., in the absence of an activation of helper T cells and their cooperative actions on the B cells). In that situation the Ig-secreting cells, which are generated to mainly produce IgM, remain short-lived and do not generate memory B cells. It remains also possible that gut-associated B1 cells can be flexible enough to respond in a T-cell-dependent fashion, form germinal centers, and generate long-lived, Ig class-switched plasma cells and memory B cells. In such cases they are, or become, conventional, B2-type B cells. Germinal center-like lymphoid aggregations have been seen to form near the flat epithelium M cells.

PERIPHERAL B CELLS WITHOUT Ig

In normal B cell development, B-lineage cells that cannot express Ig molecules on their surface are restricted to the primary lymphoid organs and will die there, usually with half-lives of 2 to 4 days. Experimentally induced ablation of surface Ig expression in peripheral mature B cells induces their rapid death (Lam et al., 1997). Therefore, neither immature, mature, memory B cells, nor plasma cells without Ig expression can ever be detected in the peripheral lymphoid organs, and even most B-cell tumors appear to be selected to express Ig. However, several observations, most of them made in vitro, suggest that B-lineage cells from as early as the Blineage committed progenitor B cells and pre-BI cells can differentiate all the way to memory-phenotype and plasma cell-like stages without ever expressing Ig. In kL-chainrearrangements-deficient bone marrow, over 90% of the small pre-BII cells have the proper phenotype, but no Lchain rearrangements, since rearrangements at the L chain loci are so slow that they can only be detected in the remaining 10% of the small pre-BII cells (Yamagami et al., 1999a, 1999b). Most striking is the development of RAG-deficient pre-BI cells (hence with IgH and L-chain loci in nonrearranged, germline configurations) in vitro in the stimulatory presence of CD40-specific monoclonal antibody and IL4 to sμ-sg-switched cells with a mature phenotype (Rolink et al., 1996). Hence, the differentiation of B-lineage cells is a stepwise process driven by cell–cell contacts and cytokines that can occur without the synthesis or surface deposition of Ig. The roles of the pre-BcR and BcR also become apparent from these experiments. They control, positively and negatively, proliferation, anergy, and apoptosis of B-lineage cells and, thereby, ascertain the production of appropriate numbers of cells with selected specificities. Viral infection of B cells by EBV (Epstein-Barr virus) may be another way, by which the requirement of surface Ig deposition on B cells for their survival and use in the peripheral lymphoid organs may be circumvented. The EBV encodes latent membrane proteins (LMP), of which LMP 2A, a multispanning transmembrane protein, expressed in B lymphocytes, can associate with Iga and Igb and share with these two anchor proteins an ITAM motif (Gross et al., 2005; Fruehilng & Longnecker, 1997; Caldwell et al., 1998). In fact, the phosphorylated form of the ITAM

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motif of LAMP 2A recruits tyrosine kinases and adaptor proteins, which are also used by the BcR complex for signaling. In this way, LMP 2A acts as a BcR analog, supporting the selection into the peripheral mature B-cell compartments of B cells lacking BcRs. High-level transgenic expression of LMP 2A selectively recruits cells into the BI compartments (e.g., into the gut and intestine) while low level expression promotes development of conventional, B2-type, and marginal zone B cells (Casola et al., 2004). Neither of the high-level or low-level LMP2A expressing BcR-negative B-cell populations can be stimulated by T-cell-dependent antigens to develop germinal centers in peripheral lymphoid organs, indicating that BcRs on the surface of B cells are needed for peripheral germinal center responses. By contrast, the low-level, but not the high-level (i.e., the conventional) B2 (and not the B1) cell-directed LMP 2A expression allows germinal centers to be formed in the gut. Hence, the role of BcRs in germinal center formation in the gut might be replaced by an antigen-unspecific mechanism of antigen uptake, which allows processing and presentation by MHC class II molecules to helper T cells. Alternatively, these gut-associated BcR-deficient B cells might be stimulated by bystander helper T cells activated with bacteria of the gut or by bacteria-derived polyclonal activators, such as LPS and lipoprotein (Kawai & Akira, 2009), in the latter case via TLR2, TLR4, and other receptors (Akira & Takeda, 2004), or by other T-lineage cells or NK cells not restricted by the recognition of MHC class/ peptide complexes. In summary, T-cell (or NK-cell) dependent, BcR-independent (and perhaps MHC class II-independent) activation of B cells in germinal centers can lead to GC responses in which the activated B cells (as well as T or NK cells) can be expected to secrete pro-inflammatory cytokines such as IFN-g, IL-1, IL-4, IL-5, IL-6, and TNFa as essential mediators of immune responses. Furthermore, it has been observed in pre-BI cell-transplanted RAG-deficient mice, which contained only pre-BI cell-derived BI cells but no T cells, that one-third of the normal levels of IgA can be produced—apparently T-cellindependently—mainly by cells in the intestinal lamina propria without the formation of germinal centers. It is likely that these cells are also present in BcR-deficient, high-level LMP 2A-expressing lamina propria BI cells. Obviously they cannot produce IgA, but, again, they can be expected to respond to bacteria by the secretion of cytokines that might be of pro- or anti-inflammatory types. In conclusion, the GALT-associated B-cell compartments can develop even when the B cells do not express BcRs, or even MHC class II (i.e., can function in cellular responses of proliferation and differentiation to effector functions in an antigen-independent, and perhaps bacteriadependent way, leading to pro-inflammatory (and maybe also anti-inflammatory) responses. From all of these results, it becomes evident that peripheral mature B cells have important functions beyond their capacity to produce, secrete, class switch, and hypermutate antibodies in the regulation of immune reactions.

LINKING THE DEFENSIVE POWER OF THE INNATE IMMUNE SYSTEM WITH ANTIGEN SPECIFICITY OF THE ADAPTIVE IMMUNE SYSTEM

Since B lymphocytes can be stimulated to develop into cells that no longer deposit their BcRs (Ig) as antigen-specific receptors into the surface membrane but rather secrete this Ig as so-called plasma cells into the blood stream, antigen specificity of a B cell can be transferred to the innate

immune system in three ways. (i) Ig molecules of different IgH chain classes can find receptors for the constant regions of their H chains (so-called Fc receptors) and, thereby, can transfer this antigen specificity (by so-called opsonization) to antigen-nonspecific cells (e.g., cells of the innate immune system, such as macrophages or dendritic cells). (ii) The antigen-unspecific cells of the system can bind antigen–antibody (Ig)-complement immune complexes via complement receptors, again gaining antigen specificity by opsonization. (iii) As mature B cells displaying Ig on their surface, they can attract “their” specific protein antigens with high avidity, take it up, process it, and present it as peptides complexed with MHC class II molecules to helper T cells (i.e., they can act as antigen-specific antigen-presenting cells).

THE TRANSFER OF IMMATURE LYMPHOCYTES FROM PRIMARY LYMPHOID ORGANS TO MATURE COMPARTMENTS IN THE PERIPHERY

Although VH-repertoires expressed in immature B cells in the bone marrow and in immature and mature B cells in the spleen are not significantly different, almost 90% of the newly formed immature B cells never leave the bone marrow (Rolink et al., 1999a). Therefore, they should be subject to additional deletion mechanisms. In humans, there is a further reduction of the percentage of autoreactive cells (40% to 20%) across this transition, while the percent of polyreactive cells remains at the low level (6%) already achieved during the transition from pre-B to immature B cells (Wardemann & Nussenzweig, 2007). Again, SLE and RA patients fail to establish this repertoire change at this second checkpoint. Indicating that several mechanisms for establishing recessive tolerance may contribute to these diseases (Yurasov et al., 2005). Once arrived in the spleen, hardly any cells are lost at the transition from immature, including transitional T1 and T2 cells, to mature B cells. Hence, the mature B1 and B2 cells emerge only after passing through at least two checkpoints, suggesting that several mechanisms may operate to establish tolerance. Both of the checkpoints are not functioning in SLE and RA patients and, thus, may contribute to these diseases. The BcR complex with Iga and Igb is mandatory for transition to the peripheral sIgM1B cell pools—no sIg2 B cells survive normally. The transition from short-lived to long-lived, from AA4.11 to AA4.12, CD212/CD232 to CD211/CD231 cells is controlled by a series of genes, including OBF, btk, and CD40. Thus, btk/CD402 or btk2/ OBF− double deficient mice have a strong defect at the transition from the immature, transitional T1- and T2-type B cells to mature B cells (Rolink et al., 1999a). In addition, cross-linking of surface IgM by anti-IgM-antibodies (possibly a polyclonal example of a cross-linking auto-antigen) induces immature B cells of bone marrow as well as spleen (T1 and T2) to apoptosis. This apoptosis can be prevented by the transgenic expression of the anti-apoptotic transgene bcl-2, and is circumvented by polyclonal activation with LPS and with CD40-specific antibodies or CD40 ligand. Deficiency in the expression of the TNF-family member BAFF (also called B-lys), or its receptor, BAFF-R, blocks the maturation of immature B cells into conventional, B2-type B cells, but not to B1-type B cells (Rolink et al., 2002b; Rolink & Melchers, 2002; Ng et al., 2005; Moore et al., 1999; Schiemann et al., 2001; Gross et al., 2001). In vitro BAFF induces polyclonal maturation of immature B cells from bone marrow and from the spleen (T1 or T2) without proliferation. BAFF has been found in the sera of some SLE


patients, as well as in the sera of NZBxNZW SLE autoimmune disease prone mice. In these mice, administration of BAFF-specific antibodies prevents, or at least delays the development of SLE-disease. Therefore, excessive production of BAFF, administered at the sites of negative selection of immature B cells, could “rescue” autoreactive B cells from deletion, leading to autoimmune reactivities in the peripheral mature conventional B-cell repertoires. One major site of BAFF production has been found to be dendritic cells. Hence, in order to interfere with the deletion of autoreactive immature B cells such dendritic cells would have to be localized near the sites of B-cell deletion in bone marrow or the spleen, and would have to be strongly activated to BAFF production to effect rapid maturation of autoreactive immature B cells before they are subjected to negative selection. Such a scenario still needs to be investigated, and the observed partial rescue of B-cell tolerance by T-cell independent antigens may be one experimental way to probe the molecular and cellular requirements for such competition with clonal deletion. In T-cell development, once a/b TcR-expressing thymocytes have been probed as CD41CD81 DP cells for their appropriate fitness to MHC-self-peptide complexes, their first choice, either to become a Th or a Tc cell, has been made. Their further fate, to polarize either into Th1 or Th2 or follicular B helper T or Th17 cells or to Tc1 or Tc2 cells, appears to made as a consequence of a response to MHC-foreign peptide complexes created by introduction or infection of a foreign protein antigen in the secondary lymphoid organs. While Treg cell induction begins in the thymus and appears to be guided by MHCII-self-peptide complex recognition, the rules of their initial selection and their further maturation need to be worked out in detail. The generated repertoires of lymphocytes with BcRs and TcRs, at any time in the life of an individual, are limited by the number of cells generated per day (i.e., 0.1% of the total B- and T-cell pools in a newborn and may be less than 0.001% in an adult), possibly decreasing with age as the frequencies of CLPs and their DJ-rearranged descendants (i.e., FLT3L/IL7/stromal cell-clonal precursors have been seen, at least for the B lineage, to decrease 20- to 100-fold within a few months in mice and from birth to 10 years of age in humans (Ghia et al., 1998, 2000). Since the thymus involutes after sexual maturity, this should also be the case for T-lineage progenitors. This means that in newborn mice with around 5 3 107 B cells as well as T cells, 5 3 104 cells each may be generated per day, while in adult mice, with around 5 3 108 B cells and T cells each, around 5 3 103 B cells and T cells may be generated. Humans, which show striking similarities in the generation of B cells and T cells in bone marrow and the thymus, have approximately 1,000-fold higher numbers of B and T cells and should, therefore, also generate 1,000-fold more cells with newly formed BcRs or TcRs.

FAILURES OF CENTRAL TOLERANCE B Cells

Prolongation of the short half-life of B cells brought about either by a decrease in expression and/or activity of proapoptotic genes or by an increase in pro-survival gene expression (e.g., B lymphocyte-activating factor, BAFF) favors B-cell driven autoimmunity that manifests as SLE-like syndromes (Mackay et al., 1999; Rolink et al., 2002b). Elevated expression of the genes encoding Toll-like receptors (Deane et al., 2007; Shlomchik, 2008) can promote similar autoimmune manifestations.

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Signaling defects can also impair central tolerance induction in immature B cells. For example, the NZM2410/NZW mouse strain-derived z alleles of Sle1 and its sublocus, Sle1b, as well as the orthologous human locus impair B-cell anergy, receptor editing, and deletion (Liu et al., 2007; Kumar et al., 2006). Members of the SLAM family of costimulatory molecules are candidate Sle1b gene products, among which the Ly1081 isoform of the Ly108 gene is highly expressed in immature B cells (Xie et al., 2007). The Ly108.2 allele, but not the SLE-associated Ly1081 allele, sensitizes immature B cells to deletion and RAG reexpression. Hence, Ly108 is a potential regulator of tolerance checkpoints, as it may censor self-reactive B cells by protecting from autoimmunity. When the z allele of Sle1 is combined with the Fas lpr mutation, lymphoproliferative autoimmunity induced by the phosphatidylinositol-3-kinase-AKT-mTOR pathway is induced. As the signaling pathways in immature B cells will become known in greater detail, more genes that interfere with the proper establishment of B-cell central tolerance are likely to be identified.

T Cells

Lowered expression of pro-apoptotic genes and enhanced expression of pro-survival factors (Takahama et al., 1992; Buch et al., 2002; Bouillet et al., 2002) contribute to a shift towards inclusion of self-peptide/self-MHC-reactive T cells in the mature compartments. The mode of antigen presentation appears to play a major role in human T-cell-dependent autoimmune diseases. Juvenile diabetes contributing susceptibility factors include those that affect the amount of insulin expressed intrathymically (McCaughtry et al., 2008). Consequently, thymic overexpresssion of insulin in diabetes-prone NOD mice reduced the incidence of diabetes (Baldwin et al., 2005). Like in the B-cell lineage, mutations in signal transducing molecules, such as ZAP-70, result in autoimmune disease (Smith et al., 1989). It appears that the ZAP-70 mutations alter the balance of positive and negative selection, allowing certain T cells to escape negative selection and then to be activated by their self-ligands in peripheral lymphoid tissues. It seems possible that these mutations could also disturb the balance of positive selection and Treg cell generation in the CD41 T-cell compartments. In analogy to B cells expressing more than one BCR, it was found that expression of a second TCR in addition to an autoreactive TCR can “dilute” the signaling capacity of the autoreactive TCR such that these cells can escape negative selection (Wack et al., 1996).

CONCLUSIONS

By the time newly generated lymphocytes have been deposited in their peripheral, mature compartments of all the T cells and a large part of the B1 cells have been auto-antigenligand selected (hence their repertoire purged) even before a foreign antigen can select “its” specific lymphocytes for a response. This leaves “holes” in the diversity of these repertoires (i.e., their capacities to recognize a foreign antigen). By contrast, those conventional B cells that are ignored by negative and positive selection should be more diversely reactive, although their short half-life in the system limits them. If these conventional B cells could not enter a helper T-cell-dependent response of Ig class switching and v-region hypermutation, the capacity of most of them to recognize the wide world of foreign antigens might well be much too low to elicit a clonally selected, high-avidity immune response with defensive power.

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The author has recently written two review articles (Melchers, 2009; von Boehmer & Melchers, 2010), which have covered parts of the contents of this article. He is the recipient of a Reinhard Kosellek grant of the DFG (ME 2467/1-1).

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3 The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates JIM KAUFMAN

INTRODUCTION

Thus, the essence of the adaptive immune system has been said to be wide diversity, exquisite specificity, and memory. In contrast, the so-called innate immune system described in mammals lacks the capacity to generate completely new receptors within an individual and lacks memory. There is a division of labor among lymphocytes (discussed in chapter 2 and elsewhere in this book). B cells produce antibodies (Ab), which recognize shapes as the primary defense of the adaptive immune system to extracellular pathogens. Some T cells, those that carry T-cell receptors (TcRs) composed of gamma and delta chains ( TcR), also recognize shapes although these shapes are generally found on a (cell) surface. Other T cells, which carry TcRs composed of alpha and beta chains (ab TcR), recognize only major histocompatibility complex (MHC) molecules carrying molecular pieces (peptides or lipids) from within the cell, thus revealing pathogens that have escaped from antibodies to the inside of a cell. Peptides from pathogens such as viruses replicating in the cytoplasm (or contiguous spaces such as the nucleus) are presented by classical class I molecules to T cells bearing CD8 molecules, whereas pathogens such as bacteria or parasites in intracellular vesicles (such as the endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes) as well as the extracellular space, which is topologically equivalent, give rise to peptides that are presented by classical class II molecules to T cells bearing CD4 molecules. Since the CD4 cells recognize antigens from the extracellular space, they also have a role in regulating B cells that are major effectors of the adaptive immune system dealing with extracellular antigens. Nonclassical class I molecules called CD1 molecules carry lipids from intracellular vesicles to be recognized by a special group of CD8 bearing T cells. The MHC molecules are loaded with their antigens by sophisticated pathways composed of various kinds of molecules, some of which are encoded by genes located in the MHC (discussed in detail in many other chapters of this book). Classical class I molecules bind peptides, which are produced in the cytoplasm by the proteasome (under certain conditions augmented by various inducible components), and pumped into the lumen of the endoplasmic reticulum by the transporter associated with antigen presentation (TAP, a member of the ABC transporter family). The class

The pathogens and tumors confronting an individual are so many, so various, so unrelenting, and so lethal, it is a wonder that any of us survive to discuss the issue. The fact we are here demonstrates that overall and in the long run, the defense systems do work. To do so, they are incredibly complex, with a complicated interplay of many different cell types interacting in many specific locations of the body, utilizing hundreds of cell surface and soluble ligands to bind hundreds of different receptors that respond through various signaling systems to thousands of target genes, all levels of which are controlled by many layers of regulation. This extraordinary complexity is presumably the result of the accumulation of many changes appearing step by step over millions of years, each step arising by accident and, in general, being selected by the need to defend the individual organism from the particular pathogens present at the time. Here we will consider the evolution of only one part of the immune response. The so-called adaptive immune response, examined in great detail for humans (Homo sapiens) and mice (Mus musculus), is conceptually simple. The essential cell is the lymphocyte with a cell surface glycoprotein that functions as an antigen receptor, with each antigen receptor having a binding site with a different protein sequence, which is constructed by somatic changes in the genes for the antigen receptor in the lymphocyte (mediated in part by the recombination-activating genes, RAG1 and RAG2). Each lymphocyte (or clone of lymphocytes) bears a different antigen receptor that can specifically recognize a small group of complementary shapes; but, altogether, antigen receptors of the whole population of lymphocytes can bind an extraordinarily large diversity of shapes. Upon recognition of a molecule with a particular shape, only those clones of lymphocytes that recognize that shape will be stimulated to be activated and to proliferate (so-called clonal selection). Upon a second encounter with the same shape, there will be a faster, greater, and qualitatively different immune response.

Jim Kaufman, University of Cambridge Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, Department of Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, United Kingdom.

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HOST DEFENSE: GENERAL

I molecule and TAP are part of a peptide-loading complex (PLC), which includes the dedicated chaperone tapasin (or TAP-binding protein, TAP-BP) in association with ERp57 (a member of the protein disulfide isomerase, PDI, family), the lectin chaperone calnexin and/or calreticulin, and perhaps other molecules (such as endoplasmic reticulum associated aminopeptidase, ERAAP, and PDI itself). Classical class II molecules bind to a trimer of invariant chain (Ii) in the endoplasmic reticulum and are transported (primarily via the cell surface) to a specialized endosomal compartment called MIIC, in which Ii is degraded and then antigenic peptides produced by endosomal proteases are loaded. The class II molecules are stabilized by nonclassical class II molecules that act as dedicated chaperones, DM (as well as DO in some cells) until high affinity peptides are bound. Different CD1 molecules are known to traffic to different endosomal compartments based on signals in their cytoplasmic tails and to be loaded with lipids generated by lipid hydrolases and other degradative enzymes. Of the accessory molecules in these pathways, the genes for some of the inducible components of the proteasome (LMP2 and LMP7), both TAP subunits (TAP1 and TAP2), tapasin, and both chains of DM (DMA and DMB) and DO (DZA and DOB), are located in the MHC. For all immune responses, there is an evolutionary arms race between pathogens and the host (discussed in detail in many other chapters of this book). Pathogens are under selective pressure to change the protective peptides that bind to classical MHC molecules and then stimulate effective T-cell responses, so most pathogens have many variants. In return, and more slowly, the hosts are selected to have multiple MHC molecules, both by having a multigene family and multiple alleles. The most efficient method would appear to be a large multigene family, but each additional MHC molecule increases the possibility that any given T cell will recognize a self-peptide leading to autoimmunity, which is countered (at least in part) by deletion of that T-cell clone in the thymus. Thus, if there are too many MHC molecules, the repertoire of T-cell specificities may be reduced so much that the T-cell response becomes ineffective. In addition, the total number of MHC gene loci may be limited by the number of MHC molecules that can be accommodated on the cell surface. However, there is no obvious reason why the number of MHC alleles should be limited, and, indeed, MHC molecules are among the most polymorphic known, with the human class I locus HLA-B having over 1,000 alleles worldwide. In contrast, the human Ab and TcR loci have large multigene families and further generate variability by gene rearrangement (and other processes) in somatic cells, so they have little allelic polymorphism. Also, the genes involved in processing and loading the peptides (e.g., LMPs, TAPs, tapasin, DM, DO, Ii) are relatively nonpolymorphic in typical mammals, but this becomes a crucial point in the discussion that follows. Although this all sounds quite complicated, the actual adaptive immune system elucidated in typical mammals is, in fact, significantly more complex. Even if we could, it would be beyond the scope of this review to try to explain the emergence of lymphocytes, natural killer (NK) cells, dendritic cells, regulatory T cells, follicular dendritic cells, and the like, with their many interactions, receptors, and signaling cascades. It is far beyond this review to explain the emergence of the various lymphoid tissues, including the thymus and the critical processes of self-tolerance, although some innovative work in this area has been reported recently (Boehm & Bleul, 2007; Bajoghli et al., 2009). It is also beyond our scope to consider all of the various evolutionary novelties and adaptations that have emerged

independently in each of the vertebrate taxa. In this review, we will focus on the question of how the essential components of the adaptive immune system—Ab, TcR, and MHC molecules—arose and began to interact.

THE TIME FRAME: ADAPTIVE IMMUNE SYSTEMS IN JAWED VERTEBRATES AND INVERTEBRATES

There is a long history of speculation about the origins of the adaptive immune system so extensively studied in mammals. There was certainly the possibility of a very ancient origin, given that multicellular organisms have existed for billions of years and presumably always needed protection from pathogens. Moreover, the elements of the adaptive immune system might have evolved originally for other purposes, for instance, development or the nervous system. It was also speculated that the allorecognition system of soft-bodied invertebrates was a primitive form of MHC recognition, although it is now clear that the genes involved in tunicate allorecognition are not closely related to MHC genes (Scofield et al., 1982; De Tomasso et al., 2005). A more recent suggestion is that MHC molecules arose for selection of olfactory specificity in mate recognition (Boehm & Zufall, 2006). Whatever the original function, it appears that the adaptive immune system so extensively studied in mammals arose at the base of the jawed vertebrates over 500 million years ago (Flajnik & Kasahara, 2001, 2010; Litman et al., 2005). In all jawed vertebrates examined thus far, the essential elements of the adaptive immune system are present (Fig. 1). In particular, sharks (as representatives of cartilaginous fish, the most phylogenetically primitive jawed vertebrates) have RAG1 and RAG2, IgM Ab, ab and  TcR, and an MHC containing class I and class II genes, as well as genes for processing molecules such as TAPs, tapasin, and proteasome components (Bernstein et al., 1994; Rast et al., 1997; Ohta et al., 2002; Terado et al., 2003). No such genes (with two possible exceptions discussed below) have been found in more phylogenetically primitive organisms, such as the jawless fish (the two kinds of cyclostomes, the hagfish Eptatretus stoutii and the sea lamprey Petromyzon marinus, among several species examined). With the exception of apparent RAG genes in the sea urchin (Fugmann et al., 2006), no such genes were identified in the genomes of other deuterostomes (two chordates, the cephalochordate amphioxus or lancet Branchiostoma floridae and the urochordate tunicate Ciona intestinalis, and an echinoderm, the purple sea urchin, Strongylocentrotus purpuratus), nor in the genomes of any other invertebrate (Azumi et al., 2003; Rast et al., 2006; Putnam et al., 2008; Holland et al., 2008). These data intensified the contrast between vertebrate and invertebrate immune responses, and led to the concept of a “Big Bang,” during which an adaptive immune system suddenly appeared in jawed vertebrates (Schluter et al., 1999). Despite years of functional experiments with invertebrates pointing to recognition of allo- and xenoantigens, none of the essential genetic components of the adaptive immune system in jawed vertebrates were found in the first complete genomic sequences of invertebrates (C. elegans Sequencing Consortium, 1998; Medzhitov & Janeway, 2002): an insect (the fruit fly Drosophila melanogaster) and a nematode worm (Caenorhabditis elegans). Therefore, the notion that invertebrates lack an adaptive immune system was strengthened, and the question became how invertebrates managed to survive without a system that is essential to jawed vertebrates. Early thoughts included the ideas that short lifespans and/or large numbers of offspring made an adaptive immune system

3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates

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FIGURE 1 An idealized tree, showing the relationships of the animals (and their defense systems). Phyla are indicated above the tree with thick lines; common names of species are indicated above the tree with thin lines; defense systems are indicated.

unnecessary, although many invertebrates do not fit this profile, with examples ranging from cockroaches, which live for 3 years like a mouse to some sea urchins, which live for hundreds of years. Another possibility is that invertebrates expand their innate immunity enough to counter their pathogens, which might be (part of) the answer for the purple sea urchin and amphioxus, whose genomes contain hundreds of Toll-like receptor (TLR) and other pattern recognition genes (Fig. 1) (Rast et al., 2006; Huang et al., 2008). A key concept that has relevance no matter what the defense system might be is that pathogens that overcome all of their hosts may face a bleak evolutionary future, and so, in general, the virulence of pathogens is constrained and guided by the particular defenses that their host(s) evolve. In other words, pathogens will not be selected to overcome an adaptive immune system that is not present in their host(s), and therefore, pathogens of invertebrates will not be virulent in the same way as those facing the adaptive immune system of jawed vertebrates. The idea that pathogens evolve with their host means that invertebrate immune systems are not “worse” than vertebrate immune systems, just different, with different benefits and costs. Indeed, jawed vertebrates now have many pathogens, which evade the adaptive immune system, and, on top of that, have autoimmunity that is a consequence of the adaptive immune system, prompting the characterization of “one

moment of ecstasy followed by 400 million years of agony” (Hedrick, 2004). In fact, at least some groups of invertebrates can have adaptive immune systems based on molecules other than Ab, TcR, and MHC (Fig. 1), as first described in some molecular detail for the fibronectin-related proteins (FREPs) from a mollusk, the snail Biomphalaria glabrata. The snail FREP system consists of a multigene family, whose members are composed of immunoglobulin (Ig) V (variable)-like domains and a fibronectin-like domain, apparently undergo somatic mutation within an individual, and have expression correlated with responses to parasites such as schistosomes (Adema et al., 1997; Zhang et al., 2004). In arthropods (such as the insects, the fruit fly Drosophila melanogaster and the mosquito Anopheles gambia, and two species of the crustaceans water flea, Daphnia), a gene related to the human gene Down’s syndrome cell associated molecule (Dscam) is involved in axon guidance in neurons during development, but then is expressed in adult fat body and hemocytes, the latter involved in various kinds of immune response in insects. Alternative splicing, mainly among the extracellular Ig-like domains, gives rise to a large number of variants, and transient silencing of the Dscam gene in mosquitoes has been reported to lead to decreased resistance to the bacterial pathogens and insect malaria (Watson et al., 2005; Dong et al., 2006; Brites et al., 2008).

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In jawless fish, like lampreys and hagfish, two variable lymphocyte receptor (VLR) genes, composed mainly of leucine-rich repeats (LRRs) give rise to many variants by gene conversion from nearby LRR pseudogenes. These variants are clonally expressed in lamprey and hagfish lymphocytes. Moreover, there is evidence that the VLR-A gene product is secreted by lymphocytes with some properties in common with vertebrate B cells, in response to and then binding to specific antigens. In contrast, the VLR-B gene product is expressed on the surface of other lymphocytes with some properties in common with T cells. Thus, the adaptive immune system of the jawless fish is founded on a different set of antigen-specific receptor molecules, but otherwise has many features in common with the adaptive immune system of jawed vertebrates, suggesting that they may both have descended from a common ancestor (Pancer et al., 2004b; Cooper & Alder, 2006; Guo et al., 2009). Overall, it seems likely that many, if not most, invertebrates will have some form of adaptive immunity based on different molecular systems, which may allow us at last to understand which features of adaptive immunity are essential in a way that was impossible with just a single example. The postulated “Big Bang,” the apparently sudden appearance of all the essential components of our familiar adaptive immune system, could be explained by another, still somewhat controversial phenomenon, the 2R hypothesis. On the basis of fragmentary data on genome sizes, Susumu Ohno (1970) postulated the following scenario: two rounds of genome-wide duplication at the base of the vertebrates, which, for each gene, would have led to one old gene for the previously essential function and three new

genes (so-called paralogs) available for new functions (socalled neofunctionalization). Such a large number of new genes could allow a burst of innovation, leading to new features (e.g., notochord and vertebrae). Support for this idea came from the analysis of Hox gene clusters, for which there is one in the invertebrate amphioxus and four paralogous regions in vertebrates such as mammals (Garcia-Fernandez & Holland, 1994). Analysis of gene families found in the human MHC on chromosome 6 led to the identification of MHC paralogous regions on chromosomes 1, 9, and 19 (Fig. 2), each one of which has a subset of genes thought to have been present in the primordial cluster (Kasahara et al., 1997; Flajnik & Kasahara, 2001, 2010). For instance, class I genes are found in the MHC while the genes for homologs CD1 and neonatal FcR are found on chromosome 1 and 19, respectively. Similarly, the genes for inducible proteasome components LMP2 and LMP7 are located in the MHC, while the gene for MECL1 in the MHC-paralogous region is on chromosome 9. The complement component genes for C4 are found in the class III region of the human MHC on chromosome 6, and the homologs C5 and C3 are found in the MHC paralogous regions on chromosomes 9 and 19. Many other genes, which are not necessarily involved in the immune system, are also found in these MHC-paralogous regions, including the developmental factors Notch1 on chromosome 9, Notch2 on chromosome 1, Notch3 on chromosome 19, and Notch4 on chromosome 6. Thus, the apparently sudden appearance of the adaptive immune system at the base of the vertebrates could be explained by the sudden appearance of many homologous

FIGURE 2 A model for the evolution of the adaptive immune system of jawed vertebrates, from the primordial MHC (left) through two rounds of genome-wide duplication (middle) and subsequent silencing, deletion, and break up to give MHC paralogous regions (right). Black boxes indicate genes based on data from chickens; open boxes indicate genes based on data from other species. Gga and Hsa, chromosomes from chickens and humans, respectively.


genes, which could adapt to new functions. However, the “Big Bang” might have taken as much as 70 million years, based on the apparent divergence times of jawless fish and the armored fish that led to the jawed vertebrates. Fossils of extinct intermediate forms spanning this time show no sudden jump in the body plan, but instead show a stepwise acquisition of new features (Donoghue & Purnell, 2005). Although there is little scope to observe the evolution of the immune system through fossils, it seems likely that the adaptive immune system of jawed vertebrates also evolved in steps, none of which are separated between those organisms still living. However, these steps might still be inferred by the molecular equivalent of fossil traces.

FIRST ANTIGEN-SPECIFIC RECEPTOR WITH IMMUNOGLOBIN VARIABLE REGIONS: THE  T-CELL RECEPTOR

The presence of antigen-specific receptors with highly variable regions (Ab and TcR) is the major difference between the adaptive immune system in jawed vertebrates and the

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many different kinds of innate immunity. Antigen-specific receptors in innate immunity (e.g., TLRs) can have great specificity for particular antigens, which are found in pathogens and not in the host, but in the vertebrates examined thus far they lack both the enormous diversity and the memory of adaptive immunity. The single most important event in the appearance of the adaptive immune system of vertebrates was undoubtedly the appearance of split Ig V domains with the potential of the separated genomic segments to be rejoined in an imprecise manner (Fig. 3). From the first discoveries of split V genes, it was postulated that insertion(s) by a transposable element was responsible, based on the specific recognition by RAG proteins of recombination signal sequences (RSS) flanking the initial DNA cleavage site (Sakano et al., 1979). More recently, the RAG proteins have been shown to have transposase activity, and a class of transposons have been described whose integrases and recognition sequences are closely related to RAG and RSS sequences (Kapitonov & Jurka, 2005). A (relative of a) potential target gene has been described in the sea lamprey, which encodes a molecule related to TcR

FIGURE 3 A model for the evolution of split variable region-containing antigen specific receptors by insertion of transposons (top), with cartoons indicating the location of the CDRs in a V domain (lower right) and the footprint of the CDRs on an MHC molecule (lower left). In the upper left, open circles indicate domains (Ig V type and C type), lines indicate transmembrane and cytoplasmic regions, filled circles indicate D and J segments of the protein, grey bars indicate membranes; one chain is gray to indicate that the molecule may or may not have been a dimer. In the upper right, boxes indicate genes or gene segments, and dotted lines indicate regions introduced by transposon insertion with the heavy black lines indicating RSS. In the cartoons, cylinders indicate a-helices, arrows indicate b-strands, thin lines indicate other conformations of the protein, the thick line indicates peptide bound to the MHC molecule, and the dotted ovals indicate the footprint of the CDRs from the a-chain and the b-chain of a TcR on a class I molecule.

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but with an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail like some NK cell receptors (Pancer et al., 2004a; Yu et al., 2009). Another potential relative of the target gene is the variable region-containing chitin-binding protein (VCBP), found as large diversified families in amphioxus and tunicate genomes (Dishaw et al., 2008). These examples are two of several gene families interpreted to have mixtures of the properties of innate and adaptive immunity, leading to the view of an intimate intertwining of the evolution of innate and adaptive immunity (Kasahara et al., 2004; Flajnik & Du Pasquier, 2004; Litman et al., 2005, 2007; Cannon et al., 2008). A single event that gave rise to all split V genes in jawed vertebrates would mean that one kind of variable antigenspecific receptor came first: either Abs (which recognize three-dimensional shapes and have a high enough affinity to bind soluble antigens),  TcRs (which recognize shapes located on cell surfaces), or ab TcRs (which recognize peptide or other antigens on MHC molecules located on cell surfaces). Although in principle, Ab seem like the simplest system to evolve, the fact that joining of the segments of split V genes only generates diversity at complementarity determining region 3 (CDR3) strongly indicates that ab TcRs were the original variable antigen-specific receptor in jawed vertebrates. It was long a mystery why diversity is found in three (or four) loops of variable antigen-specific receptors, but this diversity is generated by joining split V gene segments (J and D, and even multiple D) only at CDR3 (Fig. 3). As first suggested on the basis of a model (Davis & Bjorkman, 1988), many three-dimensional structures of ab TcR bound to peptide-MHC complexes show beyond a doubt that CDR1 and CDR2 contact mainly the MHC molecule, whereas CDR3 contacts mainly the peptide. The original model envisaged a scenario, in which the primordial TcR-like molecule recognized a collection of peptide antigens bound to a single monomorphic MHC class I molecule, so that the TcR needed only to generate diversity at CDR3 in order to recognize different peptide antigens. Later acquisition of MHC polymorphism and polygenism drove the acquisition of diversity in CDR1, CDR2, and elsewhere, primarily by polygenism of ab TcR. After that,  TcR evolved from ab TcR by losing the requirement for recognizing antigens in the context of an MHC molecule, and then Ab evolved from ab TcR by losing the requirement to stay bound to a cell membrane. However, an argument that Ab or  TcR came first might still be made, based on the fact that CDR3 is in the center of the antigenbinding site and thus might have been particularly important for binding molecular shapes. There are other steps in the emergence of the first variable antigen-specific receptors which remain mysteries, the sequence of events perhaps obscured by further evolution (Flajnik, 2002; Eason et al., 2004; Hsu et al., 2006). TcR locus organization seems relatively stable, perhaps due to the coevolution with the MHC. However, in cartilaginous and some bony fish, certain TcR loci are organized in clusters of V-D-D-J-C, and there are many germline-joined TcR genes expressed early in ontogeny. In contrast, Ab locus organization varies wildly throughout the vertebrates, with various heavy chain and light chain isotypes found in different vertebrate groups. There are even single-chain Ab described in cartilaginous fish (new antigen receptors, NARs) and camels, which are generally thought to be derived rather than an ancestral form. One mystery is that TcR and nearly all Ab are heterodimers of two chains with different numbers of joining segments. The establishment of this pattern must have been very early, because this feature is found in all three classes

of receptor (Fig. 3): one chain (a and  chains of TcR, light chains of Ab) only requiring joining of the V segment with a J segment, and the other chains (b and  chains of TcR, heavy chains of Ab) requiring joining of a J segment to D segment(s) before joining with the V segment. One possibility is that multiple V segments followed by multiple J segments gives the possibility of sequential rearrangements, whereas once V-D-J joining is complete, all J segments are deleted and rearrangement generally halts. Another mystery is the curious arrangement of TcR loci in most jawed vertebrates: two separate loci for b and  chains, but a combined locus for a and , with V, D and J segments as well as C exons upstream of the Ja segments and Ca exons, so that V-Ja joining would delete the D and J segments. The Ab heavy chain loci of (certain) bony fish have a somewhat similar pattern, with the IgM locus embedded in the IgZ locus (although both have J and D segments). An underlying reason for this organization (as well as for germlinejoined TcR expressed early in ontogeny) has been suggested (Danilova & Amemiya, 2009): very young animals have few lymphocytes, and these loci are organized and regulated to restrict diversity to recognition of the most dangerous pathogens during early ontogeny. A third mystery is how and why the RSS are distributed, with the heptamer-nonamer spacing of 12 and 23 nucleotides (roughly the distance of one and two turns of the DNA helix, respectively) flanking different segments in the different Ab and TcR loci. In mammals, these distributions restrict the heavy chains of Ab to one D segment, but allow TcR multiple D segments.

THE PRIMORDIAL MHC MOLECULE: SIMILAR TO A CLASS II MOLECULE

The inference that the first discernible recognition by variable antigen-specific receptors was the recognition of MHClike molecules by TcR suggests that intracellular pathogens were the first major problem tackled by the emerging adaptive immune system. In fact, there are a wide range of MHC-like molecules described in well-studied mammals, ranging from the highly polymorphic peptide-binding classical class I and class II molecules located in the MHC to a host of nonclassical molecules with a variety of polymorphism levels, genomic locations, and functions, some immunological and some not. There are many examples of MHC-like molecules with “closed grooves,” which do not bind peptide or any other ligand (Wilson & Bjorkman, 1998), and some of these molecules function as indicators of stress or infection recognized by T cells (for instance, MIC-A and MIC-B recognition by  TcR) (Li et al., 1999). So, it is easy to imagine the beginning of MHC molecules as such a monomorphic MHC-like molecule appearing on the surface of a cell to signal infection through recognition by a nonvariable TcR-like molecule. The first point at which we have some direct evidence, however slight, is the recognition of peptides on an MHC-like molecule by a TcR molecule with variability in CDR3 generated by genomic joining, and that narrows the list of examples to classical class I, classical class II, and CD1 molecules. Based on structure as well as function, these molecules have all descended from a common ancestor, so which one was first? The case for class I molecules coming first has been made repeatedly (Flajnik et al., 1991; Flajnik & Kasahara, 2001), based on the hypothesis that property of peptide binding evolved first in chaperones such as heat shock protein 70 (HSP70, an inducible gene which is located in the MHC of mammals), and that the exon encoding the chaperone domain was then shuffled to the beginning of a gene encoding a transmembrane protein with an extracellular Ig domain. Other arguments that class I molecules came before


class II molecules have included the nearly ubiquitous tissue distribution of class I molecules compared to the relatively restricted expression of class II molecules (at least in mammals), and the direct function of class I molecules in resisting viruses compared to the indirect function of class II molecules in regulating antibody production. A major argument against this scenario is that the structures of chaperones such as HSP70 have now been solved, and they have no obvious relationship to MHC peptide binding domains (Zhu et al., 1996). Moreover, the argument that class II molecules are now specialized in tissue distribution and function is not necessarily true for the original class II molecules. CD1 molecules have a structure like class I molecules but pick up their antigen lipids from deep endosomal compartments like class II molecules, so they are certainly candidates which could give rise to both. Thus far, CD1 genes have been identified in mammals and birds, but not yet in other vertebrates, and the evidence has been interpreted both for and against an ancient origin of CD1 molecules (Miller et al., 2005; Salomonsen et al., 2005). The hypothesis for class II molecules coming first was made initially on the basis of the domain organization and later on the basis of crystallographic structures and gene organizations (Kaufman et al., 1984; Kaufman et al., 1990). This scenario (Fig. 4) envisages a single gene, which encodes a homodimeric molecule with the domain structure of a class II b chain, and then this gene duplicates and diverges to encode a heterodimeric molecule with the domain structure of a class II molecule. The fact that most class II genes are arranged in a/b gene pairs in opposite transcriptional orientation allows an inversion around a central point but

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with asymmetric breakpoints, which leads in one step to a class I heavy chain gene and a gene encoding the end of a class II a chain, that is the Ig domain with a transmembrane region and a cytoplasmic tail. Finally, a stop codon or splice site mutation leads directly to a b2-microglobulin (b2m) gene. One argument against this simple molecular scenario is that the conformations of the Ig domains in the heterodimers is not the same; the class II a2 domain and b2m are oriented differently from the class II b2 domain and the class I a3 domain, which then requires some explanation that is so far not forthcoming. The class II first scenario assumes that the initial tissue distribution and function of the class II molecule was the same as the class I molecule, except that class II molecules sampled antigen from intracellular vesicles rather than the cytoplasm. Since the extracellular space is topologically equivalent to intracellular vesicles, it was a natural extension of class II function to regulate Ab production by B cells, once they appeared. Regardless of whether it was structurally like class I or class II, the origin of the first MHC-like molecule is of interest. Thus far, no molecule with distinctive feature of MHC molecules, variously described as the “MHC fold,” “peptidebinding domain,” or “open-faced sandwich” has been found in invertebrates. Even though proto-MHC genomic regions have been proposed in organisms as distant from vertebrates as protochordates, nematodes, and fruit flies (Abi-Rached et al., 2002; Danchin et al., 2003; Castro et al., 2004; Danchin & Pontarotti, 2004), no gene with the appropriate features has been identified nearby. Perhaps part of the difficulty might be the fact that there seems to be little or no sequence identity required for generating the MHC fold, as illustrated

FIGURE 4 A model for the evolution of MHC molecules, from a class II b-chain-like homodimer to a class II heterodimer by duplication and divergence, and then to a class I chain with b2m by inversion. On the left, circles indicate domains (open for class II a-chain, gray-filled for class II b-chain, with SS for intrachain disulphide bond), lines indicate transmembrane and cytoplasmic regions, gray bars indicate membranes. On the right, boxes indicate exons (open for coding regions of class II a-chain, gray-filled for coding regions of class II b-chain, black-filled for 3 untranslated region), P indicates promoter region with arrow showing direction of transcription, curved arrow around axis, and lightning bolts indicating an inversion around a central point with asymmetric breakpoints. Adapted from Kaufman, 1988.

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by the fact that such homologous domains in class I and CD1 share only 10% sequence identity, so perhaps the gene is present but just as yet unrecognized.

COEVOLUTION OF GENES LED TO THE PRIMORDIAL MHC

The mammalian MHC is a large complex region (MHC Sequencing Consortium, 1999; Kelley et al., 2005) filled with hundreds of genes, pseudogenes, and repetitive elements, typically divided by recombination into regions including a class I region with class I genes and certain other “framework genes,” a class II region with class II genes as well as proteasome and TAP genes, and in between, a class III region with complement component genes, TNF cytokine genes, and many other kinds of genes. The outer boundaries of the MHC can be extended, so that the tapasin gene is found in the “extended class II region,” while TRIM genes and olfactory receptors might be included in the “extended class I region.” However, there is much variation in the boundaries and detailed organization of the MHC between different mammals with the gene organization of the class II and class III regions most similar but with class I genes varying wildly. For instance (Fig. 5), the human classical class I genes (HLA-A, B, and C) are located in the class I region, two mouse classical class I genes (H-2D and L) are located in the class I region but one (H-2K) is located in between the class II region and the extended class II region, while in the rat all classical class I genes (RT1-A, A1, and A2) are located in between the class II region and the extended class II region. This example is mentioned because it has functional implications discussed below, but more detailed descriptions

and the concepts of the framework in the mammalian class I regions are discussed elsewhere (Amadou, 1999). Outside of mammals, the organization of the MHC is reported to be even more various (Kelley et al., 2005). The MHC of the frog Xenopus tropicalis appears to be much the same size as the human MHC, but the MHC of chickens (Gallus gallus) and many other birds seems particularly small and compact, while some passerine birds are thought to have a much larger MHC. Of particular note is the arrangement among bony fish and at least one frog (Xenopus ruwensoriensis), in which the MHC appears to have fragmented (Du Pasquier & Blomberg, 1982; Sammut et al., 2002; Stet et al., 2003). For instance, in zebrafish (Danio rerio) the class I region includes the proteasome and TAP genes and is completely separated (on different chromosomes) from the class II genes found in at least two loci and from class III region genes, which appear to be scattered throughout the genome (Bingulac-Popovic et al., 1997; Sambrook et al., 2005). In the phylogenetically most primitive jawed vertebrates, the cartilaginous fish (as exemplified by the sharks Triakis scyllium and Ginglymostoma cirratum), the class I, TAP, inducible proteasome, class II and class III region (complement components C4 and factor B) genes are found together in a single MHC locus (Ohta et al., 2000, 2003; Terado et al., 2003), so the organization in X. ruwensoriensis and the bony fish appears to be derived. A simple explanation in both cases would be genome-wide duplication followed by silencing and deletion, as discussed in the next section. Amid all of this variability of the MHC in different vertebrates, at least one general rule has emerged about the relationship of genomic organization and function: the coevolution of polymorphic interacting genes that are closely linked in the

FIGURE 5 A cartoon showing the relationship between coevolution of interacting genes and (recombinational) distance in the genome, as illustrated for the class I and TAP genes of the MHC. (Left) The TAP heterodimer and class I molecule(s) are embedded in a membrane. The number of well-expressed class I molecules ranges from three in humans to one in chickens. Similarly, the specificity of interaction with peptide (as indicated by the number and depth of indentations) increases for TAPs and may decrease for class I molecules from humans to chickens. (Right) Different regions of the MHC labelled in bold are separated by thin vertical lines, genes labeled in normal script are indicated by thick vertical lines, and distance between the TAP genes and the most distant class I gene is indicated by the horizontal arrow. Adapted from Kaufman, 1999; B. Walker et al., submitted.


genome. This idea was first suggested (Germain et al., 1985) to explain a curious situation in mouse class II molecules: both the Ab and Aa genes are polymorphic, but the Eb gene is also polymorphic while the Ea gene is not. A hotspot of recombination separates the A region with the Ab, Aa, and the 5 end of the Eb gene (including the exon encoding polymorphic peptide-binding domain) from the E region containing the nonpolymorphic 3 end of the Eb gene and the Ea gene (as well as other genes). The Ab and Aa chains within one haplotype (but rarely from different haplotypes) will interact to make a class II heterodimer, whereas the nonpolymorphic Ea chain will interact with Eb chains from all other haplotypes. The fact that the Ab and Aa genes are rarely separated by recombination means that optimal combinations can coevolve and remain together, whereas the frequent separation of Eb and Ea genes means that one (in this case, the Ea gene) evolves to be an “average best fit” to interact with all of the alleles of the other (in this case, the Eb gene). Once this concept is recognized, examples of it can be seen elsewhere. For instance, of the human class II genes, the DQA and DQB genes are rarely separated by recombination and coevolve as polymorphic partners in haplotype-specific manner; the same is true of DPA and DPB, but DRA is nonpolymorphic and the DRA protein interacts with the proteins encoded by multiple DRB alleles and loci. Human b2m interacts with all classical class I alleles and loci, as well as many different MHC-like proteins including those encoded by CD1 loci gene; the b2m gene is monomorphic and located on a different chromosome from the MHC and class I-like genes. Similarly CD4, CD8, and TcR loci are all nonpolymorphic and located on different chromosomes from the MHC. However, other pressures can cause interacting proteins located on different chromosomes to become polymorphic, a situation which can lead to serious problems of interaction. For instance, NK cell receptors encoded by the killer inhibitory receptor (KIR) complex are individually polymorphic as well as the multigene family expanding and contracting to give “haplotype polymorphism,” all of which are presumably selected by interactions with pathogens. Some combinations of KIR and MHC haplotypes confer poor responses to infectious pathogens, susceptibility to autoimmunity, and reproductive difficulties (Parham, 2005; Trowsdale & Moffett, 2008). In other words, in order for two interacting genes to both become polymorphic without difficulty, in general they must be genetically closely linked so that they can be selected to coevolve. The same concept applies to the genes within the MHC, most clearly shown for the presentation system of class I molecules (Fig. 5). In typical (placental) mammals, the various components involved in peptide processing and loading are located far away from the classical class I genes and are not polymorphic. For instance, in humans, the genes for two inducible components of the proteasome and TAPs are located in the class II region, tapasin is located in the extended class II region, the third inducible proteasome component, ERp57, calnexin and calreticulin are located on different chromosomes from the MHC, and the few variants described for these genes are not reported to result in functional differences. In mice, there is one classical class I gene located in the H-2K region between the class II and the extended class II region, but there is at least one other classical class I gene far away in the class I region (the H-2D region), and the TAP, tapasin, and proteasome genes are nonpolymorphic. However, in laboratory rats (Rattus norvegicus), the only classical class I genes are located in the RT1A region between the class II and the extended class II region, and there is some haplotype-specific coevolution between the class I gene(s) and the TAP2 gene (Momberg et al., 1994; Joly et al., 1998; Hurt et

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al., 2004). There are two lineages of TAP2, with the TAP2A alleles allowing transport of peptides with any amino acid at the C-terminus, but the TAP2B alleles allowing transport of peptides with a hydrophobic amino acid at the C-terminus. This specificity is generally mirrored in the closely linked class I gene(s), and mismatch of class I and TAP2 specificity found in a natural recombinant led to changes in class I maturation and presentation of peptides to cytotoxic T cells (CTLs). The Syrian hamster (Mesocricetus auratus) may also have this structural and functional oligomorphism (Lobigs et al., 1999). In chickens, the classical MHC (the BF-BL region) has two classical class I genes, but only one is expressed at a high level, apparently due to coevolution with the TAP1, TAP2 and tapasin genes (Kaufman et al., 1995, 1999a, 1999b). Recombination across the chicken MHC is very rare, with no recombinants recovered from thousands of deliberate matings and only one “natural recombinant” known among experimental chicken lines (although there is some evidence for gene conversion or double reciprocal recombination of short stretches within the MHC). No proteasome genes have been found (yet), but the TAP1, TAP2, and tapasin genes are located very close to the two classical class I genes and are highly polymorphic and moderately diverse in sequence, with each haplotype examined (except the natural recombinant) having a unique allele differing in roughly 1% of amino acid residues (Walker et al., submitted; van Hateren et al., submitted). Moreover, they are polymorphic in function, working with a dominantly expressed class I gene in a haplotype-specific manner. Peptide translocation assays show TAP specificity at least three peptide positions, which matches or even exceeds the peptide-binding specificity of the single dominantly expressed class I molecule (Fig. 5). Although such interactions have not yet been shown directly for tapasin, the natural recombinant has been used to show that class I maturation is haplotypedependent through class I residues known in mammals to be important for interaction with tapasin. Most importantly, the coevolution between TAPs, tapasin, and the class I genes leads to a single dominantly expressed class I molecule, which, in turn, has functional consequences (Kaufman et al., 1999a, 1999b; Wallney et al., 2006; Walker et al., submitted; van Hateren et al., submitted). As an example (Fig. 6), peptides isolated from cells of

FIGURE 6 A cartoon showing that coevolution between the peptide-translocation specificity of TAP and the peptide-binding specificity of class I molecules leads to a single dominantly expressed class I molecule, as illustrated with the chicken MHC haplotype B4. Thin lines indicate peptides, a minus sign within a circle indicates negatively charged residue, a plus sign within a circle indicates positively charged residue, a circle indicates a hydrophobic residue. Adapted from Kaufman et al., 1999b.

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the B4 haplotype overwhelmingly have negative charges at anchor residues (aspartic acid and/or glutamic acid at positions 2 and 5 and glutamic acid at position 8), and models of the dominantly expressed class I molecule reveal positively charged residues in the peptide-binding domain well positioned to bind such peptides, which are totally lacking in the poorly expressed molecule. The TAP1 in B4 has positive charges in three positions, which are negatively charged in other haplotypes, and overwhelmingly, peptides bearing negative charges at positions 2 and 5 and glutamic acid at position 8 are translocated in B4 cells. In general, the peptide-binding specificity of the dominantly expressed class I molecule converges (or is exceeded by) the peptidetranslocation specificity of the TAP, which means that class I molecules encoded by other loci or haplotypes will receive few, if any, peptides, and thus, will not be utilized much by the adaptive immune system. The important functional consequence of a single dominantly expressed class I molecule is that an MHC haplotype may not be able to respond effectively to a particular pathogen if the class I molecule fails to bind a peptide that confers protection, as compared with a multigene family of MHC molecules, one of which is likely to find a protective peptide (Kaufman et al., 1995; Kaufman, 2008). In fact, the chicken MHC is known for strong associations with resistance to infectious pathogens as well as responses to vaccines, whereas the human MHC is known for strong associations with autoimmune diseases, with the few associations with infectious diseases being relatively weak, and in some cases known to involve interactions with NK cells rather than peptide presentation to T cells. Although a wide range of critical tests are required to establish that this apparent difference is real, it is at least clear for a few examples that peptide presentation by the single dominantly expressed class I molecule can determine whether a chicken lives or dies. Although the data is still patchy, many, if not most, nonmammalian vertebrates examined have the salient features for the class I system first discovered for the chicken MHC: one class I gene expressed at a high level, with polymorphic TAP and tapasin genes next to the class I gene(s), and for one fish, strong genetic associations with resistance to infectious pathogens (Kaufman, 1999). For example, the duck (Anas platyrhynchos) has five class I genes, of which only one is well expressed, adjacent to the polymorphic TAP genes (Mesa et al., 2004; Moon et al., 2005). Similarly, a single classical class I gene is located next to the TAP genes in the frogs Xenopus laevis and X. tropicalis (Ohta et al., 2006). A bony fish, Atlantic salmon (Salmo salar) has a single classical class I gene, with inducible proteasome components, tapasin, and (at least one) TAP genes close by (Grimholt et al., 2002; Lukacs et al., 2007), and cartilaginous fish as exemplified by sharks (Triakis scyllium and Ginglymostoma cirratum) have one (and in some haplotypes, two) class I genes with the proteasome components and TAP genes closely linked (Ohta et al., 2002). The situation for class II genes is less clear, although many birds have elements of the simple class II system of chickens (Jacob et al., 2000; Salomonsen et al., 2003). Thus far, there is little relevant functional data available for nonmammalian vertebrates, but for X. laevis there is an association of the MHC with resistance to a bacterial pathogen (Barribeau et al., 2008), and for Atlantic salmon there are strong genetic associations of the class I region with resistance to a virus and the class II region with resistance to a bacterial pathogen (Grimholt et al., 2003). Taking this data together, there appear to be at least two strategies of MHC genomic organization leading to differences in function. The salient features of the chicken MHC (and those of most nonmammalian vertebrates) are

most likely to represent the ancestral organization, with the typical mammalian (and some nonmammalian vertebrate) MHC organizations being derived. In this view, a large inversion occurred in the lineage leading to mammals, bringing the class III region in between what we now call the mammalian class I and class II regions, but separating the TAP, tapasin, and proteasome genes from the class I genes in the process. In fact, it appears that this event took place in the lineage leading to placental mammals, since at least one marsupial, the American opossum (Monodelphis domestica), has the MHC organization typical of nonmammalian vertebrates (Belov et al., 2006). Independent events leading to the breakup of the ancestral organization of class I, TAP, tapasin, and proteasome genes may have occurred several times. For instance, another marsupial, the Tamar wallaby (Macropus eugenii), has TAP genes in the MHC, but the classical class I genes located at the ends of several other chromosomes (Deakin et al., 2007; Siddle et al., 2009). Some passerine birds have been shown to have a multigene family of expressed classical class I genes, for which individual alleles in warblers and sparrows (great reed warbler Acrocephalus arundinaceus and house sparrow Passer domesticus) confer resistance to infectious pathogens (Westerdahl et al., 2000, 2005; Bonneaud et al., 2006). The genomic sequence of another passerine bird, the zebra finch (Taeniopygia guttata), appears to have classical class I and TAP genes on different chromosomes. The class II B genes of a bony fish, the threespined stickleback (Gasterosteus aculeatus), form a multigene family with expansion and contraction, with evidence that intermediate numbers of loci/alleles are important in mating and pathogen resistance (Wegner et al., 2003). Finally, there may be yet other strategies for MHC organization and function, of which there are two examples that express a large number of classical class I genes at a low level (Sammut et al., 1999; Persson et al., 1999; Miller et al., 2002), with only one expressed class II gene in a salamander, the axolotl (Ambystoma mexicanum), and no obviously expressed class II gene in a bony fish, the Atlantic cod (Gadus morhua). What could be the reason for the presence of a single dominantly expressed MHC molecule in so many nonmammalian vertebrates when a multigene family of MHC molecules allows a much broader response to pathogens (albeit potentially at the expense of greater autoimmunity)? The answer may be that this is the way in which the class I part of the primordial MHC first got started (Kaufman et al., 2008). The primordial class I, TAP, tapasin, and proteasome genes presumably all had functions other than the ones they now fulfill, and then evolved to interact. The simplest and most efficient way for such coevolution to take place would be for the genes to be closely linked, in much the same way that the class I, TAP, and tapasin genes have coevolved as haplotypes in the chicken MHC. Conceptually, this is the same idea proposed for the evolution of metabolic pathways at the beginning of life. The natural recombinant in the chicken MHC may be one example of catching such a coevolution in the act (van Hateren et al., submitted). The Scandinavian B19 haplotype (also known as B19var1) is a recombinant of the B12 and B15 haplotypes, with the site of recombination in the middle of the TAP2 gene. Thus far, all the genes sequenced from BG1 to the middle of TAP2 are identical between B19 and B12, while the end of TAP2 and the dominantly expressed class I gene BF2 are nearly identical between B19 and B15. Transfection of BF2 cDNAs from B15 and B19 into B15 cells showed that BF2*15 matured more rapidly and more completely than BF2*19. Given that the class I peptide motif and the translocation specificity in B15 and B19 cells have the same anchor residues, the only likely


mismatch is with tapasin: BF2*15 interacts well with the tapasin in B15 cells, but BF2*19 expects B12 tapasin. There are only eight differences between BF2*15 and BF2*19, of which five are changes back to the residues found in the BF2*12. Some of these residues are implicated in human class I molecules with tapasin interaction, and swapping these residues between the two molecules shows that they are enough to increase or decrease maturation. Thus, small changes can allow coevolution between genes in a newly formed haplotype, and it is precisely such small changes that are envisaged to allow the primordial class I, TAP, and tapasin genes to coevolve in order to work together as a team. Does this scenario of coevolution fit with the speculations outlined above that the first variable antigen-specific receptor was a TcR recognizing a class II-like molecule? If a class II-like gene came first, then the principles are likely to be the same, although there may have been much repurposing from a molecule sampling the intracellular vesicles for class II-dependent killer cells to a molecule primarily (although not exclusively) involved in regulation of antibody responses. Consistent with coevolution leading to a single dominantly expressed class II molecule in chickens, there are strong associations of the MHC with responses to inactivated vaccines, a single nonpolymorphic class II A gene located outside of the MHC, and two classical class II B genes, only one of which is well expressed, and polymorphic DM genes nearby with which to coevolve in a haplotype-specific manner (Jacob et al., 2000; Salomonsen et al., 2003; Kaufman et al., 2008). Overall, much remains to be clarified about class II genes throughout the rest of the vertebrates, but consistent with the coevolution model, the class II-dependent genetic associations with antibody responses in mammals (immune response or Ir genes) are relatively weak, while associations of the class II region in Atlantic salmon with responses to bacteria are very strong (Benacerraf & Germain, 1978; Grimholt et al., 2003). Whether TcR was the first variable antigen-specific receptor may also involve coevolution within the primordial MHC, as explored in the next section.

THE PRIMORDIAL MHC: THE BIRTHPLACE OF THE ADAPTIVE IMMUNE SYSTEM OF JAWED VERTEBRATES

What did the primordial MHC look like? In various invertebrates, many of the genes in and around the mammalian MHC, including the complement components of the class III region, are found close together in regions dubbed the “proto-MHC” (Abi-Rached et al., 2002; Danchin et al., 2003; Castro et al., 2004; Danchin & Pontarotti, 2004). However, with the possible exceptions of a TcR-like and perhaps a TAP-like gene (Uinuk-ool et al., 2002, 2003; Pancer et al., 2004a), no invertebrate genes have been identified as obvious direct ancestors of the essential components of the adaptive immune system. Nevertheless, genomes in vertebrates provide clues, which suggest that the genes for these essential components were originally all in the same region, allowing them to coevolve into a functioning system. As described above (Fig. 2), MHC paralogous regions have been identified in mammals and are presumed to have arisen from two rounds of genome-wide duplication at the base of the vertebrates (Kasahara et al., 1997; Flajnik & Kasahara, 2001, 2010). They are far from perfect copies of each other, with much evidence of regions breaking up, pieces breaking off, genes deleted, and families expanded, and most importantly, members of gene families with new functions. In humans, the classical class I (and some nonclassical) class I genes are located in the MHC on chromosome 6, the nonclassical class I

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genes for CD1 molecules are located in the MHC-paralogous region on chromosome 1, and the nonclassical class I gene encoding an Fc receptor is located in the MHC-paralogous region on chromosome 19, but no class I-like gene is located in the MHC-paralogous region on chromosome 9. Also, homologs of certain complement component genes are found in the MHC-paralogous regions (C3, C4, and C5 on chromosome 9, 6, and 19), but the last homolog (the protease inhibitor a2-macroglobulin) is found in the NK receptor cluster (NKC) on chromosome 12, rather than in the MHCparalogous region on chromosome 1. These are just a few of the many examples taken to illustrate regions breaking up and/or pieces breaking off (a2-macroglobulin in the NKC), genes deleted (class I-like genes on chromosome 1), families expanded (many class I genes on chromosome 6, several CD1 genes on chromosome 1), and genes with new functions (Fc receptor gene on chromosome 19). Examination of the chicken MHC shows some of the same genes (Fig. 2), but many expected genes are located elsewhere in the genome (or deleted from the genome entirely) and other unexpected genes are present, overall giving an alternative view of the dynamics of the MHC paralogous regions and allowing some insight into the primordial MHC (Kaufman et al., 1999a; Salomonsen et al., 2005; Rogers et al., 2005, 2008). For instance, a pair of lectin-like genes were identified in the original sequence of the chicken MHC (the BF-BL region) and, on the basis of genomic organization and expression patterns, shown to correspond to pairs of genes in the NKC (particularly the NK receptor gene NKR-P1 and its ligand LLT1 in humans, or Clr in mouse and rat). In contrast, only two lectin-like genes were identified in the chicken region corresponding to the NKC. Later, two genes encoding chicken CD1 were located immediately adjacent to the sequenced portion of the chicken MHC. The simplest explanation for the presence of both NK receptors and CD1 in the chicken MHC is that they were both present in the primordial MHC before the two rounds of genome-wide duplication, and were silenced in different genomic regions in the lineage leading to birds versus the lineage leading to mammals. With this idea in mind, traces of other gene families present in the MHC become understandable. An example recognized early on (Kasahara et al., 1997) is the presence of lectin-like genes CD23 and DC-SIGN in the MHC-paralogous region on human chromosome 19 and many other lectin-like genes, expressed in NK/T cells and myeloid cells, located in the NKC on human chromosome 12. Similarly, the lymphocyte receptor region (LRC) on human chromosome 19 contains many homologous genes, including the KIR genes expressed primarily on NK cells, and a variety of homologs (including an Fc receptor) expressed on cells of the myeloid lineage (Martin et al., 2002). A homolog called NKp30, a member of the natural cytotoxicity receptor (NCR) genes, is located in the human MHC (Moretta et al., 2001). Conversely, two class I-like Mill genes are found just outside the LRC in mice (Kasahara et al., 2002). This fits well with the suggestion that the regions syntenic to the MHC, the NKC and the KIR genes were originally next to each other based on the arrangement in the tunicate Ciona intestinalis (Olinski et al., 2006; Zucchetti et al., 2009). A most intriguing example is a gene related to TcR genes found on the edge of the MHC of the frog X. tropicalis (Ohta et al., 2006). Such examples suggest that all of these gene families were located together in the primordial MHC before the two rounds of genome-wide duplication. The two concepts of paralogous regions and coevolution taken together make a powerful prediction (Kaufman et al., 2008). The presence of MHC, NK receptor, and TcR gene

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families in the same genomic locations suggests that genes for receptors and ligands originated in the same genomic region (Fig. 2), presumably to allow coevolution so that they would function together, much in the same way as was outlined above for coevolution of disparate genes to form the class I presentation pathway. In this view, the adaptive immune system of jawed vertebrates arose in a single genomic region, the primordial MHC, which has been breaking apart ever since.

CONCLUSIONS

Some 50 years of research have shown that the adaptive immune system of jawed vertebrates appeared once at least 500 million years ago. Many important events involve drastic changes in the genome, including transposon insertion to create split V genes, horizontal transfer of recombination activating genes (RAGs), inversions and translocations of DNA to create MHC genes, genome-wide duplications to create genes for new functions, and further reorganizations to alter MHC function. Throughout the evolution of this adaptive immune system, coevolution of linked genes was important for different genes to begin to work together, a process still evident in the coevolution of antigen presentation systems in most nonmammalian vertebrates. The best guess is that the birthplace of this adaptive immune system was the primordial MHC, which has been falling apart ever since. Thanks to John Chattaway, Aimee Parker, Andrew Chan, and Hannah Siddle for reading the manuscript, and to the Wellcome Trust (089305/Z/09/Z) for support.

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4 Host Defense (Antimicrobial) Peptides and Proteins LAURENCE MADERA, SHUHUA MA, AND ROBERT E. W. HANCOCK

DEFENSINS

INTRODUCTION

Defensins are a family of small cationic peptides ranging from 3 to 5 kDa in size and possessing a -sheet structure containing three intramolecular disulfide bonds between six conserved cysteine residues (Froy, 2005). Beyond this pattern, peptides within the defensin family are highly variable. In mammals, over 50 defensin members have been identified (Yang et al., 2001). Defensins members are categorized into three families based on their sizes and patterns of disulfide bonding: the -, -, and (among the higher primates only) -defensins (Brown et al., 2007). Humans only possess the - and -defensin subfamilies (Brown et al., 2007). To date, six -defensins, HNP1–4 and HD5–6, and six -defensins, hBD1–6, have been identified in humans (Yamasaki & Gallo, 2008; Brown et al., 2007).

The innate immune response is an ancient defense system against pathogens that is evolutionarily conserved among plants, invertebrates, and vertebrate animals. It is characterized by a rapid, highly effective response against foreign microbial invaders. The effectiveness of innate immunity stems from many multifaceted processes working in concert to rapidly eliminate a threat. The detection of pathogens by sentry cells immediately leads to the triggering of inflammation, the effector arm of innate immunity. Inflammation leads to increased trafficking of immune cells, including phagocytes, antigen-presenting cells, and mast cells, toward the site of infection where they initiate microbial killing or further potentiate the inflammatory response, reinforcing cell recruitment. Pathogen clearance is mediated by a complex set of strategies, ranging from the ingestion of microbes by phagocytes to the production of antimicrobial molecules, including reactive chemical species or lytic compounds. Another strategy is the production of cationic host defence (antimicrobial) proteins and peptides, compounds that play an important role in innate immunity, not only as antimicrobial agents, but also as immune regulators.

Localization and Regulation

Defensins are found widely within the body depending on the family and type of defensin involved. In general, defensins are found in the granules of leukocytes and in the epithelia of the human body (Menendez & Finlay, 2007). HNP1–4 -defensins are prominent in the granules of neutrophils, from which they were first isolated (Yang et al., 2001), and also exist in the granules of other leukocytes including monocytes, lymphocytes and natural killer (NK) cells, and in intestinal Paneth cell granules, where they are secreted into intestinal crypts (Froy, 2005). The enteric -defensins HD5 and HD6 are found in the granules of epithelial cells, including Paneth cells and the cells of the urogenital tract. In contrast, -defensins are expressed in epithelial cells that line the esophagus, intestines, skin, kidney, pancreas, and gut (Brown et al., 2007), whereas hBD5 and hBD6 are found in the epididymis and airway epithelia (Froy, 2005). Unlike -defensins, epithelial -defensins are not stored in cytoplasmic granules (Brown et al., 2007). The production of defensins by the body is variable and depends on many factors, including the defensin class, cell type, and environmental stimuli. -Defensins are constitutively expressed in cell granules (Menendez & Finlay, 2007). In contrast, many -defensins are inducible by a variety of environmental factors, including microbial components and/or host inflammatory mediators, indicating that -defensins might play a role in the host antimicrobial or inflammatory response (see Table 1).

CATIONIC HOST DEFENSE PEPTIDES

Cationic host defense peptides (HDPs) encompass a diverse family of peptides that play a role in the antimicrobial innate immune response of many species. They are generally defined as peptides 12 to 50 amino acids long, containing up to 50% hydrophobic residues and 2 to 9 positively charged arginine or lysine residues (Brown & Hancock, 2006). The two major classes of HDPs found in mammals are the defensins and cathelicidins, which are produced by immune cells, including leukocytes and epithelial cells. Originally thought to play a direct antimicrobial role against pathogens, it has become evident that cationic peptides also modulate the many functions of innate immunity (Fig. 1). Laurence Madera, Shuhua Ma, and Robert E. W. Hancock, Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.

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FIGURE 1 The multiple roles of host defense peptides in the immune response.

Numerous studies have shown -defensin induction in epithelial cells in response to bacterial infections (Froy, 2005; Menendez & Finlay, 2007). Specific bacterial components, including lipopolysaccharide (LPS) of gram negative bacteria and peptidoglycan of bacterial cell walls, lead to production of -defensins (Menendez & Finlay, 2007). In addition, host factors produced in response to infections play a large role in -defensin induction, especially cytokines that are cell-to-cell signalling mediators essential in the coordination of the immune response. Interleukin 1 (IL-1) and tumor necrosis factor  (TNF-), cytokines that promote acute inflammation, are strong inducers of -defensin expression (Yamasaki & Gallo, 2008). Supporting the link between -defensin induction and infection is the finding that regulation of -defensin expression is dependent on signaling pathways activated in an immune response. In epithelial cells, hBD2 induction is dependent on the transcription factors NF-B and AP-1, both essential for the transcription of many inflammatory mediators (Froy, 2005). This is supported by the finding that hBD2 expression in an atopic dermatitis model is downregulated by IL-10, a suppressor of NFB activity (Menendez & Finlay, 2007). The induction of hBD3 is also dependent on the activation of the mitogen activated protein kinase (MAPK) signalling pathways involving p38 kinase and ERK-1/2, central mediators of many cellular processes including inflammation (Menendez & Finlay, 2007). -Defensins can be induced by TLRs, which are a major subset of pathogen recognition receptors that detect microbes. For example, in the lung, activation of TLR2, TLR3, TLR4, and TLR9 can lead to hBD2 and hBD3 expression, whereas in the intestine,

activation of TLR4 and the complex of TLR2 and TLR6 leads to hBD2 induction (Froy, 2005). There is a great deal of evidence that -defensin regulation is linked to the detection and progression of a microbial infection. However, the variety of roles that defensins play in the immune response is far less understood.

Antimicrobial Activity

Defensins have generated a large amount of interest due to their modest in vitro antimicrobial activity against a wide range of microorganisms, including enveloped viruses, fungi, and bacteria (Brown et al., 2007). This direct antimicrobial is likely very significant under circumstances where defensins are found in high concentrations, such as the crypts of the intestine and the granules of neutrophils where milligram per milliliter concentrations are found. Defensins also possess antiviral properties, such as against adenovirus, papilloma virus, herpes simplex virus (HSV), and human immunodeficiency virus (HIV) (Yamasaki & Gallo, 2008), and have modest antimicrobial activity against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (Chen et al., 2006). hBD3 is more potent and has a broader spectrum of antimicrobial activity against organisms including fungi and gram-positive bacteria, such as Staphylococcus aureus, possibly due to its higher net charge relative to hBD1 and hBD2 (Brown et al., 2007). Although the mechanism of antimicrobial activity by defensins has not been fully elucidated, it is believed that the positive charge of defensins allows them to bind to negatively charged molecules on microbial membranes, such as LPS and lipoteichoic acid (LTA) in gram-negative

4. Host Defense (Antimicrobial) Peptides and Proteins

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TABLE 1 Human cationic host defense peptides and their regulation and functions in the immune response Host defense peptide

Regulation

Immune functions

-Defensins

Constitutive

Binds and neutralizes bacterial toxins

-Defensins

Inducible in epithelia by bacterial infection: S. aureus, Campylobacter jejuni, H. pylori, and Mycobacterium bovis Inducible by LPS and peptidoglycan Inducible by inflammatory cytokines IL-17, IL-1, and TNF- Suppressed by anti-inflammatory cytokine IL-10

Chemotactic for T-lymphocytes, immature dendritic cells, mast cells, and monocytes Promotes chemokine production and mast cell degranulation Promotes production of inflammatory cytokines Promotes T-cell proliferation Promotes wound healing and regulation of the extracellular matrix

hCAP18/LL-37

Constitutive in leukocyte granules; Inducible in epithelia by bacterial infection H. pylori Inducible in epithelia by inflammatory cytokines IL-1 and IL-6 Suppressed by Shigella.

Chemotactic for monocytes, neutrophils, and T-lymphocytes Promotes chemokine production and induces mast cell degranulation Inhibits neutrophil apoptosis Suppresses of LPS-induced inflammation Regulates dendritic cell maturation and TH polarization of adaptive immunity; Promotes wound healing and angiogenesis

and gram-positive species respectively. This defensin interaction with the membrane leads to translocation across the outer membrane and access to this membrane where either the disruption of membrane integrity results in increased membrane permeability or action on other targets leads to death. The importance of defensin-mediated bacterial killing during an immune response is a point of contention, except under the previously mentioned circumstances when defensin concentrations are very high, since it has been demonstrated that in vitro defensin antimicrobial activity is dramatically reduced in a physiological environment. For example, the addition of 100 mM NaCl, approximately equivalent to the concentration found in airway fluid and blood, reduces hBD2 activity 5-fold to 500-fold and completely abolishes hBD1 activity against gram-negative bacteria (Bowdish et al., 2006). In addition, defensins are sensitive to the presence of divalent cations. It has been shown that -defensin activity is compromised by Mg21 and Ca21 at concentrations as low as 0.5 mM (Bowdish et al., 2006) even though many bodily fluids (including airway fluids, plasma, and sputum) contain Mg21 and Ca21 at concentrations of around 1 mM. Under many circumstances the physiological concentrations of defensins are too low to manifest any significant antimicrobial activity. hBD1 displays antimicrobial activity at concentrations around 1 μg/ml only under low ionic strength conditions that do not exist in vivo; however, the concentrations of hBD1 found in bronchoalveolar fluid is 10-fold lower, at 0.1 μg/ml (Bowdish et al., 2005). Thus, it is likely that defensins play a direct antimicrobial role in a limited number of environments, such as in the phagocytic granules of neutrophils and intestinal crypts where defensin concentrations can reach up to milligrams per milliliter, overcoming any ionic antagonism, or under circumstances where the ionic strength is relatively low (Froy, 2005). The antiviral effects of human defensins are far less understood. Defensins have been postulated to bind to viral

surface molecules, potentially inhibiting their entry and invasion mechanisms. Human -defensins have been shown to bind to viral glycoproteins, molecules integral to the viral invasion process, such as HSV glycoprotein B and HIV gp120 (Yamasaki & Gallo, 2008; Klotman & Chang, 2006). However the antiviral activity of defensins also varies with the physiological environment. For example, HNP-1 is able to directly neutralize HSV and HIV virions in a serum-free environment, an effect that is abolished with the addition of serum (Klotman & Chang, 2006). Interestingly, in the presence of serum, HNP-1 acts on the host cell, inhibiting HIV viral growth following the invasion and reverse transcription stages. Indeed, defensin antiviral activity can be attributed to many cell-mediated effects, including the regulation of host surface receptors used by viruses for invasion, or modulating cellular signaling pathways required for viral growth (Jenssen et al., 2006; Klotman & Chang, 2006). Although in physiological conditions and peptide concentrations the ability to act directly against pathogens is limited, recent studies show that defensins are still able to coordinate many aspects of the host immune response.

Immunomodulation

The pathogenesis of a microbe can be divided into a series of stages, which include the initial invasion by the microbe, the inflammatory innate immune response, the transition into the adaptive immune response, and the resolution of infection (or the establishment of chronic infections, or death of the host). It is becoming evident that defensins play a major role in coordinating each of these processes (see Table 1). Defensins have potent neutralizing functions against certain bacterial toxins, limiting pathogenesis at the early stages of infection. For example, HNP1–3 are able to bind and sequester the lethal factor (LF) of Bacillus anthracis from host cells, thus preventing potential cell death, and can also competitively inhibit diphtheria toxin and P. aeruginosa exotoxin A, preventing the ADP-ribosylation of host elongation factor-2 (Brown et al.,

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2007). HNP1-3 is also able to inhibit the binding of S. aureus staphylokinase to host plasminogen, preventing fibrinolysis and the spread of infection (Brown et al., 2007). Defensins also play a role in cell recruitment, influencing the ability of immune effector cells to migrate to the site of infection, which is a mainstay of the innate immune system and impacts all subsequent responses. The -defensins are directly chemotactic for naïve CD4 and CD8 T-lymphocytes as well as immature dendritic cells and monocytes (Yang et al., 2001). hBD1 and hBD2 are also chemotactic for immature dendritic cells and memory T-lymphocytes, while hBD3 is capable of attracting immature dendritic cells, mast cells, and monocytes (Menendez & Finlay, 2007; Yang at al., 2001). Similarly, hBD4 can directly chemoattract mast cells and monocytes (Froy, 2005). As defensins are secreted (e.g., through phagocyte degranulation) or induced at infectious sites, it is likely that defensins enhance the ability to chemoattract immune effector cells to the site of infection. In addition to directly promoting cellular mobilization, defensins induce the production of cytokines and chemokines, which potentiate cell recruitment. For example, -defensins can induce the production of neutrophil chemokine IL-8 in alveolar macrophages and lung epithelial cells as well as cause the degranulation of mast cells and histamine release, promoting permeabilization of blood vessels and further cell recruitment (Yang et al., 2001). hBD3 and hBD4 can also cause degranulation of mast cells (Menendez & Finlay, 2007). Defensins also induce the production of inflammatory cytokines by immune cells. For example, -defensins induce the production of proinflammatory cytokines IL-6 and IFN (gamma interferon) by T lymphocytes and IL-1 and TNF- by human monocytes (Froy, 2005). The -defensins hBD1 and hBD4 induce keratinocytes to produce IL-18, a cytokine that promotes the recruitment of neutrophils and the activation of cell-mediated immunity (Yamasaki & Gallo, 2008). Defensins can also promote the production of regulatory cytokines to balance the inflammatory response. In particular, -defensins stimulate the production of the immunosuppressive cytokine IL-10 in T lymphocytes (Froy, 2005). Defensins also contribute to the adaptive immune response by influencing lymphocyte activity. For example, in murine models, HNPs enhance the proliferation of CD4 T-lymphocytes stimulated with CD3 activating antibodies (Lillard et al., 1999). Correlated in part with this finding, HNPs enhance antigen-specific humoral and cellular responses in mice treated with antigen, indicating an adjuvant role for defensins (Bowdish et al., 2006). Defensins also contribute to wound healing; in particular, hBD2–4 promote the migration of keratinocytes and enhance their proliferation, suggesting that -defensins promote reepithelialization of a wound area, while HNP1 promotes extracellular matrix formation by increasing levels of pro-alpha collagen and decreasing levels of matrix metalloproteinase-1, an enzyme that cleaves the extracellular matrix (Yamasaki & Gallo, 2008). Thus it is known that defensins extensively regulate many immunological processes, but the overall importance of defensins in immunity during an infection or inflammation is still inadequately understood.

Defensins in Human Disease

Attempts to determine the exact roles of each defensin in the overall immune response by knockout studies have not been successful due to the functional redundancy by other defensins and immune mechanisms. Indeed, mice for example contain at least 54 -defensins and 44 -defensins (Amid et al., 2009). However, a substantial number of immunological disorders are correlated with defensin dysregulation, providing tantalizing clues as to the roles of defensins in immunity.

Defensins are upregulated in inflammatory diseases of the bowel, lung, and skin. For example, hBD2 is significantly upregulated in cases of psoriasis, an autoimmune disease of the skin (Yamasaki & Gallo, 2008). Although no causal relationship has been established between defensin dysregulation and inflammatory disease, either defensins or their regulation may be essential for inflammatory homeostasis. Interestingly, HD5–6 downregulation correlates with ileal Crohn’s disease, an inflammatory disorder of the gastrointestinal tract, while a genetic polymorphism leading to low copy number of hBD2 associates with colonic Crohn’s disease. It is hypothesized that the enteric defensins are involved in maintaining control over the normal flora and thus establishing homeostasis in the gut (Menendez & Finlay, 2007), although it is equally possible that their immunomodulatory role is influential. Altered defensin levels are also correlated with differential susceptibility to infections. Psoriatic patients with elevated defensin levels have decreased susceptibility to infection relative to atopic dermatitis patients with reduced defensin levels (Yamasaki & Gallo, 2008). Animal studies also support the role of defensins in the clearance of pathogens. (i) In a mouse model of peritoneal Klebsiella pneumoniae infection, very small doses of HNP1 (4 ng to 4 μg) caused protection that was more likely due to an increase in leukocyte accumulation than to direct antimicrobial activity. Similar results were observed in S. aureus thigh infections (Welling et al., 1998). (ii) Administration of porcine -defensin BD-1 in piglets protected them against a Bordetella pertussis challenge (Menendez & Finlay, 2007). (iii) Overexpression of rat -defensin BD-2 in the lungs increased protection against P. aeruginosa induced pneumonia (Menendez & Finlay, 2007). (iv) BD-1 knockout mice showed delayed clearance of Haemophilus influenzae in the lungs and increased levels of Staphylococcus sp. in the bladder (Menendez & Finlay, 2007).

CATHELICIDINS

Cathelicidins are a family of peptides produced from a precursor polypeptide containing a conserved N-terminal signal and propeptide sequence encompassing approximately 100 amino acids (termed the cathelin domain), as well as a highly variable C-terminal peptide domain (Zanetti, 2004). The N-terminal signal sequence allows for targeting of the polypeptide to the endoplasmic reticulum for intracellular storage (Zanetti, 2004). The cathelin domain is essential for the storage of the polypeptide, preventing its degradation by proteases and inhibiting the biological activity of the Cterminal peptide domain (Zanetti, 2004). During excretion of the precursor polypeptide from cells, the C-terminal peptide domain is cleaved, resulting in a mature cathelicidin of 12 to 80 amino acids in length (Zanetti, 2004). In mammals, more than 50 cathelicidin members have been identified according to UniProt, a comprehensive database of protein sequence and function (Uniprot Consortium, 2008). Among the different members of the cathelicidins family, there is very little structural homology in the mature cathelicidin peptide domain. The mature peptide can fold into a variety of secondary structures, including -helices, -hairpins, and looped structures (Brown et al., 2007). In humans, only one cathelicidin has been found, an 18-kDa precursor human cationic antimicrobial protein, or hCAP18, which is processed into a variety of molecules, including prominently the 37-amino-acid peptide LL-37 (Zanetti, 2004).

Localization and Regulation

In the human body, hCAP18 is found in a range of cellular environments. Although originally isolated and identified in the granules of neutrophils, it can also be found in the


granules of many leukocytes including NK cells, monocytes, mast cells, B-lymphocytes, and  T-lymphocytes (Dürr et al., 2006). It is also constitutively expressed in epithelial cells, including those of the airways, mouth, esophagus, and intestine, and is inducible in keratinocytes (Zanetti, 2004). In addition, LL-37 is a component of most secreted bodily fluids, including sweat, gastric juices, airway fluids, seminal plasma, and breast milk (Dürr et al., 2006). Although hCAP18 is normally expressed in many intracellular environments, the levels of hCAP18 can vary and are regulated during inflammation and infection. Regulation of hCAP18 can occur at different stages, including the expression and production of the precursor polypeptide and the cleavage of the mature C-terminal peptide domain. Increased expression of hCAP18 is regularly observed during inflammation (its production during infection may indeed be related to this); hCAP18 can be modestly induced in response to the presence of host inflammatory cytokines, IL-1, and IL-6. Also, LL-37 is substantially induced in myeloid cells by the hormonal form of vitamin D3 [1,25(OH)2D3] and, interestingly, critically ill septic patients had significantly lower plasma 25(OH)D concentrations compared to healthy controls (Jeng et al., 2009). Processing of hCAP18 into the active peptide LL-37 can also be regulated. In humans, the enzymes responsible for hCAP18 processing vary between cellular environments. In neutrophil granules, hCAP18 is processed by the serine protease proteinase 3, whereas in seminal fluid and under acidic concentrations such as those found in vaginal fluid, gastricsin (Pepsinogen C) is responsible for cathelicidin processing (Zanetti, 2004). On the skin surface, the stratum corneum tryptic enzyme SCTE processes hCAP18 into LL-37 (Yamasaki & Gallo, 2008). In vitro studies using bovine neutrophil cathelicidins Bac5 and Bac7 demonstrate that the addition of small amounts of elastase results in the complete processing of their pro-forms within minutes (Zanetti, 2004). The addition of elastase inhibitors prevents the maturation and activation of these cathelicidins (Zanetti, 2004). This suggests that the maturation of cathelicidin precursors is intricately linked to the regulation of their cognate processing enzyme. Interestingly, cathelicidin levels can also be directly influenced by microbial pathogens. Cleavage of hCAP18 into LL-37 can be triggered by elastase-producing pathogens such as P. aeruginosa (Zanetti, 2004). In contrast, gut epithelial biopsies of Shigellainfected patients show low expression of hCAP18, suggesting a possible Shigella virulence strategy (Zanetti, 2004). In vitro studies support this finding by demonstrating that hCAP18 expression is downregulated in monocytes and epithelial cell lines infected with Shigella (Zanetti, 2004). It is evident that microbial infections directly regulate hCAP18/LL-37 levels via many complex avenues. In turn, hCAP18/LL-37 plays a multifunctional role in the immune response against pathogens.


In a dilute medium such as phosphate buffer, the mature human cathelicidin peptide LL-37 exhibits broad-spectrum in vitro antimicrobial activity against many pathogens, although it is arguable as to whether this activity is relevant in vivo. LL-37 is antimicrobial against gram-positive bacteria, including Streptococcus sp., Listeria monocytogenes, and S. aureus, and gram-negative bacteria, including E. coli, P. aeruginosa, and Salmonella enterica serovar Typhimurium (Dürr et al., 2006), and the fungus Candida albicans (Dürr et al., 2006). LL-37 also displays moderate antiviral properties, being able to slow the growth of vaccinia virus, but having only a mild effect on HSV infection (Dürr et al., 2006). However, as

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with the defensins, LL-37 activity is sensitive to the presence of monovalent and divalent cations and its direct antimicrobial effects are severely abrogated under physiological conditions. In the presence of 100 mM sodium, LL-37 antimicrobial activity is reduced by twofold to eightfold, whereas in cell culture medium containing physiological concentrations of salts, 1 mM Mg21 and Ca21 and 150 mM NaCl, LL-37 at concentrations of up to 100 μg/ml showed no effect against S. aureus or serovar Typhimurium (Bowdish et al., 2006). To put this in context, LL-37 is normally secreted in the bronchoalveolar fluid of infants at approximately 5 μg/ml, or 10 to 15 μg/ml in children with severe inflammatory disorders (Bowdish et al., 2005). Other host factors can also interfere with the antimicrobial activity of LL-37. Apolipoprotein 1, a human serum protein, has been shown to inhibit the antimicrobial actions of LL-37 (Bowdish et al., 2006). Glycosaminoglycans like heparin strongly inhibit activity. Indeed, the direct antimicrobial role of LL-37 is likely limited to conditions where its concentration is sufficient to overcome the previous limitations, for example, in seminal fluid or in neutrophil granules (Zanetti, 2004). Although many physiological environments do not harbor the optimal in vitro conditions for LL-37 antimicrobial activity, under these discussed conditions, LL-37 is a multifunctional modulator of the host immune response.

Immunomodulation

LL-37 plays multiple roles during an immune response (see Table 1). It is a potent enhancer of immune cell trafficking. LL-37 is directly chemotactic for monocytes, neutrophils, and T-lymphocytes by engaging the G-protein coupled receptor, FPRL-1 (Yamasaki & Gallo, 2008). LL-37 also enhances immune cell recruitment by other routes (e.g., mast cells are directly chemoattracted through a separate Gprotein coupled receptor). LL-37 also induces chemokine production. Murine models have demonstrated that the administration of LL-37 increases levels of the monocyte chemoattractant protein 1 (MCP-1) (Dürr et al., 2006). In vitro studies show increased production of chemokines such as CXCL-1, MCP-1, MCP-3, and IL-8 (among others) in LL-37-stimulated human peripheral blood mononuclear cells (Bowdish et al., 2006; Mookherjee et al., 2009a). LL-37 can also play a balancing role in the immune response, having both pro- and anti-inflammatory functions. LL-37 can promote inflammation and cell-mediated immunity by inducing IL-18 in human keratinocytes, as well as by enhancing the processing of IL-1 in LPS-stimulated human monocytes (Yamasaki & Gallo, 2008). The presence of LL-37 also leads to mast cell degranulation resulting in the release of chemoattractants, prostaglandins, and histamine release (Dürr et al., 2006). LL-37 can also work in tandem with host mediators to selectively modulate inflammation and cell trafficking. Synergy between LL-37 and IL-1 in human peripheral blood cells has been observed, leading to a significant increase of cytokine production, including IL-6 and IL-10, and production of chemokines MCP-1 and MCP-3 (Yu et al., 2007). LL-37 also potentiates the production of proinflammatory cytokines TNF- and IL-6 in peripheral blood cells stimulated by unmethylated CpG DNA, a DNA motif frequently found in the bacterial genome and an activator of human TLR9 (Yu et al., 2007). In contrast, LL-37 is a strong suppressor of LPS-induced inflammation, preventing host damage from a systemic inflammatory response. It inhibits LPS-mediated induction of inflammatory mediators, including TNF- and reactive oxygen species in macrophages, and IL1-, TNF-, IL-6, and IL-8 in human peripheral blood cells (Mookherjee et al., 2006). Consequently, LL-37 is able

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to increase the survival of rats in LPS-induced sepsis models (Dürr et al., 2006). Although the LPS-suppressing effects of LL-37 were originally attributed to its capacity to bind LPS, various studies have shown that LL-37 inflammatory regulation is much more complex and likely cell mediated, especially since it can act 30 to 60 min after LPS treatment of cells. Indeed, LL-37 can also limit the production of IL-1, TNF-, IL-6, and IL-8 in TLR2-stimulated human peripheral blood cells (Mookherjee, 2006). These observations suggest that LL-37 might play an important role in coordinating the inflammatory reaction in response to different pathogens. LL-37 also plays a role at the transition between the innate and adaptive immune system and has been demonstrated to have adjuvant activity. It can increase coreceptor CD86 expression and endocytic activity (Brown & Hancock, 2006). LL-37 promotes the polarization of human monocyte-derived dendritic cells to Th-1 type responses by enhancing the secretion of the Th-1 cytokines IL-12 and IL-6 and decreasing the levels of the Th-2 cytokine IL-4, as well as proinflammatory cytokines, in LPS-stimulated dendritic cells (Brown & Hancock, 2006). In addition to its immune regulatory effects, LL-37 is a promoter of wound healing. Epithelial wounds typically display increased levels of local LL-37, which is inducible by various growth factors, leading to increased attraction of keratinocytes (Dürr et al., 2006). Indeed, LL-37-specific antibodies inhibited epithelial healing in a human skin wound model (Yamasaki & Gallo, 2008). LL-37 also promotes angiogenesis in epithelia, which may play a role in reepithelialization (Yamasaki & Gallo, 2008), while its ability to modulate inflammatory activity may also assist in wound healing.

hCAP18/LL-37 in Human Disease

Efforts to characterize the overall role of LL-37 in the immune system have been based on in vivo models and correlational studies linking LL-37 dysregulation to immune disorders. Some immune disorders suggest LL-37 is required for the proper clearance of pathogens, although it is uncertain whether these studies reflect direct antimicrobial activity, as several authors assert, or immunomodulatory activities. As with hBD2, lower expression of hCAP18 is found in atopic dermatitis patients relative to psoriatic patients, which may explain their differential susceptibilities to infection. Patients with morbus Kostmann, a neutropenic disorder, are treated with cytokine G-CSF to restore circulating neutrophils with normal oxidative responses. However, these neutrophils are deficient in LL-37 and this may correlate with the chronic periodontal disease and excessive oral inflammation observed in these patients (Brown et al., 2007). Murine models show that knocking out CRAMP, a murine cathelicidin structurally related to LL-37, increases the susceptibility to skin infections by group A Streptococcus (Zanetti, 2004).

MECHANISMS OF ANTIMICROBIAL KILLING

HDPs possess in vitro antimicrobial activity, ranging from modest (many defensins and LL-37) to potent (e.g., pig protegrin), against an extensive range of microbes including bacterial, viral, and fungal pathogens. However, the mechanisms utilized by HDPs to elicit their direct and broad-spectrum actions are quite complex and likely involve multiple targets. For bacteria, it is known that HDPs interact with negatively charged components on microbial membranes, such as LPS or LTA, and then translocate to the cytoplasmic membrane. Although it is known that HDP interaction with the membrane is a prerequisite for killing, there are many possible results leading to bacterial death, namely the disruption of membrane integrity, interference with essential processes

dependent on membrane-bound enzymes (cell division, cell wall synthesis), or translocation across the membrane to access cytosolic targets. Four models have been proposed to address peptide-membrane interactions, as reviewed by Jenssen et al. in 2006: (i) the toroidal pore, (ii) barrel-stave, (iii) carpet, and (iv) aggregate models. In the toroidal pore model, peptides are thought to intimately associate with the head groups of lipids and reaggregate into the bacterial membrane in a perpendicular orientation, resulting in the formation of a peptide-lipid pore, thus increasing membrane permeability. Certain peptides that form -helical structures in a membrane setting, such as LL-37, have been proposed to act in this fashion. In the barrel-stave model, peptides cluster on the membrane, also in a perpendicular fashion. In this case, the hydrophobic regions of the peptides interact with fatty acyl chains of membrane lipids, and their hydrophilic regions face one another, forming a pore in the microbial membrane. The carpet model, unlike the previous two, suggests that peptides remain aggregated on the membrane surface in a parallel fashion, resulting in localized peptide clusters. At high peptide concentrations the aggregates form micelles with areas of the membrane, leading to a disruption of membrane integrity. None of these models, however, really considers peptide translocation, which is more common than previously supposed, having been experimentally observed with many HDPs. One model to explain both membrane disruption and translocation is the aggregate model. In this model, peptides aggregate on the membrane surface and at a critical concentration form complexes (mixed micelles) with the membrane lipids, permitting increased membrane permeability (which for some peptides might cause death) and allowing for the translocation of peptides across the bilayer as the aggregates collapse. It must be noted that only some of these models may be relevant to antimicrobial killing depending on the peptide and the cellular environment. The mode of action against fungi is quite poorly understood; however, it has been observed that peptides interact with fungal cell walls and membranes as well as acting on internal targets including respiratory processes (Jenssen et al., 2006). A wide variety of studies have revealed mechanisms of antibacterial killing that are unrelated to membrane disruption. Indeed, it seems likely that many peptides have multiple targets, explaining in part the low rates of resistance development to these HDPs. Some peptides inhibit microbial growth without any major disruption of the membrane by acting on intracellular targets. The -helical peptides of some species, such as fish pleurocidin and frog dermaseptin, apparently disrupt bacterial growth by interfering with nucleic acid and/or protein synthesis (Jenssen et al., 2006). The -sheet defensin HNP-1 inhibits nucleic acid synthesis in E. coli, and other peptides, including bovine indolicidin, also inhibit protein synthesis (Jenssen et al., 2006). It is hypothesized that the cationic properties of the peptides allow for their interaction with a range of negatively charged enzymes and molecules leading to disruption of their functions. Binding to membrane components of enveloped viruses and host cells is a major antiviral mechanism of peptides, but a wide range of cellular targets or actions on different stages of the viral life cycle have been reported for many viruses (Jenssen et al., 2006).

MECHANISMS OF IMMUNOMODULATION

HDPs, in addition to their direct but often weak microbial killing ability, regulate an immense range of host immunological functions during an infection. Through selective stimulation of innate immune mechanisms, they likely have a strong impact on the effectiveness of these processes in killing


microbes. Due to limitations on the direct microbial killing activities of peptides in a physiological environment at moderate concentrations (e.g., on the mucosa), it seems likely that the primary function of HDPs during an infection is modulation of the immune response. The question is thus how HDPs exert their far-reaching control over the host immune system. Although immunomodulation by HDPs is a relatively new field, researchers have begun to elucidate their regulatory and immune effector mechanisms. Nevertheless, the mechanisms utilized by peptides to regulate immunity may be as numerous as their downstream effects, and it is now clear that there is enormous complexity involved in the selective action of peptides on immune cells, as revealed in a recent systems biology study of LL-37 (Mookherjee et al., 2009a). HDPs are potent modulators of cellular signalling cascades, particularly those that are involved in the regulation of immunity. HDPs regulate immune signalling pathways to promote chemotaxis, through the production of chemokines, as well as regulating downstream immune responses by altering cytokine levels and other cellular functions. MAP-kinase pathways are induced by HDPs. For example, LL-37 induces the phosphorylation of MAP-kinase constituents to modulate the production of chemokines and cytokines (Mookherjee et al., 2009a). Inhibitor experiments show that LL-37-induced transcription of chemokines IL-8, MCP-1, and MCP-3 in human peripheral blood cells is dependent on MAP-kinase signaling while phosphorylation of p38 and ERK-1/2 in response to LL-37 has been directly demonstrated (Bowdish et al., 2006; Mookherjee et al., 2009a). hBD3 and hBD4 also induce the phosphorylation of p38 and ERK-1/2 in human mast cells, correlating with increased mast cell degranulation (Menendez & Finlay, 2007). HNPs also induce the production of IL-8 in monocytes in a p38-dependent manner, and in lung cells, dependent on the activity of the phosphatidylinositol-3-kinase (PI3-K) pathway, another regulator of cell growth and inflammation (Syeda et al., 2008). In keratinocytes, hBD2-4 also induced the phosphorylation of STAT-1 and STAT-3, signalling mediators utilized by cytokines to induce further downstream immune responses (Menendez & Finlay, 2007). Other pathways engaged include the transient but obligate engagement of the NF-B/IB and PI3-K pathways. HDPs are also able to act as relatively weak chemokine analogues, binding to chemokine receptors of host cells at micromolar concentrations, as opposed to nanomolar concentrations for most conventional chemokines to initiate chemotaxis. hBD1 and hBD2 chemoattract immature dendritic cells and memory T-lymphocytes by engaging the chemokine receptor CCR6 (Yamasaki & Gallo, 2008; Yang et al., 2001). LL-37, in a similar fashion, attracts monocytes, neutrophils, and T-lymphocytes by binding to the receptor FPRL-1 (Yang et al., 2001). LL-37, in addition to promoting cell recruitment, acts to modulate inflammation by antagonizing or acting cooperatively with various inflammatory signalling mediators. LL-37 is a potent suppressor of TLR4 activation by LPS and inhibits the downstream nuclear translocation of NF-B subunits p50 and p65 in LPS-stimulated human monocytes, which form a transcription complex essential for the expression of many inflammatory mediators (Mookherjee et al., 2006). It demonstrates rather mixed effects with molecules that signal through other TLRs, suppressing stimulation of TLR2 by LTA, demonstrating mixed responses (antagonism or enhancement depending on the read-out) with CpG oligonucleotides (a TLR9 agonist) and flagellin (a TLR5 agonist) while acting synergistically with IL-1 through the related IL-1 receptor (Mookherjee et al., 2006). HDPs also use growth-related signalling pathways to mediate wound

63

healing. LL-37 is an activator of the epidermal growth factor receptor (EGFR) in keratinocytes, leading to increased keratinocyte migration at wound sites (Yamasaki & Gallo, 2008). Analogously, hBD2-4 increases EGFR activity, correlating to an increase in keratinocyte proliferation (Menendez & Finlay, 2007). LL-37 also utilizes FPRL-1 signal activity to induce angiogenesis (Yamasaki & Gallo, 2008). LL-37 is also a potent inhibitor of human neutrophil apoptosis, signaling through P2X7 receptors and G-protein coupled receptors other than FPRL-1 and involving the activation of PI3-K but not ERK-1/2 MAP-kinase. Of note, although a number of extracellular receptors have been described, LL-37 and the synthetic innate defense regulator peptides are able to freely penetrate into host cells, in a process that mirrors that of so-called cellpenetrating peptides like HIV-1 Tat. Importantly, uptake into cells is absolutely required for chemokine induction by monocytes and epithelial cells. The recent findings of intracellular receptors including GAPDH (Mookherjee et al., 2009b) and others (unpublished data) indicates that LL-37 acts intracellularly through hub proteins to impact on signaling pathways. Although headway is being made in discovering how HDPs regulate cellular signaling, many uncertainties remain. For example, as mentioned above, LL-37 exerts paradoxical effects on the inflammatory response, limiting the activity of certain inflammatory stimulants, such as LPS, but acting synergistically with other stimulants, such as IL-1. Gene expression studies of monocytes show that LL-37 itself induces hundreds of genes while leading to the suppression of a portion of LPS-induced gene expression (while a significant portion remains unchanged) (Mookherjee et al., 2006; Mookherjee et al., 2009a). Thus LL-37 is an authentic modulator of immunity as opposed to an antagonist or immune stimulant. It seems that HDPs balance inflammatory responses, selectively strengthening protective immune response while preventing excessive inflammation. The ability of peptides to utilize multiple receptors and signaling mechanisms to maintain that balance is a major topic of interest in current host defense peptide research.

HOST DEFENSE PROTEINS

Host defense and antimicrobial proteins are another important component of the innate immune response. We utilize the term protein here to describe larger molecules of more than 100 amino acids with a well-defined tertiary structure. Several of these can be digested down to active fragments, some of which have the above-described features and activities of HDPs and thus are likely related to these peptides. Host defense proteins include more than 700 structurally diverse members expressed extensively in both plants and animals (Hall et al., 2002). Humans possess a wide range of host defense proteins that perform many antimicrobial and immunomodulatory functions (see Table 2).

BACTERICIDAL PERMEABILITY-INCREASING PROTEIN (BPI)

BPI is a highly positively charged, 55- to 60-kDa protein that is mainly found in the primary (azurophilic) granules of neutrophils in humans, rabbits, and cows (Canny & Levy, 2008). In humans, BPI is expressed and synthesized in the bone marrow in the granules of neutrophil precursors, called granulocytes, prior to neutrophil maturation (Weiss, 2007). To a lesser extent, BPI is also expressed in eosinophils and the epithelial cells of the gastrointestinal and respiratory tracts, where it acts as a local defense agent against gram-negative bacteria (Schultz, 2007;

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HOST DEFENSE: GENERAL TABLE 2 Human host defense peptides and proteins Polypeptide -Defensins HNP1–4

Mass (kDa) 3–4

Granules of neutrophils, monocytes, lymphocytes, NK cells, and Paneth cells Granules of various epithelial cells

HD5–6 -Defensins hBD1–4 hBD5–6 Cathelicidin: hCAP18 (LL-37)

Bactericidal permeabilityincreasing (BPI) protein

Localization

4–5 Various epithelial cells Paneth cells; epithelial cells of epididymis and airways 18 (4.5)

55–60

Granules of neutrophils, monocytes mast cells, B-lymphocytes, and  T-lymphocytes; various epithelial cells; bodily fluids (sweat, gastric juice, airway fluids, seminal plasma, and breast milk) Granules of neutrophils, eosinophils, epithelium

Lactoferrin

80

Neutrophils; various epithelia; bodily fluids (airway fluid, cervical mucus, breast milk, tears, and saliva)

Lysozyme

14

Neutrophils, monocytes, macrophages, keratinocytes, Paneth cells, pneumocytes, secretory cells of the apocrine gland, various epithelia; bodily fluids (tears, saliva, breast milk, respiratory fluid)

Peroxidases

150

Neutrophils, monocytes, eosinophils, epithelia, milk, saliva

Secretory leukoprotease inhibitor (SLPI)

12

Macrophages, various epithelia

Serprocidins (cathepsin G, elastase, proteinase 3, azurocidin/CAP37) Psoriasin (S100A7)

25–30

11

Neutrophils

Keratinocytes

Brown et al., 2007). BPI is also found on the surfaces of neutrophils and monocytes. However, it is hypothesized that this may be due to the degranulation of adjacent activated neutrophils. The level of BPI remains relatively unchanged. In the peripheral blood of humans, neutrophils store BPI in their primary granules with minimal de novo protein expression. In dermal fibroblasts, the expression of BPI is only slightly increased by IL-4, whereas it was not elevated by other cytokines including TNF-, IFN-, IL-4, and IL-1 in CaCo-2 and other epithelial cells (Schultz, 2007). Interestingly, the pattern of expression of BPI is different from that of other azurophilic granule proteins. This is presumably due to the observation that the BPI promoter binds transcription factors CCAAT/ enhancer binding proteins  and  (C/EBP, C/EBP) rather than C/EBP, which regulates the expression of other azurophilic granule proteins (Weiss, 2007). Like other lipid-binding proteins, BPI has high binding affinity to LPS. Structurally, human BPI is very similar to LPS-binding protein (LBP). BPI has two similar-sized domains separated by a proline-rich linker. The C-terminal domain is responsible for the binding of BPI to bacteria and facilitating its phagocytosis. The N-terminal domain is responsible for the interaction between BPI and LPS. The cationic N-terminal region of BPI also contributes to BPI-LPS binding through electrostatic interactions with the anionic lipid A region of LPS (Brown et al., 2007). It is thought that the ability of BPI to bind LPS is the basis for its observed antibacterial and endotoxin-neutralizing effects.


BPI has modest activity against gram-negative bacteria and has been presumed to act primarily via its interaction with

LPS on the bacterial outer membrane (Schultz, 2007). BPI is thought to mediate its antimicrobial effects by increasing membrane permeability via a process analogous to selfpromoted uptake, involving binding of LPS by BPI leads to its insertion into the outer membrane of intact gramnegative bacteria, stimulating the rearrangement of outer membrane lipids and displacing LPS-bound divalent cations Mg21 and Ca21 (Canny & Levy, 2008). However, since this cannot per se lead to the killing of cells, we assume that it must translocate across the outer membrane and attack other targets in a manner analogous to HDPs. Disruption of the outer membrane increases the susceptibility of microbial phospholipids in the membrane to the metabolic enzyme, phospholipase A (Schultz, 2007). Some gram-negative bacterial species are very resistant to the antimicrobial effects of BPI, specifically those with structural modifications of their LPS, such as those with long LPS chains or those with structural diversity within the lipid A region of LPS (Brown et al., 2007).

Immunomodulation

The ability of BPI to bind LPS is probably even more influential in the host innate immune response. BPI, in contrast to its structural relative LBP, neutralizes the inflammatory response elicited by bacterial endotoxin. BPI is able to reduce LPS-mediated endothelial damage and the production of nitric oxide and inflammatory cytokines, including TNF-, IL-1, IL-6, IL-8, and IL-10 (Van Amersfoort et al., 2007). It also inhibits the binding of LPS to blood vessels, thus preventing circulatory disturbances during infections caused by gram-negative bacteria (Brown et al., 2007). In addition, BPI also exhibits other immunomodulatory prop-


erties, including the suppression of IL-1-induced nitric oxide synthase (iNOS) expression in endothelial cells. BPI can also promote the induction of T-cell-dependent immunity by presenting bound gram-negative bacterial components, such as outer membrane vesicles, to dendritic cells. These dendritic cells in turn trigger adaptive immunity (Schultz, 2007).

Therapeutic Potential

Efforts to harness the LPS-neutralizing effects of BPI as potential therapeutics against sepsis models have not proven effective. Recombinant BPI has been tested as an experimental treatment for meningococcal and other forms of sepsis caused by gram-negative bacteria. Unfortunately, efficacy was not demonstrated in Phase III clinical trials. In contrast, in a rabbit model of peritoneal inflammation, extracellular BPI, secreted into inflammatory exudates, showed potent bactericidal activity against many gram-negative bacteria. In addition, some defensins, cathelicidins, and low concentrations of complement potentiated the activity of BPI (Brown et al., 2007).

LYSOZYME

Lysozymes, also called muramidases, are small, abundant cationic enzymes that are widely distributed in plants and animals. They primarily cleave the 1, 4-glycosidic linkage between N-acetyl muramate and N-acetyl glucosamine residues in peptidoglycan, an essential component in bacterial cell walls (Brown et al., 2007; Niyonsaba & Ogawa, 2005). Different species have varying copy numbers of the lysozyme gene, ranging from 1 in humans to 10 in sheep and cows. In humans, lysozymes are expressed in polymorphonuclear leukocytes, monocytes, macrophages, keratinocytes, Paneth cells of the small intestine, tracheal epithelial cells, pneumocytes, and apocrine gland secretory cells (Niyonsaba & Ogawa, 2005). In addition, lysozyme is abundantly found in many bodily secretions, including tears, saliva, breast milk, and respiratory fluid (Brown et al., 2007). The lysozyme protein is divided into two domains connected by a long -helix with six active sites, termed A, B, C, D, E, and F. These active sites are required to bind six consecutive sugar residues, facilitating peptidoglycan binding and processing (Niyonsaba & Ogawa, 2005).


Between different species, lysozymes have conserved functions including bacteriolytic and antifungal activities and antitumor and anti-inflammatory properties (Brown et al., 2007). Lysozymes by themselves are able to kill a very restricted range of bacteria by increasing osmotic stress via its disruption of peptidoglycan. Peptidoglycan is a structural glycosidic polymer and is most prominent in the cell wall of gram-positive bacteria, where it maintains shape, stability, and osmotic resistance. Gram-negative bacteria are generally more resistant to lysozyme activity due to the stabilizing effect of the outer membrane (Masschalck & Michiels, 2003). While certain gram-positive bacterial organisms, such as Micrococcus lysodeikticus and Bacillus species, are susceptible to low lysozyme levels, most pathogens can persist in environments with low lysozyme concentrations (Brown et al., 2007). Higher levels of lysozyme are antibacterial, but it has been suggested that this is due to its cationic nature rather than its enzymatic activity (Brown et al., 2007). The antibacterial activity of lysozyme is much more effective in combination with other host defense factors, including complement and cationic peptides, suggesting that lysozyme acts in concert with other host defense mechanisms to aid in pathogen clearance.

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LACTOFERRIN

Lactoferrin is an 80-kDa iron-binding plasma protein that belongs to the transferrin protein family, which includes serum transferrin, ovotransferrin, melanotransferrin, and the inhibitor of carbonic anhydrase (González-Chávez et al., 2009). It is produced by many animals including humans, cows, goats, horses, dogs, several rodents, and fish (González-Chávez et al., 2009). In humans, lactoferrin is expressed in the secretory granules of neutrophils and has been detected in epithelial cells and mucosal secretions including airway fluid, cervical mucus, breast milk, tears, and saliva. It is the second most abundant protein in milk after casein. Lactoferrin consists of two similar ferric-ion-binding domains; these permit lactoferrin to deprive iron from bacteria and inhibit bacterial growth (Brown et al., 2007). Lactoferrin can be proteolyzed to the cationic peptide lactoferricin by gastric pepsin in the milk of animals. Lactoferricin has moderate activities against bacteria, protozoa, viruses, and fungi due to its cationic bactericidal nature, but not iron absorption, in analogy with cationic host defense peptides (Jenssen & Hancock, 2009). Other host defense functions of lactoferrin unrelated to iron absorption include blocking biofilm development by P. aeruginosa, binding to specific surface receptors, inhibiting the attachment of Helicobacter pylori to gastric epithelial cells, and facilitating clearance of infected host cells by enhancing pro-apoptotic signals (Brown et al., 2007).

CHEMOKINES

Chemokines are an extensive family of small chemotactic cytokine proteins of 7 to 10 kDa in size and include approximately 50 members in humans. Most chemokines are also cationic proteins and thus possess modest antibacterial properties in dilute medium. However, chemokines play an essential role in the host immune response primarily by directing the movement of immune effector leukocytes (including neutrophils, lymphocytes, macrophages, dendritic cells, and monocytes) to the site of infection. According to their functions, chemokines can be divided into two classes: inflammatory and homeostatic. Homeostatic chemokines are constitutively expressed, whereas the expression of inflammatory chemokines is only induced upon activation by the triggering of circulating leukocytes by inflammatory mediators such as TNF-, IFN-, microbial components, or trauma and other danger signals (Allen et al., 2007). Chemokines direct cell movement and migration by acting as soluble chemoattractants or adhering to tissues by binding to their receptors. There are about 20 different chemokine receptors, all of which are G-protein coupled receptors. Based on the patterns of cysteine residues in their ligands, chemokines and their receptors are classified into four families: CXC, CC, C, and CX3C, where C represents cysteine and X/X3 stands for one or three noncysteine amino acids (Allen et al., 2007). Following chemokine receptor binding, a series of events, including cytoskeletal rearrangement and the regulation of cell surface adhesion molecules, lead to cell motility and chemotaxis (Moser et al., 2004). In response to the presence of foreign antigens, tissue damage, and other physiological insults, chemokines play important roles in attracting and activating specific leukocytes to the affected sites and in stimulating strong immune responses against pathogens. Recently, chemokines, in addition to their roles in inflammation and infection, were shown to be involved in autoimmune diseases and cancer (Ward & Marelli-Berg, 2009). It should be noted that chemokines possess characteristics similar to a group of mediators that have been termed alarmins. Alarmins are secreted in response to inflammation and

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act as warning signals of tissue damage or invading pathogens (Oppenheim & Yang, 2005). Alarmins possess adjuvant properties, activating and maturing antigen-presenting cells, namely dendritic cells, and enhancing their antigenspecific immune responses. Similar to chemokines, alarmins utilize Gi protein-coupled receptors (including chemokine receptors) on antigen-presenting cells (including dendritic cells, monocytes, and macrophages). However, while the actions of alarmins are limited to their enhancement of antigen-presenting cell function, chemokines play a diverse role in directing the responses of both innate and adaptive immunity. It has been proposed that defensins and cathelicidins are alarmins, but we are uncertain if this adequately describes their function.

CONCLUSIONS

Host defense peptides and proteins are major components in the arsenal of our immune system. Although host defense peptides and proteins were studied initially for their direct antimicrobial effects, it is becoming clearer that they are multifunctional regulatory agents, responsible for the coordination and development of a wide range of immune response actions. The ability of host defense peptides and proteins to mediate a wide array of functions, including cell recruitment, wound healing, and inflammatory regulation to name a few, makes them exciting candidates for the development of novel therapeutics based on immunomodulation. By unlocking the functional mechanisms of these peptides and proteins, researchers will be able to design novel treatments that serve to fine-tune the host immune response. Immunomodulation will transform the way we respond to a wide range of immune-related diseases, ranging from novel, broad range anti-infective therapies to immune regulators that tame a dysfunctional immune response in disorders such as chronic inflammation and autoimmunity. The cationic peptide research of the authors is supported by grants from the Grand Challenges in Global Health Research program and the Canadian Institutes for Health Research (CIHR). S.M. is supported by a postdoctoral fellowship from Wyeth/CIHR while R.E.W.H. holds a Canada Research chair.

REFERENCES Allen, S. J., S. E. Crown, and T. M. Handel. 2007. Chemokine: receptor structure, interactions, and antagonism. Annu. Rev. Immunol. 25:787–820. Amid, C., L. M. Rehaume, K. L. Brown, J. G. Gilbert, G. Dougan, R. E. Hancock, J. L. Harrow. 2009. Manual annotation and analysis of the defensin gene cluster in the C57BL/6J mouse reference genome. BMC Genomics 10:606. Bowdish, D. M. E., D. J. Davidson, and R. E. W. Hancock. 2005. A re-evaluation of the role of host defence peptides in mammalian immunity. Curr. Protein Pept. Sci. 6:35–51. Bowdish, D. M. E., D. J. Davidson, and R. E. W. Hancock. 2006. Immunomodulatory properties of Defensins and Cathelicidins. Curr. Top. Microbiol. Immunol. 306:27–66. Brown, K. L., and R. E. W. Hancock. 2006. Cationic host defense (antimicrobial) peptides. Curr. Opin. Microbiol. 18:24–30. Brown, K. L., N. Mookherjee, and R. E. W. Hancock. 2007. Antimicrobial, host defence peptides, and proteins. In Encyclopedia of Life Sciences (ELS). John Wiley and Sons, Ltd., Chichester, UK. doi: 10.1002/9780470015902.a0001212.pub2 Canny, G., and O. Levy. 2008. Bactericidal/permeabilityincreasing protein (BPI) and BPI homologs at mucosal sites. Trends Immunol. 29:541–547. Chen, X., Z. Xu, L. Peng, X. Fang, X. Yin, N. Xu, and P. Cen. 2006. Recent advances in the research and development of human defensins. Peptides. 27:931–940.

Dürr, U. H. N., U. S. Sudheendra, and A. Ramamoorthy. 2006. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim. Biophys. Acta 1758: 1408–1425. Froy, O. 2005. Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways. Cell. Microbiol. 7:1387–1397. González-Chávez, S. A., S. Arévalo-Gallegos, and Q. RascónCruz. 2009. Lactoferrin: structure, function and applications. Int. J. Antimicrob. Agents 33:301.e1–8. Hall, S. H., K. G. Hamil, and F. S. French. 2002. Host defense proteins of male reproductive tract. J. Androl. 23:585–597. Jeng, L., A. V. Yamshichkov, S. E. Judd, H. M. Blumber, G. S. Martin, T. R. Ziegler, and V. Tangpricha. 2009. Alterations in vitamin D status and anti-microbial peptide levels in patients in the intensive care unit with sepsis. J. Transl. Med. 7:28. Jenssen, H., P. Hamill, and R. E. W. Hancock. 2006. Peptide antimicrobial agents. Clin. Microbiol. Rev. 19:491–511. Jenssen, H., and R. E.W. Hancock. 2009. Antimicrobial properties of lactoferrin. Biochimie 91:19–29. Klotman, M. E., and T. L. Chang. 2006. Defensins in innate antiviral immunity. Nat. Rev. Immunol. 6:447–456. Lillard, J. W., Jr., N. Prosper, O. Chertov, J. J. Oppenheim, and J. R. McGhee. 1999. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc. Natl. Acad. Sci. 96:651–656. Masschalck, B., and C. W. Michiels. 2003. Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria. Crit. Rev. Microbiol. 29:191–214. Menendez, A., and B. Finlay. 2007. Defensins in the immunology of bacterial infections. Curr. Opin. Immunol. 19:385–391. Mookherjee, N., K. Brown, D. M. E. Bowdish, S. Doria, R. Falsafi, K. Hokamp, F. M. Roche, R. Mu, G. H. Doho, J. Pistolic, J. P. Powers, J. Bryan, F. S. L. Brinkman, and R. E. W. Hancock. 2006. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J. Immunol. 176:2455–2464. Mookherjee, N., P. Hamill, J. Gardy, D. Blimkie, R. Falsafi, A. Chikatamarla, D. J. Arenillas, S. Doria, T. R. Kollmann, and R. E. W. Hancock. 2009a. Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells. Mol. Biosystems 5:483–496. Mookherjee, N., D. N. Lippert, P. Hamill, R. Falsafi, A. Nijnik, J. Kindrachuk, J. Pistolic, J. Gardy, P. Miri, M. Naseer, L. J. Foster, and R. E. W. Hancock. 2009b. Intracellular receptor for human host defense peptide LL-37 in monocytes. J. Immunol. 183:2688–2696. Moser, B., M. Wolf, A. Walzl, and P. Loetscher. 2004. Chemokines: multiple levels of leukocyte migration control. Trends Immunol. 25:75–84. Niyonsaba, F., and H. Ogawa. 2005. Protective roles of the skin against infection: implication of naturally occurring human antimicrobial agents -defensins, cathelicidin LL-37 and lysozyme. J. Dermatol. Sci. 40:157–168. Oppenheim, J. J., and D. Yang. 2005. Alarmins: chemotactic activators of immune responses. Curr. Opin. Immunol. 17:359–365. Schultz, H. 2007. From infection to autoimmunity: a new model for induction of ANCA against the bactericidal/permeability increasing protein (BPI). Autoimmun. Rev. 6:223–227. Syeda, F., H. Y. Liu, E. Tullis, M. Liu, A. S. Slutsky, and H. Zhang. 2008. Differential signaling mechanisms of HNPinduced IL-8 production in human lung epithelial cells and monocytes. J. Cell. Physiol. 214:820–827. Uniprot Consortium. 2008. The Universal Protein Resource (UniProt). Nucleic Acids Res. 36:D190–195. Van Amersfoort, E. S., T. J. C. Van Berkel, and J. Kuiper. 2003. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin. Microbiol. Rev. 16:379–414. Ward, S. G., and F. M. Marelli-Berg. 2009. Mechanisms of chemokine and antigen-dependent T-lymphocyte navigation. Biochem. J. 418:13–27.

4. Host Defense (Antimicrobial) Peptides and Proteins Weiss, H. J. P. 2007. The bactericidal/permeability-increasing protein (BPI) in infection and inflammatory disease. Clin. Chim. Acta 384:12–23. Welling, M. M., P. S. Hiemstra, M. T. van den Barselaar, A. Paulusma-Annema, P. H. Nibbering, E. K. Pauwels, and W. Calame. 1998. Antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation. J. Clin. Invest. 102:1583–1590. Yamasaki, K., and R. L. Gallo. 2008. Antimicrobial peptides in human skin disease. Eur. J. Dermatol. 18:11–21.

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Yang, D., O. Chertov, and J. J. Oppenheim. 2001. Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and activities of human defensins and cathelicidin (LL-37). J. Leukoc. Biol. 69:691–697. Yu, J., N. Mookherjee, K. Wee, D. M. E. Bowdish, J. Pistolic, Y. Li, L. Rehaume, and R. E. W. Hancock. 2007. Host defense peptide LL-37, in synergy with inflammatory mediator IL-1, augments immune responses by multiple pathways. J. Immunol. 179:7684–7691. Zanetti, M. 2004. Cathelicidins, multifunctional peptides of the innate immunity. J. Leukoc. Biol. 75:39–48.



5 Reactive Oxygen and Reactive Nitrogen Intermediates in the Immune System CHRISTIAN BOGDAN

INTRODUCTION

a broad spectrum of mechanisms that impede the replication or lead to the killing of the pathogen (Table 1 and Fig. 1). Some of these mechanisms, such as the phagocyte NADPH oxidase (Phox) or the type 2 nitric oxide synthase (NOS2 or iNOS), are oxygen-dependent and require pathogen recognition receptor- or cytokine-mediated gene expression, whereas others are oxygen-independent and are immediately available because of preformed proteins (e.g., defensins, proteases). In general, the different effector mechanisms are not reserved for certain pathogens and they do not operate in isolation, but are frequently interdependent. For example, the acidification of the phagolysosome enhances the activity of many proteases, which are characterized by an acidic pH optimum; the myeloperoxidase (MPO) converts H2O2 (which is generated from O22 by superoxide dismutases [SOD]) into highly toxic halides; the MPO participates in the interferon (IFN)--mediated priming of phagocytes for Phox-dependent ROI production; ROIs and RNIs react with each other, which leads to the generation of new effector oxidants (e.g., peroxynitrite); and the withdrawal of iron from the pathogen allows the phagocyte to generate ROIs via the iron-catalyzed Haber-Weiss reaction (reviewed in Bogdan, 2004; Adachi et al., 2006). It should also be noted that for certain pathogens (e.g., Listeria monocytogenes, non-tuberculous Mycobacteria, and Anaplasma phagocytophilum) the exact mechanism(s) that lead to their killing by activated phagocytes still remain to be identified (von Loewenich et al., 2004; Birkner et al., 2008; Gomes et al., 2008).

Inorganic molecules such as nitric oxide (NO), superoxide (O22), or hydrogen peroxide (H2O2) are central secretory products of activated phagocytes. A simplified view presented in most immunology textbooks considers reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNI) merely as toxic defense and effector molecules of phagocytes that either help to kill viral and microbial pathogens, or, when produced chronically or in exuberant amounts, can cause severe tissue damage during infections and antigen-driven inflammatory processes. The mechanistic basis for the potent antimicrobial capacity (and for the adverse effects) of ROIs and RNIs is their rapid and high production, their small molecular size and transmembrane diffusibility, and their avid reactivity with organic targets (Bogdan et al., 2000; Bogdan, 2004; Fang, 2004; Segal, 2005). From a broader perspective, however, ROIs and RNIs also serve as important signaling molecules in various organ systems, including the immune system (Bogdan et al., 2000; Bogdan, 2001b; Forman & Torres, 2001; Hancock et al., 2001; Reth, 2002; Bedard & Krause, 2007; Brown & Griendling, 2009). Numerous examples for beneficial as well as counterprotective regulatory functions of RNIs and ROIs have been discovered during the past 20 years (Bogdan, 2004), which not only illustrate how difficult it is to judge their true role during infections, but also raise scepticism about the proposed use of antioxidants or RNI/ROI donors for the treatment of inflammatory or infectious diseases, respectively. In this chapter, I will review the sites of production and the functional diversity of ROIs and RNIs within the immune system with a special emphasis on their host-protective effector and regulator potential.

REACTIVE OXYGEN INTERMEDIATES Enzymatic Systems for the Generation of ROIs

THE PHAGOCYTE AND ANTIMICROBIAL DEFENSE: AN OVERVIEW

ROIs, ROSs, or reactive oxygen metabolites (ROMs) are interchangeable collective terms for all inorganic oxidants consisting of oxygen, hydrogen, and possibly a halogen. Important examples of ROIs are free oxygen radicals (e.g., O22, hydroxyl radical [OH], peroxyl radical [RO2], perhydroxyl radical [HOO]); relatively stable nonradical oxidants (e.g., hypochlorous acid [HOCl], ozone [O3], and H2O2); and labile oxidants with excited electrons (e.g., singlet oxygen [1O2]). There are multiple enzyme systems that generate ROIs. These include: (i) the electron transport chain in mitochondria, the

Following an encounter, entry or uptake of viruses, bacteria, protozoa, or fungi granulocytes and macrophages can resort to Christian Bogdan, Microbiology Institute, Clinical Microbiology, Immunology and Hygiene, Friedrich Alexander University, Erlangen, Nuremberg, and University Clinic of Erlangen Wasserturmstraße 3/5, D-91054 Erlangen, Germany.

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HOST DEFENSE: GENERAL TABLE 1

Antimicrobial effector mechanisms of phagocytes Constitutive/rapidly available

Oxygen independent

Oxygen-dependent

Induced by cytokines and/or crosslinking of PRR

1. Hydrolytic and proteolytic enzymes - lysozyme C* - esterase, gelatinase , matrix metalloproteases (MMP) - serprocidins (azuricidin/ CAP37*, proteinase 3*, elastase*, cathepsin G)

1. Phagosomal acidification ATPase-dependent proton pump

2. Antimicrobial peptides - a-defensins* - b-defensins - bactericidal/permeability increasing protein (BPI)* - cathelicidin (CAP18)(*) and cathelicidin-derived C-terminal peptide (LL-37) - pentraxin (PTX-3)(*) 3. Antimicrobial proteins - histones*

2. Phagolysosomal fusion - small GTPases (47 kDa IRGs, e.g. Irgm1/LRG-47)

4. Depletion of micronutrients and trace elements - lacto(trans)ferrin (iron) - calprotectin (also called MRP8/14 or S100A8/A9 complex)* (Zn21, Mn21) 1. Oxidases - NADPH phagocyte oxidase (Phox, NOX2) - myeloperoxidase (MPO)* 2. Iron catalyzed formation of ROI - Haber-Weiss reaction - Fenton reaction 3. Antibody catalyzed formation of ROI - catalytic antibodies

3. Amino acid depletion - tryptophan depletion (IDO) - arginine depletion (arginase, iNOS/NOS2) 4. Depletion of micronutrients and trace elements - lipocalin-2 (iron)

Oxidases/Oxidoreductases - NADPH phagocyte oxidase (Phox, NOX2) - inducible or type 2 nitric oxide synthase (iNOS or NOS2)

CAP, cationic protein; IDO, indoleamine-2,3-dioxygenase; IRG, immunity-related GTPase; Irgm1, immunity-related GTPase family M member 1; LRG-47, lipopolysaccharide- and interferon-regulated GTPase of 47 kDa; MRP, myeloid-related protein; PRR, pattern recognition receptor; S100 proteins, family of calcium-binding proteins of low molecular weight (9–13 kDa) * These molecules are part of the proteome of “neutrophil extracellular traps” (NETs), which are web-like structures consisting of chromatin (DNA and histones) and granule proteins and are extruded during the death of granulocytes (Urban et al., 2009).

monooxygenases, and oxidases in peroxisomes, and the cytochrome p450 oxidoreductases, all of which generate ROIs as by-products; (ii) the widely expressed xanthine oxidase system, which transforms hypoxanthine to xanthine, and further to uric acid and thereby generates H2O2 from H2O and 3O2; (iii) the generation of ROIs by the pseudoperoxidase or phenoloxidase activity of hemoglobin or hemocyanin, respectively, after contact with microbial pathogens; (iv) the family of NADPH-dependent oxidases (NOX)/dual oxidases (DUOX), which transport electrons from cytoplasmic NADPH to generate O22 (NOX1, 2, 3, 4, and 5) or H2O2 (DUOX) (Bedard & Krause, 2007; Kawahara et al., 2007); (v) and the MPO in the primary (azurophilic) granules of neutrophils and the lysosomes of blood monocytes, which converts H2O2 in the presence of halide anions (Cl2  Br2  I2) to a variety of toxic

products including hypohalous acids (e.g., HOCl), halogens (e.g., Cl2), and other oxidants (e.g., chloramines, hydroxyl radical [OH2], singlet oxygen [1O2]) (Klebanoff, 1999).

Generation of ROIs by Phenoloxidases and Respiratory Proteins

An ancient and highly conserved system for the generation of ROIs is the pro-phenoloxidase (pro-PO)/phenoloxidase (PO)/semiquinone/quinone system that is already found in primitive invertebrates. Inactive pro-PO, which is present in the hemolymph of many arthropods, is cleaved into active PO by a serine protease, which is triggered by pathogenassociated molecular patterns (PAMPs) interacting with the respective pattern-recognition receptors (PRR). The PO converts phenols into quinones, which either polymerize

5. ROIs and RNIs in the Immune System

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FIGURE 1 Schematic overview of antimicrobial effector mechanisms of neutrophils and other phagocytes. In resting neutrophils gp91phox (NOX2) and p22phox are located in the plasma membrane (depicted) as well as in the membrane of secondary and tertiary granules (not depicted), all of which can contribute to the formation of phagosomes. Upon stimulation (e.g. exposure to pathogens [black oval], crosslinking of Fc-receptors by opsonized particles) the Rho-GTPase Rac (Rac1 or Rac2 depending on the cell-type and species) and p47phox in the cytosol become activated by CARD9-mediated release of GDP-dissociation inhibitor (GDI) and subsequent guanine nucleotide exchange factor (GEF)-mediated exchange of GDP for GTP and by phosphorylation (asteriks), respectively, and translocate to the membrane. Subsequent translocation of p67phox and p40phox leads to the enzymatically active phagocyte NADPH oxidase complex, which generates O22 on the cell surface or within the phagosome. The O22 is converted into H2O2, either spontaneously by the acidic pH in phagolysosomes, or by host- or pathogen-derived superoxide dismutases (SOD). The iron-dependent Haber-Weiss-reaction (HWR), catalase (Cat) and myeloperoxidase (MPO) help to generate further species of ROI, all of which contribute to the killing of the pathogens (dashed oval). Inducible nitric oxide synthase (iNOS or NOS2), which is found in the cytosol (not depicted) as well as in a vesicular compartment (nitroxosomes) of macrophages and granulocytes, generates citrulline and nitric oxide (NO) from the amino acid l-arginine and molecular oxygen. Neutrophils contain large numbers of primary, secondary and tertiary granules, which are loaded with a broad spectrum of antimicrobially active compounds (see Table 1 for abbreviations and details).

into melanin or enter redox cycles with the highly reactive semiquinone, leading to the generation of O22. The deposition of melanin impedes the replication of microbes, whereas the formation of semiquinones and O22 contributes to the killing of microbes (Cerenius & Söderhall, 2004; Park et al., 2007). Recently, it was discovered that organisms of the subgenus Chelicerata (e.g., horseshoe crabs, ticks, and mites), which lack the hemolymphatic pro-PO system, utilize the oxygen-carrier protein hemocyanin for the generation of ROIs. Exposure of purified hemocyanin, first to microbial proteases (e.g., elastase of Pseudomonas aeruginosa, subtilisin A of Staphylococcus aureus) and then to PAMPs (e.g., lipopolysaccharide [LPS], lipoteichoic acid [LTA]), led to a substantial induction of PO activity and O2− production (Jiang et al., 2007). A similar observation was

made with the mammalian respiratory protein hemoglobin, which is frequently released from erythrocytes during bacteremic infections with pathogens that express hemolysins. When human Fe31 hemoglobin (methemoglobin) interacted with microbial proteases and PAMPs, its pseudoperoxidase activity (which leads to the formation of O22 from H2O2) was strongly enhanced (Jiang et al., 2007). The advantages of generating antimicrobially active ROIs by respiratory proteins are obvious. First, the ROIs release is instantaneously available because it does not rely on the activation of immune cells, enzymes, or signaling cascades of the hosts. Second, it is unlikely to cause widespread tissue damage, because it will only occur in close proximity to the pathogen due to the need for direct activation of the respiratory proteins by microbial proteases and PAMPs (Bogdan, 2007a).

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Generation of ROIs by the NOX/DUOX Family

The NOX/DUOX family of NADPH-dependent oxidases is evolutionarily highly conserved. In humans, it consists of five different isoforms of NOX (NOX1 to 5) and two isoforms of DUOX (DUOX1 and DUOX2). The NOX/DUOX isoforms differ in their cytosolic subunits, their primary tissue expression pattern and their known key functions in physiology and pathophysiology (Table 2). Independent of the exact enzymatic source, ROIs generated by NOX/DUOX exert three different categories of effects (i) modulation of cellular functions; (ii) regulation of cell differentiation and growth and of tissue repair; and (iii) induction of cell death and tissue destruction. From a molecular point of view these effects result from the oxidation of proteins, lipids, carbohydrates, and/or nucleic acids that either alter intra- or intercellular signaling processes, including gene transcription, or cause direct irreversible damage of cell membranes, organelles, cytoskeletal structures, or enzymes (Bedard & Krause, 2007; Brown & Griendling, 2009). With the exception of DUOX, which was recently implicated in the antimicrobial defense of mucosal epithelia cells (Geiszt et al., 2003; Donko et al., 2005) and the recruitment of leukocytes to wounds (Niethammer et al., 2009), the NOX2/Phox is the only member of the NOX/DUOX family for which a strong antimicrobial and regulatory function in the innate and adaptive immune system has been documented. Therefore, the following summary of the production and function of ROIs will focus on NOX2/Phox.

Enzymology, Subcellular Localization, and Regulation of Phox

NOX2, also termed gp91phox or CYBB (b-subunit of the flavocytochrome b558) is a NADPH:O2-oxidoreductase that transfers electrons from the substrate NADPH (electron donor) to the substrate molecular oxygen (electron acceptor) and generates NADP1, protons, and superoxide anions (O22) (see Fig. 1). 2 O2 1 NADPH → 2 O22 1 NADP1 1 H1 The rapid oxygen consumption that leads to the prompt production of large amounts of ROIs has been termed “respiratory burst” or “oxidative burst.” It results from the assembly of a multicomponent protein complex, which occurs in the plasma membrane or in intracellular membranes (Clark, 1999).

Formation and Localization of Active Phox

The NOX2 itself, which forms the catalytic core of the multiprotein Phox complex, contains two hemes as prosthetic groups and binding sites for the substrate NADPH and the cosubstrate FAD. NOX2 is constitutively associated with a 22 kDa protein (p22phox, a-subunit of flavocytochrome b558 [CYBA]), which stabilizes NOX2 and is thereby indispensable for NOX2 expression. The NOX2/p22phox heterodimer is termed flavocytochrome b558 (Nauseef, 2008b; 2008a). In resting human neutrophils, 70% to 90% of the flavocytochrome b558 is localized in the secondary (specific) and tertiary (gelatinase) granules, and 10% to 30% in the plasma membrane (Jesaitis et al., 1990; Kjeldsen et al., 1994). In mouse bone marrow-derived macrophages, flavocytochrome b558 is found in Rab11-positive recycling endosomes, in Rab5-positive early endosomes, and in the plasma membrane (Casbon et al., 2009). Phox is also detectable in certain lysosome-related organelles and phagosomes of mouse and human dendritic cells (Vulcano et al., 2004; Savina et al., 2009). Upon stimulation of phagocytes by phorbol ester, chemoattractants, pathogens, microbial products, or antibody-opsonized particles, at least four cytosolic proteins(p40phox, p47phox, p67phox, and the small Rho-GTPase Rac1 or Rac2) are

translocated to the membrane, where they associate with the flavocytochrome to form the active Phox (see Fig. 1). The membrane translocation of the p40-p47-p67phox complex and its interaction with p22phox and NOX2 requires the phosphorylation of a number of proteins, notably of p47phox, which, in its nonphosphorylated form, assumes an autoinhibited state (Lambeth et al., 2007; Nauseef, 2008a). Different isoforms of phosphoinositide 3-kinase (PI3K) are critical for the early (PI3K) and late phase (PI3Kd) production of phosphatidylinositol-3,4,5-trisphosphate (PtdIns[3,4,5]P3) in phagocytes following priming and stimulation. PtdIns(3,4,5)P3 regulates various protein kinases (e.g., AKT, PDK1, and certain protein kinase C [PKC] isoforms), which then phosphorylate the autoinhibitory domain of p47phox (Rommel et al., 2007). The membrane translocation of the GTPase Rac1 requires the caspase-recruitment domain (CARD) containing protein 9 (CARD9), which, upon phagocytosis, associates with the GDP-dissociation inhibitor (GDI) LyGDI, and thereby interrupts the blocking effect of LyGDI on GDP-Rac (Wu et al., 2009). The Ras-related GTPases Rap1a and 1b (not depicted in Fig. 1) can also associate with the Phox complex, but they are not essential for the oxidative burst (Li et al., 2007). Depending on the cell type, the active Phox complex assembles on the plasma membrane, on the early or recycling endosomes, or on secondary or tertiary granules. Each of these compartments can supply membrane to the forming phagosomes, which, therefore, are prone to express a strong activity of Phox. O22 can be released by activated neutrophils through exocytosis of intracellular granules carrying active Phox (Kobayashi et al., 1998). Also, following cell death, Phox subunits are found on so-called neutrophil extracellular traps (NETs) (Munafo et al., 2009), which are formed by webs of DNA decorated with histones and various antimicrobial effector proteins (Urban et al., 2009). It is evident that the release of O22 into phagosomes is suitable to combat phagocytosed pathogens, whereas the extracellular accumulation of ROIs might also cause severe tissue damage.

Regulation

The oxidative burst of professional phagocytes (neutrophils and macrophages) is efficiently triggered by Fc- or complement-receptor-mediated phagocytosis of pathogens, by internalization of immune complexes, by chemoattractants (e.g., IL-8, leukotriene B4, C5a, platelet-activating factor), exogenous arachidonic acid, and by certain microbial products such as bacterial peptides (e.g., N-formylmethionyl-leucyl-phenylalanine [fMLP]) and fungal cell wall components (e.g., zymosan). Experimentally, phorbol esters (e.g., phorbol 12-myristate 13-acetate [PMA]), IgG-coated latex particles, or sheep red blood cells are used to activate Phox (Babior, 1999; Yamauchi et al., 2004; Kim & Dinauer, 2006). Although resting phagocytes constitutively express the membrane-bound and cytosolic components of Phox, the activity of Phox is modulated by multiple factors, such as the micromilieu in the tissue, soluble mediators of the immune system, or microbial organisms. Known regulators of Phox are: (i) the concentrations of molecular oxygen, protons (pH value), and NADPH, as seen by the strongly reduced ROI production of phagocytes under hypoxic or acidic conditions (Gabig et al., 1979) or in the presence of an inhibitor of the NADPH-generating hexose-monophosphate shunt (Borregaard et al., 1984; Adachi et al., 2006); (ii) the store-operated calcium entry (SOCE), where the initial phospholipase C- and inositol-triphosphate-dependent depletion of intracellular calcium store causes the subsequent influx of extracellular Ca21 ions, which then mediate the

TABLE 2

NADPH-dependent oxidases (NOX/DUOX family) (Bedard and Krause, 2007; Kawahara et al., 2007; Lambeth et al., 2007)

Family member (catalytic core)

Membrane-bound subunits

NOX1

Cytosolic subunits

Cell/tissue distribution (selective examples) prototypic cell type in bold

Functions in physiology (selective examples)

Functions in pathology (selective examples)

NOXO1 3), NOXA1 4)

Colon epithelium, prostate, vascular smooth muscle cells, endothelial cells, osteoclasts, keratinocytes, microglia

Proliferation of smooth muscle and colonic epithelial cells (?); angiogenesis; osteoclast differentiation

Prostate cancer (?), colonic cancer (?), inflammatory bowel disease (?), neurotoxicity (?)

p22phox

Rac1 or Rac2, p40phox, p47phox, p67phox

Phagocytes (neutrophils, monocytes, macrophages, dendritic cells, B and T cells, hepatocytes, endothelial cells, keratinocytes, neurons

Antimicrobial defense, anti-inflammation, signaling in immune cells; cognitive functions (memory, learning); osteoclast activity; tissue repair

Apoptosis, tissue damage; hypertension; inflammatory diseases; atherosclerosis; neurodegeneration; liver injury/ cancer

NOX3

p22phox

NOXO1 NOXA1(?) Rac (?)

Inner ear (cochlear and vestibular sensory epithelia), fetal tissues

Formation of otoconia crystals in the endolymph of the inner ear

Age-, noise- or drug-induced hearing loss?

NOX4

p22phox

Rac (?)

Kidney cortex and tubular epithelium, osteoclasts; endothelial, smooth muscle and hematopoietic stem cells, hepatocytes, keratinocytes, fibroblasts; neurons, ovaries

Growth of smooth muscle and endothelial cells (?); enhanced adipocyte differentiation (?); osteoclast activity; wound healing; oxygen sensing (?)

Cellular senescence; diabetic nephropathy (?); pulmonary hypertension and fibrosis; growth of melanoma cells (?)

NOX5

None

None (regulation by Ca21 fluxes)

Testis (spermatocytes), spleen (mantle zone [B cells] and periarterial lymphoid sheath [T cells]), ovaries

Sperm development and function; meiotic maturation of oocytes (?)

Prostate cancer (?)

DUOX1

p22phox (?) DUOXA11)

None

Epithelial cells of the thyroid (thyrocytes) and airways, prostate

Prerequisite for thyroid hormone synthesis; mucosal antimicrobial defense; wound healing

DUOX2

p22phox (?) DUOXA22)

None (regulation by Ca21 fluxes)

Epithelial cells of the thyroid, salivary glands, airways, and gastrointestinal tract

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1), 2) DUOX maturation proteins located in the endoplasmatic reticulum (ER), which are crucial for preventing the ER-retention of DUOX1 and DUOX2. 3) NOXA1 (NOX activator 1 5 p67phox homolog). 4) NOXO1, NOX organizer 1 (p47phox homolog).


NOX2

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translocation of Rac, enhance the formation of the active Phox complex (by recruiting the calcium-binding myeloidrelated proteins MRP8 and MRP14), and activate PKC for the phosphorylation of cytosolic Phox components (Brechard & Tschirhart, 2008); (iii) the cytokines tumor necrosis factor (TNF) and IFN- and the LPS of gramnegative bacteria (Ding et al., 1988; Amezaga et al., 1992; Adachi et al., 2006); or (iv) intracellular pathogens such as A. phagocytophilum, which reside in neutrophils and partially shut down the oxidative burst (Garcia-Garcia et al., 2009; Bussmeyer et al., 2010). Pretreatment of macrophages or neutrophils with TNF, IFN-, or LPS (so-called “priming”) potentiates the production of O22 in response to a triggering agent. Part of the enhanced oxidative burst of IFN-- or TNF-primed phagocytes is likely to result from an increased mRNA expression of gp91phox, p47phox, p67phox, and/or p22phox. The transcription factors PU-1, activated protein (AP)-1, interferon-regulatory factor (IRF)-1, and interferon consensus sequence-binding protein were shown to participate in the upregulation of p67phox and/or gp91phox mRNA (reviewed in Bogdan, 2004). Recently, an entirely novel mechanism for the priming, as well as the activation of neutrophils by TNF for enhanced Phox activity was proposed. Yazdanpanah et al. (2009) reported that in mouse and human fibroblasts riboflavin kinase (RFK), which causes the conversion of vitamin B2 (riboflavin) into flavin mononucleotide (FMN) and—via the flavin adenin dinucleotide (FAD) synthetase—further into FAD, is a TNF responsive enzyme. Stimulation by TNF had three major consequences. First, RFK was rapidly recruited to the TNF receptor 1 (TNFR1) complex. Second, within the same time frame, p22phox, NOX1, NOX2, and Rac1 became detectable in the TNFR1 receptosome, provided RFK was present. Third, the cellular levels of FMN and FAD increased. RFK was found to bind to the TNFR1 death domain and to p22phox. This bridging function of RFK was essential for TNFinduced ROI production. Yazdanpanah et al. (2009) argued that the increased activity of RFK in close proximity to Phox leads to greater FAD saturation of NOX1 and NOX2 and accounts for the priming and activating effect of TNF. For a long time, the mechanism(s) by which microbial ligands of Toll-like receptors (TLR)—such as the TLR4agonist LPS—prime and activate Phox have been unclear. Detailed biochemical analyses and the study of patients with defined immunodeficiencies revealed that the IL-1 receptor-associated kinase (IRAK) 4 and the NF-kB essential modulator (NEMO or IkB kinase ), two kinases downstream of TLR4, are critical for the phosphorylation and/or membrane translocation of p47phox or p67phox, respectively (Pacquelet et al., 2007; Singh et al., 2009). In contrast, the TLR9-induced oxidative burst of neutrophils was normal in the absence of IRAK4, most likely because cross-linking of TLR9 led to PI3K-mediated phosphorylation of cytosolic Phox components (Hoarau et al., 2007).

Function of ROIs: Antimicrobial Activity

One of the key functions of ROIs in the immune system is their contribution to the control of infectious pathogens by neutrophils, macrophages, and other phagocytic cells (Bogdan et al., 2000; Bogdan, 2004; Fang, 2004; Segal, 2005). The antimicrobial activity of Phox can be due to five different categories of mechanisms. •

ROIs might exert direct toxic effects on viruses, bacteria, protozoa, and fungi, which can be damaged via oxidation, peroxidation, hydroxylation, or chlorination of proteins, lipids, nucleic acids, iron-sulfur clusters, or heme prostetic groups (reviewed in Klebanoff, 1999; Bogdan, 2004; Fang, 2004).

•

•

•

•

The Phox promotes the activity of serine proteases with a neutral pH optimum (e.g., cathepsin G and elastase). The release of the anionic superoxide (O22) into the phagosomal vacuole necessitates charge compensation. This occurs partly by the influx of K1 ions, partly by the reaction of O22 with protons that are pumped into the vacuole by the H1/ATPase, or are derived from acidic granules fusing with the phagosome. These events raise the intravacuolar tonicity and pH (Segal et al., 1981; Reeves et al., 2002). The hypertonic K1 displaces the cationic granule proteases (cathepsin G, elastase) from the anionic proteoglycan granule matrix, whereas the neutral to alkaline environment provides optimal pH conditions for their proteolytic activity (Segal, 2005). ROIs, notably H2O2, can cause a novel form of cell death (“NETosis”) that is characterized by the extrusion of “neutrophil extracellular traps” (NETs) (Fuchs et al., 2007), which fulfill antimicrobial functions (see above and Table 1). ROIs help to block the phagosomal escape of intracellular bacteria (e.g., L. monocytogenes) into the cytosol (Myers et al., 2003). Several of the signaling functions of ROIs (see below) undoubtedly contribute to the development of a protective immune response and thereby also to the control of infectious pathogens.

The function of Phox for the defense against bacterial, protozoan, or fungal pathogens is best documented by the analysis of the course of infections in transgenic mouse models. The analysis of p47phox-, gp91phox-, Rac2-, or MPO-deficient mouse strains revealed that depending on the type of pathogen, ROIs were dispensable (e.g., A. phagocytophilum) or played a contributory (e.g., Leishmania donovani, L. monocytogenes) or essential role (e.g., Aspergillus fumigatus, S. aureus) for combatting the pathogen and/or for resolution of the inflammation (reviewed in Bogdan, 2004; Fang, 2004; Birkner et al., 2008).

Function of ROIs: Signaling and Immunomodulation

The signaling function of H2O 2 and other ROIs has been subject of earlier detailed reviews (Reth, 2002; Nathan, 2003; Gloire et al., 2006; Stone & Yang, 2006; Brown & Griendling, 2009). Numerous in vitro and in vivo studies have demonstrated that ROIs regulate the differentiation, proliferation, function, and death of many cell types. From a molecular point of view, ROIs alter the redox status of the cells, for instance, by lowering the content of thiols such as glutathione, which secondarily affects the function of many enzymes (Forman & Torres, 2001). Therefore, it was not surprising to see that ROIs, in particular H2O 2, modulate various signaling pathways (e.g., phosphatases, phospholipases, Janus kinases [JAK], signal transducers and activators of transcription [STATs], mitogen activated protein kinases [MAPK], and ion channels) and transcription factors (e.g., NF-kB). The activation of Phox and the production of ROI turned out be essential for certain steps in the signaling cascade of TLRs and NOD-like receptors, which sense extracellular or intracellular pathogens respectively (Ogier-Denis et al., 2008; Martinon et al., 2009). As PRR also trigger Phox (see above), the inflammatory processes following the detection of pathogens and the oxygen-dependent antimicrobial effector mechanisms are interdependent events. Some more recently recognized effects of ROIs in the immune system will follow.


Differentiation of Immune Cells

In Drosophila melanogaster, multipotent progenitor cells in the medullary zone of the primary lobe of the lymph gland give rise to three different types of mature hemocytes: plasmatocytes, which support wound healing, phagocytose bacteria, and form extracellular aggregates to entrap bacteria; crystal cells, which express phenol oxidase activity; and lamellocytes, which are crucial for the encapsulation reaction similar to the granuloma formation in vertebrates. OwusuAnsah and Banerjee (2009) observed ROI production by the multipotent progenitor cells and found that the production of ROI was critical for the development of the mature hemocytes through a signaling pathway that involved the activation of the Jun N-terminal kinase (JNK) and of the Forkhead box protein O (FoxO) as well as the downregulation of polycomb proteins. In mammals, there is compelling evidence that the ability of macrophages to fuse and to form multinucleated giant cells or osteoclasts is dependent on Phox-generated ROI (Quinn & Schepetkin, 2009).

Wound Healing

ROI and various redox-sensitive events contribute to the wound healing response (Sen & Roy, 2008), but the exact source(s) of ROI that drive the dermal tissue repair remain largely unknown. Recently, Niethammer et al. (2009) demonstrated that in locally injured zebra fish larvae, a tissue-scale gradient of H2O2 generated by DUOX acivity in epithelial cells is required for the directional migration and infiltration of leukocytes into the wound. It is currently unclear whether this phenotype results from a direct chemotactic effect of H2O2 or is due to the release of a downstream chemoattractant.

Regulation of Antigen Presentation

The activation of Phox following endocytosis of microbes or antigenic particles antagonizes the acidification of the phagosomes by acidic granules (neutrophils) or by the vacuolar ATPase-dependent H1 pump (dendritic cells) because the generated O22 anions essentially consume protons. In neutrophils, the Phox-mediated neutralization or alkalinization of the phagocytic vacuole catalyzes the activity of serine proteases delivered to the phagosome by acidic granules (see above). In contrast, in dendritic cells, a neutral or alkaline pH limits the degradation of T cell-stimulatory peptides and epitopes by lysosomal cysteine and aspartyl proteases with an acidic pH optimum (e.g., cathepsin D). Consequently, in mouse and human dendritic cells, the Rab27a-dependent recruitment of “inhibitory lysosome-related endosomes,” which contain membrane subunits of Phox, to the phagosome and the Rac2-mediated assembly of Phox in the phagosomal membrane are prerequisites for MHC class I-restricted presentation of exogenous (phagosomal) antigens (so-called cross-presentation) (Savina et al., 2009).

Regulation of T Cell Survival and Function

The activation of T lymphocytes via T-cell receptor stimulation is associated with an early onset and sustained production of H2O2 and a delayed release of O22. mRNA and/ or protein analyses revealed the unexpected presence of p22phox, p47phox, p67phox, and gp91phox in primary mouse and human T-cell blasts (Jackson et al., 2004). Whereas the early production of H2O2 and the late-phase release of O22 were independent of NADPH oxidase, the sustained generation of H2O2 required Phox components as well as Fas/FasL interactions (Jackson et al., 2004). TCR-triggered ROI release is likely to contribute to T-cell homeostasis, because p47phox- or gp91phox-deficient T cells showed an improved survival in vivo, an enhanced activation of Erk

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kinase, and a relative increase of T-helper type-1 cytokine secretion (Jackson et al., 2004; Donaldson et al., 2009; Purushothaman & Sarin, 2009). Mice deficient in p47phox developed lymph node hyperplasia (with increased numbers of B and T cells) even in the absence of infection (Donaldson et al., 2009) and an increased severity of T-cell-driven autoimmune diseases (Gelderman et al., 2007a; Gelderman et al., 2007b; Hultqvist et al., 2009). There is also evidence for the expression of NOX1 in naive CD41 T lymphocytes, which might participate in IL-4 receptor signaling and the inactivation of receptor-associated protein tyrosine phosphatases (Sharma et al., 2008).

Anti-Inflammatory Function of ROIs

Whereas the pro-inflammatory and tissue-destructive functions of ROI are known from infectious, autoimmune, or degenerative disease models (Snelgrove et al., 2006; Sorce & Krause, 2009), the anti-inflammatory role of ROIs, which was first identified as an effector mechanism of suppressor macrophages (Metzger et al., 1980) and later rediscovered in myeloid-derived suppressor cells (reviewed in Bronte & Zanovello, 2005; Nagaraj et al., 2009), has only recently received new attention (Hultqvist et al., 2009). Patients with congenital defects of Phox components suffer from chronic granulomatous disease (CGD), which is characterized by recurrent bacterial and fungal infections, but also by sterile hyperinflammatory responses and an increased incidence of autoimmune diseases. In vitro and in vivo analyses suggest that multiple mechanisms account for the enhanced inflammation that is seen in the absence of functional Phox (Schappi et al., 2008). These include (i) a reduced spontaneous apoptosis of CGD neutrophils (Brown et al., 2003); (ii) a defective externalization of phosphatidyl-serine in CGD neutrophils, which causes an impaired recognition and removal (efferocytosis) of apoptotic neutrophils by macrophages (Fernandez-Boyanapalli et al., 2009; Sanmun et al., 2009); (iii) an inefficient indoleamine-2,3-dioxygenase-catalyzed and superoxide-dependent conversion of tryptophan into 3-hydroxykynurenine, which leads to an expansion of Th17 cells and d T cells and to a lack of regulatory T cells (Romani et al., 2008); (iv) a missing oxidative inactivation of proinflammatory mediators (e.g., MRP8/S100A8 protein) (Harrison et al., 1999); (v) a general deficiency of ROI-dependent signaling, which leads to a preponderance of proinflammatory stimuli (e.g., Ca21, IL-8) and a lack of antiinflammatory mediators (e.g., adenosine, cAMP, prostaglandin D2, transforming growth factor-b) (Geiszt et al., 1997; Brown et al., 2003; Lekstrom-Himes et al., 2005; Rajakariar et al., 2009).

REACTIVE NITROGEN INTERMEDIATES Pathways for the Generation of RNIs

The terms RNI, RNS, or reactive nitrogen metabolites (RNM) are used for all inorganic or organic compounds that consist of nitrogen and oxygen, exhibit a nitrating or nitrosating capacity and may carry a halogen or sulfur group. Examples are the radicals nitric oxide (also called nitrogen monoxide, NO) and nitrogen dioxide (NO2), the nitrosonium cation (NO1), the anions nitroxyl (NO2) and peroxynitrite (ONOO2), the dinitrogen trioxide (N2O3), nitryl chloride (NO2Cl) and various S-nitrosothiols (e.g., S-nitrosocysteine). The term RNI also covers higher oxidation products of NO such as nitrite (NO22) and nitrate (NO32). The key enzyme system to generate RNIs are the three different isoforms of nitric oxide synthase (NOS). All NOS isoforms are homodimeric enzymes that, in the presence of

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molecular oxygen, convert the amino acid l-arginine into Nv-hydroxy-l-arginine and further into citrulline and NO, which initially is complexed to ferric heme (Fig. 2). The formation of the NOS homodimer and the complex oxidoreductase reaction require the binding of calmodulin, the incorporation of iron protoporphyrin IX (heme) and zinc, and the presence of multiple cofactors and cosubstrates ([6R]-tetrahydrobiopterin [BH4], NADPH, FMN, FAD, thiol) (Stuehr et al., 2009). Two of the NOS isoforms (neuronal NOS, nNOS, or NOS1; endothelial NOS, eNOS, or NOS3) are constitutively expressed in many cell types and tissues. They are mainly but not exclusively subject to posttranslational regulation by Ca21 fluxes and primarily fulfill homeostatic functions in the cardiovascular system (eNOS) and the musculoskeletal and central nervous system (nNOS). Due to their Ca21-dependency and constitutive expression these two isoforms are frequently termed cNOS. There is evidence for the expression and function of nNOS and eNOS in inflamed tissues (Rabelink & Luscher, 2006; Zhou & Zhu, 2009) and in cells of the immune system (e.g., T lymphocytes) (Bogdan, 2001a; Ibiza et al., 2008). A third isoform of NOS has been termed inducible nitric oxide synthase (iNOS or NOS2), because it is not expressed in strictly resting cells, but only after exposure to pathogens, microbial ligands, and/or cytokines. iNOS is readily inducible in mouse and human cells that typically encounter microbial pathogens and respond to pro-inflammatory stimuli (e.g., macrophages, dendritic cells, hepatocytes, and epithelial and endothelial cells) and is widely expressed during infectious, autoimmune, malignant, and other inflammatory processes. Whereas the expression of iNOS by human macrophages in vivo is indisputable (Fang & Nathan, 2007), its inducibility in human monocytes and macrophages in vitro is a matter of debate (Bogdan, 2000, 2001a). The expression of iNOS in primary T lymphocytes is highly controversial because most studies lack the analysis of iNOS protein in single T cells (Bogdan, 2000, 2001a; Vig et al., 2004; Choy & Pober, 2009).

Another enzyme that can participate in the generation of RNI is MPO. NO22 converts the inactive form of MPO (compound II, MPO-Fe[IV]) into the native enzyme (ground state, MPO-Fe[III]), thereby releasing the potent nitrating  NO2 species. Furthermore, HOCl can also react with NO22, leading to the formation of NO2Cl, which shows a strong chlorinating reactivity towards aromates (Eiserich et al., 2002). RNI can also originate (i) from the chemical reaction between O22 and NO, which leads to the generation of peroxynitrite (ONOO2) (Ferrer-Sueta & Radi, 2009); (ii) from nitrite anions (NO22), which under acidic conditions or in the presence of desoxyhemoglobin release NO; or (iii) from the nitrite reductase and anhydrase reaction of methemoglobin, which catalyzes the reaction between NO22 and NO to form N2O3 (Basu et al., 2007). With the exception of the generation of peroxynitrite, the footprint of which (i.e., tyrosine nitration) is frequently seen in inflamed tissues, the (patho)physiological role of these iNOS-independent routes of generation of RNI remains to be established. The rest of this chapter will therefore exclusively deal with the expression and function of iNOS.

Subcellular Localization and Regulation of iNOS

The expression of iNOS mRNA and protein has been extensively studied in macrophages and other phagocytes, but also in hepatocytes, cardiac myocytes, endothelial cells, epithelial cells, and keratinocytes (Bogdan, 2000). In macrophages, iNOS protein and activity has been detected in three different locations: in the cytosol; in a membranous (particulate) compartment consisting of 50 to 80 nm vesicles, which were operationally termed “nitroxosomes” and await further characterization; and in the cortical actin cytoskeleton immediately beneath the plasma membrane (reviewed in Bogdan, 2000, 2001a, 2004). In human embryonic kidney cells, primary human bronchial epithelial cells, bladder papilloma cells, cardiac myocytes, and vascular smooth muscle cells, iNOS mRNA or protein was not only diffusely distributed in the cytosol, but also found in perinuclear inclusion-like bodies which were coined “physiologic aggresomes” (Jones et al., 2007; Pandit et al., 2009).

Positive and Negative Regulators of iNOS

FIGURE 2 Generation of nitric oxide from l-arginine by nitric oxide synthases (NOS). Only those cofactors are depicted, which are directly involved in the flux of electrons. The initial product of the reaction is not free NO, but a ferric heme-NO complex (not depicted). For further details see text.

iNOS is strongly and rapidly induced by proinflammatory cytokines, notably by IFN-ab and IFN-, in combination with a TLR ligand (e.g., LPS, flagellin, mycobacterial proteins and lipoarabinomannan, H. pylori urease, hemozoin, Trypanosoma cruzi, filarial cystatins; viral, bacterial, or protozoan DNA, unmethylated CpG desoxyoligonucleotides, double stranded RNA) or with a ligand or cross-linker of a costimulatory cell surface receptor (e.g., CD40 ligand, antiCD8, anti-Fc receptor IIb [anti-CD23]). Additional factors that have been reported to coinduce iNOS in macrophages are environmental and milieu factors (e.g., hypoxia, acidic pH, hyperthermia, ionizing radiation, monosodium urate crystals), hormones (e.g., estrogens), and the activation of certain ion channels (e.g., potassium channel). Conversely, a number of cytokines (e.g., transforming growth factor-b, IL-4, IL-13), nonproteinaceous soluble mediators (e.g., peroxisome proliferator-activated receptor- agonists), glucocorticoids, infectious pathogens, or their products (e.g., LPS, leishmanial lipophosphoglycan, Candida albicans, Francisella tularensis) downregulated or counteracted the induction of iNOS in macrophages (Bogdan, 2000; Nathan & Shiloh, 2000; Bogdan, 2001a, 2004; Fang, 2004; Dai et al., 2008; Parsa et al., 2008; Fernandez-Arenas et al., 2009). From a mechanistic point of view, the expression of iNOS in macrophages is regulated at the level of gene transcription, mRNA stability, mRNA translation, protein stability, and enzyme activity.


iNOS Gene Transcription

Potent regulators of iNOS gene transcription in macrophages are STAT1 and STAT2, IRF-1, NF-kB and AP-1 (activated protein-1), which are activated by IFN-, IFN-ab, TNF, or TLR ligands (Bogdan, 2001a). In many cell types, additional transcription factors (e.g., octamer factor, peroxisome proliferator-activated receptors, hypoxia-inducible factor-1, nuclear factor IL-6, zinc finger Kruppel-like factor 4) contribute to the activation of the iNOS promotor. Furthermore, the induction of iNOS is modulated by the various enzymes that intersect with the signaling cascades of the JAK/STAT, the TLR, and/or the NF-kB pathway (e.g., PKC; PI3K; protein tyrosine phosphatases; phosphatase and tensin homologue deleted on chromosome 10 [Pten]; MAPK, such as extracellular signal-regulated kinase 1 and 2 [ERK1/2], suppressors of cytokine signaling [SOCS]) (Bogdan, 2001a; Kleinert et al., 2004; Feinberg et al., 2005).

iNOS mRNA and Protein

The analysis of this level of iNOS regulation was pioneered by Vodovotz et al. (1993), who found that TGF-b downregulates the stability of iNOS mRNA as well as the synthesis and stability of iNOS protein. Since then, several proteins have been described that bind to AU-rich elements within in the 3-untranslated region of the iNOS mRNA and either stabilize (e.g., tristetraprolin; embryonic lethal abnormal vision-like protein, HuR; human polypyrimidine tract binding protein, PTB [also termed heterogenous nuclear ribonucleoprotein I hnRNP I]) or destabilize the iNOS mRNA (e.g., KH-type splicing regulatory protein [KSRP], mouse PTB) (Korhonen et al., 2007; Soderberg et al., 2007, and references therein). The reduced iNOS protein stability in TGF-b-treated macrophages most likely results from enhanced ubiquitination and proteasomal degradation (Kolodziejski et al., 2002; Mitani et al., 2005; Takaki et al., 2006). Conversely, analogs of cyclic adenosine monophosphate (cAMP) or activators of cAMP synthesis inhibited the ubiquitination of iNOS in macrophages and thereby enhanced the stability of the iNOS protein (Won et al., 2004). In inflammatory mouse macrophages, in a mouse keratinocyte cell line, and in neonatal rat astrocytes, it was observed that the synthesis of iNOS protein is dependent on the availability of l-arginine. A deficiency of extracellular arginine ( 10 M) led to a striking reduction of iNOS protein without altering the level of iNOS mRNA (reviewed in König et al., 2009). Arginine depletion can result from the IL-4-, IL-10-, IL-13-, or TGF-b-mediated induction and activation of arginase, which converts arginine into ornithine and urea (Morris, 2009) and might underlie the counterprotective effect of type-2 T-helper cell responses during infections with pathogens that are controlled by iNOS. The molecular mechanism underlying this unique form of posttranscriptional regulation of iNOS is currently being investigated in the laboratory of the author. If the arginine depletion results from a hyperstimulation of arginase, it is conceivable that spermine, a product of the ornithine metabolism, accounts for the inhibition of iNOS protein synthesis (Bussiere et al., 2005). Recently, the first evidence was presented that small noncoding RNAs (so-called microRNAs), which are known to suppress gene expression at the posttranscriptional level, might be involved in the regulation of iNOS (Dai et al., 2008). MicroRNAs might directly block iNOS protein translation and/or degrade iNOS mRNA, which has not yet been demonstrated, or modulate the expression of iNOS indirectly via downregulation of iNOS regulatory genes (Wang et al., 2009).

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iNOS Enzyme Activity

Considering the known enzymology of iNOS (Stuehr et al., 2009), the activity of iNOS to generate NO is dependent on the presence of the substrate l-arginine and of multiple structural and enzymatic cofactors (see above). Like all heme-containing flavoproteins, iNOS is quite susceptible to inhibition by oxidative or nitrosative stress (Sun et al., 2009). In the presence of high amounts of NO, iNOS loses its capacity to generate NO but instead functions as a NADPH-dependent NO-oxygenase (Stuehr et al., 2009). The downregulation of iNOS activity in the presence of exogenous NO, peroxynitrite or ROI might serve as a feedback control in inflamed tissues. Enzymatically inactive iNOS protein can be sequestered in cytoplasmic inclusion-like bodies (aggresomes) (Pandit et al., 2009).

Functions of iNOS: Conceptual Framework

The functions of iNOS in the immune system result from the three possible consequences of the iNOS reaction: (i) the depletion of arginine; (ii) the intermediate generation of Nv-hydroxy-l-arginine, a known inhibitor of host cell as well as parasite arginases; and (iii) the generation of NO (Fig. 3). The highly reactive NO and the  NO-derived congeners have received the greatest attention. Due to their multiple reaction partners (see below) they exert a great variety of effects on immune cells, host organs, and on infectious pathogens, ranging from benign signaling events to overt toxicity, such as microbial death, tissue damage, or vasodilatory shock (Bogdan, 2001b, 2001a; Nathan, 2003). As arginine is a semiessential amino acid for the mammalian host organism and is strictly required for the growth of some pathogens (Gaur et al., 2007; Reguera et al., 2009), iNOS-mediated arginine depletion could not only impair the function of immune cells (Rodriguez et al., 2007), but also contribute to the control of infectious pathogens (see below). The question, whether the iNOS intermediate Nv-hydroxy-l-arginine is relevant as an inhibitor of host or parasite arginases in vivo, is a matter of debate. Considering the fact that Nv-hydroxy-l-arginine does not accumulate, but rapidly enters the second step of the iNOS reaction (Stuehr et al., 2004), strongly argues against this possibility. However, independent of the underlying mechanism, iNOS/NO routinely assumes a bipartite role (i.e., cytotoxic effector function versus signaling and immunomodulatory function) in all major disease categories (i.e., in infectious, autoimmune/inflammatory, malignant, or cardiovascular diseases) (Fig. 3).

Functions of iNOS: Antimicrobial Activity

The antimicrobial effects of iNOS/NO fall into four major categories.

Direct (Cyto)toxic Effects of NO

NO acts on (i) structural components; (ii) the genome and the replication, transcription, and translation machinery; (iii) metabolic products and nutrient pathways; and (iv) virulence factors of infectious pathogens. Major molecular targets of NO are DNA molecules, where NO can cause mutations, oligonucleosomal fragmentation and inhibition of synthesis and repair; structural proteins, which are altered by S-nitrosylation, ADP-ribosylation, or tyrosine nitration; enzymes and metalloproteins, which can be inactivated by disruption of Fe-S clusters, zinc fingers, or heme groups; and membrane lipids that can be modified by peroxidation (Bogdan, 2001b, 2004; Fang, 2004; Bogdan, 2007b; Jones-Carson et al., 2008). Paradoxically, the toxic effects of NO can be expedited by microbial porins, which facilitate the penetration of NO through the bacterial cell wall (Fabrino et al., 2009). 

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FIGURE 3 Conceptual framework for iNOS-dependent effects in the immune system (for details see text).

NO is capable of reacting with O22 generated by Phox or by the pathogen itself, which leads to the formation of peroxynitrite (ONOO2), an RNI species with major nitrating capacity (Ferrer-Sueta & Radi, 2009). The cytotoxic function of NO and ONOO2 is not only restricted to pathogens, but may also extend to host cells. This explains why iNOS-deficient mice might exhibit less severe disease and a higher survival rate after infection with certain pathogens (reviewed in Bogdan, 2000, 2001a). 

NO-Independent Antimicrobial Effect of iNOS

In the absence of l-arginine or arginase (which generates ornithin from l-arginine and, via the ornithin decarboxylase, the growth-promoting polyamines) pathogens such as trypanosomes, Leishmania, Giardia lamblia, or Schistosoma mansoni fail to proliferate or survive (Bogdan, 2004; Gaur et al., 2007; Muleme et al., 2009). The presence of an arginine biosynthetic pathway in obligatively intracellular bacteria such as Ehrlichia chaffensis might rescue these bacteria after arginine depletion by iNOS (Hotopp et al., 2006). Nv-hydroxy-l-arginine is a potent inhibitor of arginases, including arginases of eukaryotic pathogens (Kropf et al., 2005), which has led to the suggestion that it might exert antileishmanial activity (Iniesta et al., 2001). To date, however, there is no in vivo evidence for this hypothesis.

NO-Mediated Inhibition of Microbial Evasion and Resistance Mechanisms

Microbial pathogens express a number of virulence and resistance factors, which protect them against host effector molecules. Some of these evasion mechanisms, such as the expression of RNI or ROI detoxification systems, are specifically upregulated in the presence of oxidative or nitrosative

stress or by NO derived from microbial NOS (Nathan & Shiloh, 2000; Nathan, 2003; Bogdan, 2004; Fang, 2004; Shatalin et al., 2008; Das et al., 2009; Laver et al., 2009; Purwantini & Mukhopadhyay, 2009). On the other hand,  NO and its congeners also convey signals to the host cells, which allow them to overcome at least some of the defense strategies of the microorganisms. Paradigmatic examples for this kind of NO-mediated counterstrike are (i) the dispersal of biofilms formed by Pseudomonas aeruginosa, which otherwise protect these bacteria from the action of phagocytes, antimicrobial peptides, and antibiotics (Barraud et al., 2006); (ii) the inhibition of the phagosomal escape of Listeria monocytogenes into the cytosol (Myers et al., 2003); (iii) the reversal of the blockade of phagosomal maturation and phagolysosomal fusion in macrophages infected with Leishmania donovani or Mycobacteria tuberculosis (Winberg et al., 2007; Axelrod et al., 2008); and (iv) the inhibition of the gene expression, protein synthesis, and function of effector molecules and toxins of Salmonella enterica serovar Typhimurium or enterohemorrhagic Escherichia coli (McCollister et al., 2005; Vareille et al., 2007).

Paving of a Protective Immune Response by NO

NO that is released by iNOS-positive phagocytes feeds back on the producer cell as well as on other immune cells. Some of the ensuing effects contribute to the development of a protective immune response against infectious pathogens and can therefore be viewed as a fourth category of antimicrobial effects of NO (Bogdan, 2000, 2001a, 2004). Notable activities of NO along this line of thinking are the proposed type 1 T-helper cell-stimulatory effect of NO (Niedbala et al., 2002), the enhancement of antigen processing and presentation by dendritic cells (Duan et al., 2008; Huang et al., 2008), 


and the possible protection of immune cells from premature death due to the antiapoptotic function of NO (Brune, 2003; Choy & Pober, 2009).

Functions of iNOS: Signaling and Immunomodulation

As already indicated above NO and other RNI have a strong regulatory effect on cells of the immune system. This is due to the fact that immune cells are phenotypically highly versatile and undergo phases of lineage differentiation, proliferation, functional diversification, and regulated cell death during inflammatory processes. Many of the known molecular targets of NO have been studied in detail in immune cells and play an important role in shaping of the immune response. These include soluble guanyl cyclase; ion channels; heterotrimeric GTP-binding proteins; phospholipases; NOTCH1 receptor; TLR adaptor molecules (e.g., MyD88 [myeloid differentiation molecule 88]); JAK and MAP kinases; Src kinases; members of the STAT family; caspases; protein phosphatases; metalloproteases; transcription factors (e.g., NF-kB, AP-1, Sp1, HIF-1[hypoxia inducible factor-1]); cell-cycle proteins (e.g., cyclin D1); histone (de)acetylases; DNA methylases; regulators of mRNA stability and translation (e.g., iron regulatory protein-1 and -2); and enzymes that catalyze the posttranslational processing of cytokines (e.g., IL-1b converting enzyme, TNF-converting enzyme) (Bogdan, 2000, 2001b, 2001a; Nathan, 2003; Bogdan, 2004; Into et al., 2008; Kim et al., 2008). In the following, some more recently published effects of iNOS/NO on dendritic cells and T lymphocytes will be summarized.

Dendritic Cells and Antigen Presentation

The ability to express iNOS in response to cytokines and TLR ligands is a general feature of mouse myeloid dendritic cells in vitro and during chronic inflammations in vivo (De Trez et al., 2009) and does not constitute a separate subpopulation of dendritic cells as previously suggested (Serbina et al., 2003). In dendritic cells, endogenous or exogenous NO promotes the depolymerization, processing, and presentation of bacterial carbohydrate antigens by the MHC class II pathway and the induction of MHC class II and of costimulatory molecules (CD80 and CD86) on the plasma membrane. iNOS interacted with the MHC class II-associated invariant chain (CD74) and NO inhibited the caspase-dependent degradation of CD74, which supported the traffic of MHC class II molecules to the cell surface (Duan et al., 2008; Huang et al., 2008). An interesting issue is the question of whether RNI (e.g., NO, ONOO2) and ROI (e.g., HOCl) can alter the immunogenicity of antigens via chemical modification (e.g., oxidation of carbohydrate side-chains, tyrosine nitration, formation of protein aldehydes or chloramines). Recent data suggest that RNI and ROI might function as natural adjuvants causing the generation of neoantigens, which are more efficiently endocytosed (Prokopowicz et al., 2009). It is possible that RNI- or ROI-generated neoantigens contribute to epitope spreading in chronic inflammatory and autoimmune diseases (Griffiths, 2008).

T Lymphocytes

Numerous studies have shown that the proliferation and cytokine production of T lymphocytes in response to specific antigens or mitogens is controlled by the expression of l-arginine-metabolizing enzymes (iNOS and arginase) in myeloid cells. Three different mechanisms were reported: (i) NO derived from iNOS suppressed the IL-2 receptor signaling pathways (JAK/STAT, Ras/MAPK, PI3K/Akt) and the production of IL-2 (Bronte & Zanovello, 2005); (ii) the activity of iNOS or arginase depleted l-arginine, which subsequently caused a strong downregulation of

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CD3z, the main signal-transduction component of the T-cell receptor (Rodriguez et al., 2003); and (iii) the consumption of l-arginine by arginase deprived iNOS of its substrate, which led to a reduced production of NO. As  NO normally scavenges O22, the arginine depletion resulted in an accumulation of H2O2 (derived from O22), which suppressed T-cell proliferation (Kusmartsev et al., 2004). These and other data demonstrated that not only Phox, but also iNOS and arginase are key effector pathways of suppressor macrophages and myeloid-derived suppressor cells (Marigo et al., 2008; Nagaraj et al., 2009). They form a feedback control of T-cell activation, contribute to the resolution of inflammatory processes, and account for the suppression of T-cell responses in certain nonhealing infectious diseases and malignancies (Marigo et al., 2008; Modolell et al., 2009). A down regulation of T-cell responses was also observed in the presence of dendritic cells that expressed increased amounts of iNOS and NO in response to IFN- after exposure to apoptotic cells (Ren et al., 2008). Inflamed endothelium blocked the adhesion of Th1 cells in an iNOS/NO-dependent manner, which might represent another mechanism to terminate a T-cell response in situ (Norman et al., 2008).  NO has the ability to deviate the differentiation of T lymphocytes. Depending on the concentration of NO and the presence of cofactors, it was reported to drive the development of Th1 cells (Niedbala et al., 2002), Th2 cells (Daniel et al., 2006), or regulatory T cells (Niedbala et al., 2007).

PERSPECTIVE

The role of ROI and RNI in the immune system (as well as other organ systems) is unpleasantly ambiguous: They function as aggressive oxidants that kill microbial intruders and, at the same time, cause collateral damage to host tissues; and they serve as signaling molecules that not only control and tune the immune system, but also alarm the infectious pathogen to switch on mechanisms for protection against the host defense machinery. Therefore, ROI and RNI are potentially beneficial and detrimental at the same time, depending on their concentration, on the tissue microenvironment and the time course of generation, and certainly also on the chemistry of the individual products. Consequently, the frequently advertized consumption of antioxidants for protection against aging, carcinogenesis, or tissue degeneration has to be judged as equally problematic or ineffective as the systemic application of nonselective RNI- or ROI-donors for the treatment of infectious or autoimmune diseases (Bailey et al., 2007; Gaziano et al., 2009; Lippman et al., 2009). On the other hand, host cellor pathogen-specific targeting or activation of inhibitors of iNOS, Phox, or SOD, or of RNI- or ROI-donors might open up new avenues for future therapeutic approaches. Progress in this direction has recently been made (Singh et al., 2008; Valdez et al., 2008). I wish to apologize to all researchers whose work could only be cited in form of review articles due to space restrictions. The preparation of this chapter and the conduct of some of the studies reviewed were supported by grants to C. B. from the Deutsche Forschungsgemeinschaft (Bo996/3-3, SFB643 A6) and from the IZKF Erlangen (Project A24).

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6 Complement in Infections WILHELM J. SCHWAEBLE, YOUSSIF MOHAMMED ALI, NICHOLAS J. LYNCH, AND RUSSELL WALLIS

INTRODUCTION

THE COMPLEMENT SYSTEM

The complement system provides a fundamental component of the body’s immune response to invading microorganisms. The description of the bacteriolytic activity of serum or plasma by Jules Bordet led to the discovery of the complement system nearly 120 years ago. Bordet observed that bacteriolytically active serum essentially requires two components, one of which is present prior to immunization and is heat-labile, which he called alexion, and the other component, which is generated during the immunization process and is heat-stable. This component was later described as “amboceptor” by Paul Ehrlich and is now known to be an immunoglobulin, of the subclasses Igm, or IgG1, or IgG2 or IgG3. He demonstrated in 1898 that the same basic mechanisms that compose the bacteriolytic activity of immune sera are responsible for the hemolytic activity of serum towards erythrocytes of other species, a methodology that was used to analyze the biological activities of complement for decades to come. The observation that the bacteriolytic activity of immune serum is lost following heat inactivation (56°C for 30 min), but can be restored upon addition (complementation) of smaller quantities of fresh nonimmune serum, prompted Paul Ehrlich in the late 1890s to give the complement system its present name. Up to the present day, the bactericidal (or hemolytic) activity of complement mediated through the formation of a lytic membrane attack complex (MAC) (following the activation of the terminal complement components C5 to C9), is still the most widely known biological activity of the complement system. It is, however, by far not the most effective physiological mechanism by which complement contributes to the clearance of invading microorganisms from the body. This chapter highlights the various roles of the complement system in the orchestration of the immune response towards microbial infections, gives examples of microbial strategies to evade complement-mediated clearance, and discusses how acquired and inherited complement deficiencies may predispose an organism to infectious disease.

Composition and Organization of the Complement System

The complement system is composed of more than 32 components in plasma or on the cell surface, including positive and negative regulators. Activation of the complement system encompasses a series of carefully regulated initiation, amplification, and deactivation steps to adapt to specific physiological requirements (Whaley & Schwaeble, 1997). Complement activation acts through a cascade of sequential activation steps. Many complement components are zymogens that are present in plasma in their proenzymatic form and may be cleaved during the activation step and thereby converted into their enzymatically active state to form part of a multimolecular enzyme complex that cleaves and activates other complement components further downstream. Complement is activated by three pathways (Fig. 1): the classical pathway, the alternative pathway, and the lectin pathway (Schwaeble et al., 2002), all of which lead to the activation of C3, the key component of complement, and the subsequent formation of the cytolytic MAC. Following complement activation, the biologically active peptides C3a and C5a (released as a result of activation of C3 and C5) elicit a number of proinflammatory effects such as chemotaxis of leukocytes; degranulation of phagocytic cells, mast cells, and basophils; smooth muscle contraction; and increase of vascular permeability. In response to complement activation products, the inflammatory response is further amplified by subsequent generation of toxic oxygen radicals, induction of synthesis, and release of arachidonic acid metabolites and cytokines (Kirschfink & Mollnes, 2003).

The Classical Pathway

The classical pathway is triggered by C1, a multimolecular initiation complex. There is strong evidence that C1 is a major player in both the innate and the adaptive antimicrobial host defense and for the role of C1 in the maintenance of immune tolerance. The C1 complex (approximately 790 kDa) comprises a recognition subcomponent, C1q, and a heterotetramer

Wilhelm J. Schwaeble, Youssif Mohammed Ali, Nicholas J. Lynch and Russell Wallis, Department of Infection, Immunity and Inflammation, University of Leicester MSB, University Road, Leicester LE1 9HN, United Kingdom.

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FIGURE 1 A simplified diagram of the three complement activation pathways: the classical, the lectin pathway, and the alternative pathway. Both the classical and the lectin pathway share the identical C3 and C5 convertase complexes formed after cleavage of C4 by either activated C1s (classical pathway) or activated MASP-2 (lectin pathway) (see 1) and subsequent cleavage and activation of C4b-bound C2 through activated C1s or MASP-2 (see 2). Activation of the alternative pathway is tightly controlled by membrane associated complement regulatory components and the competition of the main fluid phase antagonists factor B and factor H for binding C3b or hydrolsed C3. The affinity of factor B to bind C3b is higher on “activating “surfaces, and the half-life of C3bB, C3Bb, and C3Bb(C3b)n complexes, significantly increased by the action of properdin, allows the alternative pathway amplification loop to be formed. The alternative pathway activation loop allows C3b to be used to (i) generate more alternative pathway C3 convertases and (ii) maximize complement opsonization of activating (microbial) surfaces, or (iii) switch substrate specificity of the C3 convertases to cleave C5 through deposition of multiple C3b molecules in close proximity. This flow diagram summarizes the synergisms between the three activation pathways and points out that the initial C3b required for the alternative pathway activation loop to form can either be provided via the classical or the lectin pathway, or via spontaneous hydrolysis of C3 (C3(H2O), which, like C3b, can bind to factor B in the absence of activation of the latter two pathways.

of the C1r and C1s zymogens forming a C1q:C1r2:C1s2 complex (Fig. 2). C1q is composed of six identical subunits joined together through their collagen-like stalks that end in globular heads. Each subunit is a heterotrimer of three polypeptide chains, the C1qA-, C1qB-, and C1qC-chains, encoded by tandem genes forming the C1q cluster. Classical pathway activation is initiated either by binding of C1q to a

bacterial surface component or indirectly by binding to immune complexes. Binding of C1q to complement activators causes a conformational change in the collagenous region of C1q, leading to the autoactivation of C1r, which subsequently activates C1s. C1s then cleaves C4 and C4b bound C2, leading to the formation of the C3 convertase (C4b2a) (Arlaud et al., 2002).

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FIGURE 2 Structure of the classical and lectin pathway recognition components. The basic subunits are trimers, with N-terminal collagen-like and C-terminal globular domains. In C1q, the subunit is a heterotrimer of C1qA-, B-, and C-chains and the globular heads are antibody Fc binding domains. For MBL and the ficolins, the subunits are homotrimers and the globular domains are C-type lectin and fibrinogen-like domains, respectively. Subunits assemble, via their collagenous domains into higher order oligomers, typically trimers and tetramers for MBL and the ficolins, and hexamers for C1q.

Activation of C3 exposes its reactive thiol ester group, which covalently bonds the larger cleavage product (C3b) to the activating surface. Accumulation of C3b results in the formation of C4b2a(C3b)n, the classical pathway C5 convertase.

The Lectin Pathway the lectin Pathway recognition Molecules

The lectin pathway is activated by carbohydrate recognition molecules that bind to polysaccharide on the surface of a pathogen. Two kinds of recognition molecules have been described, mannan-binding lectin (MBL) and the ficolins. The structure of these macromolecules is very similar to that of C1q. The basic subunit is a homotrimer, with an N-terminal collagen-like domain and a C-terminal carbohydrate recognition domain (CRD). These homotrimers associate via their collagenous regions to form the higherorder oligomers found in plasma. In MBL, the CRD is a C-type lectin domain, whereas in the ficolins the CRD is formed by a fibrinogen like domain (Fig. 2). MBL is present in serum as a mixture of higher oligomers (trimers, tetramers, and hexamers), and the biological activity of MBL increases with the number of oligomers (Wallis et al., 2005). Rodents and other mammals produce two different types of MBL, MBL-A and MBL-C, encoded by two separate genes, MBL1 and MBL2. In humans, MBL1 is a pseudogene and not translated, while MBL2 encodes the human orthologue to murine MBL-C.

Two of the human ficolins (L-ficolin and H-ficolin) are serum proteins that activate the lectin pathway by direct recognition of acetylated sugar moieties on the surface of pathogens. The third type of human ficolins that may activate the lectin pathway is M-ficolin, which is found on the surface of neutrophils and monocytes where it may act as a phagocytic receptor. In addition, M-ficolin forms complexes with the lectin pathway serine proteases, MASP-2 and MASP-1, and activates the lectin pathway of complement on N-acetyl glucosamine coated microtiter plates. M-ficolin was also shown to bind to certain types of bacteria such as S. aureus (Liu et al., 2005). Unlike humans, mice have only two types of ficolins, ficolin A (which is found in the serum and resembles human L-ficolin) and ficolin B (which is expressed by phagocytes and their precursors in bone marrow). In contrast to human M-ficolin, murine ficolin B does not bind to MASP-2 and does not trigger complement activation via the lectin pathway (Endo et al., 2005).

the Classical Pathway and the lectin Pathway specific serine Proteases

The protease subcomponents of the classical and the lectin activation pathway are homologous proteins that are composed modular subunits or domains, an N-terminal CUB domain (CUBI), followed by an EGF-like domain, followed by another CUB domain (CUBII), followed by two complement control protein domains (CCP1 and CCP2) and

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the C-terminal serine protease (SP) domain (Schwaeble et al., 2002). In the presence of Ca21, the C1r subcomponents form a centrally located C1r dimer via binding interactions between their C-terminal SP domains and the CCP domains of their respective C1r binding partner. The N-terminal CUBI, EGF, and CUBII domains of C1r bind to the corresponding CUBI, EGF, and CUBII domains of C1s to form the heterotetramer (C1s-C1r-C1r-C1s) of approximately 340 kDa. The interaction with C1q is also Ca21-dependent. When C1 binds to a target via its globular heads, C1r activates through autocleavage. C1r then cleaves and activates its only substrate C1s. To ensure only localized activation, the SP domains of C1s remain attached to the complex via single disulfide bonds to their respective N-terminal fragments (see Color plate 3). The first substrate of C1s is C4. Once cleaved it rapidly attaches to the pathogen surface via a reactive thioester bond. C2 then binds to the C4b fragment and is also cleaved by C1s to form the C3 convertase (C4b2a), the next component of the pathway. Thus, C1 activation and subsequent steps in the reaction cascade are limited to the surfaces of pathogens and other targets. The organization of the lectin activation pathway differs considerably from that of the classical pathway. Three lectin pathway specific serine proteases, termed mannanbinding lectin-associated serine proteases (MASPs) have been described (i.e., MASP-1, MASP-2, and MASP-3) (Color plate 3). In addition to these serine proteases, an enzymatically inactive truncated form of MASP-2 of 19 kDa, called MAp19 or sMAP, is a component of the multimolecular lectin pathway activation complexes (Schwaeble et al., 2002; Stover et al., 1999). MASP-1 was described as a C1s-like serine protease associated with MBL by the pioneering work of Matsushita and Fujita (1992). In 1997, the second MBL-associated serine protease (named MASP-2) was isolated from the MBL/MASPs complex (Thiel et al., 1997). All MASPs form homodimers, which associate through binding interactions between the CUBI, EGF, and CUBII domains of each partner. In contrast to the essential requirement for the classical pathway serine protease C1s to be activated by C1r, MASP-2 on its own was shown to be sufficient to form a complex with MBL and cleave C4 and C4b-bound C2 to generate a C3 convertase (C4b2a). As in the classical pathway, the C4b2a complex converts C3 into C3a and C3b and accumulation of covalently bound C3b in close proximity subsequently leads to the formation of a C5 convertase complex C3bBb(C3b)n. Unlike MASP-2, MASP-1 is not able to cleave C4, but may cleave C2 (Rossi et al., 2001). This may explain why MASP-1 can significantly accelerate the turnover rate of lectin pathway activation (Takahashi et al., 2008). A third lectin pathway specific serine protease, MASP-3, was shown to be generated by an alternative splice process as an alternative gene product of a single structural MASP-1 gene. The substrate and physiological role of MASP-3, however, remains a mystery and since it shows no enzymatic activity that may support activation via the lectin pathway, it is considered to regulate lectin pathway functional activity by competing with MASP-2 for the binding sites of the lectin pathway recognition subcomponents (Dahl et al., 2001). A similar regulatory function has recently been demonstrated for an enzymatically inactive protein of 19 kDa, called MAp19 or sMAP (Stover et al., 1999), encoded by an alternative splice mechanism of the MASP2 gene. Like MASP-3, MAp19 binds to the serine protease interaction sites of the lectin pathway recognition complexes and may regulate lectin pathway functional activity (Iwaki et al., 2006).

MBl/MasPs Complexes

The binding between MBL and MASPs was described by several studies. Analysis of the MBL/MASPs complex from human serum revealed that MASP-1 and MAp19 have high binding affinity with trimeric MBL oligomers, whereas MASP-2 and MASP-3 mainly associated with tetrameric MBL oligomers (Dahl et al., 2001). In contrast to this finding, Teillet et al. (2005) postulated that MASPs and MAp19 have the same affinity towards trimeric and tetrameric MBL oligomers. This observation was confirmed by another study, which showed that MASPs can bind efficiently with a dimer form of rat MBL-A. MASP-1, MASP-2, MASP-3, and MAp19 form homodimers through binding via CUBI-EGF-CUBII domains in a calcium-dependant manner. The MASPs and MAp19 binding to MBL or ficolins occurs through a CUBI-EGF moiety and the strength of binding increased by the CUBII domain (Chen & Wallis, 2001). Both the serine proteases of the classical pathway as well as the serine protease of the lectin pathway bind to the same amino acid motif within the collagenous region of their respective recognition molecules. The C1 complex, however, readily dissociates at high salt concentrations, while the MBL/MASPs complex stays intact even at salt concentrations as high as 500 mM, a characteristic physiological feature used to discriminate between the two activation routes.

The Alternative Pathway

Factor B, factor D, and properdin (factor P) are specific components of the alternative pathway of complement activation. Unlike the classical and the lectin pathway, the alternative pathway is initiated through a spontaneous steady state hydrolysis of C3 to form C3(H2O), which in turn binds to factor B to form a C3(H2O)B zymogen complex. In this complex, factor B is cleaved by factor D releasing a Ba fragment while Bb remains attached to the complex formed with C3(H2O). The newly formed complex C3(H2O) Bb forms a C3 convertase enzyme and cleaves C3 into C3a and C3b. Once C3b is generated, it will bind to the surface of pathogens where it can bind to another molecule of factor B and form a new alternative pathway C3 convertase C3bBb. A new viewpoint is that properdin can bind to the surface of pathogens and initiate the alternative pathway activation by stabilization of C3bBb convertase (Kemper & Hourcade, 2008). The alternative pathway can also act as an amplification loop where C3b generated by either the classical or the lectin pathway binds to factor B, which (upon binding to C3b) is subsequently cleaved by factor D to form the alternative pathway convertase C3bBb (Schwaeble & Reid, 1999). Similar to the C3 convertase of the classical and the lectin activation pathway (i.e., C4b2a, see above), the alternative pathway convertase will switch its substrate specificity from cleaving C3 to cleaving C5 upon binding of multiple C3b molecules in close proximity.

The Membrane Attack Complex

C3 convertases produced by either of the three complement activation pathways convert C3 into C3b and C3a. Accumulation of C3b in close proximity of either C4b2a or C3bBb complexes switches the substrate specificity of these C3 convertases from C3 to C5 forming the C5 convertase complexes C4b2a(C3b)n or C3bBb(C3b)n, which cleaves C5 into C5b and C5a. C5b then binds to the cell surface and reacts with C6, C7, and C8 to form a C5b to 8 complex that leads to polymerization of C9, which inserts into the lipid bilayer of the bacterial cell membrane and initiates the MAC formation leading to cell lysis (Podack et al., 1982).


CONTROL OF COMPLEMENT ACTIVATION

The complement activation is tightly regulated by membrane-bound and fluid-phase regulatory components to avoid runaway activation of the enzymatic cascade that could lead to excess host tissue damage, inflammation, and depletion of complement components. Key events at the center of the cascade are carefully controlled by five closely related complement control proteins, all of which are encoded by genes located in the RCA (regulator of complement activation) cluster on chromosome 1q32 in humans. The complement regulators encoded in the RCA cluster are all composed of structural subunits of approximately 60 amino acids, called short consensus repeats (SCRs) or complement control protein (CCP) motifs. These include the membrane-bound regulators, complement receptor types 1 and 2 (CR1 and CR2), decay-accelerating factors (DAF or CD55), and membrane cofactor proteins (MCP or CD46). CR2 only binds the inactive forms of the C3 protein (i.e., C3d and C3dg); it has minimal complement regulatory functions but functions primarily as a member of the B-cell coreceptor complex and regulates the cell cycle progression during Bcell activation. CR1, MCP, and the fluid-phase regulators, factor H and C4 binding protein (C4bp), act as cofactors for factor I-mediated conversion of membrane-bound C3b and C4b into their inactivated forms, which no longer serve as components of active convertase complexes. In addition to factor I cofactor activity, CR1, C4bp, and factor H shorten the half-life of the C3 and C5 convertases by binding to complex-bound C3b and C4b and dissociating these complexes (decay-accelerating activity). This function is also mediated by DAF, which in contrast to the above, has no cofactor activity for factor I (Table 1). A truncated, 43kDa alternative splice variant of the factor H gene, now called factor H-like protein 1 (FHL-1), is also found in serum and has similar regulatory activity to factor H (Schwaeble et al., 1987). Activation of the classical and lectin pathways is controlled by C1 inhibitor (C1-INH), a serine protease inhibitor (SERPIN) that binds to C1r and C1s complexes and MASP-1 and MASP-2 complexes to prevent spontaneous activation of the proenzymes. C1-INH also removes activated serine proteases from the recognition subcomponents, blocking further activation (Chen et al., 1998).

TABLE 1

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Clusterin and S protein are regulators for the terminal activation cascade of complement. They bind to the C5b-7 complex and prevent the insertion of C8 and C9, leading to the inhibition of the MAC formation. CD59 is another regulator, which inhibits binding of C8 and C9 to the C5b-7 complex, preventing formation of the MAC.

THE BIOLOGICAL EFFECTS OF COMPLEMENT ACTIVATION

Complement activation leads to a multitude of biological activities, including opsonization, initiation of a proinflammatory response, immune complex clearance, and direct killing of cells via the MAC. Opsonization of pathogens is mediated by the major opsonin C3b or iC3b (the hemolytically inactive cleavage product of C3b) and, to a lesser extent, C4b. C3b coats the surface of microorganisms and enhances their phagocytosis by leukocytes via binding to complement receptor type 1 (CR1) and type 3 (CR3). L-ficolin and MBL have been reported to initiate phagocytosis directly by binding to pathogens and enhance phagocytosis by binding to collectin receptors on the surface of the phagocytes (Jack et al., 2001). In other instances, complement can mediate direct killing of bacteria, especially gram-negative bacteria via the formation of the MAC, which leads to destruction of cytoplasmic membrane and cell lysis. During complement activation, proinflammatory anaphylatoxins such as C5a and C3a are released. These increase vascular permeability and promote formation of inflammatory exudates, which in turn enhance the recruitment of inflammatory mediators and inflammatory cells to the site of injury. This ensures efficient elimination of invading pathogens or other inflammatory factors. Increased vascular permeability facilitates extravasation of leukocytes to the site of inflammation and anaphylatoxins, especially C5a, act as potent chemotactic factors that stimulate leukocyte migration. In addition, C5a was found to increase the synthesis of other chemotactic agents, like ecosanoides and chemokines. Schindler et al. (1990) reported that C5a stimulates the expression of interleukin-1 (IL-1) and tumor necrosis factor (TNF) genes. Both C5a and C3a stimulate the production of monocyte chemotactant protein-1 and macrophage inflammatory protein-2 from mouse endothelial cells (Laudes et al., 2002).

Regulatory components of complement activation and their known interaction with pathogens

Complement regulatory protein

Physiological targets

Pathogens binding complement regulatory proteins (known surface receptors in parentheses)

Fluid Phase Factor H

C3b, C3bBb, C3bBb(C3b)n

Factor H-like protein 1 (FHL-1) As above C4 binding protein (C4bp) C4b, C4b2a, C4b2a(C3b)n

Borrelia burgdorferi (BbCRASPs); C. albicans (CaGpm1p); S. pyogenes (m-protein, Fba & Scl1); S. pneumoniae (PspC) As above S. pyogenes (m-protein); S. pneumoniae (PspC); C. albicans

Membrane Bound Complement receptor 1 (CR1)

C3b, C3bBb, C3bBb(C3b)n C4b, C4b2a, C4b2a(C3b)n MCP C3b, C4b S. pyogenes (m-protein); HIV Decay-accelerating factor (DAF) C3bBb, C3bBb(C3b)n, C4b2a, C4b2a Protectin (CD59) C8, C9 HIV, CMV, vaccinia virus Clusterin C8, C9 Vitronectin (S-protein) C8, C9

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Complement also plays a major role in the clearance of apoptotic and necrotic cells as well as immune complexes. The globular head of C1q binds to the surface of apoptotic cells and facilitates the uptake of this complex by macrophages (Taylor et al., 2000). Opsonization of apoptotic cells with iC3b leads to recognition of these cells by CR3 and CR4 receptors on the surface of phagocytes with subsequent engulfment of these cells (Mevorach et al., 1998). Complement also inhibits the precipitation of immune complexes and enhances its solubility by binding to C1q, C3b, and C4b. This binding inhibits further increase in the size of the immune complexes. These complexes bind to CR1 on the surface of erythrocytes, which transfer them into the liver and the spleen where they are cleared from the circulation by the resident macrophages.

COMPLEMENT DEFICIENCIES

An intact complement system plays a major role in protection against infectious and noninfectious diseases. Complement deficiencies are associated with recurrent invasive bacterial infections and the development of autoimmune diseases.

Classical Pathway Deficiencies

A deficiency in any component of the classical pathway is associated with an increased risk of immunological disease and recurrent bacterial infections. Individuals with deficiencies of early complement components are more susceptible to autoimmune diseases. For example, C1q and C1s deficiency is associated with an increased risk of developing autoimmune diseases, such as systemic lupus erythematosus (SLE), mainly due to the impaired clearance of immune complexes and removal of apoptotic cells from the circulation (Carroll, 2004), and C2 deficiency was found to be a predisposing factor for atherosclerosis. Children deficient in C2 are susceptible to recurrent pneumococcal infections (Jönsson et al., 2005). C1q deficiency increases the risk of recurrent invasive bacterial infections and polymicrobial peritonitis (Celik et al., 2001). C3 and factor B deficiencies significantly increase the risk of Streptococcus pneumoniae and Pseudomona aeruginosa infection (Brown et al., 2002).

Lectin-Pathway Deficiency Deficiencies of the lectin pathway activation molecules are associated with an increased risk of recurrent bacterial infections and nonbacterial disorders. MBL deficiencies are due to three different mutations in exon 1 of the mbl2 gene located on human chromosome 10. These mutations cause single amino acid substitution at codons 52, 54, and 57. A single nucleotide polymorphism (SNP) at codon 52 leads to the substitution of glycine with aspartic acid at position 34 of the mature protein. Another point mutation at codon 54 leads to substitution of glycine with glutamic acid at position 37 of the mature protein. These two mutations disrupt the collagenous region and impair the interactions between MBL subunits during biosynthesis (Wallis & Cheng, 1999). The third mutation at codon 57 is due to the substitution of cysteine with arginine at position 32 of the mature protein. This mutation leads to the disruption of the oligomerization of MBL polypeptide chains (Wallis et al., 2004). Several polymorphisms within the promoter region of the MBL2 gene were shown to contribute to the large interindividual variations in serum MBL levels. Of those, three polymorphisms H/L, X/Y, and P/Q (at positions 2550, 2221, and 14, respectively) were the most frequently occurring promoter SNPs (Turner, 2003).

Inherited MBL deficiencies are common and most individuals with these deficiencies are healthy and show no obvious predisposition to recurrent infections or morbidity when compared with MBL-sufficient control populations (Dahl et al., 2004). Nevertheless, MBL deficiencies are found to be associated with an increased susceptibility for severe bacterial infections in children and adults especially when combined with other immunodeficiencies such as HIV infection or post-chemotherapy. MBL deficiency was also associated with high risk of developing arterial thrombosis and other cardiovascular disorders. MBL deficiency is also associated with a high risk for developing sepsis in pediatric patients. Patients with MBL deficiencies are more prone to develop rheumatoid arthritis and persistent inflammatory conditions (Turner, 2003). For L-ficolin, another recognition component of the lectin pathway, there is some evidence that low plasma levels play a significant role in recurrent respiratory tract infections in children (Atkinson et al., 2004).

Alternative Pathway Deficiency

Deficiencies of factor H and/or factor I (inherited or acquired) are associated with a typical hemolytic-uremic syndrome (aHUS), which results in thrombotic microangiopathy associated with diarrhea. Deficiencies of properdin, the only positive regulator of complement activation (which ensures the amplification of complement activation by increasing the half-life of the alternative pathway C3 and C5 convertases), result in an increased risk of infection, in particular, meningococcal infections (Schwaeble & Reid, 1999). In a mouse model of properdin deficiency, it was found that the severity of polymicrobial peritonitis was significantly increased in deficient mice when compared to their wild-type littermates (Stover et al., 2008).

THE ROLE OF COMPLEMENT IN BACTERIAL INFECTION Streptococcus pneumoniae as a Model Organism

S. pneumoniae infection is the major cause of pneumonia, otitis media, septicemia, and meningitis in the United Kingdom and it causes substantial morbidity and mortality, especially in young children and elderly patients . 65 years old (Kyaw et al., 2003). Historically, in 1900, S. pneumoniae was the major cause for children’s death in the United States, killing more than 47 of every 1,000 children per year. The increase in living standards and the improvement of nutrition limited the number of deaths even before the prevalence of effective antibiotic treatment. However, in developing regions such as Africa and Asia, pneumonia is still a major cause of the high mortality seen in children, accounting for approximately 25% of deaths in children under the age of 5 years, with more than 1.2 million infant deaths every year (Kadioglu et al., 2008). This high rate of mortality is associated with very poor living standards, malnutrition, and ineffective medical care. Older people are also subjected to the risk of S. pneumoniae infection, with a mortality rate of approximately 20% in patients older than 65 years and increasing to 40% in patients older than 85 years (Kadioglu et al., 2008). Pneumococci colonize the nasopharynx of young children, and the incidence of colonization may reach up to 40%. Several studies have reported a link between a high risk of acute and recurrent otitis media and the rate of colonization in the nasopharynx (Marchisio et al., 2003). Certain diseases like diabetes, asplenia, chronic lung diseases, and AIDS can increase the risk of pneumococcal infection.


Capsular Polysaccharides of S. pneumoniae

The capsular polysaccharides of S. pneumoniae (CPS) forms the outermost layer surrounding pneumococci. The thickness of the CPS is about 200 to 400 nm. To date, 91 different serological CPS serotypes have been identified (Kadioglu et al., 2008). The capsule is the major virulence factor of S. pneumoniae and encapsulated strains are generally more virulent than noncapsulated strains. Moreover, pneumococcal mutants that lack the polysaccharides capsule were less virulent than the parent strains, indicating that virulence is determined by the capsule type (Kelly et al., 1994). The thickness and the chemical structure of the CPS determine the differential ability of various serotypes to survive in blood and to cause invasive diseases. Pneumococci from different serotypes differ in their ability to cause diseases. This difference between serotypes in pathogenesis is often due to the chemical structure of CPS. Capsular polysaccharides that activate the complement system and allow the deposition of C3b, but prevent its further processing into iC3b and C3d, are more susceptible to phagocytosis and are poorly immunogenic as they can be easily cleared. In contrast, serotypes that have C3b deposited on their capsules and are quickly processed into iC3b and C3d are more resistant to phagocytosis and induce a strong humoral immune response.

Pneumococcal Cell Wall Components

Cell wall peptidoglycans and cell wall polysaccharides (CWPS) are potent inflammatory components and induce inflammatory reactions similar to those associated with S. pneumoniae infection. For example, mice injected with purified peptidoglycans or CWPS showed an inflammatory response similar to that observed after infection with S. pneumoniae and typical symptoms of pneumococcal disease, such as otitis media. These cell wall components activate the complement system and contribute to the generation of the complement anaphylatoxins C3a and C5a, which in turn enhance vascular permeability, induce mast cell degranulation, and lead to neutrophil recruitment into the inflammatory site, as described above.

Pneumolysin

Pneumolysin is a potent virulence factor produced by all serotypes of S. pneumoniae. Pneumolysin is released as a 52 kDa soluble monomer. It binds cholesterol-containing membranes and the monomeric subunits oligomerize to form a pore in the target cell membrane that leads to cell death. Pneumolysin also inhibits ciliary beating of respiratory epithelium and phagocytosis, induces cytokine synthesis, and mediates CD41T cell activation and chemotaxis (Kadioglu et al., 2004). Pneumolysin activates the classical pathway of complement in the lung after intranasal infection and induces an inflammatory response that delays the onset of bacteremia in mice. Intranasal infection of mice with the S. pneumoniae D39 mutant strain deficient in pneumolysin production showed better clearance of the pneumolysin mutant strain after 24 hours in comparison to the wild-type strain. This finding suggests that early activation of complement facilitates the clearance of pneumococci from the lung directly after infection (Jounblat et al., 2003). In contrast, prolonged activation of complement via pneumolysin during the course of pneumonia leads to severe inflammatory response, which exacerbates lung tissue damage (Kadioglu et al., 2000).

Pneumococcal Cell-surface Proteins

Amongst the cell surface proteins of S. pneumoniae, the pneumococcal surface protein A (PspA) and C (PspC) play key roles in the microbial defense against complement attack (Bergmann & Hammerschmidt, 2006).

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PspA is expressed by all clinical isolates and appears to play a major role in the pathogenesis of S. pneumoniae. PspA interferes with complement activation and C3 deposition on the pneumococcal surface and hence it protects the bacteria from complement-mediated phagocytosis. Mice infected with PspA-deficient S. pneumoniae showed a longer survival time when compared to mice infected with the wild-type strain. Furthermore, immunization of mice with PspA prior to intravenous infection with S. pneumoniae protected the mice from the lethal sepsis and prolonged the survival time, in comparison to the nonimmunized controls (Roche et al., 2003). PspA conjugated with capsular polysaccharides (CPS) from pneumococcus serotype 23F significantly increased the immunogenicity of the capsular polysaccharides, resulting in a high antibody titer response against both CPS and PspA. When challenged with S. pneumoniae expressing PspA, mice showed a significant improvement in survival time in comparison to mice challenged with PspA alone or in combination with CPS. Increased protection in immunized mice was due to the high capacity of the produced antibodies to bind S. pneumoniae and enhance complement deposition (Csordas et al., 2008). PspC is a major virulence factor of S. pneumoniae and contributes to many different biological functions. It binds to IgA, complement component C3, and factor H. Binding to factor H protects the bacterium from complement attack and complement-mediated phagocytosis. PspC was also shown to bind to the classical and lectin pathway regulatory component C4-binding protein to protect the bacterial surface from complement attack. In addition, PspC is involved in the adherence of pneumococci to lung tissues and enhances colonization of the nasopharynx. PspC is upregulated when pneumococci adhere to the host epithelial cells in the respiratory tract and its expression is required for subsequent tissue invasion. Immunization of mice with purified PspC protects the mice from bacterial colonization and S. pneumoniae infection. Furthermore, mutants deficient in PspC have a reduced ability to colonize the nasopharynx and cause lung infection when compared to the wild-type strains. In a model of pneumococcal sepsis, mice infected with serotype 2 and serotype 3 mutant strains deficient in PspC showed a significant increase in survival as compared to the wild-type strains (Iannelli et al., 2004).

host defense against S. pneumoniae

An effective immune defense against S. pneumoniae infection depends on the collaboration between humoral and cell-mediated immunity. Mucosal immunity plays a key role in the protection against respiratory pathogens; the mucous covering the epithelial cells prevents adhesion of microbes and facilitates their removal by the ciliary movement. Extracellular killing of inhaled bacteria has been reported by several antimicrobial factors in the lung lavage, including lysozymes, iron-binding proteins, and fibronectin with subsequent clearance of bacteria by phagocytosis. Breakdown of mucosal defenses results in S. pneumoniae colonization of the upper respiratory tract and nasopharynx, increasing the risk of pneumococcal infection, which may eventually lead to bacteremia (Lamblin & Roussel, 1993). Many complement components are synthesized by alveolar macrophages and lung epithelial cells, and complement activation plays a major role in the host defense against invasive S. pneumoniae infection. Activation of complement C3 and its deposition on the surface of S. pneumoniae significantly facilitates phagocytosis by alveolar leukocytes. Opsonophagocytosis and killing of pathogens by resident alveolar macrophages are the major mechanisms leading to the clearance of invading bacteria from the lung. Because resident macrophages cannot phagocytose most strains of S. pneumoniae,

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recruited PMNs (polymorphonuclear leukocytes) play the main role in clearing all strains of S. pneumoniae from the alveolar area. Opsonization of bacteria is initiated by antibodies against pneumococcal capsular polysaccharides, complement deposition, or soluble lung surfactant proteins such as surfactant protein A (SP-A), which enhance phagocytosis of S. pneumoniae in nonimmunized subjects (Kuronuma et al., 2004). Complement activation enhances bacterial clearance through opsonization and phagocytosis. C-reactive protein (CRP) is a human acute phase protein that is mainly produced in the liver. Its levels are dramatically increased in response to infection or tissue damage. CRP binds to phosphocholine residues on S. pneumoniae capsular polysaccharides in a Ca12-dependant manner and appears to activate the classical pathway of complement in human serum lacking specific S. pneumoniae antibodies (Kaplan & Volanakis, 1974). In contrast to human CRP, murine CRP is not an acute phase protein so transgenic mice expressing human CRP have been used as a model for testing the role of the human CRP in infection studies. Such transgenic mice had a significant lower mortality than nontransgenic mice. The higher resistance to infection was also associated with a significant reduction in bacteremia (Szalai et al., 1995). In another mouse model of infection, passive administration of human CRP showed a significant protective role against S. pneumoniae, with improved survival times and a significantly lower bacteremia. Interestingly, although C1q binds to CRP, leading to complement activation, the protective role of CRP appears to be complement independent. This finding was confirmed by using a mutant form of CRP that does not bind to C1q and so cannot activate the classical pathway. Passive administration of the recombinant mutant or wild-type CRP showed the same level of protection in mice following experimental S. pneumoniae infection (Suresh et al., 2006). Serum amyloid protein (SAP) is another acute phase protein that plays an important role in protection against S. pneumonia infection. SAP binds to pneumococci, increases complement deposition via the classical pathway, and thus facilitates phagocytosis. Mice deficient in SAP are defective in clearing S. pneumoniae and have significantly higher rates of mortality. Reconstitution of these mice with human SAP reduced the disease severity during the course of infection and the mice showed a clear improvement in controlling the infection with a significant increase in survival (Yuste et al., 2007).

the role of Complement in fighting S. pneumoniae Infection

As soon as pneumococci invade host tissues, the innate immune response is activated, leading to deposition of opsonins on the surface of S. pneumoniae and inducing phagocytosis. Complement activation is one of the first host defense mechanisms and the role of complement in the defense against S. pneumoniae infection has been known for nearly 100 years. During that time, numerous studies have characterized the roles of the complement system in pneumococcal infection. Recently, several gene-targeted mice with deficiencies in one or more complement components have been used to assess which pathway of the complement system is most important for protection against S. pneumoniae. C1q-deficient mice were found to be more susceptible to infection with S. pneumoniae after intranasal infection than the wild-type mice, confirming that the classical pathway has a protective role against S. pneumoniae infection. Natural IgM antibodies against capsular polysaccharides can activate the classical pathway. Mice deficient in IgM are more susceptible to

infection, with a higher bacterial load in blood and in lung tissues in comparison to the wild-type controls, and mice deficient in factor B also showed a significantly higher level of bacteria in lung tissues and in blood in comparison to the wild-type. However, when compared to the classical pathway, the degree of protection provided by the alternative activation pathway appeared to be of lesser significance. Studies are currently under way to assess the role of the lectin pathway in S. pneumoniae infection. As expected from its central role in complement activation, mice lacking C3 are most severely compromised in fighting infections. This deficiency affects the innate as well as the adaptive immune response to S. pneumoniae throughout the course of infection (Brown et al., 2002). Regardless of which pathway contributes most toward complement activation, deposition of C3b on the surface of bacteria is a key step in the downstream complement activation cascade leading to C3b-mediated phagocytosis and clearance of pathogens, as well as the formation of MAC, leading to a lysis of bacteria. As a consequence, the pneumococcus has developed several mechanisms to resist complement attack. (i) The capsular polysaccharides are a major factor in protecting pneumococci from complement attack and opsonophagocytosis (Jarva et al., 2002). (ii) The pneumococcus modulates alternative pathway activation by recruitment of factor H onto its surface to prevent complement-dependent opsonization using host factor H to shift the balance in the favor of the inhibitory regulatory mechanism. (iii) Pneumococcal surface protein A (PspA) was recently identified as a key player in the inhibition of complement deposition. This protein is highly electronegative and has an inhibitory effect on C4b deposition to the bacterial surface (Li et al., 2007). (iv) Pneumolysin also has a protective role against complement-mediated clearance. Mutagenic strains deficient in pneumolysin showed increased C3 deposition on the bacterial cell surface, highlighting the important role of pneumolysin in the pathogenesis of S. pneumoniae infection (Yuste et al., 2005).

The Roles of Complement Activation in the Pathophysiology of Polymicrobial Peritonitis

Intra-abdominal infection is a severe medical complication associated with a high level of morbidity and mortality. A breakdown of the barriers between the gut lumen and the peritoneum subsequently may lead to polymicrobial infections of the peritoneum with mixed aerobic and anaerobic commensal bacterial flora in the intestine. Polymicrobial peritonitis is a life-threatening condition frequently occurring after ruptured appendicitis or rupture of colonic diverticulum (Stover et al., 2008). Polymicrobial peritonitis is also one of the major complications of continuous peritoneal dialysis. Sepsis is characterized by massive infiltration of neutrophils into the peritoneum where they help in the clearance of the invading pathogens. When the innate immune defense fails to overcome the bacterial infection, the pathogens find their way into the blood stream and disseminate through host organs, inducing an exaggerated inflammatory response. During sepsis, macrophages, neutrophils, lymphocytes, and endothelial cells produce powerful inflammatory mediators, including TNF-a, IL-6, IL-1, and IL-8, in addition to acute phase proteins such as Creactive protein. At the same time, the complement system is shifted towards activation and produces potent proinflammatory mediators such as C5a, which stimulate the production of the cytokines and chemokines. The proinflammatory mediators produced in the early stages of sepsis recruit and activate the phagocytic cells (neutrophils and macrophages), which become hyperactive and produce reactive oxygen species (e.g., H2O2), which are effective in killing bacteria, but at the same time may cause tissue injury. The next step of sepsis


is characterized by production of anti-inflammatory mediators such as IL-10, transforming growth factor-b and IL-13, which down regulate the production of the proinflammatory mediators. The innate immune response then becomes hypoactive, and finally, the immune defense is suppressed, leading to the final stage of septic shock (Riedemann et al., 2003). The treatment of sepsis depends on blocking the complement anaphylatoxin C5a and its receptor C5aR. The anaphylatoxin C5a is continuously produced during complement activation and promotes potent proinflammatory effects. C5a activates phagocytic cells to release the granular enzymes and stimulates superoxide anion release from neutrophils. In addition, it causes vasodilation, increases vascular permeability, and causes T-cell apoptosis. Excessive production of C5a can lead to uncontrolled proinflammatory response and tissue damage that can lead to multiorgan failure. C5a receptors are highly expressed by many cell types in all organs such as the lungs, the liver, the kidney, and the heart. C5 mRNA expression is up regulated during sepsis. In a mouse model of CLP, blocking C5a receptors with specific polyclonal antibodies significantly improved the survival of mice in sepsis. In addition, the serum level of TNF-a and IL-6 and the bacterial loads in the blood and in different organs were significantly lower than in control mice (Riedemann et al., 2003). Mice deficient in C3 and C5 were found to be more susceptible to sepsis when compared to mice deficient in C3 only, with a significant higher rate of mortality and septicemia in mice with combined deficiency. C3 deficient mice suffered from impaired bacterial clearance due to the absence of C3b (which is the major opsonin) resulting in an absence of opsonophagocytosis. A recent report demonstrated that C3-deficient mice were able to activate C5 in the absence of C3 through a new complement activation pathway where thrombin could directly cleave C5 and release biologically active anaphylatoxin, C5a. On the other hand, mice deficient in C5 were found to have a significantly higher degree of bacteremia when compared to wild-type control. This septicemia may be due to the inability of C5-deficient mice to generate C5b, which is important for the formation of the MAC (Flierl et al., 2008). In a rat model of CLP, preimmunization of rats with antibodies against C5a improved the survival of septic rats and decreased the bacterial load in the blood and in different organs. Based on the previous data, blocking C5a and/or C5aR could be a useful therapeutic intervention during sepsis, as this treatment would still allow formation of the MAC. At the same time, blocking C5a or its receptors may decrease the inflammatory storm during sepsis, especially after successful clinical trials showed that anti-C5a treatment can decrease complement-mediated inflammatory responses following ischemia and reperfusion of the heart. In addition, reduced tissue damage in a model of intestinal ischemia/ reperfusion in rats was also observed after pretreatment of rats with anti-C5a (Wada et al., 2001).

Inhibition of Complement by Microbial Virulence Factors

Numerous microbial pathogens express virulence factors that sequester host complement control proteins to the microbial surface, effectively disguising the organism as host tissue and so avoiding complement-mediated killing. Well-documented examples include Borrelia burgdorferi, the causative agent of Lyme disease, and the human pathogenic yeast, Candida albicans, which bind factor H and FHL-1 via borrelial complement regulator-acquiring proteins (BbCRASPs) and C. albicans phosphoglycerate mutase (CaGpm1p), respectively (Siegel et al., 2008; Poltermann et al., 2007). C. albicans also sequesters host C4bp, which retains its complement regulatory activity, although the C4bp receptor has not yet been identified.

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Streptococcus pyogenes (group A Streptococcus or GAS), the most common cause of acute pharyngitis, binds multiple complement regulatory proteins, including factor H, FHL-1, C4bp, and MCP. Binding is normally attributed to the streptococcal M-protein, although factor H, FHL-1, and C4bp will bind to GAS in the absence of M-protein. Other bacterial surface receptors have been identified for factor H and FHL-1, including fibronectin-binding protein a (Fba) and streptococcal collagen protein 1 (Scl1). Interaction of FHL-1 and Fba promotes intracellular invasion of epithelial cells by GAS (Oliver et al., 2008).

Complement Opsonization of Intracellular Bacterial Parasites

The surface of Mycobacterium avium activates complement via each of the three pathways and the deposition of C3b significantly enhances the uptake of M. avium by macrophages in vitro. It was therefore considered that complement opsonization may provide a critical entry route for mycobacteria to infect and survive in host macrophages. A recent in vivo study, however, has shown that mice deficient of C3 show a similar rate of macrophage ingestion of M. avium when compared to their C3 sufficient littermate controls (Bolhsson et al., 2001). This points to the conclusion that complement opsonization may not be essential for the infectivity of M. avium and possibly other mycobacteria in vivo.

THE ROLE OF COMPLEMENT IN VIRAL INFECTION

Over the past 80 years, there have been numerous reports indicating that the complement system, either independently or in the presence of an antibody, plays an important role in the immune defense against viral infection. The two main mechanisms by which complement may contribute to viral neutralization are either enhancement of viral phagocytosis through complement opsonization or through direct viral lysis. Many viruses have lipoprotein membranes and are therefore susceptible to complement-mediated lysis, which neutralizes viral infectivity. Viruses have, therefore, developed several strategies to protect themselves from complement attack, which contribute to viral virulence. For example, glycoprotein C of herpes simplex virus 1 (HSV-1) blocks the interaction of C5 and properdin with C3b, preventing the formation of C5 convertase (Friedman et al., 2000). Complement deposition on HIV-1 is generally thought to counteract the immune response by enhancing viral entry into host cells. However, HIV-1 is susceptible to complement-mediated lysis, and whether complement deposition is beneficial or detrimental to the virus depends upon the extent of opsonization. HIV-1 sequesters host complement-control proteins, including MCP and CD59 to regulate opsonization, and therefore protecting itself from lysis (Stoiber et al., 2008).

COMPLEMENT RECEPTOR TYPE 2 AND MEMBRANE REGULATORS AS ENTRY PORTS OF VIRAL INFECTION

The demonstration that the Epstein-Barr virus (EBV) utilizes CD21 or complement receptor type 2 (CR2), a complement receptor with a very distinct expression profile, explains why EBV exclusively infects specific cell types, such as B cells and follicular dendritic cells, where CR2 is expressed (Jacobson & Weis, 2008). Other viruses were shown to utilize membrane-attached regulators with much wider tissue distribution as ports of cellular entry and infection, such as coxsackievirus B3 and hantavirus binding to DAF or the measles virus binding to MCP or CD46 (Maisner et al., 1994).

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6. Complement in Infections Li, J., D. T. Glover, A. J. Szalai, S. K. Hollingshead, and D. E. Briles. 2007. PspA and PspC minimize immune adherence and transfer of pneumococci from erythrocytes to macrophages through their effects on complement activation. Infect. Immun. 75:5877–5885. Liu, Y., Y. Endo, D. Iwaki, M. Nakata, M. Matsushita, I. Wada, K. Inoue, M. Munakata, and T. Fujita. 2005. Human M-ficolin is a secretory protein that activates the lectin complement pathway. J. Immunol. 175:3150–3156. Maisner, A., J. Schneider-Schaulies, M. K. Liszewski, J. P. Atkinson, and G. Herrler. 1994. Binding of measles virus to membrane cofactor protein (CD46): importance of disulfide bonds and N-glycans for the receptor function. J. Virol. 68:6299–6304. Marchisio, P., L. Claut, A. Rognoni, S. Esposito, D. Passali, L. Bellussi, L. Drago, G. Pozzi, S. Mannelli, G. Schito, and N. Principi. 2003. Differences in nasopharyngeal bacterial flora in children with nonsevere recurrent acute otitis media and chronic otitis media with effusion: implications for management. Pediatr. Infect. Dis. J. 22:262–268. Matsushita, M., and T. Fujita. 1992. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J. Exp. Med. 176:1497–1502. Mevorach, D., J. O. Mascarenhas, D. Gershov, and K. B. Elkon. 1998. Complement-dependent clearance of apoptotic cells by human macrophages. J. Exp. Med. 188:2313–2320. Oliver, M. A., J. M. Rojo, S. Rodriguez de Cordoba, and S. Alberti. 2008. Binding of complement regulatory proteins to group A Streptococcus. Vaccine 8:I75–I78. Podack, E. R., H. J. Muller-Eberhard, H. Horst, and W. Hoppe. 1982. Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants. J. Immunol. 128:2353–2357. Poltermann, S., A. Kunert, M. von der Heide, R. Eck, A. Hartmann, and P. F. Zipfel. 2007. Gpm1p is a factor H-, FHL-1-, and plasminogen-binding surface protein of Candida albicans. J. Biol. Chem. 282:37537–37544. Riedemann, N. C., R. F. Guo, and P. A. Ward. 2003. Novel strategies for the treatment of sepsis. Nat. Med. 9:517–524. Roche, H., B. Ren, L. S. McDaniel, A. Hakansson, and D. E. Briles. 2003. Relative roles of genetic background and variation in PspA in the ability of antibodies to PspA to protect against capsular type 3 and 4 strains of Streptococcus pneumoniae. Infect. Immun. 71:4498–4505. Rossi, V., S. Cseh, I. Bally, N. M. Thielens, J. C. Jensenius, and G. J. Arlaud. 2001. Substrate specificities of recombinant mannan-binding lectin-associated serine protease-1 and -2. J. Biol. Chem. 276:40880–40887. Schindler, R., J. A. Gelfand, and C. A. Dinarello. 1990. Recombinant C5a stimulates transcription rather than translation of interleukin-1 (IL-1) and tumor necrosis factor: translational signal provided by lipopolysaccharide or IL-1 itself. Blood 76:1631–1638. Schwaeble, W., M. R. Dahl, S. Thiel, C. Stover, and J. C. Jensenius. 2002. The mannan-binding lectin-associated serine proteases (MASPs) and MAp19: four components of the lectin pathway activation complex encoded by two genes. Immunobiology 205:455–466. Schwaeble, W., J. Zwirner, T. F. Schulz, R. P. Linke, M. P. Dierich, and E. H. Weiss. 1987. Human complement factor H: expression of an additional truncated gene product of 43 kDa in human liver. Eur. J. Immunol. 17:1485–1489. Schwaeble, W. J., and K. B. Reid. 1999. Does properdin crosslink the cellular and the humoral immune response? Immunol. Today 20:17–21. Siegel, C., J. Schreiber, K. Haupt, C. Skerka, V. Brade, M. M. Simon, B. Stevenson, R. Wallich, P. F. Zipfel, and P. Kraiczy. 2008. Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1. J. Biol. Chem. 283:34855–34863.

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7 Immune Defense at Mucosal Surfaces MARIAN R. NEUTRA AND JEAN-PIERRE KRAEHENBUHL

INTRODUCTION

GENERAL ORGANIZATION OF THE MUCOSAL IMMUNE SYSTEM

The mucosal surfaces of the body together represent a vast surface area separated from the outside world only by delicate epithelial barriers. The epithelia covering mucosal tissues face a daunting task. This is illustrated dramatically in the intestine, whose luminal microbial populations form complex ecosystems that are essential for digestion, epithelial maintenance, normal immune function, and protection against pathogens (Backhed et al., 2005; Pédron & Sansonetti, 2008). In the human colon, for example, hundreds of commensal bacterial species are present, at densities up to 1012 per gram of luminal contents (MacPherson & Harris, 2004). Thus, it is not surprising that mucosal tissues are extremely immunologically active; for example, the number of antibody-producing B cells in the lamina propria of the intestine is estimated to be greater than in any other organ in the body including the spleen, thymus, and lymph nodes (Brandtzaeg et al., 2005). Although antigen-specific effector and memory lymphocytes are present throughout mucosal tissues, the initial inductive events that lead to their formation occur primarily in organized mucosa-associated lymphoid tissues (MALT), located either within the mucosa or in the lymph nodes that drain the mucosa (McGhee et al., 2005). The basic mechanisms of antigen processing and presentation and lymphocyte differentiation that underlie mucosal immune responses are essentially the same as in the systemic immune system. However, the mucosal immune system faces unique challenges. Foreign antigens must be sampled by immunologically competent cells without compromising the epithelial barrier. In addition, epithelial and antigenpresenting cells must distinguish and respond to potential antigenic and pathogenic threats in an environment rich in commensal microorganisms and nutrients. This chapter will review our current understanding of how this complex task is accomplished, with particular focus on mucosal immunity in the intestine.

Organized Mucosa-Associated Lymphoid Tissues: Local Inductive Sites

Organized MALT, recognized microscopically by the presence of lymphoid follicles, are sites in which immune responses are generated. The best-known example is the Peyer’s patches of the intestine, where follicles are aggregated in large clusters that are visible to the naked eye. The composition and function of these tissues have been described in detail and extensively reviewed (Brandtzaeg et al., 2005; MacPherson et al., 2008; Cerutti, 2008). Mucosal follicles, like those of lymph nodes, consist primarily of a cluster of immature B cells, often including a germinal center supported by a network of follicular dendritic cells. Mucosal follicles are flanked by interfollicular T-cell areas that contain a distinct population of mature, antigen-presenting dendritic cells, naive or antigen-sensitized T cells, and high endothelial venules that allow entry and exit of migrating cells (Kelsall, 2008). Each mucosal follicle is separated from the overlying epithelium by a subepithelial “dome” region filled with T and B cells, along with numerous dendritic cells (Brandtzaeg et al., 2005; Kelsall, 2008). Cells of the dome region function in close collaboration with the follicle-associated epithelium (FAE), which delivers antigens and microorganisms from the lumen into the subepithelial tissue. These sites are organized to allow antigen sampling in an environment where incoming foreign materials and pathogens can be immediately captured, processed, and presented for induction of appropriate immune responses (Neutra et al., 2001). Major organized MALT structures appear during fetal life at predetermined sites positioned to respond to incoming antigens and pathogens that challenge the mucosa immediately after birth. They persist throughout life in the distal small intestine (Peyer’s patches), tonsils and adenoids, appendix, and (in some species) the large intestine. In humans, the oral pharynx is monitored by a ring of mucosal lymphoid tissues that include the palatine tonsils, lingual tonsils, and adenoids. In rodents (but not humans) a strip of organized lymphoid tissue lies along the base of the nasal cavity. Solitary or isolated lymphoid follicles (ILF) and

Marian R. Neutra, Department of Pediatrics, Harvard Medical School, GI Cell Biology Laboratory, Children’s Hospital, Boston, MA 02115. Jean-Pierre Kraehenbuhl, Health Sciences eTraining (HSeT) Foundation, CH 1066 Epalinges, Switzerland.

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associated T-cell clusters, only visible microscopically, occur in many mucosal locations and are common throughout the gastrointestinal tract (Wang et al., 2008). They appear only after birth, induced by exposure to antigens and microorganisms. In the human digestive tract, ILFs are most frequent where microbial populations are abundant, such as in the large intestine, cecum, and rectum (McGhee et al., 2005). ILFs in the trachea and bronchi are generally present only under conditions of antigenic challenge (Bienenstock & Clancy, 2005). ILFs are also common at mucocutaneous transitions such as the anal-rectal junction and near the ducts of secretory glands that empty onto mucosal surfaces. The initial event leading to an ILF is not clear, but local antigen entry or epithelial signals are likely to be involved. An early stage of ILF formation is recognizable as a small cluster of T lymphocytes near an intestinal crypt, first described as “cryptopatches” in mice. ILF formation involves a distinct sequence of events including entry of migratory dendritic cells and distinct lymphocyte populations and induction of a follicle-associated epithelium (Wang et al., 2008). The “mature” ILF structure is functionally analogous to Peyer’s patches, capable of antigen sampling and induction of immune responses.

Mucosal Tissues as Immune Effector Sites The Lamina Propria: A Diffuse Lymphoid Tissue

Most mucosal cells involved in immune defense are distributed diffusely throughout the subepithelial connective tissue and are directly or indirectly responsible for immune effector functions. Mucosal effector lymphocytes include terminally differentiated, antibody-producing B cells, helper and regulatory CD41 and cytotoxic CD81 T cells, and NK cells (Brandtzaeg et al., 2005; Lefrançois & Puddington, 2006). Memory B and T cells are also stationed in the mucosa to provide rapid effector responses to local antigen reexposure. Indeed, the great majority of T cells in the lamina propria are memory/effector cells. Mucosal macrophages phagocytose pathogens and macromolecular debris while dendritic cells monitor the mucosa for incoming antigens (Kelsall, 2008; Coombes & Powrie, 2008). Collectively, these cells control the populations of beneficial commensal microorganisms associated with the mucosa, prevent colonization and entry of pathogens, and destroy pathogens and infected cells if invasion occurs.

Intraepithelial Lymphocytes and Immune Surveillance

Throughout the small and large intestines, a specialized population of lymphocytes resides in the lateral intercellular spaces between epithelial cells. These intraepithelial lymphocytes (IELs) are primarily T cells that express Tcell receptors (TCRs) capable of recognizing foreign antigens presented on epithelial cell surfaces (Kunisawa et al., 2007; Ishikawa et al., 2007). IELs form a heterogeneous cell population that consists of both CD41 and CD81 cells (Lefrançois & Puddington, 2006). In the upper airways most IELs are CD41 cells, while in the gut CD81 cells expressing either a or  T-cell receptors represent the major IEL population. In rodents,  T cells predominate, but in the human intestine most IELs express a TCRs; the frequency of these subtypes varies among species and between the small and large intestine. The TCRs of IELs detect antigens presented on epithelial cells, either by classical MHC I molecules (Das & Janeway, 2003; Shastri et al., 2005) or nonclassical MHC molecules such as CD1 (Cohen et al., 2009). Intestinal IELs are oligoclonal and, like skin dendritic T lymphocytes, they probably participate in epithelial monitoring and repair processes rather than in immune

defense. Infection of epithelial cells may result in presentation of peptides on MHC I and/or presentation of microbial lipid antigens on CD1d (Nieuwenhuis et al., 2009). Some IELs also express receptors typical of NK T cells that recognize a nonclassical MHC molecule called MICA/B (MHC class I-related gene A/B) expressed on damaged epithelial cells (Kunisawa et al., 2007). There is evidence that stimulated IELs can induce epithelial apoptosis, dampen the responses of mucosal CD4 cells, and secrete factors that stimulate epithelial cell proliferation. IELs, like other mucosal lymphocytes, migrate into the intestinal mucosa by adhering to adhesion molecules on mucosal venules and by following chemokine gradients generated by epithelial cells, as discussed below. Additional adhesion molecules are involved in the retention of IELs in their intraepithelial niche. IELs express the integrin aE 7 that binds to E-cadherin, an adhesin that holds the lateral membranes of epithelial cells together though calcium dependent, homotypic interactions (Agace et al., 2000). In E–cadherin-deficient mice, IELs are reduced but not absent, indicating that alternate docking mechanisms exist. Although the functions of IELs are not completely understood, they appear to play an important role in epithelial maintenance by eliminating damaged or infected epithelial cells and promoting epithelial cell replication and replacement.

SAMPLING OF ANTIGENS AND PATHOGENS AT MUCOSAL SURFACES Barriers to Antigens and Microorganisms at Mucosal Surfaces

Luminal microorganisms are generally excluded from close contact with epithelial cell surfaces by the interplay of mucous and fluid secretions, antimicrobial peptides, and mucosal clearance mechanisms such as ciliary activity and peristaltic movements. Secretory mucins, the products of genes such as muc2, are large, heavily glycosylated, and negatively charged glycoproteins that are secreted onto mucosal surfaces by goblet cells. Mucins form highly hydrated, loose gels that allow diffusion of large molecules including immunoglobulins (Cone, 2005). However, microorganisms coated with antibody are immobilized in mucus gels due to multiple low-affinity interactions of immunoglobulins with mucins, which promotes entrapment and clearance. The luminal side of the mucus layer is continually eroded and dispersed into the lumen as it is replenished from the epithelium below. Defensins, and other antibacterial proteins such as lysozyme, are secreted by Paneth cells in the crypts (Bevins, 2006; Ouellette, 2006). In addition, S-IgA exported by crypt cells provides nonspecific defense by adhering to lectin-like determinants on certain microorganisms through its carbohydrate side chains or through other binding mechanisms, to block adherence and to entrap potential pathogens (Phalipon & Corthesy, 2003). Surface specializations of epithelial cells are also important for mucosal protection against microorganisms. In the intestines, the apical plasma membranes of enterocytes have closely packed microvilli coated with a thick layer of integral membrane mucins called the filamentous brush border glycocalyx. This coat serves as a diffusion barrier that prevents contact of most microorganisms with integral components of the enterocyte plasma membrane and impedes access to the small intermicrovillar membrane domains involved in endocytosis (Frey et al., 1996; Neutra & Kraehenbuhl, 2005). These membrane-associated mucins adsorb pancreatic proteases and contain enterocyte enzymes involved in terminal digestion, as well as intestinal alkaline phosphatase; together, they protect against microbial contact

7. Immune Defense at Mucosal Surfaces

and degrade microbial products such as lipopolysaccharide (LPS). Membrane mucins and enzymes are continually shed from the surfaces of enterocytes and replaced, presumably to release attached microbes and renew the epithelium’s protective coat.

Epithelial Cells as Sensors of the Mucosal Environment

Epithelial cells are active participants in both innate and adaptive mucosal immune responses, providing the mucosal immune system with a constant stream of information about the contents of the lumen. Epithelial cells respond to microbes and toxins by sending cytokine and chemokine signals to underlying mucosal cells to trigger innate, nonspecific defenses and to promote adaptive immune responses in a variety of ways (Backhed et al., 2005; Rimoldi et al., 2005; Kagnoff, 2006; Rakoff-Nahoum & Medzhitov, 2008). Epithelial cells can detect the presence of microbial components through receptors that recognize common molecular components unique to microorganisms such as flagellin, proteoglycans, lipopolysaccharides, and viral or bacterial nucleic acid sequences. These “pattern-recognition receptors” include Toll-like receptors (TLRs) on epithelial cell membranes and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) in the enterocyte cytosol (Rakoff-Nahoum & Medzhitov, 2008). In intestinal epithelial cells, TLRs and NLRs (NOD1 and NOD2) influence gene expression through a complex system of intracellular signaling pathways governed by adaptors and enzymes that activate specific transcription factors, which in turn promote expression of proinflammatory and/or immunomodulatory cytokines and chemokines. While maintaining the ability to respond vigorously to pathogens, normal intestinal epithelial cells prevent unwanted inflammatory responses to commensal organisms and nutrients by negatively regulating intracellular signal-

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ing pathways through cytoplasmic factors such as A20 (a ubiquitin-modifying enzyme), SOCS1 (a suppressor of cytokine signaling), and PPAR gamma (an inhibitor of NFB activity) (Honda & Takeda, 2009). Thus epithelial cells can mount diverse responses to microbial components including inflammation, enhancement of adaptive immunity, or epithelial cell proliferation. Indeed, normal maintenance and renewal of the intestinal epithelium is dependent on controlled TLR-mediated responses (Rakoff-Nahoum & Medzhitov, 2008) and disruption of the flora (e.g., by antibiotics) leads to increased vulnerability to pathogens. Epithelial cells also respond to TLR ligands by releasing chemokines such as CCL20 and CCL25 that attract DCs and lymphocytes into the mucosa (Sierro et al., 2001; Vijay-Kumar & Gewirtz, 2008) and cytokines such as BAFF and APRIL that induce B-cell proliferation and IgA class switching at mucosal effector sites (Shang et al., 2008; Cerutti, 2008). Thus, the epithelium actively contributes to the maintenance of the mucosal barrier and promotes innate and adaptive mucosal protection.

Collaboration of Epithelial Cells with Dendritic Cells in Antigen Sampling

Induction of mucosal immune responses is complicated by the fact that antigens and microorganisms on mucosal surfaces are separated from cells of the mucosal immune system by epithelial barriers. Thus, the mucosal immune system must obtain samples of the external environment without compromising the integrity and protective functions of the epithelium (Neutra et al., 2001). The cellular organization of the local epithelial barrier determines the way in which samples are obtained, but in all cases, a close collaboration between epithelial cells and DCs is essential (Fig. 1). In stratified epithelia that lack tight junctions, such as those in the oral cavity, vagina, or anal canal, motile dendritic (or Langerhans) cells invade the epithelium to capture

FIGURE 1 Antigen sampling across epithelial barriers. Antigen sampling strategies at mucosal surfaces involve collaborations between epithelial and dendritic cells (DCs). The diverse mechanisms involved are adapted to the nature of the local epithelial barrier. At most mucosal surfaces where the epithelium is stratified, pseudostratified, or simple columnar, DCs are stationed immediately under the epithelium, migrate into the epithelial layer, and may extend dendrites into the lumen to capture antigens. These DCs generally travel to the nearest draining lymph node to present antigen to T cells. At sites of organized mucosal lymphoid tissues, specialized M cells in the lymphoid follicle-associated epithelium deliver antigens by transcytosis across the epithelial barrier, directly to intraepithelial and subepithelial DCs. These DCs then migrate to adjacent mucosal Tcell areas to present antigen. (Reproduced from Neutra & Kozlowski, 2006).

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foreign macromolecules or microorganisms in the intercellular spaces. Antigen-loaded DCs then migrate to T-cell areas of local mucosal lymphoid tissues (as in the tonsil) or distant lymph nodes (such as those that drain the genital tract). In simple epithelia such as those lining the intestine where intercellular spaces are sealed by tight junctions, samples of luminal material are obtained using multiple strategies. In the follicle-associated epithelium overlying organized MALT, epithelial M cells efficiently transport samples of luminal material directly across the barrier. In other locations, epithelial cells may take up small amounts of antigen, or DCs may extend dendrites through the tight junctions to capture particles or microbes on the mucosal surface. These strategies are described in more detail below.

Specialized Antigen Sampling at Organized Mucosal Inductive Sites

Throughout the intestines, multiple crypts provide a continuous supply of fresh epithelial cells to each villus or intercrypt area. Where mucosal lymphoid follicles occur, epithelial cells emerging from the adjacent follicle-associated crypts migrate onto the dome formed by the underlying lymphoid follicle to form the FAE (Kraehenbuhl & Neutra, 2000). Gene expression in cells of the FAE is strongly influenced by factors produced by cells of the underlying follicle, with the result that the FAE differs dramatically from the villus epithelium (Debard et al., 2001). New sites of FAE and M cells appear in vivo where ILFs assemble in response to microbial challenge (Wang et al., 2008) or after injection of Peyer’s patch cells into the mucosa, and enterocytes in intestinal adenocarcinoma cell monolayers grown in vitro acquire M–cell-like features when cocultured with B cells or certain cytokines (Kernéis et al., 1997; Kraehenbuhl & Neutra, 2000). Thus, the distinctive patterns of FAE gene expression and M cell differentiation appear to be dependent on cells of the underlying follicle. The entire FAE presents a distinct biochemical “face” to the lumen and allows macromolecules, particles, and microorganisms relatively easy access to these infrequent areas (Neutra & Kraehenbuhl, 2005). Whereas the villus epithelium is dominated by absorptive enterocytes, mucin-secreting goblet cells, and enteroendocrine cells, the FAE contains few or no goblet or enteroendocrine cells but does contain M cells and enterocytes displaying a FAE-specific phenotype. Follicleassociated cells produce very low levels of digestive enzymes and little or no mucus. Follicle-associated crypts lack defensinand lysozyme-producing Paneth cells and do not secrete IgA into the lumen, as they lack polymeric immunoglobulin receptors. Gene expression profiling studies have revealed that the FAE expresses distinctive receptors, extracellular matrix and matrix-interacting proteins (Lo et al., 2004; Anderle et al., 2005; Hase et al., 2006), and chemokines. Gene expression in the FAE may also be modulated by the microorganisms that contact the FAE or are transported by M cells into the mucosa. For example, CCL20, a chemokine that attracts DCs and lymphocytes that express the chemokine receptor CCR6, is constitutively expressed in the small intestine only in the FAE in mice and humans, but expression is upregulated by TLR ligands (Sierro et al., 2001) and becomes more widespread during intestinal infection (Kelsall, 2008). Cells of the mouse FAE also express CCL9 (analogous to CCL23 in humans) that attracts CCR1-expressing myeloid DCs (Kelsall, 2008) and CXCL16 that attracts CXCR6-expressing B and T lymphocytes into Peyer’s patches (Hase et al., 2006). The major function of M cells in the FAE is to deliver samples of particulate foreign material and microorganisms by transepithelial transport from the lumen to organized mucosal lymphoid tissues (Neutra et al., 2001; Neutra & Krae-

henbuhl, 2005). M cells create an intraepithelial “pocket” that provides a sequestered space for activated or memory B and T lymphocytes and occasional DCs. The pocket dramatically shortens the transcytotic pathway and provides for rapid delivery of luminal samples to intraepithelial and subepithelial cells. The apical membranes of M cells are designed to allow adherence and uptake of antigens and microorganisms by phagocytosis or endocytosis. In addition, certain pathogens can induce macropinocytotic engulfment involving disruption of the apical cytoskeletal organization, although this may lead to M-cell death. Access of microbes and particles to M-cell membranes is facilitated by the lack of an organized brush border and absence of the thick, protective glycocalyx of integral membrane mucins that blankets the microvillus tips on enterocytes (Frey et al., 1996). The enhanced accessibility of M-cell membranes promotes M-cell-specific adherence of viruses and particles, whether or not the participating receptors are unique to M cells (Mantis et al., 2000). In some species, M cells express proteins that could potentially be exploited as receptors for microorganisms, including beta 1 integrin, ICAM-1, annexin, and peptidoglycan-binding protein. They also display carbohydrate structures that differ from those of neighboring epithelial cells, although M-cell glycosylation patterns vary among species, between different mucosal regions, and even within the same FAE (Neutra & Kraehenbuhl, 2005). Luminal IgA or S-IgA (but not IgG or IgM) adheres specifically to apical membranes of M cells in experimental animals and humans via an unknown mechanism (Mantis et al., 2002), an interaction that may promote immune sampling of IgA-coated microbes or luminal antigen-IgA complexes (Kadaoui & Corthesy, 2007). Antigens and pathogens released into the M-cell pocket immediately contact intraepithelial B and CD41 T cells that display the antigen CD45RO typical of activated or memory cells. The B cells express MHC class II, suggesting that they are capable of antigen presentation (Brandtzaeg et al., 2005). DCs also migrate into the pocket from the subepithelial dome region, and DC migration toward and into the FAE is enhanced when the mucosa is exposed to TLR ligands or enterotoxins, most likely due to release of chemokines from the epithelium (Sierro et al., 2001; Chabot et al., 2008). These DCs, along with the intrapocket and subepithelial DCs and lymphocytes, are well-positioned for efficient exposure or reexposure to incoming antigens, but whether the cells within the M cell pocket have unique functions is unknown.

Antigen Sampling at Widespread Mucosal Effector Sites

Although the tight junctions that join cells of the absorptive intestinal epithelium are impermeable to macromolecules, soluble antigens and bacteria are routinely sampled by lamina propria DCs. This was previously thought to be due to limited, nonspecific transcellular transport by villus enterocytes. It is now clear that more specific mechanisms are in place for widespread antigen sampling. For example, epithelial cells in the intestines and airways of human adults express the neonatal Fc receptor FcRn that recognizes IgGantigen complexes, providing a potential mechanism for sampling of luminal antigens or microorganisms that have been previously encountered (Yoshida et al., 2006). In addition, subepithelial DCs can directly sample luminal contents by sending extensions that reach between epithelial cells, through the tight junctions, and into the intestinal lumen (Rescigno et al., 2001; Neiss & Reinecker, 2006). Although the frequency of this phenomenon is not clear, the fact that noninvasive Salmonella enterica serovar Typhimurium and


Escherichia coli were taken up in this way after oral administration to mice suggests that this may be the way that lamina propria DCs routinely sample commensal microorganisms (MacPherson & Uhr, 2004). Indeed, commensal bacteria have been detected within LP DCs. However, commensal bacteria are also observed in the DCs of Peyer’s patches and it is likely that sampling of the endogenous flora occurs in both locations (Kelsall, 2008).

EXPLOITATION OF ANTIGEN SAMPLING MECHANISMS BY PATHOGENS

Mucosal mechanisms for sampling of antigens and pathogens, intended by the host for immune protection and surveillance, creates pathways across the epithelial barrier that can be exploited by pathogens to initiate infection (Neutra et al., 2002). Although the rigid, glycocalyx-coated brush border of enterocytes presents a barrier to endocytosis of particles, certain bacterial and parasitic pathogens have evolved elaborate mechanisms for establishing infection on the epithelial surface, within enterocytes, or in the mucosa. Some adhere and form dense colonies on enterocyte apical surfaces (e.g., Vibrio cholerae), and others co-opt the host cell’s membrane and cytoskeletal machinery to induce novel structures on enterocyte apical poles (e.g., attaching/effacing E. coli and Cryptosporidia) or induce macropinocytosis (e.g., Salmonella). M cells, in contrast, are easily exploited as they are designed to constitutively transcytose adherent particles such as bacteria and viruses and deliver them to phagocytic DCs for degradation and antigen presentation. Although the transport vesicles in M cells can acidify their content and contain proteases, M-cell transport is rapid and pathogens can survive and go on to infect the epithelium itself; target cells in the mucosa; or cells in draining lymph nodes, spleen, or liver. Enteric bacteria that exploit M-cell transport have diverse pathogenic strategies that are described in detail elsewhere in this volume. Briefly, M–cell-transported Shigella invades local epithelial cells through their basolateral surfaces, and phagocytosis of Shigella by macrophages and DCs causes release of proinflammatory cytokines, massive inflammation, and widespread disruption of the epithelial barrier (Sansonetti, 2006). Enteric Yersinia selectively adheres to M cells via its outer membrane protein invasin that recognizes apical M-cell beta1 integrins. After transport, it avoids phagocytosis by injecting a set of Yop proteins directly into the phagocyte cytoplasm via a type III secretion system, which disrupt the cytoskeleton and lead to phagocyte apoptosis (Handley et al., 2005). Salmonella adheres preferentially (but not exclusively) to M cells and injects proteins via a type III secretion system to disrupt actin networks and induce macropinocytosis (Ellermeier & Slouch, 2007). It is phagocytosed by subepithelial DCs, but survives by inducing formation of a nondegradative phagosome and uses migration of phagocytes out of the mucosa to facilitate systemic dissemination (Vasquez-Torres et al., 1999). Studies in experimental animals have shown that the M-cell pathway can be exploited for intestinal invasion by other bacterial pathogens, including Campylobacter jejuni in rabbits, Mycobacterium paratuberculosis in calves, and BCG (bacillus Calmette-Guerin) in rabbits (Neutra et al., 2002). Transport of several pathogenic viruses by M cells has been documented. Viruses lack elaborate signaling or injection systems and generally depend on key surface proteins to adhere to host cells. Enteric viruses are designed to survive the proteolytic environment of the intestine, and adherence to M cells is sufficient to assure endocytosis and transcytosis, as M cells constitutively transport particles of this size. For

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example, two closely related viral pathogens—reovirus in mice and poliovirus in humans—have been shown to selectively adhere to M cells and to use this as an invasion route (Neutra et al., 2002). Reovirus exploits proteases in the intestinal lumen for reconfiguration of the viral outer caspsid and extension of the attachment protein sigma 1, which mediates M-cell adherence. The sigma 1 protein of reovirus type 1 recognizes a specific sialic acid-containing trisaccharide that is present on all intestinal epithelial cell apical surfaces but is accessible to virus-sized particles only on M cells (Helander et al., 2003). Mucosal transmission of SIV (simian immunodeficiency virus) in nonhuman primates, and presumably of HIV (human immunodeficiency virus) in humans, can occur without epithelial damage by the oral, rectal, and genital mucosa (Haase, 2005). The molecular basis of HIV adherence to epithelia in vivo is not established, but studies using neoplastic intestinal epithelial cell lines in culture have shown that a combination of the ubiquitous glycolipid galactosylceramide and the chemokine receptor CCR5 can mediate HIV adherence and uptake (Fotopoulos et al., 2002). Epithelial cells themselves are not productively infected by HIV. Evidence from SIV studies in nonhuman primates indicates that depending on the nature of the local mucosal epithelium, HIV may exploit multiple mucosal antigen sampling mechanisms at different mucosal sites, including vesicular transepithelial transport by M cells, direct infection of intraepithelial T cells, and capture by intraepithelial or subepithelial DCs (Haase, 2005). As mucosal antigenpresenting cells interact with local CD41 T cells, they unwittingly infect and ultimately disable the very cells needed to mount an effective immune response. Infection of local target cells and dissemination of virus to regional lymph nodes and other tissues occurs rapidly after deposition of virus on mucosal surfaces. Whether transmitted mucosally or injected, HIV and SIV preferentially replicate in mucosal tissues such as the intestinal mucosa (Veazey et al., 2001) due to the abundance of activated T cells expressing CD4, CCR5, and a4/7 integrin, all of which are exploited as receptors for the HIV envelope glycoprotein gp120 (Brenchley & Douek, 2008).

INDUCTION OF MUCOSAL IMMUNE RESPONSES

The events involved in induction and maintenance of secretory IgA responses involve coordinated interactions among many cell types (Cerutti, 2008). The sites at which these interactions can occur are distinct and have distinct outcomes. In all cases, foreign antigens must be transported across epithelial barriers and captured by mucosal dendritic cells, as discussed in detail above. Distinct subpopulations of mucosal DCs play important roles not only in capturing antigens but also in orchestrating the nature of the resulting mucosal immune responses (Kelsall, 2008).

T-Cell Dependent Responses in Organized MALT

Initiation of primary mucosal immune responses to Tdependent (such as protein) antigens requires processing and presentation of antigen to T cells by DCs in organized lymphoid tissues. DCs are influenced by the microenvironment of mucosal tissues to assume unique phenotypes and functions, distinct from DCs in nonmucosal sites. Mucosal DCs are further differentiated with multiple subtypes in organized lymphoid tissues and in the lamina propria (Kelsall, 2008). Immature dendritic cells are present in large numbers in the subepithelial dome region and in smaller numbers within the FAE. Mucosal exposure to live bacteria, bacterial toxins, and

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TLR ligands induce a rapid influx of DCs from the subepithelial dome region into the FAE, a response that enhances antigen capture. Peyer’s patch DCs (but not lamina propria DCs) express CCR6, a membrane receptor attracted by the chemokine CCL20 constitutively expressed by FAE cells. Peyer’s patch DCs rapidly capture macromolecules, commensal or pathogenic microorganisms, and particles transported by M cells, as well as virus-infected, apoptotic epithelial cells. This triggers DC maturation and expression of CCR7, a receptor that drives them to nearby interfollicular T-cells zones where the cognate chemokines are expressed (Kelsall, 2008). Here, the DCs initiate a process leading to generation of antigen-specific T- and B-cell clones, described below. The extent to which Peyer’s patch DCs migrate to MLN is not clear, but evidence indicates that induction of mucosal effector cells can occur entirely within the mucosa. In contrast, the DCs that migrate to MLN come primarily from the lamina propria (MacPherson & Uhr, 2004). In the interfollicular areas, a constant stream of naïve T cells are available for testing and possible engagement via appropriate T-cell receptors, coreceptors, and the costimulatory molecules required for T-cell activation. Activated CD41 T-helper cells in Peyer’s patches have immediate access to B cells in the adjacent follicle. Many complex interactions occur in this unique environment; in summary, both interfollicular and follicular T cells and DCs promote B-cell immunoglobulin class switch recombination by producing cytokines, including TGF-beta, IL-6, and IL-10, and by engaging CD40-expressing B cells via T-cell CD40 ligand (Cerutti, 2008; Fazilleau et al., 2009; Reinhardt et al., 2009). The follicular environment also inhibits proinflammatory Th1 responses. Antigen-specific B cells proliferate locally, clonally expanding the follicle to produce large numbers of antigen-specific IgA lymphoblasts. Proliferating B cells expressing high-affinity receptors are selected for survival by antigen-presenting follicular DCs (Cerutti, 2008).

Homing of Lymphocytes to Mucosal Tissues

Within a few days, antigen-specific B and T cells migrate out of the organized mucosal lymphoid tissues and enter the circulation. Before they do, however, mucosal DCs induce expression of mucosa-specific cell surface adhesion molecules called “homing receptors” on both B and T cells. This occurs only in mucosa-associated tissues due to the local production of retinoic acid by epithelial cells and DCs from dietary precursors (Mora et al., 2006). Homing receptors allow mucosally induced lymphocytes to selectively adhere to counterreceptors or “addressins” on mucosal endothelial cells, particularly in the region where the antigen or pathogen was initially encountered. For example, IgA1 B cells generated in organized mucosal lymphoid tissues of the intestine preferentially migrate back into the intestinal mucosa because they express the “homing receptor” a4/7 integrin that interacts strongly with MADCAM1, an addressin expressed by venules in the small and large intestine (and lactating mammary gland) but not in other mucosal tissues. In contrast, a4/1 integrin expression by lymphocytes appears to direct them to the airways (Kunkel & Butcher, 2003; Sigmundsdottir & Butcher, 2008). This system is responsible for the regional nature of mucosal immune responses demonstrated in mice, nonhuman primates, and humans (Neutra & Kozlowski, 2006). Mucosal DCs and lymphocytes are also programmed to express mucosa-specific receptors that recognize chemokines produced by epithelial cells. Chemokine gradients in the lamina propria guide the migration of effector B and T cells as well as DCs toward epithelial surfaces. Some of

these chemokine/receptor systems are common to many mucosal tissues. For example, CCR10, which is expressed by most IgA B cells that originated in MALT, recognizes the chemokine CCL28 that is secreted by epithelial cells in many locations, including the small and large intestines, salivary glands, tonsils, respiratory tract, and lactating mammary gland (Kunkel & Butcher, 2003). This recognition promotes widespread secretion of specific IgA antibodies after mucosal immunization at one site, a phenomenon originally called the “common mucosal immune system” (Woof & Mestecky, 2005). Other chemokines are organ or region-specific; for example, circulating a4/71 B lymphocytes activated in the small intestine express CCR9, which attracts them to CCL25, a chemokine secreted by epithelia throughout the small (but not the large) intestine. Conversely, B lymphocytes that express CCR10 are attracted by the chemokine CCL28 produced in by epithelial cells in the large intestine. Other receptor/chemokine pairs are adapted to the local microenvironment; for example, expression of the epithelial chemokine CCL20 is responsive to the presence of luminal flora. Mucosal immunization can also induce IgA and IgG in serum. This is due in part to migration of antigen-loaded mucosal DCs to mesenteric lymph nodes, where both mucosal and systemic homing receptors are induced, and to systemic inductive sites such as lymph nodes and the spleen (MacPherson et al., 2008). In addition, a fraction of the B cells activated in the mucosa or in draining lymph nodes express the peripheral homing receptors, a4/1 and l-selectin (Kunkel & Butcher, 2003). By contrast, systemic immunization is generally ineffective for induction of mucosal IgA antibody responses because DCs that capture antigen at injection sites are not exposed to retinoic acid locally or in nonmucosal lymph nodes and fail to induce expression of mucosal homing or chemokine receptors on lymphocytes (Mora et al., 2006).

T-Cell Independent Responses in Widespread Mucosal Tissues

From the above, it is evident that arming the mucosa with antigen-specific, high-affinity effector and memory T cells and antibody-producing B cells takes time. Five to seven days are required for initial induction in organized mucosal lymphoid tissues, directed migration, and terminal differentiation of cells in widespread mucosal sites. A more rapid but less specific immune protection mechanism is present throughout the mucosa, where a subset of B cells can rapidly produce IgA (and to a lesser extent IgM and IgG) without CD4 T-cell help (Fagarasan & Honjo, 2003; MacPherson et al., 2008; Cerutti, 2008). In mice, these B-1 cells migrate to the intestine from the peritoneal cavity, and comparable cells of unknown origin are present in humans. B-1 cells express unmutated, polyreactive surface IgA antibodies that recognize bacterial components—primarily complex carbohydrates—with low affinity. When presented with appropriate ligands, such as common carbohydrate structures of commensal bacteria, they can be induced to produce polymeric IgA antibodies that are secreted into the lumen, as described above. These antibodies control mucosa-associated populations of commensals and provide some protection against various pathogens such as Salmonella serovar Typhimurium. They also are essential for keeping bacteria and the immune responses that control them confined to the mucosa (MacPherson & Harris, 2004). When T–cell-independent antigens contact the mucosa, multiple innate immune pathways are activated to promote IgA class switching by B-1 cells. Some antigens, such as LPS, activate epithelial TLRs (Shang et al., 2008)


and thereby induce epithelial–cell-derived cytokines, such as BAFF and APRIL that promote B-cell isotype switch and antibody production, as with high-affinity B cells derived from Peyer’s patches (Schneider, 2005; Cerutti, 2008). Other microbial oligosaccharides directly activate B cells through their membrane immunoglobulins or B-cell receptors and TLRs (Peng, 2005; Bergtold et al., 2005). Such antigens also signal through the DCs that are stationed in the mucosa or reach through the epithelium. DCs endocytose T cell-independent antigens from the environment into a nondegradative endocytic pathway, recycle antigen to the plasma membrane, and present it to B cells (Trombetta & Mellman, 2005). DCs also stimulate IgA class switch and antibody production by secreting CD40L, BAFF, and APRIL (Schneider, 2005; Cerutti, 2008).

Terminal B-Cell Differentiation and IgA Secretion

B cells from Peyer’s patches and other organized MALT complete their differentiation only after arrival in the lamina propria. Here they become IgA antibody-secreting plasma cells through the influence of cytokines including BAFF, APRIL, IL-6, and IL-10 produced by epithelial cells and local DCs, as described above. On arrival in the intestinal lamina propria, most IgA1B cells in humans produce dimeric IgA1, a subclass characterized by an extended, 0-glycosylated hinge region containing proteolytic sites vulnerable to specific microbial IgA proteases (Russell & Kilian, 2005; Woof & Mestecky, 2005; MacPherson et al., 2008). Under the influence of BAFF and APRIL from epithelial cells, a final class switch recombination event occurs and IgA plasma cells switch to production of IgA2, a form that lacks the vulnerable hinge region and is more protease resistant. Commensal bacteria are required for optimal IgA production because epithelial cells express these cytokines in response to ligation of their TLRs (Shang et al., 2008). Most IgA1 and IgM1 mucosal plasma cells express a short J-chain polypeptide that mediates dimerization of IgA and pentamerization of IgM. The J chain also mediates binding to the polymeric immunoglobulin receptor (pIgR) in epithelial basolateral membranes (Woof & Mestecky, 2005). The vesicular transport process by which pIgR exports IgA and IgM into the lumen has been studied in detail and represents the classic model for transcytosis across a polarized epithelium (Kaetzel, 2005). The pIgR is a transmembrane glycoprotein with a large extracellular ligand-binding portion, a single membrane-spanning alpha helix, and a cytoplasmic tail that governs the intracellular itinerary of the molecule. The large, highly glycosylated extracellular region called “secretory component” (SC) is comprised of five Ig-like domains and a flexible region containing a protease cleavage site. pIgR is constitutively expressed by mucosal epithelial cells in the intestinal crypts and in glands of other mucosal tissues, but its level of expression can be modulated by cytokines and other mediators (Kaetzel, 2005; MacPherson et al., 2008). pIgR binds J–chain-containing dimeric IgA and pentameric IgM at the basolateral membrane, internalizes the ligand–pIgR complex (as well as unoccupied receptor) by clathrin-mediated endocytosis, and traffics it to a common endosomal compartment where it is sorted and delivered to apical recycling endosomes. Fusion of the apical recycling endosome with the apical plasma membrane and cleavage of the extracytoplasmic portion of pIgR by an apical membrane endoprotease releases the Ig-SC complex into the lumen as secretory IgA (S-IgA). SC enhances the lowaffinity interactions of Igs with mucins and increases the protease resistance and stability of these immunoglobulins

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in the protease-rich intestinal environment (Phalipon & Corthesy, 2003; Russell & Kilian, 2005; Woof & Mestecky, 2005). S-IgA is by far most abundant in normal secretions, but S-IgM can provide compensatory protection in the immature gut and in selective IgA deficiency (Brandtzaeg et al., 2005).

Regulation of Mucosal Immune Responses

Small doses of soluble foreign proteins and peptides in the gut lumen generated during normal digestion, as well as from dying epithelial cells and some commensals, are sampled by the mucosal immune system but fail to induce immune responses. Repeated, low oral doses of soluble antigens generally result in suppression of systemic immune responses to subsequent injection of the antigen, a phenomenon termed “oral tolerance.” Potential mechanisms responsible for oral tolerance include induction of T-cell anergy, T-cell deletion, and the induction of CD41 regulatory T cells (Tregs) (Faria & Weiner, 2005). High oral doses of antigen tend to lead to anergy and deletion, while low, repeated doses induce the generation of Tregs. Tregs are induced in the mucosa or in MLN by exposure to a subset of antigen-presenting DCs, present in both Peyer’s patches and lamina propria, which express aE integrin (CD103). There is evidence that DCs from Peyer’s patches are involved in the generation of Tregs, but other evidence implicates lamina propria DCs that constitutively traffic to the MLN (Worbs et al., 2006; Kelsall, 2008). Lamina propria DCs capture antigens from commensal microorganisms, apoptotic epithelial cells, or soluble proteins derived from digested food. The DCs then mature and migrate to the MLN in a CCR7-dependent manner (Kelsall, 2008). At sites of mucosal antigen uptake or in MLN, induction of Tregs occurs simultaneously with induction of mucosal effector lymphocytes, as both are activated by mucosa-specific CD1031 DCs that produce retinoic acid and TGF- (Sakaguchi & Powrie, 2007). The genetic program in Tregs that is responsible for the cells’ suppressive functions, including production of IL-10, is controlled by expression of a transcription factor called forkhead box P3 (Foxp3). Adoptive transfer of antigen-specific Tregs to naïve mice results in transfer of antigen tolerance (Faria & Weiner, 2005). A significant number of the CD41 T cells in the normal intestinal lamina propria produce the cytokine IL-17, along with IL-21 and IL-22 (Zhou et al., 2009). These cytokines have diverse effects on neighboring cells, including production of antimicrobial peptides, attraction of neutrophils, and cell proliferation. Th17 cells are important effector cells in host defense against extracellular bacteria and fungi. Th17 cells are consistently associated with the presence of commensal bacteria, and are not present in organized lymphoid tissues (Peyer’s patches, lymph nodes, or spleen). There is evidence that a subset of lamina propria DCs (CD11c1, CD11b high) can be stimulated by flagellin to induce Th17 cells. Under normal conditions these cells appear to protect the mucosa against inflammatory damage, but Th17 cells can also enhance inflammation (Louten et al., 2009).

MECHANISMS OF MUCOSAL PROTECTION Secretory IgA

S-IgA plays multiple roles in mucosal defense (Phalipon & Corthesy, 2003; Russell & Kilian, 2005). It promotes entrapment of antigens or microorganisms in the mucus, preventing direct contact of pathogens with the mucosal surface, a mechanism called “immune exclusion” (Fig. 2). Antigen-specific S-IgA may directly block or sterically

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hinder the microbial surface molecules that mediate epithelial attachment or intercept incoming pathogens within epithelial-cell vesicular compartments during pIgR-mediated transport. Within the mucosa, dimeric IgA synthesized by local IgA plasma cells may neutralize pathogens by inhibiting adherence to target cells, mediate transport of pathogens back into the lumen via pIgR, opsonize microbes and particles to promote uptake and killing by phagocytes expressing Fc u/alpha receptors, or mediate antibody-dependent cellular cytoxicity (ADCC) that leads to destruction of local infected cells. S-IgA is also important for maintaining the complex ecosystem of commensal bacteria that is crucial for digestion and mucosal homeostasis (Peterson et al., 2007).

While high-affinity S-IgAs can block or neutralize microbial toxins and pathogens, low-affinity S-IgAs keep commensal populations in balance and prevent commensal bacteria from breaching the mucosal barrier and evoking systemic immune responses (MacPherson et al., 2008; Fagarasan & Honjo, 2003). Conversely, the presence of commensal organisms is required for maintenance of the mucosal immune system (Backhed et al., 2005). Importantly, intestinal IgA protects without inducing damaging mucosal inflammation, in part because it not only prevents commensals and pathogens from contacting the mucosa, but also because of its inability to fix and activate the complement cascade (Russell & Kilian, 2005; Woof & Mestecky, 2005).

FIGURE 2 Mechanisms of immune protection at mucosal surfaces. Mucosal immune protection depends on the contributions of multiple cell types. Antigen-specific effector B and T cells that bear mucosal homing receptors recognize addressins on mucosal high endothelial venules and enter the mucosa. In response to epithelial and dendritic cell signals, the B cells terminally differentiate to become mucosal plasma cells. Most plasma cells produce dimeric IgA that is exported into secretions as S-IgA to intercept antigens and pathogens and prevent mucosal invasion. IgA, as well as IgG from local plasma cells or blood, can also neutralize pathogens within the mucosa. Local cytotoxic T cells and antibodies collaborate to kill infected cells. Pathogens are also captured by dendritic cells (DC) and macrophages (Mf), and carried to draining lymph nodes. (Reproduced from Neutra & Kozlowski, 2006).


Mucosal IgG

IgG is also present within mucosal tissues, mostly derived from serum. However, IgG synthesis also occurs locally, especially after local mucosal immunization. IgG- as well as IgAsecreting plasma cells are present in the female genital tracts of macaques and humans (Woof & Mestecky, 2005), and significant concentrations of IgG are present in human cervical/ vaginal and rectal secretions (Neutra & Kozlowski 2006). In the human intestine, 5% to 15% of mucosal plasma cells secrete IgG, but IgG is susceptible to degradation by luminal intestinal and bacterial proteases, and in large intestinal secretions, IgG concentrations are generally 30- to 100-fold lower than those of S-IgA (Woof & Mestecky, 2005). IgG was previously thought to enter the lumen only by leakage, but it is now known that adult human epithelial cells express the pH-dependent Fc receptors that mediate transepithelial IgG transport in neonatal rodents (FcRn), (Yoshida et al., 2006), suggesting a possible IgG secretion mechanism. In any case, there is evidence that IgG as well as S-IgA in secretions can block mucosal infection, neutralize pathogens that enter the mucosa, and prevent systemic spread.

Effector and Memory T Cells

Cytotoxic T lymphocytes (CTLs) in mucosal tissues cannot prevent pathogen entry, but they may play a crucial role in clearance or containment of mucosal viral infections. CD81 lamina propria T cells exhibit potent cytotoxicity against specific pathogen-infected target cells in vitro and convey effective host protection upon adoptive transfer to recipient mice that were orally or systemically infected with pathogens (including viruses and bacteria). For example, Th-1 CD41 lamina propria T cells with specific reactivity against astrovirus, a common and important gastroenteritis virus, have been identified in normal human intestines and are presumably important in intestinal defense against recurrent astroviral infections. Th-2 CD41 lamina propria T cells play a decisive role in driving productive immunity against the intracellular pathogen Toxoplasma gondii, notably by promoting the development of IgA-producing plasma cells. Cytotoxic T cells in mucosal tissues are likely to be important in limiting mucosal replication of HIV (Belyakov & Ahlers, 2008). Mucosally immunized (but not systemically immunized) mice were protected against infection after mucosal challenge with a recombinant vaccinia virus expressing HIV envelope glycoprotein 160 (gp160), but protection was abrogated by treatment of the mice with anti-CD8 antibodies. Immunologically active mucosal tissues, such as the intestinal tract, contain abundant CD41 T cells that are targets for HIV, and as a result, the intestinal mucosa becomes a reservoir of HIV infection regardless of the site of initial viral entry (Veazey et al., 2001). Both CTLs and antibodies within mucosal tissues may contribute to preventing the establishment of such reservoirs (Brenchley & Douek, 2008).

IMPLICATIONS FOR MUCOSAL VACCINES

Many mucosal vaccines and delivery systems have been developed and tested in animals but very few have been approved for human use (Holmgren & Czerkinsky, 2005). Progress has been slow in part because vaccines given orally or deposited directly on mucosal surfaces face the same gauntlet of host defenses as do microbial pathogens: being diluted in mucosal secretions, captured in mucus gels, attacked by proteases and nucleases, and excluded by epithelial barriers (Neutra & Kozlowski, 2006). Thus the exact dose of mucosally administered antigen that actually crossed the epithelial barrier cannot be precisely determined but can only be estimated (Corbett et al., 2008). An additional challenge

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is that mucosal tissues are adapted to the presence of foreign antigens, such as microorganisms and their products. As a result, vaccines that consist of soluble macromolecules and protein subunit antigens, which may produce vigorous immune responses if injected into a sterile environment such as muscle, are often “ignored” when mucosally administered. For mucosal vaccines to be distinguished from harmless substances and nutrients, they must raise alarms in the mucosa by containing adjuvants or molecular patterns that activate innate signaling pathways in epithelial cells and underlying antigen-presenting cells (Elson & Dertzbaugh, 2005). Thus, mucosal vaccines are most effective when they mimic successful mucosal pathogens in the following key respects: they are live mucosa-tropic vectors or microbe-sized particles that adhere to mucosal surfaces, preferably to the FAE, and are avidly endocytosed by mucosal DCs. Mucosal vaccine protocols also must be adapted to the receptor-mediated recognition systems described above that dictate immune cell migration. These systems serve not only to populate the mucosa with effector cells, but also to focus the immune response at the site where antigen or pathogen was initially encountered. Although region-specific cell homing mechanisms have been most extensively defined in mice (Sigmundsdottir & Butcher, 2008), the regional nature of mucosal immune responses has been clearly demonstrated in nonhuman primates and in humans as well (Neutra & Kozlowski, 2006). Responses to mucosal vaccines are greatly amplified by local boosts, due to the abundant populations of memory T and B cells in mucosal tissues that allow for local amplification of effector responses on reexposure to an antigen. Local exposure to an antigen and/or adjuvant evokes cytokines and chemokines from epithelia, DCs, and other cells and up-regulates expression of addressins on local endothelia, increasing the numbers and enhancing the functions of mucosal effector cells (Kunkel & Butcher, 2003; Sigmundsdottir & Butcher, 2008). The value of repeated mucosal antigen exposure was reinforced by the observation that CD81 T cells activated by mucosal immunization initially had a widespread migration pattern, but over the long term, memory CD81 T cells were preferentially located in the region where antigen was originally encountered (Qimron et al., 2004).

CONCLUSIONS

In conclusion, the recent and ongoing expansion of new information about the complex interactions of mucosal tissues with the commensal flora and microbial pathogens, and the mechanisms of mucosal immune protection, promise to provide the tools needed to exploit the full potential of mucosal vaccines.

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8 Regulation of Antimicrobial Immunity YASMINE BELKAID, SHARVAN SEHRAWAT, AND BARRY T. ROUSE

INTRODUCTION

represented by CD41 T cells that develop in the thymus and express the signature transcription factor Foxp3 that is involved in controlling regulatory activities of the cells (Belkaid & Tarbell, 2009) . The major role of these Foxp31 natural T regulatory cells (nTreg) is to prevent the occurrence and modulate the expression of autoimmunity, but as will be discussed, nTregs also influence responses to several pathogens. The inducible regulatory cell types are highly heterogeneous in origin, phenotype, and the means by which they exert regulation. These inducible regulators are perhaps the most influential cell types that affect the responses to pathogens, particularly those that persist and cause chronic reactions (Belkaid & Tarbell, 2009). The inducible Treg populations include IL-10 producing CD41 T cells (often referred to as Tr1 cells), TGF-b producing CD41 T cells, CD81 regulatory cells, NK T cells, as well as CD41 T cells that convert to become Foxp31 and regulatory cells (Shevach, 2006). The latter cells usually develop in appropriate environments from naïve precursors but in some instances may develop from functionally differentiated CD41 T cells. This chapter focuses on inducible Foxp31 Tregs since it seems likely that this cell type plays a dominant role in contributing to the maintenance of homeostasis and in limiting collateral tissue damage caused by chronic effector cell responses to pathogens. Moreover, unlike other subtypes of inducible regulators, the Foxp3 transcription factor expression provides a convenient means of distinguishing these cells from nonregulatory cells. However, since there are no convenient phenotypic markers to distinguish between natural and inducible Foxp31 Tregs, it is usually not possible to decide, especially in human systems, if a regulatory effect is mediated by cells derived from nTreg or if they represent converts from nonregulatory cells. Further complicating the story with studies on human tissues is that activated human T cells may temporarily up regulate Foxp31 and not adopt a regulatory function.

Yasmine Belkaid, Mucosal Immunology Unit, Laboratory of Parasitic Diseases, Division of Intramural Research, National Institute of Health, 4 Center Drive B1-28, Bethesda, MD 20892. Sharvan Sehrawat and Barry T. Rouse, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996.

HOW DOES MICROBE HOST INTERACTION TRIGGER RESPONSES OF NATURAL Treg?

Since vertebrates have developed numerous powerful defense mechanisms, they can survive over the long term in the face of constant exposure to multiple types of microbes that could damage or destroy them. However, many of these defenses are potentially damaging, particularly if they are long acting; so they must be controlled to limit the chance of causing collateral damage to host tissues. Such control becomes of particular relevance in situations where microbes have devised strategies that permit them to persist in the body for lengthy periods of time. Regulation of exuberant reactions to microbes is achieved in many ways and includes not only host control measures, but also manipulation of potentially harmful host responses by the microbes themselves in some cases (this topic is discussed in chapter 31). Clearly, the interests of the microbes are best served by not devastating nor destroying their host since these outcomes will limit their own chance for survival and transmission. Regulation and maintenance of a homeostatic environment by the host is achieved in many ways. These include the production of proteins such as the cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-b) that counteract tissue damaging inflammatory events, as well as the existence of dedicated cell types that can inhibit the activity of the effector cells that orchestrate tissue damage. We now refer to such dedicated cells as regulatory cells, which are heterogeneous in origin and phenotype, as well as with regard to the mechanisms by which they perform regulatory activities. Conceptually, there are two main subtypes of regulatory cells that affect host microbe interactions: cells that are already present and ready to operate at the time of infection and cells that become induced and adopt a regulatory function as a consequence of the microbial infection. These two categories of regulatory cells are commonly referred to as naturally occurring and inducible regulatory T cells. The former cell type is mainly

At the onset of infection, cells are already present that can act as regulators. The most abundant of these are the CD41Foxp31 nTreg, which account for around 8% to 10% 109

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of CD41 T cells in the circulation and lymphoid tissues. The nTreg are especially abundant at sites of initial infection, such as the skin and mucosal surfaces. In fact, it is estimated that around 80% of the total nTreg population is present in the skin. As already mentioned, the majority of nTreg appears to be self-reactive and mainly serves to prevent and control autoimmunity. A minor fraction of cells, however, could express T-cell receptors (TCRs) that recognize microbial antigens especially if these are cross-reactive with selfantigens. The issue of what proportion of nTreg have TCRs that recognize microbial antigens has not been fully evaluated and merits further study. However, it is well-known that infection with at least some microbes can expand and activate the nTreg population, and if this population is removed prior to infection, the pattern of events that occur and the magnitude of the immune response generated may differ dramatically (Belkaid et al., 2002; Suvas et al., 2003). Such is the case, for example, with herpes simplex virus (HSV) and some parasitic infections as is further discussed later in this chapter. Assuming TCR stimulation may be an unusual step during Treg responses to microbes, the fact that nTreg can be expanded and activated following infection means that alternative forms of stimulation must be occurring. Logical candidates for this are the pathogen-associated molecular patterns (PAMPs) expressed by microbes that engage receptors either on nTreg themselves or on intermediary cells such as those that compose the innate immune system (Belkaid & Tarbell, 2009). The latter, in turn, will activate nTreg directly or will generate nTreg activators. The PAMPs that have received the most study are ligands for TLRs, most of which are present on nTreg. The source of TLR ligands may be the pathogen but some ligands can also be endogenously derived as a result of tissue damage at the infection site. Examples include self-DNA/RNA, some heat shock proteins, and the extracellular matrix component hyaluronidase. Various studies have shown that several TLR ligands can act on nTreg, but the outcome is variable and somewhat controversial. This topic has been recently reviewed by others (Conroy et al., 2008). It seems that in some instances, TLR stimulation results in activation and expansion of nTreg, whereas engaging other TLRs result in an inhibitory effect on nTreg function. Many studies have focused on TLR2 and TLR4 stimulation. For example, with TLR2, the majority opinion is that TLR2 and TLR4 stimulation causes nTreg to proliferate, although the suppressor functions of individual cells may be diminished (reviewed in Conroy et al., 2008). With TLR4 stimulation, some have reported that the proliferative and suppressive function of nTregs are enhanced, whereas others indicate that the main effect of TLR4 engagement is to enhance the activity of IL-10, producing (Foxp32) inducible Treg (Conroy et al., 2008). The TLR5 stimulating molecule, flagellin—usually derived in the body from enteric bacteria—was also shown to enhance Foxp3 expression and to increase the suppressive function of nTreg (Crellin et al., 2005). In contrast, other TLR ligands appear to mainly suppress regulatory T-cell activity. This has been reported for both TLR8 and TLR9 stimulation. As will be further discussed, it seems that activation of TLR9 by DNA derived from gut flora can also inhibit the conversion of conventional T cells into Foxp31 Treg (Hall et al., 2008). We must conclude that the role of TLR stimulation of nTreg is complex and remains an unfolding story. For instance, few (if any) studies have addressed neither the issue of the quantity of microbe-derived TLR activators that would be available in vivo, nor the outcome of combined stimulation with multiple TLR ligands, as many microbes may provide. Clearly the topic requires more investigation.

There are also other non-TCR mediated mechanisms by which microbes could trigger regulation by nTreg. In some instances, this could include the production of proteins by the pathogen that activate nTreg by binding to a receptor, which the cells express. Recently, for example, the HIV (human immunodeficiency virus) protein gp120 was shown to activate human nTreg via its ability to bind to the CD4 molecule (Becker et al., 2009). Additionally, cytokines generated in the microenvironment of infection could also boost nTreg activity. These include IL-2 (which is essential for nTreg survival and expansion), TNF-a, and TGF-b. Curiously, an injection of IL-2 complexed to anti-IL-2 antibody happens to be a potent means of expanding nTreg in vivo, although such a situation would not be expected to occur in physiological circumstances (Webster et al., 2009). Under conditions of microbial infections, there are many host-derived molecules as well as related molecules derived from microbes themselves that have potent effects on many cells involved in both innate and adaptive immunity. Of particular interest are the family of galectin molecules that are up regulated or secreted during inflammatory responses. Galectins either form lattices on the surface of cells by binding to cell surface glycoproteins and glycolipids or to specific receptors. The outcome is variable but includes apoptosis, proliferation, and changes of function (Rabinovich & Toscano, 2009). Perhaps of most potential interest in terms of this review are the galectins-1 and -9, both of which are secreted during microbe-induced or other inflammatory response. It is not clear if the infected cells themselves are the source of the galectins or if they come from other cells that respond to cytokines induced by the infection, such as IFN-g, which are produced by responding NK cells or later on by T cells. Galectin-9 was shown to modulate the function of T cells that are involved in inflammatory reactions such as those caused by HSV infection of the eye (Sehrawat et al., 2009). Galectin-9 mainly acts by binding to its receptor TIM-3, which is up regulated on activated cells but is also expressed by a high proportion of nTreg (Sehrawat et al., 2009; Zhu et al., 2005). Whereas galectin-9 binding to activated effector cells causes them to undergo apoptosis, its binding to TIM-3 on nTreg may lead to the expansion and activation of these cells, as we have shown with HSV-induced ocular lesions. Both events serve to resolve the ocular lesions. Galectin-1 functions in a similar way to galectin-9, although different recognition systems are involved and it is not clear if galectin-1 similarly acts to expand and activate nTreg. However, galectin-1 has been reported to expand IL-10-producing Treg (Rabinovich & Toscano, 2009). We can conclude that microbial infections could trigger the expansion and activation of nTreg in several ways, some of which may not involve engagement of the nTregspecific TCR, although this latter event might facilitate responses. These various stimulatory processes are summarized in Fig. 1.

ROLE OF INDUCIBLE REGULATORY CELLS DURING INFECTIONS Induction of Tr1 Cells

One of the first types of regulatory cells observed to influence the outcome of infections were called Tr1 cells. These were derived from nonregulatory precursors by antigen stimulation in the presence of the immunoregulatory cytokine, IL-10. The cytokine alone can inhibit the immune responses (by both Th1 cells and Th2 cells) to many pathogens in experimental models and in human infectious

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FIGURE 1 Microbes or their products may modulate Treg responses in multiple ways. A few Treg with TCR specificity to pathogen antigens proliferate in response to the infection. Alternatively, cross-reactive pathogen antigens or self-antigens, released as a result of tissue damaging inflammatory reactions, could stimulate self-reactive Treg. Stimulation independent of TCR could be provided by the microbial products such as TLR ligands or other host-derived products, such as cytokines (TGF-b, IL-2, IFN-g, TNF-a, etc.), galectins (galectin-1, 9, and 10), heat shock proteins (Hsp-60, Hsp-60 peptide p277, Hsp-gp96, etc.), cellular metabolites (retinoic acid) as a result of infection.

diseases such as tuberculosis, malaria, hepatitis C, filariasis, leishmaniasis, and schistosomiasis (Moore et al., 2001). The most remarkable example of this control is illustrated by its crucial role during acute infection of mice with Toxoplasma gondii. In this model, IL-10 production by T cells is the key regulator of effector-cell responses, as IL-10-deficient mice can control parasite number, but they succumb to lethal immunopathology driven by unrestrained effector-cell responses (Gazzinelli et al., 1996). Besides T cells, IL-10 can be produced by numerous cell types, including B cells, NK cells, macrophages, and DCs (Moore et al., 2001). IL-10 can also be produced by nTreg and, in some cases, is associated with their function; however, in most cases, the inducible Tr1-cell population is the relevant source of this cytokine during infection. During various infections, Tr1 cells develop from conventional T cells after encountering certain signals, such as exposure to deactivated or immature APCs (antigen-presenting cells), or repeated exposure to antigen or IL-10 itself. Of note, these conditions prevail during chronic infection in which APC functions are often targeted by the pathogen, and cells of the immune system are chronically exposed to microbial antigens. Consistent with a role for these cells in human disease, Tr1-cell clones can be isolated from patients who are chronically infected with HCV (hepatitis C virus) (MacDonald et al., 2002). Interestingly, these regulatory clones had similar viral antigen specificity to protective Th1-cell clones isolated from the same patient (MacDonald et al., 2002). Defined microbial products can manipulate DCs in a way that induces Treg populations (Mills & McGuirk, 2004). For example, filamentous hemagglutinin (FHA) from Bordetella pertussis was shown to induce IL-10 production by DCs; these DC favored the differentiation of naïve T cells into Tr1 cells. Similarly, Tr1 cells can be generated from naïve T cells in the presence of DCs stimulated with phosphatidylserine from Schistosoma mansoni. Although Tr1 cells define a population of T cells that can produce IL-10 and/or TGF-b, some IL-10-producing T cells can also produce IFN-g. In the context of an infectious disease, IFN-g/IL-10 double producers were first described in the bronchoalveolar lavage of patients with tuberculosis and

in individuals chronically infected with Borrelia burgdorferi. Indeed, in many chronic infections, in humans and experimental animals, the presence of CD41 T cells that produce high levels of both IL-10 and IFN-g have been documented. Recently it was shown that IFN-g and IL-10-producing CD41 T cells emerge during experimental infection with T. gondii and in a model of nonhealing leishmaniasis (Anderson et al., 2007; Jankovic et al., 2007), and that these cells share many features with Th1 cells and were the main source of protective IL-10. These T cells were identified as activated T-bet1 Th1 cells and were distinct from Th2 cells, nTregs, or other subsets of inducible regulatory T cells. Unlike IFN-g production, IL-10 production was transient, observed in only a fraction of the IFN-g-producing cells and was produced more rapidly by recently activated T cells than by resting T cells (Jankovic et al., 2007). The instability of IL-10 synthesis, which was noted only when the Th1 cells were fully activated, is probably necessary to prevent sustained suppression of effector functions. Thus, it appears that, in some cases, cells with regulatory properties could arise from fully differentiated Th1 cells as a negative feedback loop. It is likely that numerous previous studies of Tr1 cells were, in fact, incriminating similar populations. These IFN-g and IL-10-producing T cells may represent a dominant regulatory response to infections that induce highly polarized Th1-cell responses. Chronic infections may require an additional layer of regulation, which would be provided by converted Foxp31 regulatory T cells. This hypothesis is supported by the observation that during infection, the downstream effects of inflammatory responses are also often associated with anti-inflammatory processes, including TGF-b production. Furthermore, some pathogens target sites in which TGF-b is highly produced, such as the gastrointestinal tract, the skin, and the eye, which may assist in the conversion in vivo. TGF-b can be also produced by infected cells or by cells the microorganisms are in contact with, or can arise as a result of an inflammatory process. For example, the trypomastigote stage of Trypanosoma cruzi induces TGF-b and IL-10 secretion by DCs. Compelling data in a mouse model of malaria suggest that TGF-b and regulatory T cells are central

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regulators of immunopathology and parasite expansion. During late infection with Plasmodium yoelii infection, DCs migrate to the spleen of infected mice and secrete TGF-b together with IL-10 and PGE2 (Ocana-Morgner et al., 2007). Following experimental malaria infection of human volunteers, enhanced TGFb and Foxp31 Treg responses in PBMCs (peripheral blood mononuclear cells) correlate with a faster parasitic growth rate (Walther et al., 2005). Cells with nTreg characteristics are rapidly induced following blood stage infection and are associated with a decrease of proinflammatory cytokines and antigen-specific responses. Monocytes are a likely source of the early TGF-b production in this infection (Walther et al., 2005). Some nematodes can themselves express homologs of TGF-b (Gomez-Escobar et al., 1997). Compelling experimental data support the idea that Foxp31 Treg can be induced during Heligmosomoides polygyrus infection. The relative contribution of these converted Tregs to peripheral tolerance and the outcome of infections as well as how pathogens can utilize or interfere with this pathway to favor their own survival remains to be addressed. Currently, in the absence of definitive markers to distinguish endogenous versus converted Foxp31 regulatory T cells, these questions will remain difficult to answer.

ROLE OF Treg DURING ACUTE INFECTIONS

In this chapter, we consider acute infections to be those where clinical responses occur within a few days of infection with the disease course short ending in death or full recovery. Acute infections often cause lesions at entry sites such as mucosal surfaces and, if the agent persists, it does so silently. Only a few reports have focused on Treg during acute infections and some have concluded that regulatory cells have little influence on the outcome. Most notably, lymphocytic choriomeningitis virus, the favorite agent of viral immunologists, does not seem to involve any participation by nTreg (Rouse et al., 2006). With some acute infections, however, the Foxp31 Treg population is expanded and activated. Moreover, if this response is suppressed prior to infection, the outcome is different and the magnitude of the resulting immune response is increased. This is true for experimental HSV, respiratory syncytial virus (RSV), and influenza virus infections in mice, as well as with several acute parasitic infections (Belkaid & Tarbell, 2009). Many bacterial infections are acute in nature, but there is scant information as to the relevance of Treg responses in such infections. With Listeria monocytogenes, some results report no effect mediated by Tregs (Fontenot et al., 2005), whereas others indicate that this infection may cause the induction of pathogen-specific Treg responses (Ertelt et al., 2009). Why so few acute infections appear to induce Treg responses has not been adequately explained. It could be, in part, because the rapid disease course provides no time for activated or induced Treg to influence the course of events. This scenario might be especially relevant for the responses of regulatory cells that need to be generated from conventional precursor populations. Additionally, many acute infections induce strong inflammatory reactions that include Th1, Th2, and Th17 polarizing cytokines, all of which may interfere with the induction, expansion, and effector function of Foxp31 Treg. Furthermore, inflammatory cytokines may cause dendritic cell maturation, which renders them ineffective activators and inducers of regulatory T cells. In those acute infections where a dominance of IL-10 and TGF-b prevails, a more common circumstance in some locations, and perhaps under certain doses of infection, Treg stimulation is favored. This scenario may be more common with microbes involved in chronic diseases.

An acute infection that does cause activation and expansion of Foxp31 Treg is HSV infection of mice and humans. Initial studies showed that animals depleted of Tregs with PC61 MAb (which selectively depletes nTreg) became more susceptible to infection but developed heightened immune responses (Suvas et al., 2004, 2003). More recently, acute HSV infection was performed in a valuable new transgenic mouse model where all Foxp3 T cells could be specifically depleted and other cell types would be unaffected. Using this model, the Rudensky group not only confirmed that animals without nTregs make better HSV-specific Tcell responses, but also showed that disease (in their model, lethal encephalitis, fortunately a very rare outcome in the natural human host) was accelerated (Lund et al., 2008). They attributed the accelerated disease in the absence of Treg to changes in migration patterns of effector NK and T cells. When Foxp31 T cells were missing, there was a delay in the time when the effectors arrived at the infection site because of their prolonged retention in the lymph nodes as a result of chemokines building up. The virus progressed to the brain, causing encephalitis, a predominantly virological event. Accordingly, nTreg may facilitate the movement of effectors to infection sites by some as of yet unknown mechanism. A similar situation was reported recently for RSV infection (Ruckwardt et al., 2009). Here too, Treg depletion resulted in elevated immune responses in lymphoid tissues, but there was a delay in clearance of the virus from the lung infection site. They attributed this to the retention of protective CD81 T cells in the lymph node. Conceivably, understanding how Treg influence protective immunity to RSV could help explain the lack of CD81 T-cell responses in infants, which results in severe primary disease. It will be important to determine how acute infections trigger the expansion and activation of Foxp31 T cells in those infections where it does occur. With acute HSV infection, the expansion of the Foxp31 T cells was polyclonal rather than antigen specific. Possible mechanisms to explain this expansion were discussed in a previous section. However, in the case of HSV, an additional mechanism may come into play. Thus nTreg express the molecule HVEM (herpes virus entry mediator), a receptor involved in HSV entry into cells. Previous reports demonstrated that engagement of HVEM with some ligands causes T-cell expansion and activation (Tao et al., 2008). Curiously, the major glycoprotein of HSV, gD, also binds HVEM and this might represent a means by which Treg could be stimulated and expanded. The above discussion has focused on the possible influence of Foxp31 Treg in acute microbial infections. However, induced Treg, particularly the IL-10 producing Tr1 regulators, may also influence some acute infections. As explained previously, these cells are induced from nonregulatory precursors when antigen is stimulated under appropriate conditions, which include the presence of IL-10. These regulatory cells may be necessary to control responses to acute parasitic infections such as Toxoplasma gondii and murine malaria (Couper et al., 2009). With T. gondii, animals unable to produce IL-10 (and hence generate Tr1 cells) can control parasite growth but they ultimately succumb to lethal effector T-cell-mediated immunopathological lesions. An acute bacterial infection where Tr1 cells influence the outcome is Bordetella pertussis. In this infection, the filamentous hemagglutinin (FHA) of the bacterium induces dendritic cells to produce IL-10, which then act to elicit the Tr1 response (McGuirk et al., 2002). Some other pathogens may cause Tr1 induction by producing a homolog of IL-10, but these are chronic infectious agents and are discussed in another section.


Many infections that end up with chronicity initially have an acute phase during which critical events occur that influence the subsequent pattern of disease. One factor thought to play a part is the magnitude and efficacy of initial Treg responses. In hepatitis C infection, a minority of infected persons fully controls their infection and the virus is eliminated. Unfortunately, most suffer chronic disease that can, in some, end in liver failure. Since Foxp31 T cells are increased in patients infected with HCV, and their removal from antigen stimulated PBL results in enhanced responses in vitro, some have suggested that the magnitude of the initial Treg response (natural or induced) might impact the subsequent pattern of chronic disease (Rouse et al., 2006). Thus, conceivably, those that generate a rapid Treg response may go on to develop chronic disease because the protective components of immunity are constrained. Although HCV infection does expand the Treg population, no compelling evidence shows that the extent of one or more types of Treg responses correlates with the eventual outcome of infection. However, the necessary long-term studies measuring Treg responses in the blood, and more relevantly the liver, have yet to be performed. Investigation with acute lentivirus infections in primates is another example indicating that the Treg response could be a critical event. As is well-known, Old World monkeys resist SIV (simian immunodeficiency virus) induced disease, but New World monkeys are very susceptible (Pereira et al., 2007). Levels of virus replication in the different species is approximately the same, but susceptible animals succumb to CD41 T-cell depletion that is the consequence of their hyperactivation. Several explanations have been suggested to explain the differential outcome, but some have observed that African green monkeys develop a more rapid Treg response than do macaques, and that this may serve to inhibit the T-cell hyperactivation (Rouse et al., 2006). There are also reports that the extent of Treg induction may influence the outcome in HIV infection, although this idea is yet to be substantiated (discussed subsequently).

ROLE OF Treg DURING CHRONIC AND PERSISTENT INFECTIONS

Even when Treg successfully preserve homeostasis in the host by controlling excessive immune responses, one consequence of such control is enhanced pathogen survival and, in some cases, long-term pathogen persistence (Table 1). For example, in a resistant mouse model of Leishmania infection, mice remained chronically infected at the site of primary infection. Treg that accumulate at the site of infection regulate the function of local effector cells, which prevents efficient elimination of the parasite (Belkaid et al., 2002). Similarly, in the context of African trypanosomiasis, IL-10 producing Treg can limit the pathogenic consequences of the immune response against the microorganism without

compromising parasite persistence. In its reservoir host, Treg can also favor the persistence of various pathogens such as Mycobacterium tuberculosis, mouse mammary tumor virus, or the pathogenic Seoul virus (Belkaid & Tarbell, 2009). In some cases, Treg can control the fine balance established between the pathogen and its host, mediating an equilibrium that can become mutually beneficial. In other cases, regulatory control is too excessive, allowing the pathogen to replicate without restraint and overwhelm the host, thereby compromising host survival. In a mouse model of malaria, for example, depletion of Treg protected mice from death caused by the lethal strain of Plasmodium yoelii by restoring a vigorous effector immune response that eradicated the parasites (Hisaeda et al., 2004). Following experimental infection with a hypervirulent strain of M. tuberculosis, the early Th1 response was rapidly reduced, correlating with the rapid emergence of IL-10-producing Foxp31 cells (Ordway et al., 2007). Filarial disease, caused by infection with a filarial nematode, is associated with a profound systemic suppression of the host immune system (Belkaid & Tarbell, 2009). Related to this balance of protective versus damaging pathogen-directed immune response is the balance between immune activation and the development of autoimmunity. Immune responses in the context of infection can have varying effects on the potential development of autoimmunity. On the one hand, the damage to self-tissue, the activation of innate immunity, and the cross-reaction between self and foreign antigens can increase the probability of developing self-specific immune responses. On the other hand, pathogens, via TLR ligands and other mechanisms, can enhance immune-suppressive cell types such as Treg, which can result in an infection being negatively correlated with autoimmunity. For example, rates of both allergy and autoimmunity are lower in developing countries with higher rates of infectious disease (Bach, 2002). There is a growing body of literature supporting the idea that microorganism persistence is, in some cases, necessary for the maintenance of protective immunity. In a situation of chronic infection or high exposure to microbial antigens, such as in the case of plasmodium during the transmission season, the generation of potent effector/memory cells could lead to pathogenic consequences. In a model of Leishmania major infection, parasite persistence, as a result of immune suppression by Treg, was necessary for the maintenance of protective immunity against the parasite (Belkaid et al., 2002; Suvas et al., 2004). Another example of this entente between the host and the pathogen is provided by ocular infection of mice with HSV1. When infected with a low dose of the virus, Treg protected mice from the CD41 T-cellmediated pathology, a situation that is compatible with the establishment of immunity to reinfection (Suvas et al., 2004). Similarly, in Candida albicans infection in mice, reduction of the number of Treg led to a better control of the primary infection but enhanced pathology as well as a loss of immunity

TABLE 1 Tregs in chronic infection Host-microbe interaction parameters T-effectors vs Tregs Predominant cytokines Pathogen burden

Outcome Immunosuppression Tregs  T-effectors IL-10, TGF-b Prolonged persistence

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Immunity

Immune-mediated pathology

T-effectors 5 Treg T-effectors  Treg IL-2, IFN-g, IL-12, IFN-g, TNF-a, IL-12, IL-17 IL-17, IL-10, TGF-b Timely control Controlled

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to reinfection. Immunity to reinfection can be recapitulated when Treg are transferred back (Montagnoli et al., 2002). Thus, a role for Treg in the maintenance of immunity may be common to other chronic infections in which poor quality effectors are generated and pathogen persistence is required to sustain protective responses. A large number of studies support the idea that Treg play a role in the control of persistent infection in humans. In the human population, however, reliable identification of Treg is complicated by the fact that Foxp3 expression does not always correlate with regulatory properties, and activated T cells can transiently express it. Likewise, CD25 or other Treg markers cannot be used to discriminate reliably between Treg and highly activated T cells. Because peripheral blood is the most accessible compartment, most human studies that evaluate Treg functions or numbers are done using this compartment. However, these measurements may not be representative, because in some chronic infections Treg accumulate in infected tissues and consequently are reduced in the blood compartment. Despite these caveats, some reports provide convincing evidence for Foxp3-expressing Treg playing a role in a large number of human infections (Belkaid & Tarbell, 2009). In pulmonary tuberculosis patients, the frequencies of Treg are significantly higher in the blood and at the site of infection compared with normal individuals (Chen et al., 2007b). Furthermore, the frequency of Treg in pleural fluid inversely correlates with local M. tuberculosis-specific immunity. These Treg display an activation phenotype, and, in vitro, the addition of Treg back to cultures significantly suppresses the antigen-specific production of IFN-g by effector T cells. Conversely, the removal of Treg significantly enhances effector responses against bacteria by ab and gd T cells (Belkaid & Tarbell, 2009). Some data suggest that differences in susceptibility to infection may rely on a functional deficit of Treg. Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that the Fulani people are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated with classic malaria-resistance genes, and analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as to nonmalarial antigens, revealed higher immune reactivity in the Fulani group (Torcia et al., 2008). Microarray analysis on RNA from Treg indicated obvious differences between ethnic groups, with 23% of genes, including TGF-b, TGF-b receptors, CTLA4, and Foxp3, less expressed in the Fulani people as compared with the Mossi people and European donors not exposed to malaria. As further indications of low Treg activity, the Fulani showed lower serum levels of TGF-b. Furthermore, the proliferative response of the Fulani to malaria antigens was not affected by the depletion of Treg, whereas that of the Mossi was significantly increased. The results suggest that the higher resistance to infection in a defined ethnic group may derive from a functional deficit of Treg. Several studies also suggest that Treg may limit the ability of adaptive T-cell response to control HIV and SIV replication. In particular, anti-HIV responses were increased after in vitro removal of Treg from peripheral leukocytes and from lymph nodes of HIV-infected patients (Sempere et al., 2007). Several groups have shown that Treg frequencies are decreased in the peripheral blood of HIV-infected patients (Sempere et al., 2007). This observation could suggest that Treg, just as with conventional T cells, are progressively lost during HIV infection. Furthermore, cells from infected individuals that show strong HIV-specific Treg function in vitro had significantly lower

levels of plasma viremia and higher CD41-to-CD81 T cell ratios than did individuals with undetectable Treg activity. However, the observation that the expression of Treg is increased in lymphoid or mucosal tissues from HIV-infected patients and SIV-infected macaques suggests that the redistribution of Treg in infected tissues could account for the decreased frequency of Treg in the blood. Importantly, Treg purified from the lymph node of HIV patients maintain their suppressive capacity against antiviral responses (Sempere et al., 2007). The high frequency of Foxp31 Treg was much higher in the duodenal mucosa of patients infected with HIV as compared to healthy controls (Sempere et al., 2007). These findings suggest that Treg, by suppressing virus-specific immunity, may contribute to uncontrolled viral replication, therefore playing a detrimental role in HIV infection. However, there is a strong negative correlation between the level of T-cell activation and the frequency of Treg, which is most prominent during the early chronic stage of the disease, suggesting a role for these cells in the control of immune activation (Ndhlovu et al., 2008). Furthermore, the preservation of Treg in the PBMCs of HIV-infected elite controllers correlates with low CD41 T cell activation, supporting the idea that it may be one mechanism associated with the nonprogressive nature of HIV infection in elite controllers) (Chase et al., 2008). Thus, although there is a consensus on the capacity of Treg to suppress immune responses during HIV infection, the overall consequence on the development of the disease remains uncertain and is likely to be tightly dependent on the stage of the infection or the site targeted. Treg appear also to play a role in the control of chronic viral hepatitis (Rouse et al., 2006). Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most common causes of liver disease worldwide, and failure to control infection with either virus results in immune-mediated acute and chronic necroinflammatory liver disease. In patients with chronic infection with HBV, the frequencies of Foxp31 Treg are highly increased, both in the periphery and in the liver, and correlates with viral load (Belkaid & Tarbell, 2009; Rouse et al., 2006). More recently, a positive correlation was shown between IL-10-producing Foxp31 and Treg, which expanded in response to viral antigen and viral load (Barboza et al., 2007). Furthermore, antigen-specific suppression of effector responses in vitro suggests that the expansion of antigenspecific Treg during this infection may contribute to the associated liver pathology (reviewed in Alatrakchi & Koziel, 2009). HCV-associated liver disease also seems to involve Treg, which could impede immune defense against the virus. Individuals who are chronically infected with HCV have a higher number of Treg in the blood compared with those whose HCV infection spontaneously resolved or with those who are not infected (Alatrakchi & Koziel, 2009). Depletion of Treg enhances antigen-specific CD81 T responses in vitro (Alatrakchi & Koziel, 2009). During HCV infection, Treg display enhanced suppressive function when assessed in vitro during both the acute and chronic stages of the infection, compared with uninfected donors or with patients with spontaneous resolution. Tregs also accumulate in the liver of patients with chronic HCV. The inverse correlation between the HCV-specific TGF-b response by Treg and liver damage could support the idea that Treg also have a role in controlling chronic inflammatory responses and liver damage in HCV carriers (Alatrakchi & Koziel, 2009). Interestingly, patients who are chronically infected with HCV and go on to develop autoimmunity have fewer peripheral Treg (Boyer et al., 2004). However, the link between chronic infection, autoimmune disorders, and dysregulation of Treg function requires further analysis.


NEGATIVE CONTROL OF Treg INDUCTION BY PATHOGENS

It is becoming clear that the status of activation of DCs as well as inflammatory mediators modulates the capacity of antigen-presenting cells to induce Treg de novo. For instance, IL-6 and TGF-b in tandem can direct the production of IL-17-secreting T cells over Treg (Bettelli et al., 2006; Veldhoen et al., 2006), and Th1 and Th2 effector cytokines have an antagonistic effect on Treg conversion (Wei et al., 2007). In addition, exposure to activated DCs or strong costimulation limits the induction of Foxp31 Treg in favor of the induction of effector responses (Benson et al., 2007; Kretschmer et al., 2005; Wang et al., 2008). A correlate of this observation would be that inflammation triggered by infection can antagonize Treg induction. Indeed, acute infection in mice with Listeria monocytogenes failed to induce Foxp3 by conventional CD41 T cells (Fontenot et al., 2005). Furthermore, the constitutive induction of Treg in the gastrointestinal tract following exposure to oral antigens is inhibited following oral infection with the protozoan T. gondii (Oldenhove et al., 2009). Previous reports examining both gut and lung inflammation support the idea that restricted or defective Treg conversion can enhance immunopathology (Curotto de Lafaille et al., 2008; Izcue et al., 2008), supporting the idea that interference with constitutive Treg induction during infection may directly contribute to the pathological process. Furthermore, the observations also raise the possibility that exposure to antigen at a time of acute infection may impair the acquisition of tolerance against innocuous antigens.

CONTROL OF Treg PLASTICITY DURING INFECTION

Recent findings suggest that acquisition of transcription factors in addition to Foxp3 can confer unique functional characteristics to Treg (Zheng et al., 2009). For instance, expression of T-bet by Treg was recently shown to favor their proper control of Th1 environments (Koch et al., 2009). Similarly, in the context of a virulent T. gondii infection, Treg express high levels of the Th1 transcription factor T-bet (Oldenhove et al., 2009). Intriguingly, in the context of Th1 settings, T-bet expression by Treg leads to up regulation of CXCR3 and preferential homing to infected sites (Koch et al., 2009). Thus, the appropriation of T-effector lineage transcription factors by Treg suggests that under nonpathogenic situations, acquisition of these elements controlled by local DCs may represent a necessary layer of their regulation. On the other hand, under highly pathogenic situations such as following a lethal T. gondii infection, T-bet expression led to the production of IFN-g, a cytokine responsible for both effector and pathogenic responses during T. gondii infection (Oldenhove et al., 2009). Although T-cell lineage subsets were initially believed to represent stable populations of cells, recent evidence shows that lymphocytes maintain a certain degree of plasticity with respect to their capacity to produce cytokines (Lee et al., 2009; Wei et al., 2009). Cells expressing both Foxp3 and IL-17 can be found in mucosal tissue or in vitro cultures (Lee et al., 2009; Wei et al., 2009). Genome-wide mapping of H3K4me3 and H3K27me3 performed in ex vivo Treg revealed markers of both repression and induction at the tbx21 locus. However, the Ifng locus did not show any sign of induction or repression (Wei et al., 2009), suggesting that Ifng is poised for transcriptional activation. This would suggest that, in the presence of high levels of inflammatory mediators, T-bet expression might reach a threshold that superimposes an effector program on

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Treg. Given their high degree of self-reactivity, it is plausible that Treg can contribute to tissue damage or can lose suppressive capacity if armed with effector cytokines. Indeed, a recent report highlighted that Foxp3 instability and acquisition of IFN-g can favor the development of autoimmune diabetes (Zhou et al., 2009).

CONTROL OF ENDOGENOUS Treg DURING INFECTIONS

Overcoming regulation is a prerequisite to the expression of effector responses. Mechanisms associated with negative control of Treg include direct Treg stimulation, as well as strong costimulation of effector T cells mediated by local DCs. Over the past few years, a strong body of literature suggests that a primary function of IL-2 is to promote the expansion and survival of Treg (Malek & Bayer, 2004). Intriguingly, IL-2 production declines upon differentiation into T-effectors, implying that in the context of a highly polarized Th1 response, IL-2 may become limiting. Accordingly, following oral infection with T. gondii, the emergence of a Th1 response was associated with a dramatic reduction of IL-2 production by CD41 T cells in the gastrointestinal tract and in the periphery. Previous work demonstrated that T-bet acquisition, as well as exposure of T cells to IL-27, IL-12, or IL-2 itself, could limit IL-2 production (Hwang et al., 2005; Villarino et al., 2007). In addition, T-effectors, via their consumption of growth factor, may also deprive the host from exogenous IL-2. A link between defective IL-2 production and Treg dysfunction has been previously proposed as one of the mechanisms associated with breakdown of tolerance in nonobese diabetic mice (Tang et al., 2008).

MANIPULATING Treg TO CONTROL INFECTIOUS DISEASE

As discussed already, regulatory cell responses to microbes can be a double-edged sword. They can compromise immune efficacy or they can act to diminish the collateral tissue damage caused by exuberant antimicrobial reactivity, as commonly occurs in chronic infections. With the latter, however, the situation may be complex since several types of regulators with different functions may be involved during the course of a chronic infection. Moreover, even effector cells that formerly helped orchestrate lesions may take on a regulatory function under appropriate conditions. When it comes to devising therapies, there are circumstances where inhibiting the excessive activity of regulatory cells would favor microbial control, and other situations, such as chronic tissue damage, where expanding the influence of regulators would be beneficial. From a practical perspective, we may be closer to achieving the latter effect, and several approaches are quite well-developed to control chronic inflammatory reactions associated with autoimmune diseases and transplantation rejection. Several experimental infections are controlled more effectively if the influence of Treg is diminished. This can be achieved either by depressing Treg numbers or by blunting their function. The usual approach is to use a MAb that mainly targets Treg such as anti-CD25, but with mice that can be constructed that allow specific deletion of Foxp31 Treg, as discussed previously (Lund et al., 2008). The removal of Treg would not seem to be a practical method by which to manage the response to an ongoing infection, but it could prove useful to improve the efficacy of some vaccines. Thus, as initially shown with vaccines against HSV in mice, Treg depletion during vaccination resulted in improved primary and memory T-cell responses and increased

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immunity (Rouse et al., 2006). Subsequently, more extensive studies were performed with vaccines against murine malaria (reviewed in Belkaid & Tarbell, 2009). Confirming the HSV studies, depleting Treg at the time of vaccination resulted in heightened CD81 T-cell responses that were more broadly reactive in terms of epitope specificity. The Treg-depleted vaccinees also showed more durable immunity to a malaria challenge. Depleting Treg also resulted in better vaccination responses to influenza, vaccinia, and hepatitis B viruses, as well as to M. tuberculosis. Unfortunately, in the latter instance, although immune responses were accelerated and elevated in Treg-depleted animals, the level of protective immunity to virulent M. tuberculosis was not improved (McGuirk et al., 2002). The observation that Treg depression may improve the immunogenicity of some vaccines raises some interesting issues. These include understanding how the effect actually

occurs, how it can be facilitated, and whether or not the approach could be exploited with a vaccine regime in humans or animals. As to mechanisms of action, it is not clear if the enhancement effect is a consequence of removing Treg or of blocking their response to IL-2, so as to make the latter more available to drive the expansion of effector or memory T cells. Alternatively, the outcome may be the consequence of effects on antigen presentation. Thus, stimulated Treg, perhaps via the cytokines they produce, can influence the function of dendritic cells, causing them to down regulate costimulatory molecules, such as CD80 and CD86, and suppress their production of IL-6. The effects on DCs make them less effective inducers of the T cells involved in protective immunity. Since Treg may also influence some functions of effector cells, reducing Treg activity may contribute to enhanced effector cell activity and may perhaps influence their differentiation into more

TABLE 2 Strategies to boost Treg responses Strategy

Effect

Cytokines IL-2

Proliferation

IL-2 and anti-IL-2 complex TGF-b

Proliferation Induction

TNF-a IFN-g

Proliferation Induction

References Reviewed in Brusko et al., 2008 Webster et al., 2009 Reviewed in Belkaid & Tarbell, 2009 Chen et al., 2007a Chang et al., 2009

Other host proteins and products Galecin-1

Proliferation

Galectin-9 Galectin-10

Proliferation and induction Proliferation

FLT-3 ligand Vasoactive intestinal peptide Progesterone VEGF Retinoic acid

Proliferation Induction Induction Proliferation and induction Induction

Thrombospondin-1

Induction

Reviewed in Rabinovich & Toscano, 2009 Sehrawat et al., 2009 Reviewed in Rabinovich & Toscano, 2009 Swee et al., 2009 Pozo et al., 2009 Mjosberg et al., 2009 Wada et al., 2009 Reviewed in Belkaid & Tarbell, 2009 Grimbert et al., 2006

Anti-CD154 Activation-inducible TNFR-a-Fc (AITR-Fc) CTLA-4-Ig

Proliferation Proliferation

Rigby et al., 2008 Sonawane et al., 2009

Proliferation

Anti-CD3 F(ab)2

Proliferation

Reviewed in Brusko et al., 2008 Reviewed in Brusko et al., 2008

Antibodies and fusion proteins

Chemical compounds Rapamycin FTY720 Corticosteroids

Treg spared from killing Induction Induction

Basu et al., 2008 Sehrawat & Rouse, 2008 Esparza & Arch, 2006

HDAC inhibitors Tacrolimus Glatiramer acetate

Proliferation and induction Induction Induction

Tao et al., 2007 Wang et al., 2009 Cui et al., 2009


functionally effective memory cells. These issues have not been fully evaluated, nor has the outcome of Treg depletion of later phases of the primary or recall response on the efficiency of vaccination. Nevertheless, a take-home lesson from studies showing that Treg can interfere with the efficacy of vaccines is to choose immunization approaches that minimally stimulate one or more types of regulatory cells. In support of this notion, persons who were nonresponsive to standard hepatitis B vaccination possessed higher numbers of antigen specific Foxp31 Treg in their blood than did responder vaccines (Moore et al., 2005). Vaccine approaches that minimize the chances of inducing Treg include choosing appropriate adjuvants (preferably pro-inflammatory), avoiding multiple low dose immunization schemes, and not using sites for immunization (e.g., the gut) that favor the induction of Treg responses. The topic of practicality is also an issue for consideration. It seems highly likely that administering a MAb to the CD25 receptor, which is available for clinical use in humans to control transplant rejections, would be economically unrealistic for human vaccines even if the approach is shown to work and lacks side effects. However, if we unravel the molecular mechanisms responsible for Treg function, chemical and biological approaches could conceivably be devised that manipulate Treg activity in a negative direction and perhaps enhance vaccine efficacy. Possible targets include adenosine and cAMP (cyclic adenosine monophosphate), which are involved in the suppressor function of Treg. A major function of several types of Treg may be to reduce the tissue damage that results from a chronic inflammatory response to infections. Consequently, enhancing the number or function of Treg would be therapeutically useful. Conceptually, there are a number of ways for achieving this objective (Table 2). These include expanding Treg populations in vitro and using them for adoptive transfer in vivo, using chemical or biological procedures that expand and increase the function of Treg in vivo, or causing uncommitted T cells in vivo to differentiate to become regulatory in function. The field of expanding and manipulating Treg to control immunoinflammatory lesions is led by groups interested in autoimmune disease and transplantation, and little has been done so far to control chronic microbial-induced inflammatory lesions (Brusko et al., 2008). We have evaluated whether or not manipulating Treg activity represents a useful way to modulate immunoinflammatory lesions caused by ocular infection with HSV. Accordingly, it was shown in this system that lesion severity became enhanced if animals were depleted of Treg, and that the inflammatory effects of adoptive transfers of HSV primed T cells into RAG−/− recipients was modulated by co-transfers of polyspecific T cells (Suvas et al., 2004). Hence, in this system, increasing the participation of Treg was expected to reduce disease in an experimental model of an important cause of human blindness. One way to increase the participation of Treg is to generate such cells in vitro and adoptively transfer them into animals ocularly infected with HSV to see if this affected lesion severity. The procedure was successful, but only when transfers were given early in the disease process (Sehrawat et al., 2008). Curiously, the Treg population responsible for lesion suppression were not specific to HSV antigens, indicating that Treg can act nonspecifically in a bystander way in vivo, at least if they are activated. An adoptive transfer approach to control chronic microbial reactions represents an unlikely method to use in clinical situations. It seems more likely that procedures that cause conventional T cells to convert in vivo to become

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regulatory will be more realistic (see Table 2). One way of achieving this would be to use the fungal metabolite drug FTY720, because this causes conventional T cells to become FoxP31 and regulatory in function both in vitro and in vivo (Sehrawat & Rouse, 2008). However, this approach alone might not be that effective since, under the influence of the drug, Treg did not divide extensively. Therefore, a combination therapy such as the use of cytokines (e.g., IL-2) might prove to be an effective approach to expand the converted Treg (Ertelt et al., 2009). Another approach showing promise is the observation that certain galectin molecules may act as activators of Treg. For example, galectin-9 administration to animals with developing herpetic ocular lesions succeeds in reducing lesion severity in part because populations of FoxP31 are expanded (Sehrawat et al., 2009). Furthermore, blocking galectin-9 from binding to its receptor TIM-3 with simple sugars diminished the suppressive activity of Treg. Consequently, in situations where inhibited Treg response is desired, the use of simple sugars such as lactose might prove useful. In conclusion, although management of microbe-induced chronic lesions is still in the early stages, some results do show promise. We anticipate the rapid discovery of new approaches.

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9 Memory and Infection DAVID MASOPUST AnD MARK K. SLIFKA

INTRODUCTION

on the host immune system can be exceedingly long-lived and effective at preventing clinical disease upon reexposure. For example, in 1846, the isolated populace of the remote Faroe Islands (lying 200 miles off the northern coast of Scotland) experienced its first measles outbreak in 65 years. Remarkably, those denizens over the age of 65 years whom had survived infection during the previous outbreak were immune from disease (Slifka & Ahmed, 1996b). This observation demonstrates that protective immunity can persist for the life of the host without periodic reexposure from other infected individuals. Similar natural examples derived from epidemiological analyses of other geographically isolated communities demonstrate that protective immunity against other pathogens, including polio and yellow fever viruses, also persist for the life of the host (Slifka & Ahmed, 1996b). While immunity to particular agents will be covered in other chapters within the text, this chapter will review the basic principles of adaptive immunological memory.

The principal function of the immune system is to protect the host from infection. After an infection is cleared, the host retains a state of heightened readiness in the event of reexposure to the same or to a closely related pathogen. This “memory” of past infections, a cardinal feature of the vertebrate adaptive immune system, is mediated by longlived changes in the population of host B and T lymphocytes induced by previous pathogen exposure. In response to stimulation, B lymphocytes differentiate into antibody secreting cells, and T lymphocytes elaborate cytokines, regulate the innate and adaptive immune response, and kill infected host cells. Each naïve lymphocyte exhibits a unique specificity conferred by the restricted expression of a single lymphocyte receptor. Prior to initial infection, the number of B and T cells specific for a particular pathogen are very rare (approximately 1 specific cell per 104 to 106 lymphocytes). Upon infection, antigen-specific lymphocytes undergo a dramatic program of proliferation resulting in up to a 10,000-fold amplification in clonal abundance, and acquire effector functions that help eliminate infection. Following pathogen clearance, most specific lymphocytes die, perhaps creating space for future immune responses to unrelated infections. However, 5% to 10% of specific lymphocytes escape death, and persist in the host long after the infection is eliminated. Thus, as a result of infection, the host’s immune system retains a much higher frequency of lymphocytes specific for that particular infection. Subsets of memory lymphocytes exhibit important functional differences from their naïve counterparts and also have a more pervasive anatomic distribution. Thus, the vertebrate immune system is educated by the unique infectious experience of the individual, which results in long-lived changes in pathogen-specific lymphocyte quantity, quality, and location, all of which contribute to an increase in the speed, robustness, and efficacy of responses upon secondary exposure. This imprint of infectious history

DYNAMICS OF T-CELL RESPONSES

Primary T-cell responses are fairly slow to develop for three reasons. First, the frequency of naïve T cells specific for a particular antigen is exceedingly rare. Second, naïve T cells do not constitutively express effector molecules that promote pathogen clearance. And third, naïve T cells primarily recirculate from blood and lymph to secondary lymphoid organs; yet many infections occur outside of lymphoid tissue. Thus, before T cells can optimally contribute to eradication of primary infections, they must overcome limitations of quantity, quality, and location. Upon primary infection, rare cognate antigen-specific T cells first interact with professional antigen-presenting cells that have acquired foreign antigen and have localized within the T-cell zones of secondary lymphoid tissue. This interaction precipitates a program of activation, and is manifest by a remarkable period of proliferation, and changes in membrane organization, gene expression, homing, and the up regulation of effector molecules that promote pathogen clearance. Within days, antigen-specific naïve T cells expand between 100 to 10,000-fold, depending on the duration and density of antigen presentation, costimulation, and other variables (Masopust et al., 2004). Effector CD4

David Masopust, Department of Microbiology, Center for Immunology, University of Minnesota, 2-182 Medical Biosciences Building, 2101 6th St. SE, Minneapolis, Mn 55455. Mark K. Slifka, Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 nW 185th Avenue, Beaverton, OR 97006.

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T cells elaborate cytokines and costimulatory molecules that communicate information to cells of the innate and adaptive immune system, promoting affinity maturation and class switching among B cells, CD8 T-cell differentiation, macrophage activation, and many other functions. CD8 T cells secrete cytokines that promote an antiviral state and kill infected host cells via degranulation of perforin and granzymes. It should be noted that many T-cell effector functions are contact dependent. Most effector T cells down regulate CD62L and CCR7, the homing molecules that promote migration to secondary lymphoid organs. In turn, they up regulate receptors that allow them to migrate to nonlymphoid tissues, which often serve as the predominant site of infection. For instance, priming within the mesenteric lymph node induces 47 and CCR9 expression, which facilitates migration to the parenchyma of the small intestine. In contrast, priming within the inguinal lymph node induces expression of skin homing molecules. Thus, gut and skin homing receptor expression is partly coupled to the location of T-cell activation (Butcher & Picker, 1996; von Andrian & Mackay, 2000), which may preferentially target responses to sites of infection. This process of expansion, differentiation, and redistribution allows effectors to optimally contribute to pathogen clearance. In the event that the pathogen is eliminated, most T cells die en masse over a period of a few weeks, which allows room for future immune responses. However, a fraction of T cells escape this death program (typically 5% to 10%) and are maintained long after pathogen clearance. Thus, the population of host lymphocytes retains a permanent imprint, or memory, of past infections. Memory T-cell populations exhibit many properties that distinguish them from their naïve counterparts, including increased clonal abundance, reduced activation requirements in the event of pathogen reexposure, constitutive maintenance or more rapid up regulation of effector gene expression, and more widespread anatomic distribution (Masopust et al., 2004). Collectively, these properties contribute to protection from reinfection by promoting faster, larger, and more efficacious recall responses (Fig. 1). Defining the developmental cues that regulate (i) commitment to the long-lived memory T-cell lineage, (ii) heterogeneity in memory T-cell differentiation state, and (iii) T-cell homing remain some of the biggest challenges in understanding T-cell immunobiology.

COMMITMENT TO MEMORY LINEAGE

How does a small fraction of activated T cells form long-lived memory cells while others die en masse? When does this fate decision occur? It had long been debated whether memory T cells transit through an effector stage, or whether effector cells constitute a distinct terminally differentiated lineage fated for death. Recent data demonstrates that both effector and memory CD8 T cells can be generated from a single naïve cell, suggesting that this fate decision is made subsequent to activation (Stemberger et al., 2007). Reiner and colleagues recently observed that T cells divide asymmetrically during their first division (Chang et al., 2007). Based on elegant phenotypic analyses and cell transfer studies, they proposed that the fate decision between becoming long-lived memory cells and short-lived effector cells is made upon the first division following activation (Chang et al., 2007). However, several recent studies demonstrated that both CD4 and CD8 T cells express equivalent levels of certain effector molecules, such as granzyme B and interferon gamma, regardless of whether they will differentiate into memory T cells or are only shortlived effector cells (Bannard et al., 2009; Harrington et al., 2008; Lohning et al., 2008). While it remains possible that the memory fate decision is made shortly following activation, it apparently does not preclude the expression of these effector molecules. Indeed, disruption of the proapoptotic molecule, bim, allows most effector CD8 T cells to become long-lived memory cells, which argues against the possibility that memory T cells are selected solely on the basis of functionality (Prlic & Bevan, 2008). Cytokine availability can have a substantial impact on T-cell survival during the contraction phase. Increasing the availability of IL-2 or IL-15 prior to contraction results in higher numbers of memory T cells (Blattman et al., 2003; Yajima et al., 2002). Moreover, there is heterogeneity in expression of IL-7R prior to the contraction of a recently activated CD8 T-cell population. A small subset of IL-7Rhi KLRG1lo effector cells are present at the peak of clonal expansion, and these preferentially survive and become memory cells (Hand & Kaech, 2009). Transfer of activated T cells to an IL-7 deficient recipient results in poor memory development, but interestingly, over-expression of IL-7R on all T cells is not sufficient to rescue dying effector cells. These data are consistent with the hypothesis that the contraction may be partially regulated via a Darwinian competition for limited cytokine resources. Collectively, it appears that

FIGURE 1 Dynamics of primary versus secondary T-cell response. Primary T-cell responses are slow to develop, result in the selective expansion of pathogen specific clones, and establish a long-lived increase in the frequency of pathogen specific T cells (memory). Secondary (recall) responses are faster, larger, and more efficacious, and induce a long-lived boost in the number of memory T cells.

9. Memory and Infection

commitment to the memory lineage has at least partially occurred by the peak of clonal expansion, but that extrinsic factors also regulate survival. Dissecting the parameters that influence this process, including inflammation, the dynamics of antigen presentation and costimulation, and competition for cytokines, is still a subject of intense investigation (Harty & Badovinac, 2008).

HETEROGENEITY AMONG MEMORY T-CELL POPULATIONS

Seminal work by Lanzavecchia and colleagues revealed that memory T cells within human blood exhibited significant phenotypic and functional heterogeneity (Sallusto et al., 1999). They reported that antigen-experienced T cells that retained (or reexpressed) homing molecules required for trafficking to secondary lymphoid tissue, deemed “central” memory T cells (TCM), required an extensive period of restimulation before expressing certain effector molecules. In contrast, “effector” memory T cells (TEM) lacked CCR7 and CD62L, but constitutively expressed molecules associated with certain T-cell effector functions, such as perforin. These data suggested that memory T-cell phenotypic properties varied with trafficking potential. Papers that soon followed the Lanzavecchia study also revealed that large numbers of memory T cells were located within nonlymphoid tissues (Masopust et al., 2001; Reinhardt et al., 2001). Consistent with the prediction of Lanzavecchia and colleagues, these nonlymphoid populations expressed more rapid effector functions than their counterparts located within secondary lymphoid tissues (Masopust et al., 2001; Reinhardt et al., 2001). Collectively, these data pointed to the existence of a specialized subset of memory T cells that occupied common portals of pathogen entry, and were immediately poised to respond in the event of reinfection. In contrast, the population that had traditionally been studied in animal models more carefully, TCM within lymphoid tissue, was a resting subset that responded more slowly in the event of reinfection by undergoing a second round of proliferation and only acquiring certain effector functions over a period of a few days (Wherry et al., 2003). The developmental relationship between each subset remains unclear, and evidence supports both the notions that TCM derive from TEM and that TEM derive from TCM (Champagne et al., 2001; Sallusto et al., 2004; Wherry et al., 2003). T-cell priming is associated with many variables, including antigen quantity and duration, TCR affinity, inflammatory milieu, identity of antigen presenting cell, and degree of costimulation, all of which contribute to heritable changes in T-cell gene expression. Data now indicate that events during priming may regulate the distribution of memory T cells into each subset, and increases in the overall strength of stimulation preferentially result in the generation of TEM. This issue has obvious ramifications for vaccination, and suggests that nonreplicating or subunit vaccines may result predominantly in TCM. One of the great remaining challenges is to determine the importance of each subset in contributing to protection from reinfection. It is likely that the answer to this question will vary depending on the pathogenesis and tropism of the infection in question (Bachmann et al., 2005; Wherry et al., 2003). While TEM are often referred to as a homogenous population, it should be noted that they exhibit a broad range of phenotypes, often coupled with their anatomic location (Woodland & Kohlmeier, 2009). For instance, virus-specific TEM within the epithelium of the small intestine exhibit unique phenotypic and functional signatures that are not found in other tissues (Table 1; Masopust et al., 2006). Similar observations have been made for cells isolated

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TABLE 1 T-cell properties vary with differentiation state and anatomic locationa Property

naïve

Effector

CD44 CD62L e/7 CD69 Ly6C CTL Turnover

      /

      

Blood

IEL

TCM

TEM

TEM

      

     varies 

      /

a Virus-specific mouse CD8 T cells were examined before (naïve), during (effector), or after (memory) an acute infection (Masopust et al., 2006). Abbreviations: TCM, central memory; TEM, effector memory; IEL, intestinal epithelial lymphocytes (T cells within epithelium of the small intestine); CTL, cytotoxic T lymphocytes (T cells that kill infected targets in short-term in vitro assays).

from the brain and the lung airways (Masopust et al., 2004; Woodland & Kohlmeier, 2009). It remains likely that the tissue microenvironment itself plays a role in shaping memory T-cell differentiation state. This issue is important, because analyses of TEM isolated from blood will not account for the full phenotypic complexity of the large numbers of memory T cells residing in nonlymphoid organs.

MAINTENANCE AND LONGEVITY OF MEMORY T CELLS

Does immunological memory need constant reminding? One long-standing controversy in the field of T-cell memory was whether it could be maintained in the absence of constitutive stimulation by cognate antigen. Resolution of this argument had practical ramifications for the longevity of naturally acquired T-cell memory specific for pathogens that are eliminated, as well as implications for whether vaccines should provide persistent depots of antigen or require periodic boosting. After much debate, it was shown that memory CD8 and CD4 T cells persisted in mice that lacked MHC molecules, providing convincing evidence that cognate antigen was not required for memory T-cell maintenance (Murali-Krishna et al., 1999; Swain et al., 1999). However, there are numerous situations in which memory T cells may be constitutively stimulated by cognate Ag, most obviously in the setting of chronic infections, such as those caused by human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). What has become clear is that chronic antigen exposure has a pronounced effect on T-cell differentiation state and the requirements for survival (Wherry & Ahmed, 2004). Low levels of repeated stimulation promote the preferential development of TEM, whereas high levels of repeated stimulation lead to functional exhaustion and even deletion of T cells specific for chronic viruses. In settings wherein the infection is cleared, memory T cells are maintained through contact with IL-7 and IL-15 (Surh & Sprent, 2008). Memory CD8 T cells that are transferred to IL-15 deficient mice die rapidly. Moreover, IL-15 deficient mice have a marked reduction in memory phenotype CD8 T cells, although overexpression of IL-7 compensates for this defect. IL-15 also supports a slow basal level of antigen-independent turnover (termed homeostatic proliferation) among memory T cells. Thus, homeostatic proliferation is linked to survival. Competition for available

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cytokines may regulate the numbers of total memory T cells present within the host, ensuring that repeated infections do not result in a permanent expansion in the size of the adaptive immune system (Selin & Welsh, 2004). However, TEM undergo much less homeostatic proliferation than TCM (Wherry et al., 2003). Moreover, heterologous prime-boost vaccination can result in a tremendous expansion of the TEM pool within nonlymphoid tissues without inducing significant attrition among preexisting TCM (Vezys et al., 2009). These data raise the possibility that TCM, which largely populate lymphoid tissues, may be regulated differently from TEM, which are preferentially located within other anatomical compartments. The longevity of T-cell memory is still the subject of some debate. Experimental infection of mice with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), which is rapidly cleared from the host, results in a remarkably stable memory CD8 T-cell pool for more than 900 days. In contrast, virus-specific memory CD4 T cells decayed 50-fold over the same period (Homann et al., 2001), suggesting differences in the regulation of these subsets. Longer studies in mice are not feasible given their relatively brief lifespan. However, evidence suggests that certain heterologous infections may lead to the rapid attrition of preexisting memory CD8 T cells, and it remains possible that the longevity of CD8 T-cell memory in mice is an artifact of their maintenance under specific pathogen-free conditions (Selin & Welsh, 2004). Confounding these issues further, the longevity of memory T cells may depend on the nature of the priming environment. In one striking example, infection of CD4 T-cell-deficient mice with Listeria monocytogenes results in an effective primary CD8 T-cell response. However, this situation results in essentially no memory CD8 T cells that have the capacity to respond and protect the host in the event of a second infection (Prlic et al., 2007). With these issues in mind, it is essential to evaluate the longevity of T-cell memory to naturally acquired infections in humans. However, these studies have several limitations. They require a host population that is not periodically reexposed to the infection for which memory is being measured. Long-term longitudinal studies are difficult. Moreover, they are often restricted to analyses of blood, and many assays will only measure the frequency rather than the total number of recirculating memory T cells. With these caveats in mind, it was recently reported that primary human cytomegalovirus infection resulted in a long-lived increase in the total number of memory CD8 T cells, and induced a reduction in the frequency, but not the number, of preexisting memory CD8 T cells specific for influenza or EBV in blood (van Leeuwen et al., 2006). T-cell memory was recently analyzed in individuals infected with Puumala virus (PUUV), a member of the Hantavirus genus that causes mild hemorrhagic fever. Without evidence of viral persistence or reexposure, fairly robust levels of CD8 T-cell memory were detected in blood for up to 15 years after infection (Van Epps et al., 2002). Between 0.1% and 0.9% of CD8 and CD4 T cells were specific for measles virus among adults aged 25 to 38 years that had a natural history of childhood infection (nanan et al., 2000). In addition, a nonrandomized, cross-sectional analysis of T-cell immunity was performed on volunteers examined 1 month to 75 years after smallpox vaccination (Hammarlund et al., 2003). Remarkably, memory T cells could be detected up to 75 years following a single immunization, although the halflife of both CD4 and CD8 T cells appeared to range between 8 and 15 years, and memory CD8 T cells could not be detected in about 50% of individuals. Collectively, these data indicate that T-cell memory likely can persist for the life of an individual following a single exposure. However, it should

be stressed that certain infections may significantly erode T-cell memory, and the longevity of memory may depend on the nature of the primary infection or vaccine. More studies on the longevity of T-cell memory to different agents will be a welcome addition to the field.

INDUCTION OF T-CELL-DEPENDENT HUMORAL IMMUNITY

Development of antigen-specific antibody responses can be either T-cell-independent or T-cell-dependent. T-cellindependent responses are typically mounted against nonprotein antigens and T cell help cannot be provided because without protein, there is no peptide presentation to elicit a CD4 helper T-cell response. Under these conditions, the resulting T-independent antibody response is often shortlived, lasting only a few weeks or months. In contrast, T-cell-dependent antibody responses contain protein antigens, elicit CD4 T-cell help, and induce antibody responses that undergo somatic hypermutation/affinity maturation and are long-lived—in some cases lasting for decades (Amanna et al., 2007). The majority of vaccines and viral infections trigger high affinity, antigen-specific B-cell responses that are CD4 T-cell-dependent. This process begins in the germinal center (GC), which represents a specialized lymphoid compartment wherein B cells become activated to proliferate, somatically mutate, and differentiate into memory B cells (MBC) and antibody-secreting plasma cells. Within the GC, it is believed that B cells compete for antigen in the form of antigen-antibody immune complexes on the surface of follicular dendritic cells (MacLennan, 1994) and only B cells with the highest affinity are able to effectively compete and be selected for further proliferation and differentiation into long-lived antigen-specific B-cell populations. Although antigen must play an important role in the selection process, mice that lack the ability to form immune complexes can still form a normal GC and develop effective antibody responses (Hannum et al., 2000). This apparent paradox may be explained by other studies that indicate competition for CD4 T-cell help may be an even more important factor in selection of high-affinity B cells during the GC reaction (Allen et al., 2007; Schwickert et al., 2007). One recent study has elegantly shown that B cells that form conjugates with cytokine-secreting follicular T cells in vivo are enriched for the expression of activation-induced cytidine deaminase (AID), an enzyme required for somatic hypermutation and isotype-class switching, and these B cells showed a higher rate of IgG mutations (Reinhardt et al., 2009). Interestingly, this study also demonstrated that B cells conjugated to IL-4-secreting CD4 T cells had class-switched to IgG1, whereas B cells conjugated to IFn-secreting CD4 T cells had class-switched to IgG2a, providing direct evidence that these specific cytokines are intimately involved with IgG class-switching of B cells in the GC. This is an important observation because it indicates that class-switching events are not determined by broad diffusion of T-cell-elicited cytokines throughout the GC, but instead represent compartmentalized events that are dictated by specific B cell-T cell interactions. Interestingly, not only are B cells dependent upon CD4 T-cell help for induction of effective, long-lived humoral immunity, but there is also growing evidence that CD4 T cells depend on help from B cells for the induction of long-lived T-cell memory. Some of the first evidence indicating a role for B cells in the development of functional T cells was demonstrated through adoptive transfer experiments showing that CD4 T cells from B-cell-deficient mice were defective in antiviral activity (Homann et al., 1998).


At that time, it was unclear whether the fate of the CD4 T cells was due to a lack of an intact antibody response and immune complex deposition in lymphoid tissues or whether the B cells themselves were necessary for development of effective T-cell responses. This issue was revisited in a recent study that compared antiviral T-cell responses in B-cell-deficient mice versus B-cell-transgenic mice that had an intact B-cell compartment, but were unable to mount an antiviral antibody response. In contrast to the B-cell-deficient mice that had defective T-cell responses following acute or chronic LCMV infection, the transgenic mice with normal B-cell numbers developed normal antiviral T-cell responses (Whitmire et al., 2009). This indicates that immune complex formation is not a necessary requirement for developing effective T-cell memory. Instead, this indicates that the B cells themselves must play other important roles in host immunity besides antibody production and that these cells are directly required for optimal induction of antiviral T-cell responses.

DIFFERENTIAL LOCALIZATION OF MEMORY B CELLS AND PLASMA CELLS

During and shortly following the GC reaction, MBCs and plasma cells (PCs) migrate out of the follicle and into surrounding lymphoid tissue or into the circulation. Following vaccination or systemic viral infection, about 10% to 20% of fully differentiated PCs will reside in the red pulp of the spleen, whereas 80% to 90% of the PC population will migrate out of the lymphoid environment and settle into the bone marrow compartment (Slifka & Ahmed, 1998a; Slifka & Ahmed, 1998b). Unlike PC migration to the bone marrow, the majority of MBCs will either remain in lymphoid compartments (e.g., in the marginal zone [Sanz et al., 2008]) or will recirculate in the bloodstream during

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the course of continued immunosurveillance. At nearly a year after recovery from acute LCMV infection of mice, the spleen and lymph nodes continue to maintain the highest frequency of virus-specific MBCs, with the spleen containing approximately 100-fold more MBCs than in the peripheral blood when calculated per total organ or per total blood volume, respectively (Slifka & Ahmed, 1998a). Antibody-secreting cells (ASCs) can be found in the circulation, but only for a brief period of time after vaccination or infection (Bernasconi et al., 2002; Dilosa et al., 1991; Wrammert et al., 2008), and it is unclear if these represent fully differentiated, nondividing PCs or activated B cells (i.e., plasmablasts) that secrete antibody but retain the ability to divide (Claflin & Smithies, 1967). The differential migration of MBCs and PCs/plasmablasts is most likely accomplished by changes in the expression pattern of different chemokine receptors and other adhesion molecules (Tarlinton et al., 2008), but it remains unknown why PCs specifically migrate to the bone marrow compartment. This remains an area of active investigation.

DURATION OF ANTIGEN-SPECIFIC SERUM ANTIBODY RESPONSES

Induction of a durable neutralizing antibody response represents the basis of many successful vaccines and is important to the maintenance of protective immunity against a wide range of pathogens. The duration of serum antibody production following infection by measles, mumps, rubella, vaccinia, EBV, and varicella-zoster virus (VZV; i.e., chickenpox) was compared to the humoral immunity induced by vaccination with nonreplicating antigens, including tetanus toxoid and diphtheria toxoid by longitudinal analysis (Fig. 2; see Table 2). In this study (Amanna et al., 2007), over 600 serum samples from 45 subjects were used to measure serum

FIGURE 2 Duration of serological memory is dictated largely by the antigen under study. In this illustration (adapted from Amanna et al., 2007), the durability of antigen-specific antibody responses following infection or vaccination are compared over time. Antibody responses to EBV, measles, mumps, rubella, and vaccinia last a lifetime with little to no decrease in titer. Antibody responses to VZV decline more rapidly than antibody responses to other viral infections but remain more durable than the antibody responses to protein antigens such as tetanus or diphtheria. In general, the durability of serum antibody responses differ greatly depending on the antigen under study, and if the underlying mechanisms involved with determining the persistence of antibody can be elucidated, this will have important implications in terms of optimizing future vaccine design.

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antibody titers longitudinally for an average of 15 years, with serological memory in some subjects examined for up to 26 years. Following acute infection with measles, mumps, rubella, or vaccinia viruses, serum antibody responses were maintained with half-lives of over 90 years. This indicates that despite clearance of the pathogen within a few weeks after infection, immunological memory to these viruses is essentially maintained for life. Unlike acute viral infections in which the initiating pathogen is eventually cleared, chronic or latent viral infections have the potential to “boost” the immune system via persistent antigenic stimulation or periodic reactivation from latency. Analysis of EBV responses indicated that 4 of the 45 subjects showed evidence of serological “boosting,” but these immunological spiking events appeared to be relatively rare (0.6 events per 100 person-years). Examination of VZV-specific immunity revealed a higher frequency of reactivation/reexposure events with 10 out of 45 subjects (22%) demonstrating measurable spikes in serum antibody responses (1.6 events per 100 person-years). However, the duration of immunity to VZV during the intervening periods of time between serospikes revealed an antibody half-life of approximately 50 years, whereas immunity against EBV did not show a statistically significant decline (estimated half-life of 11,552 years). Although this latter result might be expected following infection by a virus that is never fully cleared from the infected host, the relatively short antibody half-life following VZV infection was unexpected and suggests that when this particular virus goes into latency, it may truly be sequestered from the immune system. Analysis of humoral immunity against tetanus and diphtheria toxins revealed that not all antibody responses last a lifetime. Indeed, antibody responses against tetanus were maintained with an estimated half-life of only 11 years, whereas antibody responses against diphtheria were maintained with a half-life of 19 years (see Fig. 2; Table 2). This is a sharply different outcome from what is observed following acute or chronic viral infection and indicates that the nature of the antigen can have a profound effect on the duration of immunological memory. It is unknown if these differences are simply a reflection of the different stimulatory microenvironments (nonreplicating antigen/adjuvant versus replicating antigen/inflammation) or if differences in the structural components of the antigen (repetitive versus nonrepetitive B-cell epitopes) impact the duration of immunological memory.

TABLE 2 Duration of serum antibody production after infection or vaccinationa Antigen Tetanus Diphtheria VZVb Vaccinia Rubella Mumps Measles EBVc

Antibody half-life in years (95% confidence interval) 11 (10–14) 19 (14–33) 50 (30–153) 92 (46–) 114 (48–) 542 (90–) 3,014 (104–) 11,552 (63–)

a Duration of antigen-specific antibody responses was determined using a mixed effects model of longitudinal analysis. Adapted from Amanna et al. (2007). b VZV, varicella-zoster virus c EBV, Epstein-Barr virus

The nature of the antigen or infection appears to play a critical role in determining the duration of the resultant humoral immune response, but host genetics appear to also be involved in this process. When the duration of immunity against multiple antigens was compared within specific subjects, individual variations in serological memory were identified (Amanna et al., 2007). For instance, although most subjects demonstrated uniform long-lived immune responses against viral infections and shorter immune responses to tetanus and diphtheria, there were exceptions. For instance, although one subject mounted a normal antibody response to EBV (with no evidence of decline), the antibody responses to all seven other antigens were similar and ranged from a half-life of 14 to 31 years. This and other individual variations indicate that there may be a genetic element involved in the determination of immunological memory in humans following vaccination or infection. Identification of the specific mechanisms that govern the development of durable antigen-specific antibody responses represents one of the most important aspects of vaccinology and a better understanding of these factors could lead to better optimization of vaccine design.

QUANTITATION OF ANTIGEN-SPECIFIC B CELLS

As mentioned before, humoral immunity is maintained by two types of B cells—MBCs and PCs. The duration of the PC response is most commonly measured indirectly by quantitation of antigen-specific serum antibody levels or directly through the use of the ELISPOT assay, which enumerates the number of individual ASCs/PCs in a given cell population. Although analysis of MBC populations has been performed routinely in animal models, the relatively rare frequency of MBCs in human PBMC samples have made it more difficult to measure this important B-cell population following vaccination or infection of human subjects. Fortunately, several developments in MBC quantitation have made it easier and more feasible to enumerate antigen-specific MBCs in clinical samples. One approach to MBC quantitation is to use polyclonal in vitro stimulation to induce MBC expansion and differentiation into antigen-specific ASCs that can be measured using an ELISPOT assay (Crotty et al., 2004). Advantages of this approach include the ability to measure MBCs of virtually any antigen specificity and with the brief in vitro expansion step, only a relatively small number of PBMCs are required to measure MBC responses. The main disadvantages of this approach is that it is possible that some MBCs may expand or differentiate at different rates (Slifka & Ahmed, 1996a) and the final quantitation of MBCs is determined relative to the total number of IgG ASCs. For this reason, the frequency of antigen-specific MBCs in the starting population is based on an approximation that is based on the expanded number of total IgG-secreting B cells in the sample. Another approach to measuring MBC numbers is to perform limiting dilution analysis (Amanna et al., 2007; Amanna & Slifka, 2006; Pinna et al., 2009). The advantage of this approach is that the limiting dilution step provides quantitation of proliferation or differentiation of individual MBC clones and can be used to measure rare MBC populations specific for up to 8 to 10 different antigens in a single assay (Amanna & Slifka, 2006). The main disadvantages of this approach is that it is labor intensive, involves purifying B-cell populations (in some cases [Amanna & Slifka, 2006] but not others [Pinna et al., 2009]) and this approach often requires a large number of PBMCs in order to identify very rare antigen-specific MBCs. A third approach to MBC quantitation is based on staining the B cells


with fluorochrome-labeled antigens and measuring antigenspecific MBCs by flow cytometry (Amanna & Slifka, 2006; Leyendeckers et al., 1999; Scibelli et al., 2005; Townsend et al., 2001). The main advantage of this approach is that it allows antigen-specific quantitation of MBC frequencies directly ex vivo and with multiparameter flow cytometry, it is possible to measure a large number of phenotypic markers on antigen-specific B cells at different stages of activation or differentiation following vaccination or infection. The main disadvantage is that accurate quantitation of rare antigenspecific MBCs requires a large number of PBMCs and not all antigens are amenable to flow cytometry techniques, especially if they are highly cross-reactive, have high nonspecific binding (e.g., some complex or highly repetitive antigens demonstrate more nonspecific staining), or are difficult to conjugate to available fluorochromes. However, with this toolbox of available MBC quantitation techniques in hand, we are beginning to learn more about the relationship between MBC and PC populations and can now determine how long antigen-specific MBC responses can be maintained.

LIFESPAN OF MEMORY B-CELL POPULATIONS

Although there is substantial evidence describing longterm immunity following infection or vaccination (Slifka & Ahmed, 1996b), there is relatively little known about the kinetics and duration of B-cell memory. One study demonstrated that vaccinia-specific B-cell memory following smallpox vaccination is quite durable, with MBC numbers maintained for over 50 years after infection with little to no evidence of decline (Crotty et al., 2003). Likewise, analysis of B-cell memory against vaccinia, measles, mumps, rubella, EBV, VZV, tetanus, and diphtheria also indicates that B-cell memory can be maintained for decades or possibly even for life after infection or vaccination (Amanna et al., 2007). Another study measuring MBC responses to VZV, tetanus, measles, influenza, and Toxoplasma gondii in one subject over the course of 17 years has also provided further anecdotal evidence that MBC populations can be maintained for long periods of time. For instance, measles-specific MBCs were identified 50 years after primary infection (Pinna et al., 2009). Interestingly, vaccinia-specific T-cell memory declines slowly over time with an estimated half-life of 8 to 15 years (Crotty et al., 2003; Hammarlund et al., 2003) whereas MBC responses appear to be stable (Amanna et al., 2007; Crotty et al., 2003). This leads one to question whether B-cell memory might be more long-lived than T-cell memory and further studies will be needed to determine if these differences are identified in other model systems.

MODELS EXPLAINING LONG-LIVED HUMORAL IMMUNITY

There are many models that have been proposed to describe how long-term humoral immunity is maintained. The oldest models proposed that persistent antigen (e.g., chronic or latent infection) or environmental reexposure would be required to maintain serum antibody responses by repeatedly stimulating MBCs to proliferate and differentiate into short-lived PCs, which would then maintain antibody levels (Slifka & Ahmed, 1996b). Although this could indeed be the case for some types of viral infections, it is clearly not a universal requirement since antibody responses to the vaccinia virus can be maintained for up to 75 years after infection. This occurs in the absence of persistent or latent infection or reexposure to vaccinia or other

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potentially cross-reactive Orthopoxviruses (Hammarlund et al., 2003). Another popular model proposes that persisting antigen in the form of immune complexes on the surface of follicular dendritic cells (FDC) is responsible for maintaining long-lived humoral immunity by continuous reactivation of MBCs into short-lived PCs (Slifka & Ahmed, 1996b). Although antigen can be maintained for several weeks or months on the surface of FDCs, it is unlikely that noncovalent protein interactions can be maintained in an aqueous environment for decades without some loss in protein levels over time. Since antibody responses to measles, mumps, rubella, and vaccinia are maintained with half-lives of over 90 years (Amanna et al., 2007), the persistent antigen theory of long-term humoral immunity has fallen out of favor. MBCs can be polyclonally activated in vitro to proliferate and differentiate into ASCs and another model suggests that nonspecific B-cell activation through heterologous infections or vaccinations may represent the mechanism used to sustain humoral immunity for decades (Bernasconi et al., 2002). Although an intriguing theory, it does not explain why antibody responses to different antigens have dramatically different half-lives, especially since one would assume that polyclonal activation should trigger equally long-lived responses by MBCs of all specificities. Clinical data also does not support this model. For instance, when human subjects were infected with vaccinia virus, there was a rapid anamnestic antibody response mounted against vaccinia, whereas serological memory to other viral infections remained essentially unchanged despite the presence of an active viral infection and activation of virus-specific T cells (Amanna et al., 2007). Moreover, each of the models that are based on MBC-based repopulation of short-lived PCs rely on a correlation between MBC numbers and antibody levels to be maintained. MBC numbers often do not correlate with antibody titers (Amanna et al., 2007), and booster vaccination induces higher MBC numbers without appreciable change in long-term serum antibody titers (nanan et al., 2001), indicating that it is unlikely that MBC-dependent models of humoral immunity provide an accurate depiction of the mechanisms involved with maintaining serum antibody responses. Other models of long-term humoral immunity are based on the premise that PCs are long-lived cells that are independently regulated from MBCs and do not require continuous replenishment from the MBC pool. Support for these models can be found in animal studies (Ahuja et al., 2008; DiLillo et al., 2008; Manz et al., 1997; Slifka et al., 1998), as well as limited clinical studies (Cambridge et al., 2003; Edwards et al., 2004) in which antigen-specific antibody responses are maintained for prolonged periods of time after MBC depletion. One model based on the long-lived PC theory suggests that the duration of serum antibody responses is determined by competition between newly formed PCs competing with preexisting PCs for niches within the bone marrow compartment (Fairfax et al., 2008; Radbruch et al., 2006). Similar to the MBC-based models, this particular model fails to predict the different antibody half-lives induced by different antigens since one would assume that newly developed PCs would outcompete the preexisting PCs regardless of their antigen specificity. Moreover, based on this model one would predict that as one reaches advanced age and the finite niches for space in the bone marrow become more constrained, then preexisting antibody titers to specific antigens should begin to decline more rapidly. This is not the case, since longitudinal analysis of antibody responses to childhood diseases such as measles, mumps, and rubella are equally stable regardless of whether the subjects are in

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their 20s or in their 60s (Amanna et al., 2007). A model that we propose is that the lifespan of PCs is determined during the initiation of the humoral immune response. During PC formation, the cells are programmed with a different lifespan depending on the stimulation conditions and microenvironment encountered during or just prior to their terminal differentiation into antibody-secreting PCs. Based on this model, weaker B-cell signaling would result in PCs with an intermediate or shorter lifespan, whereas strong Bcell signaling during the induction of the PC response would lead to more longer-lived PCs. An example of strong B-cell signaling would include stimulation by a highly repetitive antigen, which would enhance “capping” of surface BcR and lead to increased calcium flux. Further addition of costimulation by antigen-specific CD4 T cells via CD40:CD40L interactions might also lead to an increase in combined signal strength and the induction of long-lived PCs. Since PCs lose surface Ig (i.e., no longer able to detect antigen), down regulate MHC class II expression (i.e., can no longer obtain T-cell help), and become terminally differentiated cells (i.e., no longer able to divide), we believe that the programmed lifespan model provides the most reasonable representation of the clinical findings demonstrating antibody responses of different duration. It is as of yet unknown what specific factors are directly involved with the development of PCs with different predetermined life spans, but identification of these factors will be critical for developing improved adjuvants and vaccines.

CONCLUSIONS

Following infection, protective immunological memory in the form of memory T cells, memory B cells, and plasma cells can be maintained for years, and in some cases, for life. The remarkable durability of immune memory has been the focus of intense investigation and we have learned much about the factors involved with this process. Although memory T cells do not necessarily require cognate antigen to be maintained, they do require exposure to different cytokines such as IL-2, IL-7, and IL-15 in order to optimally survive the contraction phase of the immune response and to persist for long periods of time after an infection has been cleared. Following commitment to the memory cell lineage, memory T-cell populations demonstrate dynamic heterogeneity in terms of phenotype, function, and anatomical distribution, which together are likely to promote a broad arsenal of preexisting T lymphocytes capable of responding to a variety of pathogens and potential routes of infection. Similar to the maintenance of T-cell memory, maintenance of humoral immunity in the form of memory B cells, plasma cells, and serum antibody appears to be very longlived even in the absence of reexposure or reinfection. Induction of persistent antibody responses typically requires T-cell help and exposure to stimulatory cytokines. Moreover, the duration of the humoral immune response appears to be further dictated by the nature of the antigenic exposure. Antibody responses to antigens such as tetanus or diphtheria toxoids will result in slowly declining antibody responses that last for decades, whereas immunity against viral infections such as measles, mumps, and rubella will often last a lifetime. The underlying mechanisms involved with inducing and maintaining humoral immunity have not been fully established, but there are several models that have been proposed. Future studies on immunological memory will likely shed new light on the subject of cellular and humoral immunity, and as we learn more about the requirements for developing protective immune

responses, we will improve our ability to develop better and more effective vaccines.

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THE PATHOGENS

II



10 Overview of Viral Pathogens JONATHAN W. YEWDELL AND JACK R. BENNINK

and clues for understanding the workings of the innate immune system. Vertebrates have gone to great lengths to protect themselves from viruses (and also other pathogens), evolving intricate systems of innate and adaptive immunity. There are at least four practical reasons for immunologists to have a good working knowledge of virology. (i) Viruses remain a major cause of human morbidity and mortality, and pose a constant threat of devastating pandemics (Table 1) (i.e., imagine for a second, if HIV was efficiently transmitted by the aerosol route). (ii) Viruses are important vectors for vaccines against viral and nonviral pathogens and neoplasia. (iii) Viruses are evolution’s gift to gene therapists. (iv) Viruses are extremely useful experimental tools and probes for understanding the biology of cells and organisms. Given the brevity of this chapter, we have set only the modest goal of providing a simple introduction to viruses, with the hope of piquing the reader’s interest to study the subject in more depth. We encourage readers to explore in detail The Principles of Virology (Flint et al., 2008), a marvelous textbook with superb diagrams and figures that provides many profound insights into the biology and evolution of viruses. There are a number of other excellent texts that cover more specialized aspects of virology or virus–host interactions (see Coffin et al., 1997; Ewald, 1994; Knipe & Howley, 2006; Granoff & Webster, 1999; McCance, 1998; Plotkin et al., 2008; Richman et al., 2009). In addition, throughout the text we refer to textbooks and reviews as jumping off points for those who would like to chew on a given topic.

INTRODUCTION

Viruses are small segments of nucleic acid wrapped in a protein or lipoprotein shell with the ability to penetrate host cells and hijack the host machinery to generate progeny. At their simplest, viruses consist of a few thousand nucleotides that encode a nucleic acid polymerase and several proteins that comprise the virion. At their most complex, viruses contain several hundred genes encoding numerous viral structural proteins, proteins that alter cellular metabolism to favor virus replication (or enable virus to persist in a latent state), and proteins that modify the innate and adaptive host immune responses to favor virus transmission. Viruses are always completely dependent on cells for the production of energy, raw materials (e.g., amino acids, lipids, sugars), and protein synthesis. Viruses are an inescapable product of evolution. Every living species has its own unique set of viruses capable of infecting species members. Host–virus sets overlap considerably, and even dynamically. Viruses exhibit an extremely high mutation rate relative to their hosts, and are constantly changing genetically, with the consequence that their host range is in constant flux. Many hundreds of viruses are known to be capable of infecting humans, with probably thousands more that remain to be discovered. Viruses, like all life-forms, are selected simply to replicate, and typically bear no malice to their hosts. Indeed, efficiently killing hosts is a suicidal mechanism for viruses. Over time, evolution selects for a balance between viruses and their hosts. On the other hand, the genetic variability of viruses means that mutants with enhanced potential for causing mayhem are constantly generated. Moreover, encounters between species enable the introduction of new viruses, with the possibility of tremendous lethality for the new host until a new equilibrium is reached. Consequently, all species—from Escherichia coli to redwoods to blue whales—evolve strategies for controlling virus replication. How this is achieved by simple organisms is well beyond the scope of this chapter, except to point out that this area of research provides essential systems

STRUCTURAL PROPERTIES

Virions range in size from an 18 nm diameter (parvoviruses) to an 750 nm diameter (mimiviruses), amounting to a 40-fold difference in diameter and 72,000-fold difference in volume. They may be comprised of just a few different proteins to over 900 proteins, and their genomes range in size from 3.2 kilobases (hepadnaviruses) to 1.2 million bases (Table 2) (Chiu et al., 1997). For the purposes of the immune system, the most important structural distinction between viruses lies in their outer covering. Naked viruses possess a protein shell while

Jonathan W. Yewdell and Jack R. Bennink, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-3209.

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The paThogens TABLE 1 Leading virus-associated diseasesa Cause

Deaths

DALYsb

Respiratory (pneumonia & influenza) Diarrheac (all causes) AIDS Measles Chronic hepatitis Bd Chronic hepatitis C Dengue Japanese encephalitis virus

4,259,000 2,163,000 2,040,000 424,000 105,000 54,000 18,000 11,000

97,786,000 72,777,000 58,513,000 14,853,000 2,068,000 955,000 670,000 681,000

c

Carriers

33,000,000 350,000,000 170,000,000

a

Data compiled from WHO (1999, 2008) and UNAIDS (2008). DALYs, disability-adjusted life years (disability years due to a disease). c Includes nonviral infectious disease mortality. d Chronic hepatitis B contributes to these deaths from liver cirrhosis and cancer. b

enveloped viruses possess an envelope derived from the host cell membrane (usually the plasma membrane, but some viruses use membranes from the endoplasmic reticulum [ER] or Golgi complex, and sometimes do so in a highly complex manner). Viral envelopes are comprised of cellular lipids and one to many viral proteins. Most enveloped viruses contain minimal amounts of host proteins, but others are more promiscuous, and relatively large amounts of host proteins may be present, including class I or II molecules of the major

histocompatibility complex (MHC). The presence of such polymorphic host molecules provides additional potential targets for immune recognition. The structures of both naked and enveloped viruses are highly repetitive, providing the immune system an opportunity for a high avidity interaction on the basis of multivalent recognition by low affinity receptors. Enveloped viruses usually require membrane integrity for infection, making them vulnerable to the complement system or membrane

TABLE 2 Virus classification Virus family

Genomea

Coat

Adenoviridae Arenaviridae Astroviridae Baculoviridae Birnaviridae Bornaviridae Bunyaviridae Calciviridae Circoviridae Coronaviridae Filoviridae Flaviviridae Hepadnaviridae Herpesviridae Iridoviridae Mimiviridae Orthomyxoviridae Papovaviridae Paramyxoviridae Parvoviridae Picornaviridae Poxviridae Reoviridae Retroviridae Rhabdoviridae Togaviridae

DS DNA, 36–38 Kb NS RNA, 10–14 Kb PS RNA, 7.2–7.9 Kb DS DNA, 100 Kb DS RNA, 7 Kb NS RNA, 8.9 Kb NS RNA, 13.5–21 Kb PS RNA, 8 Kb NS DNA, 3–4 Kb PS RNA, 16–21 Kb NS RNA, 12.7 Kb PS RNA, 10 Kb DS/NS DNA, 3.2 Kb DS DNA, 120–200 Kb DS DNA, 150–350 Kb DS DNA, 1,200 Kb NS RNA, 13.6 Kb DS DNA, 45–55 Kb NS RNA, 16–20 Kb NS DNA, 18–26 Kb PS RNA, 7.2–8.4 Kb DS DNA, 170–200 Kb DS RNA, 22–27 Kb PS RNA, 3.5–9 Kb NS RNA, 13–16 Kb PS RNA, 12 Kb

Naked Enveloped Naked Enveloped Naked Enveloped Enveloped Naked Naked Enveloped Enveloped Enveloped Enveloped Enveloped Naked/Enveloped Naked Enveloped Naked Enveloped Naked Naked Enveloped Naked Enveloped Enveloped Enveloped

a

Abbreviations: DS, double stranded; PS, positive single stranded; NS, negative single stranded.

10. overview of Viral pathogens

disrupting peptides (and detergents). Enveloped viruses also tend to be more fragile than nonenveloped (naked) viruses, making their transmission more dependent on intimate contact between hosts. The extreme fragility of some enveloped viruses (like HIV) limits their transmission to body fluid exchange. The surface proteins of both naked and enveloped viruses are typically highly resistant to proteases present in extracellular fluids, and when evolution dictates, even to the harsh conditions of the upper gastrointestinal tract. The outer virion shell protects the viral nucleic acid, which is complexed to viral structural proteins that enable its packaging into the virion during virus biogenesis. Viruses are grouped into six categories based on the nucleic acid present in infectious virions and the immediate nucleic acid product produced in infected cells. They are: negative strand () RNA (noncoding) transcribed into () RNA, () RNA transcribed into () RNA, () RNA transcribed into () DNA, double-stranded RNA, single-stranded DNA, and double-stranded DNA. Many viruses carry their own nucleic acid polymerases to initiate the infectious cycle, which can occur either in the cytosol or in the nucleus. A common feature of animal viruses is their high particle-to-infectivity ratio, (i.e., the number of particles required to initiate an infection). With very few exceptions, this is greater than 10, commonly greater than 100, and occasionally even greater than 1,000. The extent to which this reflects structural defects in a high percentage of viruses versus a low probability that competent virions have for initiating a successful infection is a question that has dogged virology from its origins. As this is invariably determined in vitro, it is unclear whether the particle to infectivity ratio is lower in vivo, as seems likely. If structural defects are common, they are relatively minor ones, at least as far as the humoral immune system is concerned, as there is little evidence to suggest that distinct antibody responses are elicited by infectious versus noninfectious virions. Indeed, the induction of antibody responses by purposely inactivated viruses is a principal strategy of antiviral vaccination. The prodigious reproductive potential of viruses gives them extreme latitude in producing mutants—given hundreds of offspring each, with a generation time of less than 12 hours, it is advantageous to possess a high mutation rate—many orders of magnitude higher than multicellular organisms such as ourselves. In particular, RNA viruses encode polymerases that generate errors just north of the error catastrophe rate. High mutations rates, in conjunction with selection pressure for high packaging efficiency, makes multiple copies of genes in a virion a liability, and only retroviruses are known to deliberately incorporate more than a single copy of their genomes into virions.

STRATEGIES OF VIRUS ENTRY Organisms

Virus survival requires transmission between hosts. The route of transmission is a critical aspect of viral biology since it dictates (or vice versa) in what cells the infection of host is initiated and what cells must produce the virus for transmission to occur. The transmission of a given virus is usually restricted to a single anatomical location, which provides an opportunity to focus immunity on a given pathogen to a limited number of sites in an organism (see Table 3). For example, influenza virus and other respiratory viruses that replicate in the columnar epithelium of the

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upper airway are transmitted strictly through this portal, and a local immune response in the upper airway is sufficient to block infection. By contrast, enteric viruses like rotavirus only infect via the gastrointestinal track. Vaccines to these agents should therefore focus on generating effective immune responses in the respiratory and gastrointestinal systems, respectively. Similarly, virus dissemination can be prevented by local immunity at the site of transmission. Thus, HIV infected individuals could potentially be removed from the virus-transmission cycle without a global eradication of virus replication by immune removal of the virus from organs of dissemination (Flint et al. 2008; Mims et al., 2001). As the largest single organ in humans and the most external, the skin is an obvious target for initiation of viral infections. Sufficiently obvious, in fact, that the cornified epithelium is an extremely effective barrier against virus transmission, and only a few viruses have managed the trick of penetrating this barrier on their own. Transmission through the skin can be achieved by physical introduction past the keratinocytes either by natural (insect or animal bites) or artificial (hypodermic) means. In the former case, transmission may be a natural part of the virus life cycle, or may be unrelated to virus evolution (but still detrimental to unlucky individuals infected with West Nile virus, for example). Mammalian (or avian) cells differ considerably from insect cells, and the ability to productively infect members of both phyla strongly implies that cross-species transmission is important to viral survival.

Cells

Viral replication requires delivery of the viral nucleic acid to the cytosol, where it can either immediately initiate the replication cycle or do so after transport to the nucleus. Penetration can occur in two locales: either directly at the plasma membrane or after internalization into an endosome or other internal cellular membrane system (e.g., some viruses are thought to penetrate cells from the endoplasmic reticulum). Enveloped viruses penetrate by fusing their membranes with cell membranes, while nonenveloped viruses penetrate by disrupting cell membranes. In either case, viruses must first adhere to the target cell by binding its receptor to a cellular ligand, which can be any molecule on the cell surface. The specificity of viral receptors ranges from highly specific (e.g., a ligand expressed in a single cell type in a single tissue), to completely promiscuous (e.g., a molecule expressed by every cell; sialic acid is a receptor for many viruses). In the former case, the receptor is usually responsible for viral tropism, while in the latter, viral tropism will depend on other factors (such as route of entry into the host, ability to penetrate the cell, or replicate once inside). Viral penetration is usually a complicated affair that entails conformational alterations in viral surface proteins and interactions with additional cell surface proteins. There are ample opportunities for the innate and adaptive arms of the immune system to block penetration by preventing virus binding to host cells, or by preventing downstream events in the penetration and uncoating process (Berger, 1997; Doms, 2000; Kasamatsu & Nakanishi, 1998; Klasse et al., 1998; McDermott & Murphy, 2000; Norkin, 1995; Sodeik, 2000; Whittaker et al., 2000). The requirement of viral genomes to reach the cytosol provides an early opportunity for recognition by the adaptive immune system in the absence of viral gene expression. MHC class I binding antigenic peptides can be generated from viral proteins that accompany viral genomes into the cytosol. As peptide generation is often quite inefficient, this usually requires that thousands of copies of the source protein

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The paThogens

be delivered, which limits this pathway to high abundance of proteins from large viruses. There is evidence, however, that this skews CD8 T-cell responses in some systems.

OUTCOMES OF CELLULAR INFECTION

Viral penetration of cells has four potential outcomes (with all gradations in between). First, the cell may be completely inhospitable and the viral proteins and nucleic acids are disposed of with minimal perturbation of the host cells. Second, viral replication initiates but fails to produce infectious progeny with consequences ranging from minimal transient perturbations in cellular physiology to cell death. Third, viral replication results in the generation of infectious progeny and cells are either killed immediately or remain persistently infected and continue to function as they produce progeny viruses. Fourth, the virus enters a latent state and essentially disappears until it is triggered to reactivate with the production of infectious progeny. These outcomes are not mutually exclusive, and in a given individual harboring a virus, these processes likely occur simultaneously, depending on the nature of the cell infected and the exact conditions of infection (e.g., number of viruses infecting the cell, known as the multiplicity of infection [MOI]); exposure of the cell to cytokines; cell cycle status). As always, evolution only selects for the transmission of virus between hosts (the selection of viruses replicating within any given organism is only an intermediate step). In some circumstances, even nonproductive infection in certain cell types may be evolutionarily selected if it facilitates virus transmission. For example, a virus may nonproductively infect immune cells, disabling the cells and thereby enhancing viral growth in productively infected cells. On the other hand, most examples of nonproductive infection are not a result of evolutionary selection but simply an example of the law of biological entropy. There are infinitely more ways for things to go wrong than to go right. The means viruses use to achieve these outcomes varies tremendously with the virus, but all viruses must fulfill several basic tasks: (i) produce mRNA to generate viral proteins on cellular ribosomes; (ii) replicate their genome; (iii) assemble their genomes with viral (and sometimes cellular) proteins, and release progeny from the cells (Doms et al., 1993; Garoff et al., 1998); and (iv) modify host cell metabolism (including avoiding innate cell antiviral responses) to optimize viral replication. A productive viral infection usually results in the generation of hundreds to thousands (or more) progeny. The shortest infectious cycles can be completed in 4 hours. This capacity for replication provides a serious challenge to the immune system, since just three or four infectious cycles over the course of a single day can be sufficient to produce enough of the virus to infect every cell in a target organ. The host must therefore have an immediate response for such rapid cytopathic viruses, and this is, no doubt, one of the major selective forces for the evolution of the innate immune system, which must operate solo for several days (at least) until activated antigen-specific lymphocytes can generate sufficient numbers to mount an effective antiviral offensive.

Coordinated Gene Expression

Following cellular penetration, viruses usually have a precise plan of attack that entails the temporally coordinated transcription/expression of subsets of their genes. During any given segment of the infectious cycle, different viral gene products are often expressed in widely different quantities depending on the function of the gene product. This

plan can vary from cell type to cell type, and abortive infections often result from failing to successfully negotiate the transition from one stage to another. Often, the plan of expression is based on genome replication, and viral genes can be classified as early (pre-genome replication) versus late (post-genome replication). The time of genome replication varies widely, but typically occurs between 2 and 12 hours post-infection. At the great danger of over-simplification, the enzymes required for viral replication and for modifying cellular functions are expressed immediately upon penetration, while many of the proteins that make up virions (viral “structural” proteins) are produced late in the infectious cycle. For the adaptive immune system, the most important aspect of coordinated gene expression is that not all infected cells express the full range of viral gene products, and antigens presented by infected professional antigen presenting cells may be limited to early gene products.

GENETIC INSTABILITY OF VIRUSES

Genetic instability is the sine qua non of viruses. Indeed, the mutation rate of RNA viruses is sufficiently high that despite possessing a tiny genome relative to other life-forms, virtually every individual in a population is still unique. DNA viruses are generally more stable, but still can exhibit mutation rates several orders of magnitude higher than host cells. Therefore, it is necessary to think of viruses as dynamic populations of related genomes and not as monolithic entities (Domingo & Holland, 1997; Koonin, 1992; Shadan & Villarreal, 1996). Although virus recombination is probably a relatively infrequent occurrence, it still is probably very important for most viruses, which have a haploid genome, and can therefore profit by a mechanism that enables escape from lethal mutations. Indeed, it is often observed that serial plaque purification of viruses ultimately results in the generation of poorly replicating viruses, pointing to the importance of recombination in maintaining a functional genome. A number of RNA viruses foster recombination by possessing segmented genomes. Recombination also occurs by polymerases shifting from one genome to another (predominantly RNA viruses) and by strand breakage and rejoining (predominantly DNA viruses). The genetic instability of RNA viruses, in particular, makes them elusive targets for the immune system. For many viruses, mutants that escape neutralization by monoclonal antibodies arise with a frequency of 105 or higher. This can result in antigenic drift, in which the humoral immune system selects for neutralization-resistant mutants. This, of course, is a significant problem in influenza vaccination. The influenza virus, as a segmented virus, also undergoes antigenic shift, in which the viral surface proteins are exchanged in a recombination event. This can result in the introduction of novel surface virion proteins from animal influenza viruses and in devastating epidemics. Hepatitis C virus (HCV), an increasingly important human pathogen, is so highly variable that multiple serotypes are typically isolated from the same individual. For genetic instability to be of any use to the virus, the resulting gene products must be sufficiently plastic in order to utilize the mutations to produce a novel (and useful) phenotype. This is not uniform among viral proteins, and it is commonly found that variation rates among viral proteins vary hugely. At the risk of over-simplification, the extracellular domains of membrane proteins of envelope viruses demonstrate the greatest flexibility for accepting mutations, while proteins involved in protein–protein interactions (e.g., the coat proteins of naked viruses or viral replication proteins) are less tolerant of mutations.


Capturing Host Genes

Another potential source of genetic variability is the capture of host genes. This happens most often with retroviruses due to their unique life cycle, which entails integration into the host genome (the retroviral capture and mutation of host cell genes involved in cellular proliferation led to the identification of oncogenes as altered cellular genes). Small viruses are under heavy selection pressure for minimizing genome size, which make host genes difficult to swallow. However, for the large DNA viruses, such as Herpesviridae and Poxviridae family members, insertion of even large amounts of foreign genes has no discernible effect on virus function, so there is a large potential for capturing host genes. These viruses often possess sophisticated programs for manipulating host immunity, and clearly these genes are often derived from the host. Such genes are, however, usually distantly related to host genes, so these events are probably extremely infrequent. Thus, for all but retroviruses, the capture of host genes is an important factor in their early evolution but less important over a historical time frame (Alcami & Koszinowski, 2000; Becker, 1996; McFadden et al., 1998; Tortorella et al., 2000; Weinberg, 1997).

TISSUE TROPISM

A critical aspect of viral infections is that replication is usually limited to anatomical locations or cell types (Table 3). There are a number of contributing factors: (i) physical isolation of viruses due to anatomic barriers (e.g., enteric viruses may never have the opportunity to escape the GI system even though they would be capable of infecting other cell types), (ii) the specificity of viral receptors and limited distribution of cellular factors required for viral penetration (e.g., HIV requires cellular expression of CD4, which is limited to a small subset of cells and also requires host cells to express certain chemokine receptors to enable fusion of viral and cellular membranes), and (iii) even given entry to the cytosol, viruses can be rather finicky about replicating and very few viruses (if any) have the ability to productively replicate in every cell type in the body. This implies a requirement for nonhousekeeping cellular genes for productive infection, but identifying cellular gene products that positively (or negatively) influence virus infection is extremely difficult in practice. One aspect of viral tropism is particularly important to the immune system. If viruses are unable to infect professional antigen presenting cells, an alternative mechanism must be used for presenting viral antigens to naïve CD8 T cells. It is thought that under these circumstances presentation occurs via cross-priming, a process in which professional antigen presenting cells acquire viral proteins produced by infected cells and process them for presentation to T cells.

Kinetic Aspects of Infection

Many viruses replicate in a host by a program that requires the dissemination of virus from one organ to another. Virus dissemination can occur via either the blood or lymph. As lymphatics are more accessible to extracellular particles than capillaries, this is a more likely route of dissemination (the lymphatics function to collect foreign material from the periphery to the lymph nodes). Viruses also have an opportunity to reach the blood via the lymphatics if they manage to avoid sequestration in nodes on the way to the venous system. Virions probably have a difficult time spreading in this direct manner, and in many cases virus spread is mediated by infected white blood cells (erythrocytes cannot be infected as they lack essential biosynthetic processes, including protein synthesis). As white blood cells have the intrinsic ability to leave the blood or lymph and infiltrate tissues (diapedesis),

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they can provide a direct route for virus entry into organs. Lacking a cellular carrier, viruses must rely on other ways to breach the barriers that guard the tissues. Some viruses are capable of being transcytosed by endothelial cells into the underlying tissues. In tissues with sinusoids (e.g., adrenals, bone marrow, liver, spleen), the virus may be transcytosed by resident macrophages that line the sinusoids. In other organs (e.g., kidney, pancreas, gut), many capillaries are fenestrated, providing a direct route for viral penetration. Finally, some connective tissues, muscle, and the central nervous system (CNS), are highly resistant to direct penetration, since the endothelial cells of the capillaries are backed by a formidable basement membrane. In this case, virus penetration from the blood probably occurs by diapedesis. The pattern of virus spread can be dependent on the mechanism of virus maturation. Enveloped viruses can dictate whether they mature from the apical or lumenal surface of polarized epithelial cells by taking advantage of the signals the cells use to maintain cell polarization. Respiratory viruses often mature from the apical surface of epithelial cells, limiting their spread to the surface of the airway. Presumably this is a way of increasing their chance of transmission by maximizing their concentration in respiratory fluids. Other viruses with less restricted tissue tropism mature from the basolateral surface. This increases their chances of hematogenous spread. Viruses capable of infecting neurons (neurotropic viruses) take advantage of the transport capacity of neurons to travel from the periphery to the ganglia or the CNS. Such traffic can be bidirectional. Herpesviruses, geniuses of viral latency, persist in a latent state in ganglia or in the spine, and upon reactivation are transported to the periphery where they bud from sensory nerve termini to be transmitted to new hosts via intimate contact. Rabies virus travels to the CNS from sensory neurons in the vicinity of an animal bite, multiplying in the CNS and then leaving via efferent neurons to the salivary glands, where virus is manufactured for saliva-borne transmission. Incredibly, viral replication in the CNS is sufficiently cell specific to induce the typical rabid behavior essential to transmission. All of this is accomplished by the information encoded by just 12,000 nucleotides. Another mode of virus spread is from mother to fetus. For noncytopathogenic viruses, this can ensure virus maintenance in a species. In the case of retroviruses, which integrate into the genome, the line between virus and host becomes blurred as viral genes are transmitted in the genome. For cytopathic viruses, this mode of transmission may have catastrophic medical consequences to the host, but little evolutionary benefit to the virus, as a dead fetus or infant has little potential for transmission to another host.

SPECIES SPECIFICITY

The fact that receptor-independent tissue tropism of viruses is common indicates that virus replication in cells is often a complicated business that, in many circumstances, requires very specific factors in differentiated cells. It should come as no surprise then that most viruses have a very limited capacity to infect other species. Even in situations where this can occur, the features of infection in different hosts may differ enormously. The practical consequence for biomedical research is that it is extremely difficult to find animal models that mimic human viral diseases. For immunologists, it is crucial to recognize that different elements of the immune system may be used for combating infections with the same virus in different species. Moreover, molecules that evolve to interfere with host responses may be highly

138 TABLE 3

The paThogens Sites of viral infection

Skin Coxsackievirus (picornavirus) Human immunodeficiency virus (retrovirus) Measles virus (parvovirus) Rubella virus (togavirus) B virus (herpesvirus) Cytomegalovirus (herpesvirus) Epstein-Barr virus (herpesvirus) Herpes simplex virus (herpesvirus) Human herpesvirus 6 (herpesvirus) Human herpesvirus 8 (herpesvirus) Orf virus (poxvirus) Papillomavirus (papovavirus) Parvovirus B19 (parvovirus) Molluscum contagiosum (poxvirus) Vaccina virus (poxvirus) Varicella-zoster virus (herpesvirus) Lymphoid and Macrophages Alphavirus (togavirus) Enterovirus (picornavirus) Flavivirus (flavivirus) Human immunodeficiency virus (retrovirus) Lymphocytic choriomeningitis virus (arenavirus) Mumps virus (parvovirus) Rubella virus (togavirus) Ebola virus (filovirus) Epstein-Barr virus (herpesvirus) Cytomegalovirus (herpesvirus) Guanarito virus (arenavirus) Human herpesvirus 6 (herpesvirus) Human herpesvirus 7 (herpesvirus) Junin virus (arenavirus) Lassa virus (arenavirus) Machupo virus (arenavirus) Marburg virus (filovirus) Poxvirus (poxvirus) Sabia virus (arenavirus) Varicella-zoster virus (herpesvirus) Hemorrhagic Fever Congo-Crimean hemorrhagic fever virus (bunyavirus) Dengue virus (flavivirus) Ebola virus (filovirus) Guanarito virus (arenavirus) Hantaan virus (hantavirus) Junin virus (arenavirus) Kyasanur forest virus (flavivirus) Lassa virus (arenavirus) Machupo virus (arenavirus) Marburg virus (filovirus) Omsk virus (flavivirus) Rift Valley virus (bunyavirus) Sabia virus (arenavirus)

Gonads Ebola virus (filovirus) Marburg virus (filovirus) Mumps virus (parvovirus) Respiratory Coronavirus (coronavirus) Enteroviruses (picornavirus) Influenza virus types A and B (orthovirus) Measles virus (parvovirus) Parainfluenza 1, 2, and 3 (parvovirus) Polyomavirus (papovavirus) Respiratory syncytial virus (parvovirus) Rhinoviruses (picornavirus) Adenovirus (adenovirus) B virus (herpesvirus) Cytomegalovirus (herpesvirus) Epstein-Barr virus (herpesvirus) Herpes simplex virus (herpesvirus) Varicella-zoster virus (herpesvirus) Central Nervous System California encephalitis virus (bunyavirus) Colorado tick fever virus (reovirus) Coxsackievirus (picornavirus) Eastern equine encephalitis virus (togavirus) Echovirus (picornavirus) Influenza virus (orthovirus) Japanese encephalitis virus (flavivirus) Human immunodeficiency virus-1 (retrovirus) Lymphocytic choriomeningitis virus (arenavirus) Measles virus (parvovirus) Mumps virus (parvovirus) Murray Valley fever virus (flavivirus) Poliovirus (picornavirus) Rabies virus (rhabdovirus) St. Louis encephalitis virus (flavivirus) Tick-borne encephalitis virus (flavivirus) Venezuelan equine encephalitis virus (togavirus) Western equine encephalitis virus (togavirus) West Nile fever virus (flavivirus) Adenovirus (adenovirus) B virus (herpesvirus) Cytomegalovirus (herpesvirus) Epstein-Barr virus (herpesvirus) Herpes simplex virus types 1 and 2 (herpesvirus) Human herpesvirus 6 (herpesvirus) Polyomavirus (papovavirus) Vaccinia virus (poxvirus) Varicella-zoster virus (herpesvirus)

(Continued on next page)

10. overview of Viral pathogens TABLE 3

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(Continued)

Kidney Adenovirus (adenovirus) Cytomegalovirus (herpesvirus) Ebola virus (filovirus) Marburg virus (filovirus) Polyomavirus (papovavirus) Gastrointestinal Astrovirus (astrovirus) Calicivirus (calicivirus) Rotavirus types A, B, and C (reovirus) Adenovirus (adenovirus) Heart and Muscle Alphavirus (flavivirus) Coxsackievirus (picornavirus) Dengue virus (flavivirus) Echovirus (picornavirus) Hepatitis A virus (picornavirus) Human immunodeficiency virus (retrovirus) Influenza virus (orthovirus) Lymphocytic chroiomeningitis virus (arenavirus) Measles virus (parvovirus) Mumps virus (parvovirus) Poliovirus (picornavirus) Rabies virus (rhabdovirus) Rubella virus (togavirus) Yellow fever virus (flavivirus) Adenovirus (adenovirus) Cytomegalovirus (herpesvirus) Epstein-Barr virus (herpesvirus) Hepatitis B virus (hepatitis virus) Herpes simplex virus (herpesvirus) Varicella-zoster virus (herpesvirus)

species specific and can even interact with different target molecules in other species. An important element in virus evolution becomes apparent when viruses cross species barriers, particularly when species are closely related. Very few virus infections of natural hosts have high mortality rates. Again, this is a consequence of maximizing transmission, where lethality rarely benefits the virus. Just how easy it is for a virus to increase its lethality becomes apparent when the virus jumps to a species sufficiently related to the natural host to support virus replication. In this case, the brakes on virus replication imposed by natural selection may be inoperative, and the virus can be highly lethal. Indeed, the most virulent viruses for humans result from such species jumping. These include herpes B virus (natural host: African green monkeys), filoviruses (e.g., Lassa fever and Marburg

Liver Ebola virus (filovirus) Echovirus (picornavirus) Enterovirus (picornavirus) Hepatitis A virus (picornavirus) Hepatitis C virus (flavivirus) Hepatitis D virus (deltavirus) Hepatitis E virus Hepatitis G virus Junin virus (arenavirus) Lassa virus (arenavirus) Machupo virus (arenavirus) Marburg virus (filovirus) Rubella virus (togavirus) Rift Valley fever virus (bunyavirus) Yellow fever virus (flavivirus) Adenovirus (adenovirus) Epstein-Barr virus (herpesvirus) Hepatitis B (hepatitis virus) Herpes simplex virus (herpesvirus) Varicella-zoster virus (herpesvirus) Eye Coxsackievirus type A24 (picornavirus) Enterovirus 70 (picornavirus) Influenza virus (orthovirus) Human immunodeficiency virus (retrovirus) Measles virus (parvovirus) Mumps virus (parvovirus) Newcastle disease virus (parvovirus) Rubella virus (togavirus) Adenovirus (adenovirus) Cytomegalovirus (herpesvirus) Epstein-Barr virus (herpesvirus) Herpes simplex virus (herpesvirus) Molluscum contagiosum (poxvirus) Papillomavirus (papovavirus) Vaccinia virus (poxvirus) Varicella-zoster virus (herpesvirus)

viruses; natural host unknown), human immunodeficiency virus (HIV; natural host: chimpanzee or other primates), and hantaviruses (natural host: mice). Most of these viruses do not pose much of a public health risk (yet), since their transmission between humans is limited. However, the potential danger of a newly introduced virus is illustrated by HIV, which, in the course of adapting to a new host, can cause considerable mayhem. Tragically, the high rate of transmission of HIV in sub-Saharan Africa is providing a real-time lesson in the coevolution of virus and host. This horror is not without precedent in human history; the arrival of the Europeans in the Americas introduced a number of viral diseases (e.g., smallpox, influenza) the native peoples had not experienced in many generations, if ever. The devastating consequences of these diseases were a major factor in the rapid ascendancy of the Europeans.

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The paThogens

The balance between host and virus is most vividly illustrated by the introduction of the rabbitpox virus myxoma virus into the rabbit population of Australia. European rabbits were established in Australia from just a few individuals, and consequently, exhibited very little genetic polymorphism. Myxoma virus evolved among a different species of rabbit, where it causes relatively mild diseases, yet was remarkably lethal for the European/Australian rabbits, with a 99.8% mortality rate. A single year, however, was sufficient for the selection of both rabbits resistant to lethal infection and less virulent virus strains that persisted in nature.

PATHOGENICITY

Viruses need not be pathogenic, for, as discussed above, evolution selects viruses for maximal transmissibility between hosts. Pathogenicity can be a positive factor in transmissibility (e.g., in the case of respiratory viruses that are most efficiently spread by coughing, sneezing, and the outpouring of mucus in response to destruction of respiratory epithelium). A special case occurs when virus infection of immune cells causes a generalized immunosuppression resulting in infection with other organisms that favor the transmission of the original infecting virus (Fauci, 1996; Levy, 1998; Mims et al., 2001; Nathanson, 1997; Oldstone, 1996; Wright, 1996). Pathogenicity can be caused by direct virus destruction of cells, secretion or release of toxic substances by virusinfected cells, or by the host immune response to the virus. In the latter case, the process of inflammation itself may cause tissue destruction or direct immune recognition of virus-infected cells. Inasmuch as this is a normal protective response, it is not truly immunopathological in nature. A relatively uncommon outcome of viral infections is transformation of infected cells into benign or malignant tumor cells. This can be a direct effect of viral protein on cellular proliferation, as occurs for human papilloma virus (HPV), or an indirect effect of cellular proliferation induced by chronic virus infections, as in hepatitis B virus. Hepatitis B virus infections are one example where the immune system steps over the line of protection into the realm of immunopathology. Viral replication of hepatocytes appears to be innocuous, with the damage coming over many years from virus-specific CD8 T cells that infiltrate the liver and destroy infected cells. Hepatitis B virus infections, which feature the release of enormous amounts of virus into the plasma, also provide another example of immunopathology in the deposition of immune complexes composed of viral antigens and antibodies. This can cause havoc in the kidneys. Surprisingly, this is not known to occur for other virus infections in humans. Less well-established are cases of immune mimicry, when viral antigens induce responses that cross-react with cellular antigens, breaking the normally operative tolerance mechanisms. Tolerance to self-antigens may also be subverted by the process of inflammation that is induced by viral infections.

VULNERABILITY TO IMMUNE ATTACK

Whether by choice or necessity, viruses offer numerous opportunities for attack by the vertebrate immune system. The earliest is to prevent infection at the site of entry. This can occur by either nonantibody or antibody-mediated mechanisms. Protection against membrane viruses can be mediated by defensins (small proteins secreted by neutrophils, monocytes, and Paneth cells that compromise membrane integrity), complement components, and possibly other serum proteins. Natural antibodies (i.e., antibodies induced without exposure to the pathogen or closely related viruses

can also play a protective role against infection). Humans produce large amounts of antibodies specific for terminal galactose residues linked to a penultimate galactose by a 1–3a bond. This oligosaccharide is absent in humans and other primates, but is present in gut bacteria, which induce the antibody response. Interestingly, other mammals and insects can also generate this oligosaccharide linkage, and membrane viruses produced by these species may express surface glycoproteins with the oligosaccharide. It appears that humans are protected by infection from a number of viruses by such antibodies. Antibodies induced by prior exposure to a virus (or viral vaccine) are often the most efficient means of protection against viral infection, and can play an important role in clearing an infection. A number of features of virus replication in host cells enable detection and immune counterattack. First, cells express membrane-bound receptors for viral nucleic acids that can detect viruses attempting to enter the cell and trigger an innate immune response, including the synthesis of type I interferons and other proinflammatory cytokines. Interferon induction is perhaps the most critical early warning signal in virus infections, since it produces an antiviral state in both the infected cell and surrounding uninfected cells, and also provides perhaps the initial warning signal to the immune system. Second, the replication of RNA viruses (and even DNA viruses) results in the presence of unusually high amounts of double-stranded RNA in the cytoplasm. This rapidly triggers an innate immune response through Tolllike receptors including the production of interferons by infected cells. The presence of strange nucleic acids in the cytosol can also activate a cellular system that degrades double-stranded RNA, compromising viral replication. Third, the requirement for viral protein synthesis on ribosomes enables the cell to sample cellular ribosomes and display peptides from viral gene products on the cell surface in association with MHC class I molecules. Fourth, viruses with hit-and-run strategies usually try to maximize virus production by monopolizing the cellular biosynthetic metabolism and a resultant shutdown of host cell protein synthesis. This results in decreases in MHC class I expression that can be detected by NK cells, which can kill cells or release cytokines with antiviral activities. Cytokine release by NK cells augments the cytokines released by infected cells, and serves as an important accelerant in the immune response. Fifth, the changes in cellular metabolism induced by virus infection can trigger cellular apoptosis in an attempt to prevent the release of viral progeny—the Cartonian nobility of this gesture (“Tis far better thing, than I have ever done before. . .”) is somewhat compromised by the likelihood of cell death from the later stages of viral infection (Miller & White, 1998; O’Brien, 1998; Teodoro & Branton, 1997). The innate immune system has nearly complete responsibility for controlling infections for the first 3 to 5 days after infection with an infectious agent that the host has not previously experienced (or any antigenically cross-reactive viruses). Perhaps this is sufficient to completely contain low-level infections with some viruses, but for more serious threats the adaptive immune system must be mobilized to contain and clear the infection. The adaptive immune system has a variety of effector mechanisms for this purpose, all based on the specificity of antibodies and T-cell receptors for viral antigens. The genes encoding these remarkable molecules are the only genes in vertebrates that are routinely subject to somatic mutation and rearrangement. This enables the immune system to keep pace with genetic variability of viruses (and other pathogens), maintaining the capacity to respond specifically to virtually any virusencoded protein.


What is known about how the immune system uses the innate and adaptive immune systems to control viral infection is described in chapters 15, 16, 19, 20, 30, 31, 32, 39, 40, 43, 44, 48, and 51 of this book. This manuscript was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

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11 Overview of Parasitic Pathogens RICK L. TARLETON AND EDWARD J. PEARCE

INTRODUCTION

many that are parasitic (Adl et al., 2005). Among the parasitic protists, there are two major pathogenic groups (Fig. 1), the apicomplexans, named for the anterior complex of organelles involved in host cell invasion, and the flagellated kinetoplastids, named for the large mass of DNA contained within their single, elaborate mitochondrion. Other protists of substantial medical importance include the intestinal parasites Entamoeba histolytica and Giardia lamblia, and the sexually transmitted Trichomonas (Fig. 1). Protists may reproduce sexually or asexually. However, the mixing of distinct genotypes may be rare even in those capable of sexual reproduction, resulting in widely diverse and essentially independently evolving lineages within each species.

Parasites are generally considered to be eukaryotic pathogens that are not fungi. The term embraces both unicellular and metazoan organisms from widely divergent phyla. Most parasites of medical importance have highly complex life cycles in which the organisms differentiate through multiple morphotypes that are adapted to the various niches that the parasites live in as they move within and between hosts. This is complicated by the fact that transmission often involves infection and replication within an intermediate invertebrate host. Although parasites infect animals as well as people, for this chapter we will focus mainly on those parasites (or models thereof) that cause serious disease in humans. Many of the infections that we will focus on are considered to be tropical diseases, and there is little doubt that the impact of parasitic infection is greatest in developing countries of the tropics, where deficits in sanitation and the control of vectors of transmission combine to create a devastating effect. The genetic and morphologic complexity of parasites makes them challenging targets for immunologic studies. Nevertheless, parasitic infections are of enormous public health significance, and many excellent animal models of human parasitic diseases exist. Thus, experimental parasitic infections allow a tractable means for studying the real world impact of immunity. Moreover, parasitic infections tend to be strongly immunogenic and result in immune responses that are polarized in, for example, Th1 or Th2 directions. Thus, parasitic infections provide an excellent means for testing, in vivo, the roles of various factors, pathways, or cell types in the natural development of distinct types of immune responses.

Apicomplexans

All apicomplexans are parasitic and have evolved to live intracellularly, using secretions from their apical complex organelles to traverse and infect host cells. Life cycle complexity can be substantial in these parasites, with various host cell types being targeted for infection depending on the stage of development. Included in this group are the Plasmodium species, the biggest killer among the human parasites, as well as Toxoplasma gondii, and a number of primarily food and waterborne causes of diarrheal diseases (Cryptosporidium parvum, Cyclospora cayetanensis, and Sarcocystis spp.) (Fig. 1). The species of Plasmodium that cause disease in humans are, in decreasing order of importance, P. falciparum, P. vivax, P. malariae, and P. ovale. Malaria parasites enter the bloodstream of the host when an infected mosquito takes a blood meal. The infectious sporozoite stage of the parasite remains in the bloodstream for only minutes to a few hours before invading a hepatocyte. The dominant surface circumsporozoite protein (CSP), in conjunction with the thrombospondinrelated adhesive protein (TRAP), and TRAP-related protein (TLP), participate in host cell selection; invasion itself is driven by an actin-myosin-based motor that is unique to apicomplexans (reviewed in Baum et al., 2008). Inside host cells, Plasmodium and other apicomplexans replicate within a parasitophorous vacuole (PV), formed during the invasion process from the secretions of the rhoptries and dense granules. The merozoites released from hepatocytes then invade erythrocytes, initiating the erythrocytic phase of the Plasmodium life cycle. The synchronized rounds of release and reinvasion of erythrocytes elicits the periodic fevers characteristic of malaria. The process of host cell egress has recently

MAJOR PARASITES AND THE DISEASES THEY CAUSE Protists

Formerly classified as protozoans, nonfungal single cell eukaryotes are now organized as protists and may incorporate 50,000 or more distinct extant and extinct species, including Rick L. Tarleton, Center for Tropical & Emerging Global Diseases, Coverdell Center for Biomedical Research (Rm 310B), 500 D.W. Brooks Drive, University of Georgia, Athens, GA 30602. Edward J. Pearce, Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983.

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FIGURE 1 Protozoan infections. A summary of the major protozoan infections of humans and available experimental models. The lists of pathogens are not exhaustive.

received considerable attention in both Plasmodium and Toxoplasma gondii and has shown to involve a number of proteases released from the newly described secretory axoneme organelle (Yeoh et al., 2007). Eventually, a subset of parasites differentiate to produce gametes that, if taken up by the appropriate blood-feeding mosquito, will fuse to form a zygote, the source of sporozoites that are infective for humans. Disease resulting from malaria infection can be due to anemia associated with excessive erythrocyte loss following high parasitemia or, more seriously and usually in children, due to cerebral disease (coma) and metabolic acidosis. Major organ diseases related with infection result from changes in microcirculatory flow associated with adherence of the infected erythrocytes, clumping with noninfected erythrocytes, changes in the deformability of uninfected cells that contributes to reduced blood flow, and inflammation initiated by the interaction of the infected cells with the endothelium and the release of parasites following rupture of the infected erythrocytes. In pregnant women a similar process can occur within the placenta, especially in individuals carrying a first baby; this is also life threatening. In P. falciparum infections, parasitized erythrocytes adhere to endothelial cells primarily via the highly variant PfEMP1 proteins, which are expressed in modified areas of the infected erythrocyte membrane, called knobs. PfEMP1 binds to a variety of receptors on the endothelium including CD36, intercellular cell adhesion molecule (ICAM), thrombospondin, PECAM-1, and, especially in the placenta, hyaluronic acid and chondroitin sulfate A.

The life cycles of other medically important apicomplexans such as Toxoplasma gondii are somewhat simpler than that of Plasmodium. Unlike human malaria, toxoplasmosis is a zoonotic disease and usually arises in humans from the consumption of undercooked infected meat or through contact with the feces of infected felines, the sole hosts for the sexual cycle of Toxoplasma. The products of sexual reproduction, the oocysts are shed from the cat gut into the feces and upon digestion (by any mammal or bird or even some cold-blooded vertebrates), release the haploid sporozoites. The sporozoites invade host cells using a process analogous to that of Plasmodium, including the production of a parasitophorous vacuole within which rapid tachyzoite asexual replication occurs. Host cell destruction accompanies parasite egress and the released tachyzoites invade virtually any type of host cells. Strong cell mediated immune responses control the infection and contribute to the transformation of some of the intracellular tachyzoites into semiquiescent bradyzoites; persistent infection consisting of tissue cysts occurs chiefly in muscle or brain tissue. In immunocompetent populations, most Toxoplasma lines cause a highly prevalent but largely clinically silent infection. However, bradyzoites reactivate to tachyzoite production at a low rate in vivo and when immune pressure is released, such as when infected tissue is eaten by another uninfected mammal, or when infected hosts are immunosuppressed a highly virulent disease can occur. T. gondii has a diverse population structure, driven by infrequent sexual recombination, host population structure and biogeography and the variation in virulence among

11. Overview of Parasitic Pathogens

different lineages infective to humans is only beginning to be understood (Sibley et al., 2009a). The high accessibility of Toxoplasma for genetic manipulation has made it a useful model for cell biological and immunological studies.

Kinetoplastids

In humans, parasitic kinetoplastids live either exclusively extracellularly (Trypanosoma brucei), exclusively intracellularly within macrophages or other phagocytic cells (Leishmania spp.), or alternating between extracellular and intracellular forms, the latter of which can be observed in nearly any host cell type (Trypanosoma cruzi). Although these three groups of parasites can be morphologically similar and can share cellular and biochemical features (e.g., a corset of sub-cell membrane microtubules, a single flagellum extruding from a membrane pocket and providing motility, and glycosomes [peroxisomes modified to carry out glycolysis]), they nevertheless cause very different diseases (Stuart et al., 2008) (Fig. 1). T. brucei is endemic in sub-Saharan Africa, where it is transmitted by blood-feeding tsetse flies. Human African trypanosomiasis (HAT) is caused by two subspecies of T. brucei, T. brucei gambiense and T. brucei rhodesiense. Although morphologically indistinguishable from each other or from T. brucei brucei, T. b. gambiense and T. b. rhodesiense produce proteins that make them resistant to human serum, which rapidly kills T. b. brucei (Pays & Vanhollebeke, 2008). The parasites live within the blood and plasma wherein they replicate, and can eventually enter the central nervous system (CNS). This second, CNS stage of the disease, may be accompanied by severe neurological symptoms including sleep irregularities that give the disease its common name, African sleeping sickness. The mechanisms of disease are unclear, although both host immune responses and parasite-derived toxins have been proposed. The success of the infection is directly tied to the parasite’s ability to select for expression a single gene encoding the cell surface variant surface glycoprotein (VSG), from among the hundreds of VSG-encoding genes in the genome. The dense matrix of 1 3 107 molecules of a single specific VSG on the parasite surface not only shields other parasite molecules from immune detection, but also elicits the production of high levels of antibodies against this one variant. The rapidly rising antibody levels leads to the demise of parasites expressing that VSG variant, but parasites that have switched to express a different VSG gene now expand to fill the niche. This pattern of antigen switching and immune destruction results in a cyclical parasitemia, with each wave of parasites expressing a different antigen variant. This also results in a chronic infection as the supply of VSG types, including new ones generated by recombination, is essentially inexhaustible. This elegant mechanism of immune evasion also makes a vaccine-based approach for control of T. brucei difficult to envision. T. cruzi is transmitted zoonotically in the Americas, where it causes Chagas’ disease, a malady characterized by heart and/ or gut muscle destruction and dysfunction. Infection with T. cruzi is generally initiated by the deposition on the skin of metacyclic trypomastigotes by the blood-feeding reduviid bug intermediate host. Parasites then enter mammalian hosts via a break in the skin or via a mucosal surface. Congenital infection is also possible, although not common, and human outbreaks related to the ingestion of food or drinks contaminated with insect feces have become more frequently reported in recent years. Humans are largely inadvertent hosts, a consequence of living in suboptimal housing infested with the insect vectors. The infecting metacyclic and bloodstream trypomastigote forms are nonreplicating; entry into host cells and conversion into amastigotes is required for expansion of the parasite population in mammals. A wide range of host cell

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types can support parasite entry, first into a parasitophorous vacuole and ultimately into the host cell cytoplasm where amastigote replication occurs. Division is by binary fission and sexual recombination appears to be extremely rare. Following a 4–5 day period within a host cell, during which time nine or more divisions may occur, amastigotes convert back to flagellated trypomastigotes and mechanically disrupt the now nonfunctional host cell. Disease due to T. cruzi is the result of persistent infection of crucially important muscle cells in the heart and/or alimentary tract. The effects of direct parasite destruction of host cells and the parasite-induced immune responses accumulate over decades to yield clinical disease in a proportion of infected individuals. The more than 20 species of Leishmania cause human disease in many countries in Africa and Asia, in Central and South America, and in Mediterranean Europe. Different species provoke substantially different clinical outcomes, including self-limiting cutaneous lesions, progressive mucocutaneous lesions, and lethal (if untreated) visceralized infections. The stage of the parasite infectious for humans, the metacyclic promastigote, is introduced by the bite of a sand fly vector and targets phagocytic cells for infection. Although macrophages are considered the major host cell type for Leishmania, recent studies have also identified neutrophils, monocytes, and dendritic cells as important participants in the establishment and maintenance of the infection (Peters & Sacks, 2009). Once inside the host cell, the parasites are trafficked to the endolysosomal compartment where they transform into replicative amastigotes. Even in the cases of a self-healing disease, Leishmania often chronically persist in the skin in low numbers unless curative treatment is administered. Sand flies acquire the infection during blood feeding and the ingested parasites adhere to the midgut wall and replicate as promastigotes prior to transfer into the mammalian host at the time of subsequent feeding (Bates, 2008). In addition to being potent human pathogens, Leishmania infections have also been rich models for the discovery and study of cellular mediated immunity and its regulation (Sacks & Anderson, 2004), the role of host genetics in immune control (Blackwell et al., 2009), and the mechanisms of pathogen evasion of macrophage killing (Descoteaux & Turco, 2002).

Helminths

There has been renewed focus on the infectious diseases that affect the poorest people (Hotez et al., 2006). Typically these diseases have been neglected in terms of basic and applied research commitments. Of the neglected tropical diseases (NTDs) in sub-Saharan Africa and in Latin America and the Caribbean, over half are caused by helminths, and it is these helminthic diseases that account for most of the morbidity associated with the NTD spectrum (Hotez et al., 2008a; Hotez & Kamath, 2009). The magnitude of the disease problem associated with helminth infections is illustrated by the fact that in both of the regions mentioned above, there is a greater disease burden (measured in disability adjusted life years) due to NTDs than due to tuberculosis (Hotez et al., 2008a; Hotez & Kamath, 2009). Parasitic helminths belong to one of two major phyla, Nematoda or Platyhelminthes (Fig. 2). Well-known, free-living members of these phyla are Caenorhabditis elegans and the planaria, respectively. Generically, nematode development follows a pattern in which adult parasites living within the definitive host lay eggs that pass out of the body and hatch into L1 larvae, which then progress through a series of molts into L3 larvae, which are infectious and develop via additional molts into adult parasites. This relatively simple pattern has many highly specialized derivatives in different classes of

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FIGURE 2 Helminth infections. A summary of the major helminth infections of humans and available experimental models. The lists of pathogens are not exhaustive.

parasitic nematodes. Diversity is apparent in the various ways that infections are transmitted (e.g., directly or via a bloodfeeding insect vector), distinct sites within the body (e.g., intestine, lymphatics, subcutis) that are occupied by the adult worms of different species (see Fig. 2), the release of L1 larvae rather than eggs, and the sites within the body in which these L1 larvae reside (e.g., skin, blood). All of these variables can impact the types of immune response that develop and the immunopathological manifestations of infection. Platyhelminth life cycles are equally as bizarre. Within this phylum the cestodes and trematodes represent the two major groups of parasitic organisms (Fig. 2). Cestodes are tapeworms and these organisms live as adults within the intestinal lumen; as larval forms they live in intermediate mammalian hosts that usually are the prey or food-source of the definitive host. Trematodes live as adults in a wide variety of tissues (e.g., lung, liver, vasculature, intestine) and are transmitted via aquatic snail intermediate hosts (Fig. 2). A remarkable feature of many helminths is that the adult life stages remain relatively unaffected by the immune response, and it is the egg or larval stages that are the foci of immunopathological reactions.

Nematodes

Intestinal nematodes are the most prevalent helminth parasites. Species such as Ascaris lumbricoides, the hookworms (Necator americanus, Ancylostoma duodenales), whipworms (Trichuris trichiura), and Strongyloides stercoralis—together referred to as soil-transmitted helminths since they are acquired through contact with earth that has been contaminated with feces from infected humans (Fig. 2)—infect billions of people worldwide (Bethony et al., 2006). Infections

with these parasites lead to growth stunting and cognitive defects, and, especially in the case of the hookworms, anemia due to blood loss. Although somewhat amorphous compared to the obvious pathologies associated with some other helminth infections (see below), these conditions are now recognized to be a highly significant cause of morbidity (Hotez et al., 2008b). More severe and obvious pathological changes are caused by filarial nematodes (Fig. 2) (Hoerauf, 2008). Brugia malayi, B. pahangi, and Wuchereria bancrofti adult worms live within the lymphatics, where females release live L1 larvae known as microfilariae. These enter the bloodstream and circulate, awaiting transmission to a mosquito vector. Within the vector, the parasites develop through to L3 larvae, which are then transmitted to humans on subsequent blood feeds. Lymphatic damage due to these infections leads to defects in lymphatic drainage that can culminate in incapacitating swelling or elephantiasis. Onchocerca volvulus adults live in subcutaneous tissues, where they are encapsulated in nodules. As for the lymphatic filariases, microfilariae are released by female worms, but these remain within the skin where their migration leads to severe inflammation and irritation. More seriously, these larvae can migrate through the eyes causing inflammation that can lead to “river blindness.” Onchocerciasis is transmitted by blackflies.

Platyhelminths

The most serious platyhelminth infection is schistosomiasis, caused by Schistosoma mansoni, S. japonicum, and S. haematobium (Hotez & Fenwick, 2009). As adults these parasites live intravascularly, where female worms produce eggs that must transit through tissues to either the intestinal lumen


(Sm, Sj) or into the bladder lumen (Sh) in order to pass to the outside for transmission to occur. Eggs passing through tissues and trapped in other distal tissues, especially the liver, to which they have been carried by the circulation, induce granulomatous inflammation and associated fibrosis, which can have serious consequence such as potentially lethal portal hypertension. The most pathological cestode infections of humans are caused by Taenia solium and Echinococcus spp. (Raether & Hanel, 2003). As an adult, T. solium lives in the human intestine, where it is relatively nonpathogenic. The productive mode of transmission involves eggs from the definitive host being eaten by pigs. Within the pig, the eggs hatch and release stages that encyst within muscles. Humans acquire infection when they eat undercooked pork. However, a more serious situation arises when humans inadvertently eat eggs—the larval forms that emerge from the hatching eggs encyst in solid tissues. When this occurs in the brain, a potentially lethal condition known as neurocysticercosis develops. Echinococcus infections are normally transmitted between dogs and sheep, with the adult tapeworms living in the canine intestine and the encysted larval form being found in sheep that have become infected by eating grass contaminated with feces from infected dogs. Humans become involved when they inadvertently ingest eggs from infected dogs. In this case, the larval cysts that develop can metastasize, leading to a potentially life-threatening condition that can only be treated by surgery.

IMMUNITY TO PARASITES: GENERAL PRINCIPLES

Here we consider general characteristics of immunity to parasitic infections that deserve special attention.

Parasite Biology Determines What Constitutes an Effective Immune Response

CD8 T cells play an important role in immunity to viruses. The diversity of lifestyles seen in parasitic organisms means that it is simply not possible to make an equally straightforward statement about immunity to parasites. The site of infection and persistence; whether the parasite lives intracellularly or extracellularly, and (if the former) whether in a vacuole or not; whether the potential for replication exists; or where antigenically distinct progeny (e.g., eggs, larvae) are produced, are all determining factors in what constitutes an effective immune response. Nevertheless, some important generalizations can be made. For example, antibodies play an important role against parasites that live within the bloodstream, such as Plasmodium spp., T. brucei, and trypomastigotes of T. cruzi (Sacks et al., 2008). In contrast, for intracellular protozoan infections, Th1 and CD8 T-cell responses are particularly important (Sacks et al., 2008) (see chapter 24). This is because IFNg and TNFa, cytokines produced by these cell types, are able to activate infected cells to make mediators, such as NO that are cytostatic/cytotoxic. Additionally, IFNg induces the expression of a series of GTPases that play crucial roles in autophagy, a process that is increasingly recognized as being important for the control of some intracellular pathogens (Deretic & Levine, 2009). Moreover, cytotoxic CD8 T cells are able to kill nucleated cells infected with intracellular pathogens that live in or have ready access to host cell cytoplasm, such as T. cruzi, T. gondii, and the livers stages of Plasmodium spp. (Sacks et al., 2008). Th1 cells are particularly important in settings where pathogens infect macrophages or dendritic cells, which express MHC class II, such as Leishmania spp., since these cells are visible to CD4 T cells (Sacks & NobenTrauth, 2002). For T. cruzi, T. gondii, and L. major, in most

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infected humans and in mouse strains that are able to mount Th1 and CD8 T-cell responses against these infections, the immune response is able to bring populations of rapidly dividing parasites under control, although not to the point of clearance. Thus, in each case, parasites continue to exist within the host. The consequences of this vary from parasite to parasite. In the case of T. cruzi, ongoing inflammation in infected tissues such as the heart and alimentary tract, results in damage that can be extensive and life threatening. In contrast, in most cases of L. major and T. gondii, parasites persist without causing disease. In each case, suppression of the immune response allows parasites to begin proliferating freely again. This kind of reactivation of parasitic infections underlies the importance of organisms such as T. gondii, Leishmania, and T. cruzi as opportunistic infections during HIV/AIDS (Ong, 2008). It is generally true that Th2 responses are important for resistance to helminth infections (Anthony et al., 2007) (see chapter 25). However, the roles of Th2 cells and downstream mechanisms controlled by these cells varies greatly between infections. For example, primary infections with the intestinal nematode Heligmosomoides polygyrus (a mouse pathogen that is used broadly as a model for intestinal helminths, but which is most closely related to the human pathogen Trichostrongylus orientalis) are chronic, but drugcured mice are resistant to reinfection and this immunity is Th2 dependent. Immunity in this model is directed at the larval stages as they invade the mucosal lining of the gut prior to molting and emerging into the lumen as adult parasites (Kreider et al., 2007). The effector cells responsible for clearing the infection in this model are macrophages that are alternatively activated by IL-4/IL-13 (of which more below). For other intestinal infections, Th2 responses have been shown to mediate expulsion of worms through the effects of IL-4 on smooth muscle contraction (which may also be controlled by alternatively activated macrophages) and the production of molecules such as Relmb by goblet cells, which can have detrimental effects on intestinal helminth physiology (Anthony et al., 2007). Together these and presumably additional Th2-dependent factors can promote the expulsion of worms from the gut (Anthony et al., 2007). In schistosomiasis mansoni Th2 cells play a protective role by preventing the development of severe life-threatening disease during the acute phase of infection (Pearce & MacDonald, 2002). Schistosome eggs released by worms into portal vasculature either traffic across the endothelium and via intervening tissues head into the gut lumen, or are carried by blood flow into the liver, where they become trapped. In both settings, a Th2 response rapidly emerges in draining lymphoid organs and Th2 cells orchestrate the development of granulomatous lesions around schistosome eggs within tissues. These granulomas serve a host-protective function by effectively sequestering hepatotoxic molecules released from eggs, preventing them reaching the liver tissue. Moreover, alternatively activated macrophages play an important role in preventing the translocation of bacteria across the intestinal epithelium into the bloodstream as eggs pass in the opposite direction (Herbert et al., 2004). How exactly this is accomplished is unclear at present but may be related to their wound healing functions (Loke et al., 2007). Recent studies have shown that the same is likely to be true for mice infected with the larval life stage of the tapeworm Mesocestoides cortii (O’Connell et al., 2009). Again, this probably reflects the wound-healing properties of IL-4 -activated macrophages and other cell types in settings where parasite life stages are causing interstitial tissue damage. Antibodies also play roles in some helminth infections, and, in these cases, the types of antibodies that appear important are those such as IgG1 that are made by B cells that

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have been induced to class-switch by Th2 cytokines (see Butterworth et al., 1992; McCoy et al., 2008). A wealth of data, primarily from in vitro studies, indicate that antibodies are likely to function in resistance to helminths by focusing complement activation and effector cell degranulation at the parasite surface. A theme that has emerged from the study of parasitic diseases is that the decision by the host to make the wrong type of immune response can have disastrous consequences. This was first made clear in the Leishmania major model where mouse strains that mount a Th1 response control infection, whereas strains that make a Th2 response fail to control the parasites and develop a fatal disseminated infection (Sacks & Noben-Trauth, 2002). Other examples of this general principal include the fact that CBA mice make a Th17 response when infected with schistosomes, and this leads to fatal inflammation (Rutitzky et al., 2005). Commitment of a T cell to a particular lineage usually occurs very early in the immune response and is highly influenced by the contributions of cytokines and other signals from cells that are innately responsive to the pathogen. Consequently, there has been great interest in understanding which cell types contribute to the early innate response to different types of pathogens (see chapter 18). This has led to fundamental observations regarding: (i) the activation of dendritic cells to make IL-12/IL-23 by TLR agonists encoded by many protozoans, but not by helminths, and the role of this process in commitment, or not, to Th1 and Th17 response development (Gazzinelli & Denkers, 2006; Kane et al., 2004); (ii) the enrollment of basophils and other granulocytes such as eosinophils (previously considered to be the downstream of Th2 responses) in early responses to helminths, and the possible contribution of these cell types to Th2 response development (Perrigoue et al., 2009; Voehringer et al., 2004); and (iii) the ability of nonhematopoietic cells, such as intestinal epithelial cells, to respond to infection and play roles in initiating the adaptive immune response through the production of mediators such as TSLP that can affect dendritic cell biology (Artis & Grencis, 2008).

Immune Control of Parasitic Infections Is Complex

Many parasites go through a series of developmental changes as they establish and grow or proliferate within the host. The net result is that the host, though infected with one species, is exposed to sets of antigens that may be specific to each life stage. An example of this is provided by infection with Plasmodium spp., where the host is exposed to at least four distinct extracellular life stages: the sporozoite, the merozoite, and the male and female gametocytes, along with additional forms that live with hepatocytes or erythrocytes (Tsuji et al., 2001). While each of these stages expresses a basic set of genes that defines the Plasmodium life form, they each also express genes that encode proteins that serve stage-specific functions. Thus, primary exposure to malaria involves immune responses to antigens that are expressed consistently and to others, which are expressed for only distinct windows of time. Within the latter group, some antigens are expressed only transiently during infection, as would be the case for sporozoite or liver stage antigens, whereas others are expressed cyclically as the infection proceeds through rounds of erythrocyte infection and lysis. The complexity of this infection is reflected in the all-encompassing nature of the response it induces, which includes CD8, CD4, and B cell components, all of which can play a role in resistance (Wipasa & Riley, 2007). The immunodominance of particular antigenic epitopes has been well documented in bacterial and viral infections. In parasitic infections there has been a general sentiment that the genetic complexity of the pathogens will lead to highly divergent polyclonal responses. However, recent

studies with parasites as different as T. gondii and S. mansoni have revealed that single antigens do tend to play dominant roles in generating immune responses, especially when they have intrinsic adjuvant activity, as is the case for TgProfilin (Yarovinsky et al., 2006) and SmOmega-1 (Everts et al., 2009; Steinfelder et al., 2009). The example of the CD81 T cell response to T. cruzi is perhaps the most remarkable one in this respect, wherein at the peak of the T-cell response up to 30% of the entire CD81 T cell population in mice may be dedicated to the recognition of a few T. cruzi transsialidase-encoded peptides (Martin et al., 2006). We can expect greater understanding of the issue of immunodominance and cell-mediated immunity in control of parasitic infections in the near future, as more T-cell epitopes are discovered (Blanchard et al., 2008; Frickel et al., 2008), more widespread use is made of MHCI and II tetramers in the study or parasite-specific T-cell responses and as transgenic mice with T cells specific to (Sano et al., 2001) or tolerant to (Kumar et al., 2006) these epitopes become available. Complexity in immune responses coupled with a lack of clarity about the target antigens of protective immune responses presents special challenges for vaccine development against parasitic infection. Additionally, many of the identified dominant immunological targets are members of large and variable gene families, thus calling into question their utility as viable vaccine candidates. The situation is further confounded by the fact that immunity, when it is expressed in human populations or in experimental models of human parasitic infections, is, in many cases, concomitant immunity (immunity to superinfection) in which persistent primary infection can play a role in the maintenance of immunity (see below) (Fig. 3). All of these issues may have contributed to the renewed interest in vaccines based upon live, attenuated parasite lines (Beattie et al., 2008; Gigley et al., 2009; Kedzierski et al., 2008; Pinzon-Charry & Good, 2008; Selvapandiyan et al., 2009), efforts that have been immeasurably helped by accumulating post-genome transcript and proteome data that are key in identifying target genes for knockout/knockdown and, as well, the development of techniques to more rapidly produce these attenuated lines (Damasceno et al., 2009; Xu et al., 2009). While this work has progressed most dramatically in protozoa, there is reason to be optimistic that similar advances will come for helminth parasites (Brindley & Pearce, 2007; Lok & Artis, 2008).

FIGURE 3 Concomitant immunity. A primary immune response fails to clear the primary infection, (— ∙ —, – – – –) but is able to prevent the establishment of a superinfection following secondary or subsequent exposures to the same pathogen. This contrasts with the more conventional notion of protective immunity in which the primary infection is cleared by the primary immune response and the emergent memory immune response confers strong resistance to secondary infection with the same pathogen (——).


PERSISTENCE, CHRONICITY, AND EVASION

Most parasitic infections are chronic in nature (Fig. 4); presumably chronicity provides the selection advantage of increasing the potential for transmission in many cases. From the perspective of the immune response, chronicity is evidence that the immune response has failed to eradicate the infection and implies that immune responses to most parasites are to some extent ineffectual. This logic indicates that parasites have evolved to resist the immune response either by evading it (through antigenic variation or other mechanisms), or by preventing the development of appropriate effector mechanisms (as discussed in Belkaid et al., 2006), and it is clear that both strategies are utilized. A further consequence, or perhaps cause, of chronicity is the presence of regulatory mechanisms that develop to modulate immunologically mediated tissue damage associated with infection. An example here is provided by toxoplasmosis, where the absence of IL-10 leads to complete clearance of the parasite, but the development of lethal immunopathology (Gazzinelli et al., 1996). Despite the fact that many parasites can evade the immune response and establish chronic infections, there is evidence for the development of concomitant immunity. In some cases, this may be related to the fact that different life stages of parasites express stage-specific genes. Thus hosts can respond strongly to antigens that are expressed only transiently on incoming life stages. Responses against these antigens may be ineffectual against the life stages that exist throughout chronic infection, but capable of targeting secondary infections as they are acquired; such a situation appears to be the case in schistosomiasis (Smithers & Terry, 1976). In some cases it is clear that persistent infection is crucial for maintaining concomitant immunity. An informative illustration of this is provided by the Leishmania system, where, in genetically resistant mouse strains, blocking IL-10 activity leads to complete clearance of infection but with the accompanying loss of resistance to reinfection (Belkaid et al., 2001). For several protozoan pathogens, immune evasion depends on the ability of organisms to express antigens selected from a family of related but variant genes (see chapter 36). In this scenario, typified in the bloodstream forms of the African trypanosome T. brucei, the host makes a strong

FIGURE 4 Chronic infection. Many bacterial and viral infections are completely cleared within a relatively short time after acquisition (——). In contrast, many parasitic infections are chronic. Chronicity can take many forms, including the establishment of a population of organisms that is long-lived and stable, as is the case for schistosomes (which do not replicate within the definitive host), (– – – –), the control, but not clearance, of a population of replicating parasites, as is the case in certain types of leishmaniasis (—∙ —) and the cyclical expansion and contraction of replicating parasite populations that is consistent with the emergence of new antigenic variants and their sequential control by the immune response, as is the case for Trypanosoma brucei infection (∙∙∙∙∙∙).

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and effective immune response to antigens expressed on parasites introduced by the vector. However, in the case of T. brucei, as the parasites replicate within the blood, variants expressing different VSGs arise that are unaffected by the antibodies directed at the original variant (Taylor & Rudenko, 2006). The T. brucei genome encodes an archive of at least 800 VSG sequences, and this essentially means that the host is faced with the need to make hundreds of effective sequential immune responses in order to defeat the infection. In practice this is almost never possible and the infection is ultimately lethal. A similar situation occurs in malaria, where the P. falciparum genome encodes multiple families of variant antigens (e.g., VAR, STEVOR, RIFIN) (Scherf et al., 2008). The case with T. cruzi is distinct, with many representatives of large gene families (e.g., transsialidases, mucins, mucin-associated proteins) apparently being expressed simultaneously rather than sequentially (Tarleton, 2007). How such a mechanism for variant antigen expression may contribute to immune evasion has yet to be clearly defined. It is clear that helminth parasites can persist in the face of strong immune responses but there is no evidence from empirical studies or fully sequenced genomes that this is a reflection of antigenic variation. Indeed, the data indicate that infected animals are mounting responses that worms are evading through mechanisms that may include the production of antioxidants (Kuntz et al., 2007), the masking of surface antigens (Pearce & Sher, 1987), the secretion of cytokine homologs, and the production of molecules that have anti-inflammatory effects (Harnett & Harnett, 2006; Hewitson et al., 2009).

RELATIONSHIP BETWEEN IMMUNITY AND DISEASE

As in any infection, immune responses to parasites can cause pathological changes that lead to disease (see chapter 28). In acute bacterial and viral infections, symptoms associated with the inflammation such as fever and pain subside as the infection is cleared by the immune response. In many parasitic infections, however, the agents are not cleared and, in the face of a resilient pathogen, the immune response may be regulated not only by the loss of antigen, but also by the development of potent regulatory mechanisms (see chapter 35). Immunoregulation can act at two levels, either on the adaptive arm of the immune system through the regulation of lymphocyte responses, and/or by inhibiting the activation of innate cells such as macrophages. In many cases regulation is mediated by IL-10, a potent anti-inflammatory cytokine (Couper et al., 2008a). The importance of this cytokine is illustrated by the fact that in some normally chronic protozoal (T. gondii) and helminth (T. muris) infections, lack of IL-10 can lead to lethal acute disease associated with exacerbated inflammation (Gazzinelli et al., 1996; Schopf et al., 2002). The importance of IL-10 in immunoregulation during infection (Couper et al., 2008a) has focused attention on the sources of this cytokine. There has been particular interest in the contribution of Treg cells in this regard, since IL-10 is a primary product of these cells. In protozoal infections where IL-10 is clearly preventing parasite clearance and/or limiting the development of immunopathology, it has emerged that IL-10 is contributed by a variety of CD41 T cell subtypes. In self-healing Leishmania major, classical Foxp31 T reg cells are the major source of IL-10 (Belkaid et al., 2002). However, in more severe leishmaniasis and in toxoplasmosis, the crucial source of IL-10 is effector Th1 cells that have acquired the ability to make IL-10 in addition to IFNg (Anderson et al., 2007; Jankovic et al., 2007). In contrast, in malaria, the balance between a successful immune response and the

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development of immunopathology is regulated by adaptive Foxp32 Treg cells that make IL-10, but not effector cytokines (Couper et al., 2008b). The ability of CD41 T cells to make IL-10 can be promoted by the cytokines TGFb, IL-6, and IL-27 (McGeachy et al., 2007; Stumhofer et al., 2007). Thus, in settings of chronic infection, IL-10 production may broadly reflect the acquisition of anti-inflammatory function by effector T cells (Stumhofer & Hunter, 2008). In nematode infections, natural (Foxp31/CD251) Treg cells have been shown to contribute to chronicity, such that the depletion of these cells from mice chronically infected with the mouse filarial parasite Litomosoides sigmodontis (Fig. 1) allows clearance of infection (Taylor et al., 2005). However, in this case, Treg cell function is independent of IL-10. The regulation of effector T-cell responses during chronic infection can be mediated by a variety of regulatory T cells (as considered above), through lymphocyte-intrinsic hyporesponsiveness that can develop in response to repeated stimulation with antigen (Choi & Schwartz, 2007), and by the development of inhibitory/regulatory macrophages. Inhibitory macrophages, which tend to develop in Th2dominated immune responses and possess the characteristics of IL-4/IL-13 alternatively activated macrophages, are capable of inhibiting the proliferation of CD4 T cells in, for example, L. sigmodontis infected mice (Taylor et al., 2006). Alternatively activated macrophages express a defining set of genes including Arginase-1, the chitinases YM-1/1 AMCase, and the resistin-like molecule RELMa/FIZZ-1 (Kreider et al., 2007; Nair et al., 2006). The inhibition of T-cell proliferation by these cells is contact dependent and to some extent mediated by TGFb, a well-known antiproliferative cytokine. Moreover, RELMa produced by alternatively activated macrophages can inhibit the production of cytokines by Th2 cells and thereby regulate inflammatory effects downstream of these molecules (Nair et al., 2009). On the other hand, chitinases appear to play an important regulatory role by degrading invertebrate chitins, which themselves act to induce Th2-related inflammation (Reese et al., 2007). Intrinsically, hyporesponsive T cells have been identified in chronic helminth and protozoal infections, and recent work has linked the development of hyporesponsiveness in schistosomiasis to repetitive antigen stimulation and the upregulation of GRAIL, an E3 ubiquitin ligase that has previously been implicated in T-cell anergy (Taylor et al., 2009). Although apparently unrelated mechanistically, loss of responsiveness within the effector T-cell pool in chronic helminth infections is analogous to the “exhaustion” of CD8 responses in chronic viral infections. However, this relatively rapid and dramatic exhaustion of T-cell responses typified by lymphocytic choriomeningitis virus (LCMV) infection in mice is clearly not observed in all chronic protozoan infections, as evidenced by the long-term maintenance of competent CD81 T cell responses in chronic T. cruzi infections in mice and the development of a stable memory population following parasitologic cure (Bixby & Tarleton, 2008; Bustamante et al., 2008) In some settings immune responses that play pivotal protective roles early in infection become problematic and pathological in the chronic setting. An example of this is provided by the Th2 response in schistosomiasis, which, as discussed above, has clear and important protective functions during acute infection. However, the downside of this response is that Th2 cells make IL-13 in addition to IL-4, and IL-13 is pro-fibrotic, being able to induce the production of collagen by somatic cells through activation pathways that are closely related to those that underlie alternative macrophage activation (Wynn, 2004). This can be problematic in the livers of infected mice where the repetitive cycles of granuloma development and resolution as eggs

are deposited and subsequently die can lead to frustrated tissue repair and excessive fibrosis. This in turn can lead to portal hypertension, a potentially lethal condition that is apparent in the most severe cases of schistosomiasis. There continues to be great interest in the effects of chronic parasitic infections on the development of other diseases. In particular, the inverse relationship between diseases of westernized countries, such as diabetes and inflammatory intestinal conditions, and the prevalence of helminth infections has led to much speculation about the ability of immunoregulatory networks induced by parasitic helminths to modulate pathological conditions such as autoimmunity and asthma/allergy (Cooke et al., 2004; David et al., 2004; Maizels & Yazdanbakhsh, 2008). Evidence supporting this notion has accumulated over the last 5 years and there are now strong data indicating that this is the case. For example, in mice, Treg cells induced by H. polygyrus infection can protect against the development of airways hypersensitivity (Wilson et al., 2005), and Trichuris suis eggs (which hatch but do not establish infection in humans) are in clinical trials for the treatment of ulcerative colitis (Elliott & Weinstock, 2009). Moreover, defined helminth molecules such as ES62, a secreted filarial phosphorylcholine containing protein, have been demonstrated to possess potent anti-inflammatory immunomodulatory properties that may see them, or derivatives thereof, entering the clinics in the future (Harnett et al., 2009).

EMERGING AREAS

The world of parasitology has changed dramatically since the last edition of this book, in part through the release of comprehensive genome sequence data for many of the most important parasitic pathogens and an accumulating volume of other “omics” information. We now have the ability to understand what it is to be a parasite in far greater detail, and therefore are more informed about how these organisms interact with their hosts and with the immune system in particular. For example, the genome data have revealed the extent of variant gene families in the protozoans and allow us to say with some certainty that helminth parasites (at least those that have been sequenced) do not possess large families of related genes that would be emblematic of antigenic variation. The expression and functional data from transcriptomic, proteomic, and metabolomic studies are helping investigators to hone in on the best potential targets for diagnostics, vaccines, and drugs—although many of the efforts are only in their infancy. In general, the translation of new knowledge acquired at the lab bench (where parasites are often models for immunological studies) to the situation in the field (where the diseases these parasites cause are major health problems) continues to be relatively slow. Another area of great progress has been in the movement into the mainstream of molecular approaches for making transgenic, gene knock-out and knock-down protozoan parasites. This has allowed studies in which immune response development to parasitic organisms can be monitored by combining, for example, parasites expressing model antigens such as ovalbumin (OVA) with adoptively transferred OVA-specific TCR-tg T cells, or tetramers that recognize OVA-specific endogenous T cells. This approach is of particular benefit where the immunodominant parasite antigens remain to be defined and mice TCR-transgenic for specific for parasite antigens have yet to be developed. In cases where dominant antigens have been characterized, MHC class I tetramers are allowing detailed analyses of T-cell responses and promise rapid new insights in this area (see Dzierszinski & Hunter, 2008). As noted above, stage-specific protein expression and metabolic


pathway data are not only allowing the functional characterization of previously uncharacterized parasite molecules, but are also guiding investigators in the construction of attenuated parasite lines with vaccine potential. An added and important advantage of transgenesis for immunologic studies is that it allows the development of parasites expressing reporter proteins that can be visualized using in vivo-imaging systems (see Chtanova et al., 2009). Tools for the genetic alteration of helminth parasites are not as developed as those for protozoan organisms, but nevertheless there have been important recent advances in transgenesis in this arena (see Castelletto et al., 2009). Moreover, tools such as RNAi are being used to suppress gene expression in trematodes (Pearce & Freitas, 2008) and have already been used to investigate the roles of molecules that are potentially involved in immune evasion (Kuntz et al., 2007). Recent studies have made significant strides in identifying major helminth antigens and, consequently, we can expect significant developments in our understanding of immune responses to helminths through the use of MHC II tetramers, which will identify CD4 T cells that recognize these antigens. For intracellular parasites, especially among the apicomplexans, there has been an explosion in the understanding of how these pathogens modify their host cell through their secretions (reviewed in Laliberte & Carruthers, 2008; Luder et al., 2009; Sibley et al., 2009b). These studies are revealing not only previously unappreciated aspects of host:parasite interactions at the cellular level, but are also identifying additional mechanisms of immune evasion and new potential targets of host immune responses.

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12 Overview of Bacterial Pathogens PHILIPPE J. SANSONETTI AND ANDREA PUHAR

WHAT IS A BACTERIAL PATHOGEN?

In contrast to obligate pathogens, accidental pathogens can exist in the environment either as free-living bacteria or in association with other organisms, yet sometimes they infect and harm humans. Importantly, infection of humans most of the times does not promote the spread of the pathogen to a new host. Onset of infection is often accompanied by production of virulence factors (e.g., in a temperaturedependent fashion—virulence factors expression is induced at body temperature, but repressed at ambient temperature). Compared to obligate pathogens, accidental pathogens constitute a much more heterogeneous group, given the wealth of their habitats and their ability to live independently. Examples of accidental human pathogens include Legionella pneumophila, Bacillus anthracis, and Clostridium tetani. L. pneumophila, the causative agent of Legionnaire’s disease and Pontiac fever, is widely distributed in both natural and man-made aquatic systems (e.g., air-conditioning systems, water supplies, fountains), mostly by parasitizing protozoa. Occasionally, inhaled bacteria can also infect humans and multiply within macrophages (Harrison, 2005). B. anthracis is a sporulating microorganism commonly found in soil. It mainly infects herbivores, but in rare cases it also infects humans. The spores can germinate in wounds, after ingestion, or after inhalation, and can lead to cutaneous, gastrointestinal, or pulmonary anthrax, respectively (Turnbull, 2002). Similarly, C. tetani is a soil organism but its spores can contaminate wounds, resulting in growth of toxinogenic bacteria and, eventually, spastic paralysis (Johnson, 2005a). Finally, opportunistic pathogens do not cause disease in healthy individuals. In some cases opportunistic pathogens are only able to infect weakened people, whereas in other cases they already colonize the healthy host, but provoke disease only once the host is injured or the immune defense compromised (e.g., Staphylococcus aureus). As a matter of fact, one of the most common causes for infection with opportunistic pathogens is skin damage, generally following burns, surgical wounds, and introduction of catheters or implants. Furthermore, immune suppression can result from another disease (either infectious or not), genetic predisposition, the use or abuse of drugs (e.g., immunosuppressing agents in transplant patients, chemotherapy, antibiotics, psychoactive drugs, tobacco, alcohol), malnutrition, old age, pregnancy, and stress. For instance, individuals infected with human immunodeficiency virus (HIV) or hit by diseases like lymphocytic leukemia, lymphoma, and

Pathogenic bacteria are capable of inflicting damage to the infected host, thereby causing disease. This stands in contrast to commensal and mutualistic bacteria, which do no harm or are beneficial to their host, respectively. It is important to note that the vast majority of prokaryotes interacting with humans are not pathogenic. Three general types of pathogens can be distinguished: obligate, accidental, and opportunistic. Obligate pathogens are incapable of surviving in the environment and can solely grow and divide in association with their host. Indeed, they are highly adapted to their host, from whom they obtain essential nutrients such as amino acids, nucleotides, cofactors, and fatty acids. Further, they lack several enzymes involved in energy metabolism and detoxification of reactive oxygen species. This host dependence reflects on their genome size, which is mostly small and characterized by gene loss. As a consequence, obligate pathogens are generally refractory to axenic culture, which makes them difficult to study. Further, some obligate pathogens express proteins with eukaryotic origin. Examples of intracellular obligate human pathogens are Mycobacterium leprae, the causative agent of leprosy (Cole et al., 2001), and Chlamydia trachomatis, which is responsible for trachoma and genital tract infections (Stephens et al., 1998). Other intracellular bacteria such as Rickettsia rickettsii and Rickettsia prowazekii, which provoke Rocky Mountain spotted fever and epidemic typhus respectively, can be considered obligate human pathogens as humans are their only significant reservoir and infection always leads to disease. Nevertheless, R. rickettsii and R. prowazekii can also be carried as commensals by their respective vectors, ticks and lice (Raoult & Dumler, 2005). Treponema pallidum subspecies pallidum, the bacterium which causes syphilis (Fraser et al., 1998), and Streptococcus pyogenes (Group A streptococci), which is associated with suppurative skin and mucosal infections, necrotizing fascitis, scarlet fever, and sequelae such as glomerulonephritis and rheumatic fever (Kilian, 2005), are examples of extracellular obligate human pathogens. Philippe J. Sansonetti and Andrea Puhar, Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, 75724 Paris Cedex 15, France.

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myeloid dysplasia, which cause depletion or dysfunction of immune cells, are frequently victims of opportunistic pathogens. Genetic disorders such as cystic fibrosis also increase the susceptibility to respiratory infections (Doring & Gulbins, 2009), whereas congenital conditions affecting neutrophil number or function lie at the base of high incidence to bacterial infections (Spickett, 2008). Debilitating diseases such as diabetes or renal and hepatic dysfunctions, which have higher incidence in the elderly, also strongly increase the risk of opportunistic infections. Antibiotic-induced colitis is an interesting example of opportunistic infection provoked by the use of drugs. The causative agent, Clostridium difficile, is normally outcompeted by the healthy gut flora. However, thanks to antibiotic resistance, C. difficile is able to colonize the intestine when antibiotics decimate the healthy flora (Borriello & Aktories, 2005). The best-studied opportunistic pathogens are Pseudomonas aeruginosa and S. aureus. P. aeruginosa is responsible both for acute infections, such as following burns, and for chronic respiratory infections, mostly in cystic fibrosis patients (Palleroni, 2005). Notably, recent evidence suggests that P. aeruginosa is capable of sensing physiological and immunological disturbances in the host and, as a consequence, increases its virulence (Wu et al., 2005). S. aureus is often hospital-acquired, although a significant proportion of healthy adults are asymptomatic carriers (on the skin and nares). This pathogen can cause a spectrum of disorders, including bacteremia, endocarditis, bone and joint sepsis, boils, pulmonary infections, food poisoning, and toxic shock syndrome, to name a few. In particular, S. aureus has the ability to form biofilms on prosthetic devices. Unfortunately, infections are often difficult to manage, due to resistance to many antibiotics (Peacock, 2005). As we will see in the course of this chapter, the distinction between pathogenic and commensal bacteria is not always so easily made. Even more so, many microbes show mixed traits and it is difficult to strictly classify their pathogenic relationship with the host using the three categories described above: obligate, accidental, and opportunistic. Much of this is due to the fact that host-pathogen relationships are dynamic and bacteria are continuously adapting to their habitats and cues from the environment.

SITES OF INTERACTION BETWEEN BACTERIA AND THE HOST

The human body is constantly exposed to bacteria. Microbes stably colonize the parts that act as boundaries to the environment, for instance the skin and scalp, the eyes, the oral cavity, the nasopharyngeal and upper respiratory tract, the gastrointestinal tract, the urethra, and the vagina and cervix. Particularly, microorganisms densely populate the mucous membranes that line some of these organs. By contrast, other parts such as the blood, the lymph, the nervous system, the lower respiratory tract, inner organs (with the noteworthy exception of the gastrointestinal tract, as mentioned above), bones, adipose tissue, connective tissue, and muscles are generally sterile in healthy individuals. This bacterial distribution pattern has important implications for the immunology of the various body compartments; the immune system must maintain the sterility of blood and muscles when these tissues are exposed to microbes because of a breach in the skin, while at the same time it must be able to tolerate harmless microorganisms in the gut or on the skin. In recent years, the use of 16S rRNA sequencing to detect prokaryotes in samples of human origin has expanded our grasp of the bacterial body flora beyond cultivable organisms (Turnbaugh et al., 2007).

Importantly, the different body habitats have distinct physical-chemical properties as to pH and abundance of molecular oxygen and water, which allow the presence and growth of specific types of bacteria. For example, only very few bacteria have adapted to grow in the strongly acidic pH of the stomach, notably Helicobacter pylori (Marshall & Warren, 1984; Bik et al., 2006), which chronically infects a high percentage of the world’s population and was linked to the development of ulcers. Lactobacilli are the most abundant bacteria in the vagina: this is due to secretion of glycogen that they ferment to lactic acid, thereby lowering the pH, which in turn inhibits the growth of other, potentially pathogenic, microorganisms (Fredricks et al., 2005; Hyman et al., 2005). Within the respiratory tract, strong oxygenation favors the presence of aerobic microbes, although anaerobic microorganisms can exist within biofilms in the oral cavity (Aas et al., 2005; Kroes et al., 1999). Conversely, the scarceness of molecular oxygen in the intestinal lumen permits the growth of anaerobic or facultative anaerobic microorganisms. The dominant phyla are the Firmicutes (gram-positive anaerobes) and the Bacteroidetes (gram-negative anaerobes), with far less bacteria belonging to the oxygen-adapted Proteobacteria and Actinobacteria (Eckburg et al., 2005). Finally, the rather dry environment of the skin favors its colonization by gram-positive bacteria (Grice et al., 2009), owing to the composition of the gram-positive cell wall (see The Bacterial Cell Wall and Other Pathogen-Associated Molecular Patterns, below; Fig. 1 and 2), which confers higher resistance to drying with respect to gram-negative microbes (or the cell wall deficient mycoplasmas). Besides the physical-chemical properties of the various body habitats, the availability of host cell surface molecules to which bacteria can specifically attach often is the key to successful colonization of a certain organ; this phenomenon is called tissue tropism. For example, uropathogenic Escherichia coli can belong to the innocuous fecal flora, but it expresses adhesins that bind specifically to epithelial cells of the urinary tract, where it can provoke both acute and persistent infections (Wright & Hultgren, 2006).

RESERVOIRS AND MODES OF TRANSMISSION OF BACTERIA

Bacteria infecting humans can issue from several distinct sources, which are known as reservoirs. First, microbes can inhabit humans and be transmitted from person to person. In the case of pathogens, transmission is often linked to disease (e.g., by production of infectious exudates, sneezing, and coughing). Notably, obligate human pathogens are transmitted from person to person, as this way they avoid being exposed to the environment, which would be fatal. In fact, person-to-person transmission largely requires intimate contact between the carrier and the newly infected host; this is especially true for microbes causing venereal diseases. An example is Neisseria gonorrheae, the causative agent of the sexually transmitted disease gonorrhea (Mietzner & Morse, 2005). Other bacteria that are more resistant to drying can be transmitted directly from one person to another or, after a short delay, from objects that were in contact with the carrier (so-called fomites). For instance, the respiratory pathogen Bordetella pertussis (Parton, 2005), the causative agents of whooping cough, can spread through droplets or aerosols that are produced by sneezing, coughing, and speaking, or through soiled objects like glasses and cutlery. Similarly, infection with S. aureus can occur following skin-to-skin contact or after touching contaminated objects (Peacock, 2005). Mother-to-child transmission (either during pregnancy or at birth) is a special case of person-to-person transmission

12. Overview of Bacterial Pathogens

known as vertical transmission. Bacteremia, meningitis, pneumonia, and conjunctivitis are typical neonatal infections, which can be caused by a number of pathogens, among which the most common are gram-negative enteric bacilli, Streptococcus agalactiae (Group B streptococci), and C. trachomatis (Osrin et al., 2004). Second, infectious microorganisms can originate from animals, which probably constitute the largest reservoir of human pathogens. These infections are called zoonoses. In some cases animals are immune to a certain pathogen and only act as carriers, whereas in others, animals also suffer from the disease. Both wild animals (e.g., apes, birds, bats) and domestic animals are sources of microbial pathogens. Nevertheless, livestock and pets stand out as a key reservoir of zoonotic diseases given their close contact with humans. Importantly, animals are also a major source of emerging diseases (Wolfe et al., 2007). Zoonotic diseases can be acquired during direct exposure to animals or animal droppings. For example, the causative agent of Q fever, Coxiella burnetii, is endemic in cattle, sheep, and goats and is transmitted among animals and from animals to humans through inhalation of bacteria-containing aerosols. Frequently, C. burnetii hits abattoir and farm workers (Lightfoot & Lloyd, 2005). Sometimes, animal bites or scratches can result in infection. This is the case of cat scratch disease, which develops following a wound infection with Bartonella henselae; likely caused by feces from fleas that have been feeding on infected cats, which then contaminate the cat’s claws and teeth (Breitschwerdt & Kordick, 2000). Alternatively, infection with zoonotic pathogens can occur upon ingestion of contaminated animal products such as raw milk, dairies, meat, and eggs. Infectious agents spreading through this very common route include enteropathogenic E. coli (Cheasty & Smith, 2005), Brucella spp. (Murray & Corbel, 2005), Campylobacter jejuni (Vandenberg et al., 2005), and Salmonella spp. (except S. enterica serovar Typhimurium) (Threlfall, 2005). Unfortunately, the extensive use of antibacterial agents in breeding has lead to the development of multiple antibiotic resistances in zoonotic pathogens in the last decades, which constitutes a major problem for treatment. Vectors constitute another very frequent mode of transmission of zoonotic infections, in particular, blood-sucking insects. The best-known example of disease spread by vectors is the bubonic plague: Yersinia pestis is able to infect rodents and is passed to humans through the bite of fleas that have previously fed on an infected animal. Other examples include Rickettsia spp. (Raoult & Dumler, 2005) and Borrelia burgerdorferi, the causative agent of Lyme disease (Postic, 2005). B. burgerdorferi is found in wildlife and is transmitted to humans through tick bites. Third, bacteria can derive from the environment. The most relevant sources are water, food and air, and to a lesser extent soil. However, the vast majority of microorganisms that naturally inhabit the environment are harmless to humans. There are a few “true” environmental pathogens among accidental pathogens found in soil (e.g., Clostridia and bacilli), and in freshwater (e.g., L. pneumophila [as discussed above] and Mycobacterium ulcerans, which causes necrotizing skin ulcers following skin lesions) (Portaels et al., 2009). Rather, man-made contamination of resources (e.g., water, food, air) with fecal material; rubbish; and industrial, agricultural, and domestic wastewater leads to the spread of noxious bacteria. In fact, one could picture these environmental pathogens as human or zoonotic pathogens with the capacity to survive outside of the host for a prolonged time span and subsequent water-, food-, or airborne transmission. Examples of food-borne transmission of human or animal

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intestinal pathogens are given by E. coli (Cheasty & Smith, 2005), C. jejuni (Vandenberg et al., 2005), Salmonella spp. (Threlfall, 2005), and Shigella spp. (Sansonetti, 2005). It is apparent that the transmission of environmental pathogens is strongly linked to hygiene, in particular to the control of microbial presence and growth in foods and drinking water. Hence, environmental pathogens are, by far, more widespread in developing countries and disease outbreaks are often linked to a breakdown in public health measures, as happens during natural catastrophes and wars. Vibrio cholerae illustrates this case. This bacterium is a normal inhabitant of marine, estuarine, and sometimes freshwater environments, but cholera outbreaks are seldom given the high infectious dose. The lack of potable water in poor areas of the world, especially during summer months and catastrophes, can result in high bacterial counts in water and subsequent infection of individuals. In case of an epidemic, propagation is accelerated by the contamination of water with infected stool (Johnson, 2005b).

THE BACTERIAL CELL WALL AND OTHER PATHOGEN-ASSOCIATED MOLECULAR PATTERNS

The first molecules of bacterial origin that the host immune system encounters during an infection are normally components of the cell wall, which are shed by growing or dying microbes. Importantly, gram-positive bacteria (Fig. 1) and gram-negative bacteria (Fig. 2) have distinct cell wall structures (Madigan, 2000). Lipopolysaccharide (LPS) and its lipidic portion, lipid A (endotoxin) in gram-negative bacteria, techoic and lipotechoic acids in gram-positive bacteria, and peptidoglycan (together with peptidoglycanderived subunits in both gram-negative and gram-positive bacteria) are potent activators of the innate immune system. Besides cell wall components, other typical microbial compounds such as lipoproteins, flagellin (e.g., in Salmonella spp. [Threlfall, 2005]), and non-methylated DNA are able to stimulate the innate immune system. Altogether, these “non-self” molecules are referred to as pathogen-associated molecular patterns (PAMPs) and are sensed by dedicated receptors called pattern recognition molecules (PRMs). PRMs include Toll-like receptors (TLRs) on the cell surface and on the luminal side of endocytic vesicles, and Nod-like receptors (NLRs) in the cytosol of host cells. Hence, TLRs and NLRs detect extracellular and intracellular bacteria, respectively. When activated, TLRs and NLRs together participate in the mounting of an inflammatory response (Kufer & Sansonetti, 2007). Apart from microbial compounds, endogenous, typically intracellular molecules like ATP and uric acid are released by cells following infection or trauma and convey a “danger” message to the immune system, thereby contributing to immune activation (Bours et al., 2006; Matzinger, 1994). In fact, the term PAMP is somewhat misleading, as the cell wall components of all bacteria, both harmless and noxious, are essentially the same. Indeed, both commensal and pathogenic microorganisms are sensed by PRMs. However, in spite of their overall identical cell wall architectures, slight molecular differences between commensals and pathogens can decrease or burst the immunogenicity of their cell wall components. For instance, in commensal gut Bacteroidetes lipid A is pentacylated, which makes it a poor agonist of its cognate receptor TLR4 in humans (Coats et al., 2007). Conversely, in both commensal and pathogenic Proteobacteria, colonizing the mucosa of the intestinal or respiratory tract lipid A is hexacylated and therefore strongly immunogenic (Munford & Varley, 2006).

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COURSE OF BACTERIAL INFECTIONS

The general course of a bacterial infection can be divided into several distinct steps. Infection is not equivalent to disease and, as a matter of fact, the first phases of an infection are common to commensals and pathogens. •

•

•

•

FIGURE 1 The gram-negative cell wall. In gram-negative microorganisms the cell wall is composed of two lipid membranes, the inner (IM) and the outer membrane (OM). The two membranes are separated by a peptidoglycan (PG) layer, the periplasm. The exterior leaflet of the outer membrane contains lipopolysaccharide (LPS) and porins, which mediate the passage of solutes (e.g., ions and sugars), from the extracellular milieu to the periplasm. Membrane and membrane-associated proteins are present in both the inner and the outer membrane. In pathogenic gram-negative bacteria, virulence determinants such as secretion systems are associated with the cell wall, as exemplified by the Type 3 secretion system (T3SS). The basis of the T3SS spans the entire cell wall, whereas the needle protrudes into the extracellular milieu and is able to form a connection with the host cell plasma membrane. This architecture allows the translocation of effectors across the cell wall from the bacterial cytoplasm to the host cell.

Hence, it appears that the molecular identity of PAMPs alone is not enough to unleash an immune response. What is of outmost importance is also where these PAMPs are found. It is fundamental to keep in mind that in a healthy person some compartments of the body are sterile, whereas others are colonized by bacteria (see above). The human gut furnishes a particularly intriguing example; here, the numerous and complex community of commensals must be tolerated, yet pathogens must be detected and eliminated (Sansonetti & Medzhitov, 2009). Several mechanisms down-regulate the susceptibility of the gut mucosa to LPS issuing from the intestinal lumen (Backhed & Hornef, 2003). Moreover, the gut epithelium keeps luminal microorganisms at a distance by secretion of mucins (Johansson et al., 2008) and soluble antibacterial factors (Vaishnava et al., 2008), thereby restricting the amount of LPS that diffuses towards the epithelium. As a result, commensals can thrive in the gut lumen. On the contrary, when invasive pathogens like Shigella penetrate into the intestinal mucosa, their LPS is sensed which elicits a strong inflammatory reaction (Sansonetti, 2005).

•

Exposure: The host gets in contact with one of the various bacterial reservoirs and microbes are transmitted (please refer to Reservoirs and Modes of Transmission of Bacteria, on page 156). Adherence: Sometimes bacteria need to firmly attach to epithelia or extracellular material, particularly in the case of mucosal tissue in the respiratory, gastrointestinal, and genitourinary tract, or else they would be eliminated immediately. In fact, mucosas are constantly cleaned and loosely adherent matter is removed. As opposed to commensals, pathogenic microorganisms often express adherence factors in order to colonize body surfaces that otherwise would be protected (e.g., the epithelium in the bladder and kidney). Growth: Bacteria reaching a niche that is suitable for their growth and that are able to persist there start dividing and form a new colony. Commensal bacteria can now resume their infectious cycle. Note that in the case of pathogenic bacteria, growth is frequently preceded by invasion. Invasion: Bacteria mostly colonize tissues that are in contact with the environment, including the mucosa of the gastrointestinal, respiratory, and urogenital tract. Bacterial penetration into other sites of the body normally indicates the presence of a pathogen. Extracellular bacteria can gain access to normally sterile compartments from breaches in the epithelium or by degradation of host tissues. Intracellular bacteria induce their uptake into host cells. Toxicity and spread: Once pathogens have established themselves on a certain site, they multiply and produce injurious substances, causing tissue damage and disease. On some occasions, pathogens may provoke a systemic infection by spreading from the original site of infection to other parts of the body (mostly through the blood and lymph).

VIRULENCE FACTORS

In order to cause the transmission to a new host, colonize the host (i.e., gain access to host tissues and interact with them), induce the release of nutrients, and survive the immune response pathogenic microorganisms produce a number of virulence factors, as will be outlined below (see also Table 1). Most virulence factors only act locally at the site of infection, whilst others (e.g., some exotoxins), are also transported to distal parts of the body, where they subvert host cell functions. As a matter of fact, secreted proteins are among the most important virulence factors. These factors include exotoxins (Pugsley, 1993) and effectors of Type 3 (Rosqvist et al., 1994) and Type 4 secretion systems (Odenbreit et al., 2000). Exotoxins are secreted by microbes into the extracellular milieu, may then be transported by the blood and lymph to distal sites, and then enter and intoxicate target cells. Specific toxin delivery is guaranteed by the presence of toxin receptors on the surface of target cells. In contrast, bacteria directly inject Type 3 and Type 4 secretion effector proteins into target cells, one cell at a time.

Transmission

Only a few virulence factors have been shown to directly participate in the transmission of pathogens. For example, provoking coughing or sneezing favors the transmission of airborne infections, but the action of virulence factors potentially implicated in this process is mostly only indirect.


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FIGURE 2 The gram-positive cell wall. In gram-positive microorganisms, the cell wall is composed of a lipid membrane that is surrounded by a thick peptidoglycan (PG) layer. The peptidoglycan layer is interwoven with teichoic and lipoteichoic acid, which is bound to the lipid membrane. Proteins are both associated with the lipid membrane and with the peptidoglycan layer. In pathogenic gram-positive bacteria, virulence factors such as adhesins are found in the cell wall, as exemplified by the presence of a pilus. Pili are covalently linked to peptidoglycan and extend into the extracellular milieu.

As an exception, pertussis toxin from B. pertussis is thought to contribute to the development of cough (Parton, 2005). In enteric pathogens, some toxins play a direct role in causing diarrhea, which increases the chances of both fecooral transmission and spreading into the environment. For example, cholera toxin induces the efflux of water and solutes from epithelial cells into the intestinal lumen (Johnson, 2005b).

Adhesion

Microbes can either nonspecifically or specifically attach to host tissues and the extracellular matrix. Nonspecific adhesion relies on reversible interactions between the bacterial envelope and host structures. Often, these interactions are mediated by polysaccharides that some types of bacteria secrete (the glycocalix). These polysaccharides can organize into diffuse slime layers or compact capsules. Both slime layers and capsules promote the formation of biofilms (i.e., bacterial communities that are attached to solid surfaces and are held together by extracellular material of microbial origin) (Hall-Stoodley & Stoodley, 2009). For instance, Streptococcus mutans is able to attach to the smooth surface of teeth and form biofilms thanks to the production of dextran from dietary sucrose. Accumulation of lactic acid inside the biofilm then leads to decalcification of teeth, and finally results in caries (Kilian, 2005). Specific adhesion is mediated by irreversible interactions between the bacterial envelope and host structures. These interactions generally form between bacterial adhesins and a variety of host cell surface receptors, which can be carbohydrates or proteins. The main adhesive structures in bacteria are different classes of pili and fimbriae. Many clinically relevant gram-negative and gram-positive species express pili and/or fimbriae, including uropathogenic,

enterotoxigenic, enteropathogenic, and enterohemorrhagic E. coli; Yersinia enterocolitica; Salmonella spp.; Neisseria spp.; V. cholerae; Haemophilus influenzae; B. pertussis; Clostridium perfringens; Streptococcus spp.; and Corynebacterium diphtheriae (Kline et al., 2009).

Invasion

Many virulence factors belong to this heterogeneous group. Some factors are implicated in the initial colonization of the host, whereas others participate in the dissemination of the pathogen into new tissues or into adjacent cells in the case of intracellular bacteria. A number of virulence factors implicated in colonization are enzymes that degrade host structures and therefore allow bacteria to gain access to new sites of infection. For example, S. aureus hyaluronidase (Peacock, 2005) and C. perfringens collagenase (Borriello, 2005) degrade the extracellular matrix. Some exotoxins (e.g., hemolysins or toxins that suppress protein synthesis, like diphtheria toxin) also contribute to invasion, as their action can lead to cell death, and thereby tissue destruction (Funke, 2005). Intracellular pathogens need to gain access to normally nonphagocytic host cells. For this purpose, Shigella spp. (Sansonetti, 2005) and Salmonella spp. (Threlfall, 2005) induce their uptake into host cells through injection of Type 3 secretion effectors. Listeria monocytogenes, the etiological agent of listeriosis, a serious infectious disease in pregnant women, expresses internalin A on its surface, which mediates invasion (Lecuit et al., 2001). Both uptake mechanisms lead to the formation of a bacteriumcontaining vacuole, which Shigella and L. monocytogenes lyse. The latter was found to produce a pore-forming toxin (lysteriolysin O) that mediates the rupture of the vacuolar

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TABLE 1 The main virulence mechanisms that allow the establishment of an infection are illustrated. Please note that the list of examples is not meant to be exhaustive. Transmission Excretion of bacteria

How Diarrhea Coughing, sneezing

Examples V. cholerae; Salmonella spp.; enteropathogenic, enterohemorrhagic, and enterotoxinogenic E. coli; Shigella spp.; L. monocytogenes B. pertussis

Colonization Adherence

Biofilm Adhesins

Invasion

Degrading enzymes Tissue destruction by endotoxin or exotoxins Uptake into nonphagocytic cells Intracellular dissemination Systemic dissemination

Uropathogenic E. coli; P. aeruginosa; H. influenzae; K. pneumoniae; S. mutans; S. pneumoniae; S. aureus Uropathogenic, enteropathogenic, enterohemorrhagic, and enterotoxinogenic E. coli; Y. enterocolitica; Salmonella spp.; Neisseria spp.; V. cholerae; H. pylori; H. influenzae; B. pertussis; C. perfringens; C. difficile; C. diphtheriae; S. aureus; Streptococcus spp. P. aeruginosa; Porphyromonas gingivalis; C. perfringens; Clostridium histolyticum; S. pyogenes; S. aureus; S. mutans; S. agalactiae Uropathogenic and enterotoxinogenic E. coli; Shigella dysenteriae; H. pylori; C. diphtheriae; C. perfringens; C. difficile; S. aureus; S. pneumoniae Shigella spp.; Salmonella spp.; Yersinia spp.; C. trachomatis; L. monocytogenes; R. rickettsii Shigella spp.; L. monocytogenes S. enterica serovar Typhi; T. pallidum; B. burgdorferi; C. burnettii; L. monocytogenes; B. anthracis; S. aureus; K. pneumoniae

Growth Obtaining nutrients

Siderophores, degrading enzymes, exotoxins Vesicular trafficking

P. aeruginosa; V. cholerae; E. coli; S. pyogenes; S. aureus

Alter host cell signaling Kill immune cells Biofilm, capsule

B. pertussis; Yersinia spp.; Shigella spp.; Salmonella spp.; H. pylori; B. anthracis Shigella spp.; M. ulcerans; S. aureus Uropathogenic E. coli; P. aeruginosa; H. influenzae; Klebsiella spp.; Staphylococcus spp.; Streptococcus spp.; B. anthracis Neisseria spp.; H. influenzae; S. aureus; S. pneumoniae; K. pneumoniae L. pneumophila; C. burnetii; M. tuberculosis; M. leprae C. trachomatis; Salmonella spp.

C. trachomatis; Salmonella spp.

Survival Modulation of immune system Prevent phagocytosis

Intracellular survival

Avoid opsonization Phagolysosome Vacuole

membrane and bacterial escape into the host cell cytosol (Bielecki et al., 1990). After successful colonization of the initial site of infection, some pathogens spread to other tissues or, in the case of intracellular bacteria, into adjacent cells in epithelia. Very little is known about the virulence factors that mediate dissemination of pathogens such as T. pallidum, B. burgerdorferi, or S. enterica serovar Typhimurium to other organs. Motility certainly plays a role. For intracellular pathogens this process is much better understood. For example, Shigella and L. monocytogenes exploit the propulsive force that is generated from actin polymerization at their surface to push into neighboring cells. These socalled actin comets are formed thanks to the activity of two bacterial surface proteins: IcsA in Shigella (Bernardini et al., 1989) and ActA in L. monocytogenes (Kocks et al., 1992).

Growth

As part of immune defense mechanisms, nutrients are not always readily accessible to bacteria in human tissues in order to restrict their growth. In particular, iron is not plentiful, since free iron would also be damaging to the host by catalyzing the production of toxic oxygen species. Therefore, many pathogens produce iron-chelating compounds

(siderophores) that are able to subtract iron from the eukaryotic proteins to which it is bound. Examples are pyoverdine and pyochelin in P. aeruginosa (Palleroni, 2005). Further, pathogens release enzymes to free nutrients. Again, in respiratory infections P. aeruginosa secretes a lipase, which breaks down the lipids that function as lung surfactants (Palleroni, 2005). Some virulence factors have pleiotropic effects. For instance, degrading enzymes that play a role in invasion of host tissues (see Virulence Factors, Invasion, on page 159) also represent a means to provide nutrients, as bacteria can use the breakdown products (amino acids, lipids, sugars) to fulfill their energy requirements. Similarly, membrane insertion of pore-forming toxins, in addition to the impact on cell death (see Virulence Factors, Survival, on page 161), can release nutrients from the target cell. Another example is cholera toxin (see Virulence Factors, Transmission, on page 158), whose activity leads to the efflux of utilizable nutrients into the intestinal lumen. Vacuole-dwelling intracellular bacteria have evolved special strategies to recruit nutrients to the scant vesicular environment. For example, C. trachomatis redirects trafficking between the Golgi apparatus, multivesicular bodies,


lipid droplets, and the bacteria-containing vacuole to ensure supply of lipids (Cocchiaro & Valdivia, 2009).

Survival

Microorganisms have devised a plethora of tools to survive under hostile conditions. The immune system constitutes the main threat to pathogens during an infection. The principal types of bacterial virulence factors relevant to infectious diseases will be outlined below. Many pathogens secrete immunomodulatory factors (i.e., they interfere with proper functioning of immune cells), whilst others simply kill immune cells. The list of immunomodulatory exotoxins is very long. The best studied examples of toxins affecting cellular signaling are probably the two anthrax protein toxins, named lethal factor and edema factor. Lethal factor specifically blocks MAPK-dependent signaling by cleavage of all MEK isoforms except MEK5. Edema toxin increases cytosolic cAMP levels. Together, these toxins hamper the activation and migration of a variety of immune cells, including macrophages, neutrophils, dendritic cells, and lymphocytes, thereby suppressing bacterial recognition and elimination (Turk, 2007). Type 3 secretion effectors from Yersinia spp. (Trosky et al., 2008), Shigella spp. (Sansonetti, 2005), and Salmonella spp. (Threlfall, 2005) inhibit the inflammatory response, mainly by targeting NF-kB dependent signaling. More drastically, pore forming toxins such as S. aureus a-toxin, can induce lysis of erythrocytes and death of macrophages and T lymphocytes (Peacock, 2005). Further, the Shigella Type 3 secretion effector IpaB kills infected macrophages (Sansonetti, 2005). Nonproteinaceous secreted factors can also contribute to virulence—M. ulcerans produces a macrolide toxin (mycolactone) that causes death in many different cell types, including leukocytes (Portaels et al., 2009). As discussed in the section on Virulence Factors, many microorganisms secrete polysaccharides that constitute capsules or biofilms. Besides their role in attachment to surfaces, the aim of these polysaccharides is to confer physical, chemical, and immunological protection to bacteria. Note that, exceptionally, the capsule of B. anthracis is composed of glutamic acid polymers (Turnbull, 2002). For instance, capsulated microbes are more resistant to drying, which can be crucial during the transmission from one host to another. Capsules are poorly immunogenic compared to other components of the bacterial envelope and prevent phagocytosis. Resistance to phagocytosis is a crucial property, as the chances of systemic dissemination are strongly enhanced. The most common capsule-producing pathogens encompass Streptococcus spp. (Kilian, 2005), Staphylococcus spp. (Peacock, 2005), Klebsiella spp. (Murray et al., 2005), and H. influenzae, which is associated with respiratory tract infections, otitis media and meningitis (Slack, 2005). Importantly, bacteria growing in biofilms are able to partially evade the immune response and are resistant to antibiotic treatment (Hall-Stoodley & Stoodley, 2009). These findings contribute to explain why biofilm-forming bacteria often cause chronic or recurrent infections, as is the case with E. coli (Wright & Hultgren, 2006) and Klebsiella pneumoniae (Murray et al., 2005) in urinary tract infections, P. aeruginosa in cystic fibrosis patients (Palleroni, 2005), H. influenzae (Slack, 2005) and Streptococcus pneumoniae (Kilian, 2005) in otitis media, S. aureus in rhinosinusitis (Peacock, 2005), and many more. S. aureus and a number of gram-negative organisms exemplify other survival tactics that imply modification of the bacterial envelope. S. aureus bears protein A on its surface, which has the capacity for binding immunoglobulin G in such a way that the antigen binding portion sticks out, thereby hampering opsonization and phagocytosis (Peacock, 2005). Further, the carbohydrate composition of LPS in Neisseria spp., C. jejuni, and H. pylori, mimics the structure of host glycosphingolipids, which reduces immune recognition (Moran et al., 1996).

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Some organisms such as Neisseria spp. (Mietzner, 2005), S. pneumoniae (Kilian, 2005), and H. influenzae (Slack, 2005) produce proteases that degrade immunoglobulin A, which hampers antibody-mediated immunity on the mucosa of the respiratory or urogenital tract. Besides the immune system, cellular degradation mechanisms represent a threat to intracellular bacteria. For instance, pathogens residing inside vacuoles (e.g., C. trachomatis) (Cocchiaro & Valdivia, 2009) and Salmonella spp. (Threlfall, 2005), divert endosomal routing, or else they would have to face lysosomal processing. The H. pylori protein toxin VacA illustrates a different strategy; VacA interferes with proteolytical antigen processing by altering the trafficking of late endosomal compartments in T cells, which likely assists the pathogen in hiding from the adaptive immune system (Molinari et al., 1998).

HOW ARE BACTERIAL PATHOGENS CONSTRUCTED?

Our understanding of the identity and the evolution of a pathogen has radically changed during the last decade, mainly thanks to extensive genome analyses. For instance, E. coli is a wonderful model organism to study these relationships, as this species comprises various intestinal and extraintestinal pathogens besides many commensal strains. Comparing whole-genome DNA arrays of pathogenic and nonpathogenic E. coli isolates showed that, stunningly, several virulence and fitness factors are present in both noxious and harmless strains (Dobrindt et al., 2003). This picture was later reinforced by the discovery that both extraintestinal and commensal E. coli variants, among which a widely consumed probiotic strain produce a peptide-polyketide toxin that provokes DNA double-strand breaks, and thereby cell cycle arrest (Nougayrede et al., 2006). But there are many more examples beyond E. coli. In fact, accumulating evidence indicates that a number of commensal microbes including nonpathogenic Salmonella spp., Bacteroides thetaiotamicron, and Lactobacillus casei, just like pathogenic species, are capable of down-regulating the inflammatory response by targeting the central, NF-kB dependent activation pathway (Sansonetti & Medzhitov, 2009). What drives the evolution of a pathogen and how does the mixing between commensals and pathogens occur? Bacteria evolve under the selective pressure exerted by their host or by the environment. Most types of microorganisms have a conserved core genome, which encodes housekeeping functions and therefore guarantees bacterial survival and growth in the wonted niche under normal conditions. Furthermore, they possess an accessory gene pool encoding flexible traits that enables them to adapt to changing conditions or to a different habitat. For example, under starvation, genes coding for alternative nutrient-degrading enzymes can provide a strong advantage in growth over other bacterial species. Similarly, resistance to an antibiotic ensures bacterial survival when this compound is introduced into the environment or the host (Dobrindt et al., 2004). Three distinct mechanisms of genome evolution have been recognized so far. First, mobile genetic elements, which encompass genomic islands, plasmids, phages, insertion sequences, transposons, and integrons encode or govern most of the accessory traits (Dobrindt et al., 2004). Note that genomic islands comprising virulence factors are generally called pathogenicity islands. Whilst the contribution of plasmids and bacteriophages to the transmission of virulence factors has been recognized long ago, the discovery of pathogenicity islands is relatively recent (Hacker et al., 1990). As the name suggests, the majority of genomic islands are located on the chromosome. They often have

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a distinct G1C content and codon usage with respect to the core genome, are flanked by direct repeats and associated with tRNA genes, and contain cryptic or functional mobility factors. Genomic islands, in particular, and also plasmids and phages can carry entire operons and therefore account for tremendous variations in gene pools (Dobrindt et al., 2004). Mobile genetic elements are acquired by horizontal (lateral) gene transfer (i.e., transmission between different species or strains, some of which may be pathogenic, and others not). Horizontal gene transfer might be the most significant mechanisms of genome evolution in prokaryotes and generally takes place in dense bacterial communities (Dobrindt et al., 2004). For example, in S. aureus, several chromosomally integrated sequences conferring resistance to methicillin were identified (Peacock, 2005). Further, S. flexneri has a 31 kb pathogenicity island on its giant virulence plasmid that contains all the genes necessary for the invasion of nonphagocytic host cells (Sansonetti, 2005). Also, C. diphtheriae (Funke, 2005) and V. cholerae (Johnson, 2005b) acquired diphtheria and cholera toxin respectively by integration of a phage. In addition to gene addition and exchange by horizontal gene transfer, spontaneous mutations and recombination with ensuing clonal selection continuously provide new gene variants that increase bacterial fitness (Dobrindt et al., 2004). Finally, gene loss also contributes to microbial evolution (Dobrindt et al., 2004). As discussed above, the most striking case is the emergence of obligate intracellular pathogens such as M. leprae (Cole et al., 2001) and C. trachomatis (Stephens et al., 1998), which underwent massive genome reduction. Nevertheless, loss of single genes can also be responsible for significant adaptation to new lifestyles or changing conditions. For example, in S. flexneri and enteroinvasive E. coli deletion of the locus coding for lysine decarboxilase greatly increased the virulence, as one of the enzyme’s reaction products inhibits the two enterotoxins that mediate fluid accumulation during infection (Maurelli et al., 1998). The causes and mechanisms of genetic variation account for the evolution of both commensal and pathogenic bacteria. The difference lies in the fact that in pathogens virulence factors are acquired. However, as exemplified at the beginning of this chapter, sometimes it is not easy to distinguish a true virulence factor from a feature that just increases the bacterial fitness without really harming the host. Certainly, taking into account the lifestyle of a microorganism will help to make the difference between commensals and pathogens (Wassenaar & Gaastra, 2001). Still, nowadays, a clear-cut distinction between commensal and pathogenic microorganisms appears somewhat obsolete. Rather, we should think of a continuum between commensals and pathogens, where only an accumulation of virulence traits eventually leads to the development of a virulent phenotype and disease. In the future, remixing of genetic material through horizontal gene transfer and the generation of new gene variants thanks to mutations will give rise to bacteria that are both able to adapt to changing or new habitats and that express new virulence factors—the emergence of new pathogens.

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13 Overview of Fungal Pathogens AXEL A. BRAKHAGE AnD PETER F. ZIPFEL

INTRODUCTION

through many physical barriers and survival of many host defense responses (Mavor et al., 2005). C. albicans is still the species causing the majority of infections (Table 1). C. albicans is a diploid yeast with a polymorphic morphology. It can grow as a budding yeast. Yeast cells can be induced to form chains of elongated cells designated pseudohyphae. For a long time, it was suggested that pseudohyphae represent an intermediate stage between yeast cells and true hyphae with no septal invaginations. Today, it is believed that pseudohyphae represent a distinct developmental form (Sudbery et al., 2004). Under certain conditions C. albicans can also produce chlamydospores. These spherical projections are generated from pseudohyphae. They have been rarely observed in vivo (Cole et al., 1991) and are only found under specific growth conditions. The first three morphological forms can all be observed in vivo, suggesting that no single cell type is the cause of infection. Each cell type produces a varying profile of putative virulence determinants, which are thought to aid infection in particular environments. It has been proposed that yeast cells are the predominant form for dissemination, while hyphae are important for tissue and organ invasion (Gow et al., 2002). Experimental data support this view such as when C. albicans strains were investigated which could be manipulated such that they either form yeast cells or hyphae, for example, by the production of a strain whose morphology could be regulated using the morphogenetic repressor (NRG1) (Saville et al., 2003). Similarly, other C. albicans mutants, which were morphogenetically locked, are avirulent in several infection models (Lo et al., 1997; Braun & Johnson, 1997). However, such studies did not consider the fact that properties other than morphogenesis affect survival or metabolisms of these strains. Thus, it is still a matter of debate whether the morphological states are required for virulence (Mavor et al., 2005). In addition, cells in each C. albicans colony type were shown to be varying proportions of yeast, pseudohyphae, and true hyphae (Slutsky et al., 1985). Cells also switched between “white” phase cells that produced white, smooth, hemispherical colonies, and “opaque” phase cells that generated gray, smooth, flat colonies. The cellular morphology is also affected in white-opaque switching. White-phase cells are very similar to the round budding yeast of most C. albicans. The genome of C. albicans is publicly available (www.candidagenome.org).

From the ca. 150,000 fungi known today, only about 150 have been reported to be pathogenic (Hube, 2006). In this review, we mainly focus on the most prominent group of fungi that cause life-threatening diseases, the Candida and Aspergillus species. The frequency of invasive mycoses due to opportunistic fungal pathogens has increased significantly over the past 2 decades. This increase in infections is associated with excessive morbidity and mortality and is directly related to increasing patient populations who are at risk for the development of serious fungal infections. These risk groups include individuals under immunosuppressive therapy such as patients who are undergoing solid-organ or blood and marrow transplantation (BMT); major surgery; those with AIDS, neoplastic disease, advanced age; and those born prematurely (reviewed in Pfaller & Diekema, 2004; Hohl & Feldmesser, 2007; Morschhäuser, 2010).

CANDIDA

The pathogenic Candida species represent a family of opportunistic fungi of major medical importance in terms of systemic infections of immunocompromised patients. In healthy individuals, certain Candida species including C. albicans, C. dubliniensis, C. parapsilosis, and C. glabrata belong to the normal microbial flora of skin and mucosal surfaces (e.g., the oral cavity, gastrointestinal tract, and vagina) (reviewed in Mavor et al., 2005). These commensal yeasts can be detected in up to 71% of the healthy population, depending on the methods of sample collection and the body sites (Ruhnke, 2001). As commensals, Candida species are harmless; however, if the balance of the normal flora is disrupted or the immune defense is compromised, these fungi can outgrow the mucosal flora and cause disease. By a variety of possible mechanisms, the organisms can first enter, then escape the bloodstream and invade many organs causing life-threatening systemic infection. Candida cells have a plethora of attributes which each aid the fungus’ passage Axel A. Brakhage and Peter F. Zipfel, Leibniz Institute for natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Friedrich Schiller University Jena, Department Molecular and Applied Microbiology and Department of Infection Biology, Beutenbergstrasse 11a, 07745 Jena, Germany.

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TABLE 1 Candida species isolated during invasive candidosis in Europe C. albicans

49–53%

C. parapsilosis C. glabrata C. tropicalis C. krusei Others

11–21% 10–12% 6–11% 1–9% 1–10%

Modified from Mavor et al. (2005) according to data from Viscoli et al. (1999) and Andrutis et al. (2000).

Risk factors for Candida infections include prior colonization with Candida species, HIV infection, cancer chemotherapy, neutropenia, organ transplantation, indwelling catheters and devices, previous infection, autoimmune diseases, burns, antimicrobial therapy, age (old age or premature infants), gender, abdominal surgery and perforation, polytrauma, heart disease, intensive care, pulmonology, radiotherapy, rheumatology, and any therapy involving prolonged exposure to steroid drugs (Maertens et al., 2001; Safdar et al., 2004). The increase in vulnerable patients has made Candida infections increasingly important, particularly in hospital settings.

ASPERGILLUS FUMIGATUS AND OTHER ASPERGILLUS SPECIES

Infections with fungal pathogens have emerged as an increasing risk faced by patients under continuous immunosuppression. Species of the Aspergillus family account for most of these infections and, in particular, A. fumigatus can be regarded as the most important airborne-pathogenic fungus. The genus Aspergillus contains more than 180 species (Samson, 1999), including A. fumigatus, a saprobic fungus, associated with decaying organic matter such as compost and hay. A. fumigatus plays an essential role in recycling carbon and nitrogen sources (reviewed in Tekaia & Latgé, 2005). Conidia of this fungus can be isolated nearly everywhere, from the winds of the Sahara to the snows of the Antarctic (Brakhage, 2005). A. fumigatus conidia often account for a surprisingly small fraction of the Aspergillus conidia in the environment (e.g., 0.3 % of the aerial Aspergillus conidia in a particular cancer hospital). A. fumigatus is characterized by gray-green echinulate conidia (spores), that are 2.5 mm to 3 mm in diameter, so that they can reach the lung alveoli. The same may be true for several other Aspergillus species (reviewed in Rüchel & Reichard, 1999). Conidia are produced in chains basipetally from greenish phialides, 6 to 8 mm 3 2 to 3 mm in size. Phialides are flask-shaped and confined to the apical part. Conidiophores are uniseriate (i.e., there is only a single row of cells [phialides] borne directly on broadly clavate vesicles [20–30 mm in diameter]). There are no metulae like in some other Aspergillus species, such as A. nidulans. The first stage in conidia formation is a constriction of an elongated portion of the phialide; the second stage is the development of a septum that separates the conidium from the phialide. Chains of attached conidia evolve as the cutting process continues at the tip of the phialide. The compact mass of conidia around the vesicle is called a conidial head and corresponds to the portion of a brush in which the bristles are set. At its opposite end, the conidiophore arises from a foot cell, an enlarged thick-walled cell of the segmented mycelium. The conidiophore is borne as a stalk

perpendicular to the long axis of the foot cell. A. fumigatus has smooth-walled stipes (reviewed in Brakhage & Langfelder, 2002). Recently, a teleomorph of A. fumigatus was discovered (O’Gorman et al., 2009). A. fumigatus can withstand both extremes of temperatures and pH. It is a thermophilic species—growth occurs as high as 55°C and survival is maintained at temperatures up to 70°C (reviewed in Tekaia & Latgé, 2005). Clinical isolates can be markedly abnormal and are often more floccose with less conidia (Samson, 1999). These variations have led to the description of several varieties of A. fumigatus, including A. columnaris, A. phialiseptus, A. ellipticus, and A. sclerotiorum, with distinctions being based on only slight morphological differences (reviewed in Tekaia & Latgé, 2005). A. fumigatus has some physiological characteristics that allow precise molecular analyses. The gray-green conidia of the fungus are uninucleate. The nuclei of A. fumigatus are haploid. Both characteristics enable the isolation of clones from conidia and facilitate the isolation of mutants using classical or molecular techniques (Brakhage & Langfelder, 2002). The genome of A. fumigatus has been fully sequenced (www.tigr.org). Because of their abundance in air, it is estimated that several hundred A. fumigatus conidia are continuously inhaled during routine daily activities. In immunosuppressed patients, the lung is the site of infection. DnA-fingerprinting studies showed that it is impossible to discriminate between clinical and environmental isolates of A. fumigatus, which also indicates that every strain present in the environment is potentially pathogenic if encountered by the appropriate host (reviewed in Brakhage, 2005; Tekaia & Latgé, 2005). In immunocompetent individuals, mucociliary clearance and phagocytic defense normally prevent the disease. A. fumigatus can be regarded as the primary mold pathogen. Specific diagnostics are still limited as are the possibilities of therapeutic intervention, leading to the fact that invasive aspergillosis is still associated with a high mortality rate that ranges from 30% to 90%. For example, during the past 15 years, invasive aspergillosis has become the main cause of death in patients with acute leukemia and who have had a liver transplant (reviewed in Kappe & Rimek, 1999). Other Aspergillus species are increasingly isolated from patients with invasive aspergillosis, such as A. terreus, which was therefore designated an emerging pathogen (reviewed in nucci & Marr, 2005). A. fumigatus has certain physiological characteristics that enable the fungus to avoid or suppress the residual immune system in immunocompromised patients, making it an aggressive opportunistic pathogen. Such determinants of virulence determinants and their interplay seem to distinguish A. fumigatus at least from nonpathogenic species. Furthermore, all data obtained so far suggests that the virulence of A. fumigatus is a multifactorial process. An overview about putative virulence determinants is found in recent reviews by Brakhage (2005), Rhodes and Brakhage (2006), Askew (2008), Hohl and Feldmesser (2007), Brakhage and Zipfel (2008), and Tekaia and Latgé (2005).

INTERACTION WITH THE IMMUNE SYSTEM

There is a strong and intense interplay between the host immune system, which attacks invading pathogenic yeast or fungi, and immune escape responses on the fungal site, which forms a counterstrike aiming at the inactivation of damaging host immune effector components. Both host immune defense, as well as pathogen immune evasion, utilize an immense and broad arsenal of effector mechanisms and diverse components that initiate multiple host immune reactions and fungal immune escape mechanisms (Zipfel, 2009).

13. Overview of Fungal Pathogens

The host immune response attacks invading yeast or fungi and this attack is countered by immune escape mechanisms resulting in survival of the pathogen in an immunocompetent host. In general terms, microbes and microbial yeasts are attacked, damaged, and eliminated by the host immune defense, whereas pathogens as well as pathogenic yeast have developed means to inactivate the host immune defense and, consequently, do survive and replicate in the host.

IMMUNE RECOGNITION

The host immune defense against pathogenic yeast and fungi acts in multiple layers and the first cellular defense line is mediated by the activated innate immune system, which is comprised of a large number of important components and effector cells. Macrophages and neutrophilic granulocytes play a major role in killing fungal pathogens like C. albicans and Aspergillus conidia (reviewed in Mavor et al., 2005; Brakhage, 2005). Conidia of A. fumigatus escaping alveolar macrophages grow out and are attacked by neutrophils, as are hyphae from C. albicans (COLOR PLATE 4). Polymorphonuclear leukocytes (PMns) are also able to kill resting or swollen conidia of A. fumigatus (reviewed in Tekaia & Latgé, 2005). A severely depressed immune system provides an opportunity for opportunistic fungi to invade the tissue (reviewed in Brakhage, 2005; Mavor et al., 2005). The central relevance of the immediately acting innate immune response is evident. Moreover, it is also emerging that components and effector molecules of the innate immune system activate and shape the adaptive immune responses (Romani, 2008; Palm & Medzhitov, 2009). The host uses a broad spectrum of pattern recognition receptors, which are mostly expressed on the surface of phagocytic and antigen presenting innate immune cells, including granulocytes, neutrophils, macrophages, and dendritic cells. These pattern recognition molecules of the host identify specific fungal surface molecules, most of which are expressed on the cell wall and shape the host immune response. Once activated, innate immune cells of the host

secrete effector proteins, such as chemokines and cytokines, that orchestrate the immune reactions and attract additional effector cells to the site of action. Upon phagocytosis of the fungal target and degradation in the phagosomes, antigens are processed and ultimately presented to antigen specific T lymphocytes or B cells, which then induce the adaptive immune response.

Innate Immune Recognition of Fungal Pathogens

The innate immune system is important to sense and respond to fungal infections. Multiple host innate immune and pattern recognition receptors recognize fungal pathogens such as C. albicans, A. fumigatus, and Cryptococcus neoformans.

Host Pattern Recognition Receptors Sensing Human-Pathogenic Yeast

Innate immune cells use pattern recognition receptors that react and recognize specific moieties and structures on the yeast or fungal surface, including carbohydrates integrated into the fungal cell wall. This binding and interaction induce a direct cellular reaction. Several of such pattern recognition receptors have been identified which sense, respond, and mediate the host immune reaction against invading microbes. However, until now, it had been unclear how exactly pathogenic yeast or fungi interfere, control, and inhibit this host immune attack. Human pattern recognition receptors sensing yeast and fungal cell wall components (e.g., of C. albicans and A. fumigatus) include TLRs (Toll-like receptors), Galactin 3, DC-Sign (C-type lectin receptors), dendritic cell specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non integrin), Dectin 1, SCARF1, and CD36 (Table 2).

TLRs

TLRs—in particular, TLR2 and TLR4—are expressed on human macrophages and mediate immune response to both C. albicans and A. fumigatus as well as C. neoformans. These important receptors recognize components on the surface of a pathogen including cell wall associated molecules and sugar moieties such as mannan. Apparently, TLR2 and TLR4

TABLE 2 Pattern recognition receptors on the surface of human phagoycytic cells reacting with fungal cell wall componentsa Yeast

Fungal ligand

Host receptor

Phospholipomannan Zymosan (b-glycan and mannan) a-linked mannose

TLR2

b-1,2 mannosides

Galactin-3

a-linked mannose

DC-SIGn

b-glycan

Dectin (C-type lectin receptor)

Candida albcians, C. neoformans

b-glycan

SCARF1

C. albcians, C. neoformans

b-glycan

CD36

C. albicans

pH regulated antigen (Pra1)

CR3 (CD35)

Several fungi

167

Function Uptake and endocytosis of yeast cells

TLR4

TLR-dependent and TLR-independent signalling

a Multiple innate immune effector proteins are expressed on the surface of human phagocytic cells that specifically sense and bind surface components of human pathogenic fungi and act as pattern recognition receptors. b-glycan is the major component carbohydrate of the fungal cell wall and is recognized by multiple host receptors.

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THE PATHOGENS

have the ability to differentiate between C. albicans yeast cells and C. albicans hyphae. Candida cells are recognized by both TLR4 and by TLR2, but hyphae are only recognized by TLR2 (Trinchieri & Sher, 2007). TLR2 interacts with the glycolipid, phospholipomannan, and TLR4 recognizes linked mannose units from C. albicans. TLR2 and TLR4 together mediate the cellular response to yeast cells forming a proinflammatory milieu due to generation of the cytokines TnF-a and IFn-g. In contrast, TLR2 alone mediates response to hyphae, triggers induction of anti-inflammatory cytokines like IL-10, and induces anti-inflammatory effects.

CD36 define an evolutionarily conserved pathway for the innate sensing of fungal pathogens (Means et al., 2009). One important future aspect on host recognition of fungi is to identify and characterize the exact function of the known pattern recognition receptors and their interplay and cooperation in directing the immune response. In addition, the functional role of additional pattern recognition receptors, such as lectins (e.g., pentraxin-3 and members of the collectin family), surfactant protein A, and surfactant protein D, in host immune defense against fungi needs to be defined in detail (Brummer & Stevens, 2009).

Galactin-3

Complement

Galactin-3 is another important pattern recognition receptor that binds pathogen-specific oligosaccharides, delivers antimicrobial activity, and directs fungicidal activity to opportunistic fungal pathogens. Galactin-3 is expressed on the surface of human macrophages, immature dendritic and epithelial cells, and binds b-1,2-linked oligomannans on the Candida surface. Galactin-3, which lacks a classical signal peptide, is processed via a nonclassical secretion mechanism and is present on the cell surface and in the surrounding medium. This molecule binds to multivalent saccharide ligands on the cell surface and also to extracellular matrix components. Galactin-3 mediates binding to fungi, such as C. albicans b-1,2-linked oligomannosides, but not to Saccharomyces cerevisiae. This pattern recognition molecule induces direct antifungal activity and can also act in combination with TLR2 (Jouault et al., 2006).

DC-SIGN

DC-SIGn (dendritic cell-specific ICAM-3 grabbing nonintegrin) and SIGnR1 (SIGn-related 1) represent mannanbinding receptors, which contribute to the recognition of the human pathogen C. albicans (Taylor et al., 2004).

Dectin

Dectin is one central pattern recognition receptor for recognition and innate immune response to fungal infections, and this surface protein plays a major role in antifungal immune defense (Herre et al., 2004b; Luther et al., 2007). Dectin-1 is expressed on the surface of macrophages and other innate immune cells, such as dendritic cells and neutrophils. Upon ligand binding, tyrosine residues in the cytoplasmic tail contained in ITAMs (immunoreceptor tyrosine-based activation motifs) contribute to signaling. Dectin-1 and TLRs cooperate in immune recognition and signaling, which then results in the induction of proinflammatory cytokines and the production of reactive oxygen species. Dectin-1 is the principal C-type lectin, which recognizes fungal b-glucan, the major structural component of the Candida and Aspergillus cell wall. Dectin-1 is a type II transmembrane protein with a C-type lectin-like carbohydrate recognition region linked to a transmembrane region that is expressed on the surface of human phagocytes (e.g., monocytes or macrophages), dendritic cells, and also a subset of T cells (Herre et al., 2004a). Upon binding and internalization of b-glucans on the yeast surface, Dectin-1 generates intracellular signals via the nF-kB signaling pathway and induces an immune response via production of proinflammatory cytokines. Apparently host cells use multiple surface receptors to sense pathogenic fungi. Two additional innate immune pattern recognition receptors, the scavenger receptors SCARF1 and CD36, have recently been identified. They were shown to mediate host defense against C. albicans and C. neoformans. SCARF1 and CD36 bind to b-glycan moieties on the surface of the fungi and this interaction mediates cytokine production by macrophages. Both SCARF1 and

Complement is another major defense line of innate immunity and actually represents an intrinsic self-activated and controlled pattern recognition system of the host (Zipfel & Skerka, 2009). This general and broadly acting immune surveillance system attacks any invading microbe, including fungi, immediately upon contact with human body fluids and plasma. Intrinsic activation of this defense system via the alternative pathway occurs in a random continuous and nondiscriminatory manner. The alternative pathway of complement acts as a true pattern recognition system, as during the initial phase, this pathway does not discriminate between “self” and “foreign.” Thus, it is activated on both self-surfaces and dangerous foreign surfaces (Zipfel et al., 2007). However, the cascade is regulated at a second phase. If activation proceeds and is not controlled or regulated, the reaction is amplified, and new activation products are generated and deposited in the form of C3b and C5b on the surface of a foreign infectious particle. Consequently, the particle is marked for recognition and damage. Opsonization with these activation split products C3b and C5b on the surface of the target allows recognition by host phagocytic cells, particularly via complement receptors in the form of CR1 (CD35), CR2 (CD21), CR3 (CD11b, CD18), CR4 (CD11x-CD18), and CRiG. In addition, the activated complement cascade generates powerful anaphylactic fragments in the form of C3a and C5a, which recruit additional immune effector cells to the site of infection. These then further direct and coordinate the host immune response and also the adaptive response by directing T- and B-cell function (Kemper & Atkinson, 2007). The same activation-split products (in particular C3a and C4a) also display antimicrobial activity (Sonesson et al., 2007). Similarly, the activated cascade generates surface damaging molecules such as the terminal complement complex TCC.

IMMUNE EVASION AND COMPLEMENT ESCAPE OF PATHOGENIC FUNGI Immune Evasion

In order to survive within an immunocompetent host, any fungal pathogen has to control each single host defense reaction and inactivate most or all of the toxic and damaging effects of newly generated host immune effector proteins. The understanding of the multiple layers of host immune defense against fungal pathogens has substantially increased during the last years. For example, it was recently shown that in A. fumigatus, surface hydrophobin prevents immune system recognition of conidia (Vishukumar et al., 2009). At the same time, a more detailed understanding on the fungal immune escape and also of fungal proteins mediating such an immune escape has emerged (Fig. 1).

Fungal Complement Evasion

Apparently all pathogenic fungi, similar to other pathogenic microbes, have found means to evade a host complement attack. C. albicans, A. fumigatus, and also the dermatophyte

13. Overview of Fungal Pathogens

169

FIGURE 1 Immune escape strategies used by human pathogenic fungi. Human pathogenic fungi use similar and related immune escape strategies as shown here for C. albicans. Human pathogenic fungi produce surface proteins that bind host plasma proteins such as plasminogen, complement Factor H, and Factor H like protein (FHL-1). Attached to the fungal surface, host plasminogen can be activated to the active protease that degrades IgG and ECM components. In addition, attached host complement regulators Factor H and FHL-1 control surface opsonization with C3 activation products, thereby resisting phagocytosis and cell lysis. Moreover, human pathogenic fungi secrete proteases like SAPs (secreted aspartyl proteases), which degrade host immune effector proteins and thus aid in immune evasion. The thick cell wall provides an additional mechanical shield, which protects the vulnerable fungal membrane from activated host immune effector compounds.

Arthroderma benhamiae bind soluble host complement regulators to their surface (Meri et al., 2004; Behnsen et al., 2008; Vogl et al., 2008). The picture that is emerging is that human pathogenic fungi express surface receptors that bind and acquire host complement regulators, such as Factor H, FHL1, CFHR1, C4BP, and also the protease plasminogen. Four complement regulator acquiring surface proteins (CRASPs) have been identified from C. albicans and two CRASPs have been identified from A. fumigatus. Candida CaCRASP1, also termed phosphoglycerate mutase (gmp1) and CaCRASP2 (pH-regulated antigen, Pra1), were cloned, recombinantly expressed, and specific antisera generated. Both Candida proteins are expressed on the fungal surface. CRASP1/gpm1 is a moonlighting protein located in the cytoplasm and on the cell surface. Cytoplasmic CRASP1/gpm1 controls the glycolytic pathway, and on the cell surface it acts as host regulator binding protein (Poltermann et al., 2007). Candida CRASP2 binds multiple host immune regulators (Luo et al., 2009). The protein is also released and interacts with human monocyte integrin receptor CR3 (CD11b/CD18). Thus, it mediates a cellular response from these human monocytes (Soloviev et al., 2007).

PLASMINOGEN BINDING TO HUMAN-PATHOGENIC FUNGI

Pathogenic fungi similar to numerous other pathogens acquire host proteases in the form of plasminogen and attach plasminogen to their surface. The inactive protease plasminogen

is activated either by pathogen-encoded proteases or by the host activators, urokinase plasminogen activator (uPA) or tissue type PA (tPA). Activated plasmin at the yeast surface displays proteolytic activity (Fox & Smulian, 2001). Attachment of plasminogen to the surface of a pathogenic fungus has been reported for C. albicans, A. fumigatus, and Pneumocystis carinii. For C. albicans, nine different plasminogen-binding proteins have been described: seven proteins by a proteome approach and two additional by studies based on direct interaction (Crowe et al., 2003; Luo et al., 2009). These Candida plasminogen-binding proteins include CaCRASP-1 and CRASP-2, as well as alcohol dehydrogenase, thioredoxin peroxidase, catalase, transcription elongation factor, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, and fructose bisphosphate aldolase. Plasminogen-binding proteins of A. fumigatus have not been cloned yet (Behnsen et al., 2008). For the opportunistic pathogen P. carinii, enolase was identified as a plasminogenbinding protein (Fox & Smulian, 2001). Attached to the surface of the pathogenic fungi, plasminogen can be activated by the host proteases uPA and tPA and proteolytic active plasmin is formed, which not only cleaves extracellular matrix components (ECM) like fibrinogen but also aids in the inactivation of immunoglobulins. Thus, surface attached and activated plasmin modulates the immune response by inactivating antigen specific IgGs and aids in tissue integration and extravasation by degrading ECM components and proteins.

170

THE PATHOGENS

SECRETED ASPARTYL PROTEASES

Human pathogenic fungi like C. albicans secrete proteases such as aspartyl proteases, which assist and direct immune evasion. For C. albicans, 10 related proteases that form the secreted aspartyl protease (SAP) family have been identified. SAPs are associated with infection and each of the 10 secreted aspartyl proteins (SAP1–SAP10) is expressed during infection; however, they are expressed at different times and at different levels (naglik et al., 2008). C. albicans SAPs degrade a variety of host defense proteins such as lactoferrin and immunoglobulins, ECM components as well as surface proteins such as keratin, collagen, vimentin, fibronectin, laminin mucin, and E cadherin (Hube, 1996; Villar et al., 2005). Furthermore, these proteases damage host structures and components and facilitate tissue penetration by hyphae and infection of host cells. They also act on components of the host immune system. SAPs have additional functions in infection as they degrade and inactivate salivary antimicrobial peptides (Meiller et al., 2009). SAPs also directly interfere with the host complement. SAP1, SAP2, and SAP3 cleave the human complement proteins C3b, C4b, and C5b (Gropp et al., 2009). This inactivation of central components blocks complement activation, the release of anaphylactic peptides, and the formation of the terminal complement complex. The secreted SAPs form an additional, second layer of innate immune defense. Subsequent to recruitment of the host complement regulators, C. albicans secretes endogenous proteases that cleave and inactivate host complement.

FUNGAL IMMUNE MODULATORS

Pathogenic fungi have the ability to modulate the host immune response since proteins present on the surface and released proteins bind to host immune receptors. For example, the pH-regulated antigen 1, which is released by C. albicans cells and hyphae, binds to the human integrin receptor CR3(aMb2). Once bound to the human receptor, this fungal protein prevents immune attack and neutrophil-mediated response (Soloviev et al., 2007).

ADAPTIVE IMMUNE RECOGNITION

Human pathogenic fungi are also sensed and recognized by the adaptive immune system. T cells and antigen-mediated response are also important for defense against fungal pathogens (Romani, 2008).

CONCLUSIONS

A large number of host pattern recognition molecules have been identified which sense fungal pathogens and at the same time multiple fungal proteins that bind and block host immune attack. One striking feature of the various fungal proteins is that single fungal proteins bind multiple host ligands and serve multiple functions not only in immune escape but also beyond. The large number of such immune evasion proteins and the simultaneous expression of multiple proteins by one single fungal pathogen already indicate the complexity of fungal immune escape. In addition, these examples show that immune interaction (i.e., immune control of the host or immune escape of the fungal pathogen) occurs on multiple levels and layers. Therefore, the new concept of a virulence repertoire versus single virulence determinants that is currently emerging indicates the complexity of the interaction and the multiple reactions occurring at the pathogen host interface.

Judith Behnsen is gratefully acknowledged for providing critical discussions. Research in the authors’ laboratories was supported by the Deutsche Forschungsgemeinschaft (Priority Program 1160, Excellence Graduate Schools, Jena School for Microbial Communication, and the International Leibniz Research School for Microbial and Biomolecular Interactions), the ERA-NET PathoGenoMics, the European Union, and the Pakt für Forschung und Innovation of the BMBF, the Leibniz Association, the Thuringian Ministry of Cultural Affairs, and the HKI and the Grundlagenfond of the HKI.

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14 Prionoses and the Immune System JÜRGEN A. RICHT AND ALAN YOUNG

INTRODUCTION

localized primarily to Europe, has been directly linked to BSE infection (Aguzzi & Heikenwalder, 2006). This transient outbreak provoked a worldwide, major food crisis with severe economic consequences for the regions or countries affected (mainly Europe, the United States, Canada, and Japan). The prion agent accumulates in the central nervous system (CNS) and in secondary lymphoid organs, stimulating questions concerning the interface between prions and the immune system (Aguzzi & Heikenwalder, 2006). This chapter discusses the role of the immune system in prion pathogenesis and prion diagnostics, as well as active and passive therapeutic approaches to prion diseases. Prion infections still represent a fascinating biological phenomenon and stimulate interdisciplinary research efforts at the interface between neuroscience, biochemistry, immunology, and other related disciplines.

Transmissible spongiform encephalopathies (TSEs), prion diseases (or prionoses) are fatal neurodegenerative diseases of humans and animals (Prusiner, 1998). They are characterized by long incubation periods—ranging from months to years—prior to the onset of a progressive clinical course. The TSEs are unique in that they can have three distinct etiologies. They can occur sporadically; be inherited in an autosomal dominant fashion; or, as in the majority of ruminant TSEs, be transmitted by an infectious agent. In each case, the central pathogenic event is the misfolding of a hostencoded glycoprotein called the cellular prion protein (PrPc) into the disease-associated prion protein (PrPd). This process is a posttranslational event that involves a conformational change and has been proposed to occur by a seeding mechanism in which an aggregate or fibril of PrPd converts a PrPc monomer into a subunit of the PrPd fibril (Fig. 1) (Aguzzi & Heikenwalder, 2006). The protein-only nature of the TSE agent is one characteristic that can explain its high resistance to physical or chemical inactivation and the persistence of TSEs in the environment (Aguzzi & Heikenwalder, 2006). Prion diseases continue to be inevitably fatal, as no effective therapy is currently available. Human TSEs are relatively rare and include CreutzfeldtJakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), kuru, and fatal familial insomnia (FFI) (Aguzzi & Heikenwalder, 2006). In animals, several distinct TSE diseases are recognized, including scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and transmissible mink encephalopathy (TME) in farmed mink. Despite centuries of human exposure to scrapie, epidemiological evidence indicates that it is not transmissible to humans. In contrast, BSE, which was first detected in 1986 in the United Kingdom, was accidentally transmitted to several mammalian species, including humans, several feline species, and exotic zoo ungulates (Aguzzi & Heikenwalder, 2006). Approximately 200 cases of a new human TSE called variant Creutzfeldt-Jakob disease (vCJD) have been diagnosed since 1996. This disease,

THE CELLULAR FUNCTION OF PRPC IN THE IMMUNE SYSTEM

The cellular prion protein, PrPc is widely expressed on the surface of hematopoietic cells. Although the normal function of PrPc within the immune system is unknown, as is the potential effect of PrPc conversion on immune cells during the course of prion infection, a number of studies have demonstrated that the expression of PrPc differs between animal species and between individual leukocyte subsets (Linden et al. 2008). Nonetheless, certain expression patterns that may relate directly to function appear to be conserved. In sheep, PrPc expression on B cells tends to correlate directly with CD21 expression (Eaton et al., 2007; Young et al., unpublished). This developmentally linked expression of PrPc on B cells may, in part, explain the particular sensitivity of young animals for prion infection, as the maturing humoral immune system may become more resistant to disease (Ryder et al., 2009). With respect to cellular immunity, activation of T cells tends to correlate with increased transcription and expression of PrPc, suggesting a role for PrPc in regulating activation of the cellular immune response (Ingram et al., 2009; Linden et al., 2008). The transfection of PrPc into PrP-knockout transgenic mice resulted in an enhanced response to mitogen (Linden et al., 2008), which further supports this concept. In contrast, detailed analysis of PrP-knockout mice has suggested that PrPc deficiency results in a significant decrease in the ability of T cells

Jürgen A. Richt, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, K224B Mosier Hall, Manhattan, KS 66506-5601. Alan Young, Department of Veterinary Science, South Dakota State University, Box2175, ARW168F, Brookings, SD 57007.

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FIGURE 1 Prion replication. The infectious agent in prion diseases contains no nucleic acid and information appears to be stored in the structure of PrPd. Prion aggregates can grow by incorporating new prion protein (PrPc) and inducing it to refold into the pathological prion form, PrPd. The precise stoichiometry of the replication process remains uncertain.

to produce TH1, TH2, and TH17-associated cytokines, but not tumor necrosis factor alpha (TNF-a) or interleukin 9 (IL-9) (Ingram et al., 2009). This would imply an important role for PrPc in directing specific, but not innate, immune responses. In addition, recent data has indicated that resting CD41 CD251 FoxP31 regulatory T cells express increased constitutive levels of PrPc relative to CD41 CD252 cells (Isaacs et al., 2008). This increased expression did not correlate, however, with a change in the number or phenotype of regulatory T cells in these mice, suggesting a role for PrPc in the regulation of regulatory T-cell function and potentially also in self-tolerance (Isaacs et al., 2008). Together, these data imply a potential role for PrPc in regulating latestage effects of specific immunity of T cells, and may point to as-yet-undefined immunological deficiencies associated with disease progression in infected animals.

THE ROLE OF THE IMMUNE SYSTEM IN PRION INVASION Prion Infections in Lymphoid Tissues

Significant titers of prion infectivity can be detected in the lymphoreticular system (LRS) in most transmissible spongiform encephalopathies (Aguzzi & Heikenwalder, 2006). Exceptions are the majority of human TSEs (not vCJD) and BSEs, excluding an early and persistent invasion phase into Peyer’s patches of the cow ileum (Espinosa et al., 2007). However, many fundamental questions regarding the tropism of prions for lymphoid tissues remain unanswered. Whereas high prion titers in lymphoid organs are not accompanied by significant histopathological changes, prion infections in the central nervous system (CNS) cause characteristic neuropathological lesions with spongiform vacuolation, accumulation of abnormally folded PrPd, and neuronal cell loss accompanied by proliferation of astrocytes and microglial cells (Aguzzi & Heikenwalder, 2006). Nonetheless, the immune system remains a primary target for existing and experimental diagnostic techniques, while mechanisms and consequences associated with lymphoid PrPd accumulation remain unclear. During TSE progression in most species, PrPd and associated infectivity is initially localized to B-cell follicles of gut-associated lymphoid organs; however, PrPd rapidly spreads to include normal lymph nodes and extranodal follicles associated with chronic inflammation. B-cell follicles are key components of the lymphoid germinal center (Fig. 2), an immune structure key to the initiation and development of any adaptive immune response (Allen et al., 2007). The deposition of PrPd to these tissues appears to occur in the absence of any significant immune response or

the generation of specific immunity to the infectious agent, presumably due to the fact that the primary sequence of PrPd is identical to PrPc and therefore self-tolerant (Aguzzi & Heikenwalder, 2006). Nonetheless, PrPd appears within germinal centers significantly earlier in the disease process than in other tissues, and is therefore the target of many antemortem diagnostic techniques.

Lymphoid Organs and Prion Replication

Within lymphoid organs, B cells localize to morphologically and functionally distinct compartments depending on the status of their development. A majority of B cells can either be found in the splenic white pulp or in B-cell zones of Peyer’s patches and lymph nodes. There, B cells not only initiate and control immune responses but also provide cytokines for the maturation and maintenance of follicular dendritic cells (FDCs) (Aguzzi & Heikenwalder, 2006). The histogenesis of FDCs is still unclear, but since they express high levels of PrPc (Brown et al., 1999), they seem to be important in the pathogenesis of prion diseases. Maintenance failure for mature FDCs through the impairment of signals provided by B cells leads to the abrogation of germinal centers and to a generally disorganized microarchitecture (Aguzzi & Heikenwalder, 2006). FDCs are bifunctional cells supporting the formation and maintenance of the lymphoid microarchitecture. They also play a role in antigen trapping (i.e., in the capturing of immune complexes by Fcg receptors) and in the binding of opsonized antigens via the CD21/CD35 complement receptors (Tew et al., 2001). Most importantly, they are the cell type in the LRS where PrPd accumulates (Aguzzi & Heikenwalder, 2006).

Delivery of Antigen and PrPd to Germinal Centers

While the molecular mechanisms of PrPd targeting to the germinal centers of lymph nodes remain unclear, it is likely that normal germinal center function plays a major role in this apparent tropism of PrPd for lymphoid organs. Importantly, PrPd uptake and transport appears to be handled by these immune organs like any other protein antigen. Until recently, understanding of the generation of humoral responses and formation of germinal centers was limited to either in vitro observations of B cell function, or static observations of their in vivo generation in response to infection. As a result, the in vivo functions of these critical components of the immune response remained poorly understood; therefore, their potential involvement in prionoses was equally unclear. Due to a significant advancement in the technology of in vivo imaging and confocal microscopy, dynamic observation of the initiation of the immune response has been recently possible (Allen et al., 2007; Jenkins, 2008) (see Fig. 3A). The first step in any specific

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FIGURE 2 Accumulation of prions in germinal centers. PrPd concentrates and amplifies on the surface of follicular dendritic cells (FDCs), where it may serve as a continued source of systemic infection of the lymphoreticular system. During this process, PrPd complexes may interact both with FDCs and the surrounding B cells, although there are no documented defects in B-cell proliferation associated with prion infection. In contrast, effects of prion infection on germinal center architecture are documented, although there is no apparent dysfunction within the immune system.

immune response is the delivery of the antigen from the site of antigen exposure to the regional lymph node, either via the afferent lymphatics or, in the case of gut exposure, by direct uptake from the intestinal lumen via specialized “M cells” of the Peyer’s patches. Data clearly indicate that these mechanisms are also used by PrPd during initial infection in ruminants, which is also likely true in other species (Jeffrey et al., 2006b). In general, uptake and delivery to lymph node germinal centers is believed to occur very rapidly, with antigen bound to either complement components or preformed antibodies (Tew et al., 2001). Recent data indicates that antigen may be delivered to the germinal center of the regional lymph node as quickly as 1 hour following local injection (Pape et al., 2007). Complement activation apparently plays a key role in targeting these antigens to FDCs (Allen et al., 2007). Additional data suggests that antigens may also be transported via specialized “antigen transport cells,” although this process may take up to 24 hours following antigen inoculation (Tew et al., 2001). Detailed analysis using 2-photon confocal microscopy indicates that antigen delivery to germinal centers is a multistep process involving passive transport via the lymph to the boundaries of the germinal center where it is then “shuttled” to the FDCs by B cells (Allen et al., 2007). FDCs are specialized, nonhematopoietic cells that specifically bind antigen via the cellsurface complement receptors CD21 and CD35, and present whole antigen complexes to B cells during maturation of the immune response. Complement therefore plays a central role in both the initiation and maturation of the antibody response in mammals and is also inextricably involved in

the targeting and amplification of PrPd in germinal centers and the resulting pathogenesis of prionoses. The question remains as to the precise mechanism that transports PrPd to regional lymph nodes. Efforts to define similarities and differences between PrPd and other conventional antigen transport and delivery mechanisms remain elusive (see Fig. 3B). Experimental data have implicated two cellular components of germinal centers in TSE progression—FDCs and tingible body macrophages (TBMs) (Jeffrey et al., 2000). In addition, both M cells and migratory dendritic cells have been directly found to be involved in the translocation of PrPd from the intestinal lumen to the regional lymphoid organs (Huang et al., 2002; Jeffrey et al., 2006a). Although no detailed in vivo data similar to that observed with other antigens exist to demonstrate a specific role for complement in PrPd delivery to germinal centers, significant experimental data exist to support a role for this innate immune mechanism in PrPd accumulation. First, transgenic mice deficient in either the receptors for complement C3 components or the complement components themselves exhibit significantly elongated incubation periods without any noticeable disease (Mabbott et al., 2001). This would indicate that complement is involved in the amplification of PrPd during the asymptomatic period of disease. Second, PrPd can directly activate complement C1 to induce production of C1q, the first step in the classical complement cascade (Dumestre-Perard et al., 2007). This activation would be expected to result in C3 opsonization of PrPd fibrils and in delivery to FDCs, as has been reported for other antigens.

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FIGURE 3 Comparison of the delivery of protein antigen and PrPd to lymph node germinal centers. (A) Antigen is delivered to the follicular dendritic cells (FDCs) of germinal centers in at least one of two ways. Early delivery appears to occur as a cell-free, soluble antigen, likely complexed with complement; it is delivered passively via the afferent lymph to the germinal center, where it binds to complement receptors on the surface of FDCs. Over the first day, antigen may also be delivered by specialized “antigen transport cells” (dendritic cells) to the margins of the germinal center, where it is then shuttled to the surface of the FDCs. These cell-surface complexes then directly stimulate B-cell proliferation and maturation of the immune response, and some are released in the form of membrane vesicles or iccosomes where they may further stimulate B cells and tingible body macrophages. (B) PrPd is delivered to the FDCs of germinal centers either cell-free or associated with dendritic cells; it directly stimulates activation of the classical pathway of complement. It may then follow normal delivery mechanisms to reach the FDCs of germinal centers. Unlike other antigens, however, PrPd appears to be retained and potentially amplified on the surface of the FDCs, where it may serve as a continued reservoir of infection.

This, along with the characterized localization of PrPd to the surface of FDCs, would imply a role for complement in the delivery and uptake of PrPd in germinal centers of lymph nodes. It should also be noted that PrPd-dependent activation of C1q stimulates uptake of prions by dendritic cells. Therefore, C1q activation might result in increased dendritic-cell-dependent delivery of prions to local lymph nodes (Flores-Langarica et al., 2009). Regardless of the precise mechanism, it appears that the earliest stage of prion infection in lymphoid tissues involves mechanisms designed to activate the immune response and clear infectious PrPd from the immune system, as is the case with any other antigen. The ultimate result of either antigen exposure or PrPd exposure is uptake and delivery to FDCs in germinal centers.

Antigen Maintenance in Germinal Centers

While germinal centers contain B and T lymphocytes as well as macrophages, the central cell type that defines B-cell follicles in lymph nodes and ectopic lymphoid accumulation are FDCs (Tew et al., 2001). In many respects, the FDC appears similar to fibroblasts, leading some investigators to speculate that the FDC is derived from a fibroblast precursor

(van Nierop & de Groot, 2002). Nonetheless, FDCs can be differentiated from fibroblasts through their relatively high expression of complement receptors CD21 and CD35, as well as their exceptionally high expression of PrPc (Brown et al., 1999; Tew et al., 2001). Expression of complement receptors, particularly CD21, permits FDCs to accumulate antigen complexes on their cell surface, thereby contributing to B-cell proliferation and differentiation (Tew et al., 2001). While FDCs support B-cell maturation and function, activated B cells have been shown to be critical for the development of FDC networks (specifically, knockout mice deficient in B cells lack functional FDCs). Failure to maintain mature FDCs via B-cell signal impairment leads to the abrogation of germinal centers and to a generally disorganized microarchitecture (Aguzzi & Heikenwalder, 2006). Once bound to FDCs, opsonized immune complexes may be secreted as membrane-bound microvesicles, called iccosomes (Tew et al., 2001). These iccosomes appear to have the capacity to encapsulate and “shed” antigen from the surface of the FDC to the regional tissue, where they have been shown to be endocytosed by TBMs and regional B cells. The role of this antigen shedding is unclear, but it presumably participates in an amplification of immune


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B

FIGURE 3 (Continued)

responses through additional antigen presentation and Tcell stimulation. Intriguingly, the distribution of complement-opsonized antigen within germinal centers parallels observed histological localization of PrPd within affected lymph nodes.

PrPd Maintenance in Germinal Centers

A number of studies have identified PrPd accumulation within germinal centers to be associated with both FDC cell-surface complexes and iccosomes. Early experiments clearly demonstrated a necessary role for PrPc-expressing FDCs in the lymphoid accumulation of PrPd during disease pathogenesis (Aguzzi & Heikenwalder, 2006). The potential mechanisms whereby PrPd is apparently retained for extended periods of time on the surface of FDCs—and its potential effects upon the immune system—remain unclear. Data obtained by studying the disruption of germinal centers by HIV may be relevant to the potential ramifications of prion infection of germinal centers. Similar to some other persistent viral pathogens and PrPd, marked accumulation of HIV was noted on the surface of FDCs (Thacker et al., 2009). It now seems clear that this accumulation was not due to HIV infection of the FDCs, but rather due to the complement-mediated accumulation of HIV virus on the surface of FDCs. Nonetheless, this accumulation led to additional T-cell infection, so germinal centers therefore served as a maintenance reservoir for the virus (Thacker et al., 2009). It is likely that a similar scenario may exist in prion diseases, such that continued maintenance of PrPd on FDCs leads to a continued reservoir of infectivity for immune cells. FDCs express a high level of PrPc, the template for the conversion of PrPc to PrPd, and it is likely that PrPd replication may take place during this close association of PrPd with FDCs (Aguzzi & Heikenwalder, 2006). As a result, FDCs could

therefore serve as a continued reservoir for the production of new infectious particles and may play important role(s) in the dissemination of the prion agent throughout the immune system. Recent studies have indicated that both macrophages and FDCs are capable of localized production of the complement components C1 and C4, which may be activated by the increased PrPd to result in further deposition on FDCs in a cyclical amplification pattern.

The Role of Macrophages in Lymphoid Tropism of PrPd

M-cells, macrophages and dendritic cells may be one mechanism by which infectious prions are delivered to germinal centers (Fig. 3B), and may also play a role in maintenance of PrPd within affected germinal centers. Recent studies have implicated germinal-center resident phagocytes in the clearance of PrPd from affected tissues (Herrmann et al., 2003). Macrophages effectively phagocytose PrPd-containing iccosomes released from affected FDCs in prion-infected mice and ruminants (Herrmann et al., 2003). Elimination of macrophages was found to result in significant increases in PrPd within affected tissues, suggesting that they may play an important role in balancing PrPd amplification and clearance (Beringue et al., 2002). More recent studies have indicated that macrophages may also contribute to PrPd propagation. Although resting macrophages appear to have the capacity to engulf, digest, and clear PrPd, in vitro activation of macrophages with either CpG or LPS results in a decreased ability to phagocytose and clear PrPd (Gilch et al., 2007). Further evidence from this study also implied that activated macrophages may themselves serve as a means to increase PrPd levels by providing an additional PrPc template for conversion. The human prion peptide PrP 106-126 has been found to activate macrophages in vitro, stimulating both

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chemotaxis and TNF-a production and potentially resulting in further macrophage activation (Zhou et al., 2008). It is therefore possible to envision a continuous feedback loop where continued PrPd production is facilitated and enhanced by stimulation of local TBMs, while PrPd degradation decreases. Together, these studies imply an active role for tissue macrophages in regulating the local levels of PrPd. As with all in vitro studies, however, it should be noted that some lines of evidence imply that immune activation could also inhibit prion disease progression in vivo. First, generalized activation of the innate immune system with complete Freund’s adjuvant significantly prolongs survival in rodent models (Tal et al., 2003). Second, targeted activation of Toll-like receptor 9 (TLR-9) results in a slower developing disease, whereas animals deficient in TLR-4 demonstrated enhanced disease progression (Spinner et al., 2008). The precise role(s) of immune activation during disease progression, however, are unclear.

The Role of Blood Cells in Prion Dissemination

While a focus of prion science has been on the deposition and amplification of PrPd within lymphoid tissues, it is important to note that cells of the immune system are migratory, continually recirculating between the blood and the tissues via the lymph. Evidence suggests both indirect and direct effects of PrPd on the migratory immune system, although specific identification of any migratory leukocyte subsets directly affected with PrPd has not been possible yet. In an attempt to define specific mechanisms of disease transmission, a number of tissues have been tested for potential infectivity. Sisó and colleagues (2010) clearly demonstrated that both BSE and scrapie could be effectively transmitted between susceptible sheep by blood transfusion (Aguzzi & Heikenwalder, 2006). Mathiason and colleagues (2006) similarly demonstrated blood transmission between deer affected with Chronic Wasting Disease (Aguzzi & Heikenwalder, 2006). In rodent models, in vitro amplification technology has suggested that blood infectivity is associated with buffy-coat leukocytes, rather than within cell-free plasma (Castilla et al., 2005). Attempts to further isolate specific cell populations within the peripheral blood specifically associated with PrPd have proven futile to date; however, specific effects of TSE infection on individual leukocyte subsets have been demonstrated. Following scrapie infection in sheep, there is a measurable and significant down regulation of PrPc expression on the surface of peripheral blood B cells (Halliday et al., 2005). Furthermore, there are observed changes in B-cell migration through lymph nodes exposed to the scrapie agent, resulting in decreased numbers of lymph node CD211 lymphocytes (Eaton et al., 2009). While the significance of these observations is still unclear, one implication is that scrapie infection results in local changes to B-cell homeostasis in lymph nodes, potentially as a result of PrPd deposition and amplification in germinal centers. In both sheep and murine models, scrapie infection has been reported to result in localized changes in germinal center architecture, although these disruptions do not appear to be associated with any systemic immune deficiency (McGovern & Jeffrey, 2007). Specifically, scrapie infection of sheep appeared to result in FDC hypertrophy and the accumulation of excess immune complexes indicative of FDC dysfunction (McGovern & Jeffrey, 2007). Only direct measurement of B-cell migration and differentiation during scrapie pathogenesis will define mechanisms and potential immune consequences associated with these observed changes.

ROLE OF IMMUNE SYSTEM IN TREATMENT OF PRIONOSES

Two major long-term goals need to be achieved in order to treat prionoses: (i) post exposure prophylaxis of prion infections, and (ii) therapeutic interventions. The former will be discussed in detail, whereas the latter will only be mentioned briefly.

Prion Vaccination

Strategies for immunization against prion diseases continue to represent a field of intense efforts. Since prion-specific antibodies are not shown to be generated during the course of prion infections, artificial induction of humoral immune responses to PrPc and/or PrPd is needed (Gregoire et al., 2005). However, efficient vaccination against the prion protein is rather challenging for three major reasons. First, wild-type mice are highly self-tolerant to PrP as an immunogen, as the primary structure of PrPc and PrPd are identical (Gregoire et al., 2005). Second, a generation of antibodies specific for PrPc could potentially lead to systemic immunopathological disease due to the widespread expression of PrPc on various cells of the body. Third, PrP-specific antibodies may not cross the blood-brain barrier in therapeutic concentrations. These concerns are similar to the pursuit of adequate vaccination strategies for Alzheimer’s disease, making interdisciplinary experience in this field relevant. Initial experiments in tissue culture showed that anti-PrP serum and antigen-binding fragments (Fabs) directed against PrP were able to inhibit prion replication (Wisniewski & Sigurdsson, 2007). This initial work was then extended to laboratory animal models. To date, several successful interventions are described in the literature. First, mice transgenic for the m-chain of a PrP-specific antibody (6H4m mice) were found to resist prion disease after exposure by the peripheral route (Heppner et al., 2001). Later attempts showed that active immunization with recombinant PrPc delayed the onset of prion disease in wild-type mice, though the therapeutic effect was relatively modest and, eventually, all the mice succumbed to disease (Wisniewski & Sigurdsson, 2007). Follow-up experiments in a study using passive anti-PrP immunization confirmed the importance of the humoral antiPrP response, and showed that anti-PrP antibodies were able to prolong the incubation period significantly (Wisniewski & Sigurdsson, 2007). Using a much higher antibody dosage, disease was prevented in mice infected with PrPd when passive immunization was initiated within 1 month of exposure (Wisniewski & Sigurdsson, 2007). This kind of approach might be used immediately following accidental exposure in humans to prevent infection, but is unfeasible in animals where the time of infection is uncertain. Unfortunately, passive immunization has not been found to be effective in later stages of the incubation period, closer to the clinically symptomatic stages of prion infection. Immunotherapeutic approaches targeting the self-antigen PrP have to be designed in a way such that local immunity is induced, but systemic autoimmunity is avoided. Therefore, one immunotherapeutic approach to prevent prion infections could be mucosal immunization. Goni and colleagues (2005) evaluated this approach by expressing PrP in an attenuated Salmonella strain as a live vector for oral vaccination; this method has resulted in prevention or significant delay of prion disease in mice (Wisniewski & Sigurdsson, 2007). Salmonella vaccines have previously been used in ruminants and could be an inexpensive vaccine formulation to prevent the spread of prion disease, particularly in animals at risk. As the gut is the major route of entry for many prion diseases, mucosal immunization could be designed to induce primarily a local humoral immune response on mucosal surfaces, while avoiding systemic effects


and damage in the central nervous system. This avoidance of neuropathology is critical since a recent report describes a potentially serious obstacle to prion immunotherapy. After intracerebral injection of anti-PrP antibodies, a degeneration of hippocampal and cerebellar neurons was observed with bivalent but not monovalent IgG antibodies (Solforosi et al., 2004). This would suggest either that cross-linking of surface PrP receptors is toxic to neurons, or that local activation of complement-based immunity by divalent antibody results in local neurotoxicity. Approaches to develop active immunity have also been attempted. The above described studies led to a series of additional approaches using either bacterially expressed full-length PrP (Koller et al., 2002; Wisniewski & Sigurdsson, 2007), dimeric PrP (Gilch et al., 2003), synthetic PrP peptides or polypeptides with various adjuvants (e.g., Freund, Montanide IMS-1313, TiterMax, wild-type filamentous bacteriophage, CpG-oligonucleotides), or combinations thereof (Heppner & Aguzzi, 2004). However, anti-PrP titers described in those studies were low and, more importantly, the in vivo effect of these immunization approaches was not protective. In summary, a series of experiments suggest the feasibility of antiprion immunoprophylaxis, which could be implemented as passive immunization (transfer of antibodies) or active immunization (administration of antigens as vaccines). In general, active immunizations are effective, but more difficult due to selftolerance to PrPc. In contrast, passive immunization failed to confer protection if treatment was started after the onset of clinical symptoms; therefore, it might be a better candidate for postexposure prophylaxis rather than for therapy of TSEs.

Antiprion Therapy

Any successful therapy in prion disease should prevent the formation or block the actions of the neurotoxic PrPd compound. No efficacious therapeutic options against prion diseases are available to date. However, various drugs show possible prion-curing properties in vitro (Brazier et al., 2009). Substances such as Congo red, amphotericin B, anthracyclines, sulfated polyanions (Caughey & Raymond, 1993), porphyrins, branched polyamines (Supattapone et al., 2001), “b-sheet breakers,” the spice curcumin, RNA aptamers, siRNAs downregulating PrPc, dominant-negative mutants of PrPc as well as compounds targeting both forms of the laminin receptor (Vana et al., 2007) have all shown some effectiveness at inhibiting prion amplification in vitro (Brazier et al., 2009). Most of these molecules exert their biological effects by directly or indirectly interfering with conversion of PrPc to PrPd, however none have proven very effective for actual therapy. Drug candidates have been administered before, during, and after inoculation with prions. None of the compounds tested in animal models so far were effective when administered peripherally after onset of clinical symptoms. However, when infused intraventricularly, pentosan polysulfate at high levels extended the survival of mice and decreased PrPd deposition even when administered late after infection, while antimalarial drugs such as quinacrine showed no significant effect (Brazier et al., 2009). In humans, intraventricular application of pentosan polysulfate to CJD patients resulted in a mean survival period similar to or greater than in historical controls (Brazier et al., 2009).

THE ROLE OF THE IMMUNE SYSTEM IN PRION DIAGNOSTICS

Current diagnostic assays for TSEs are limited to postmortem analysis of CNS and lymphoid tissues for the presence of characteristic lesions, and the presence of PrPd in these

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tissues by immunohistochemistry, ELISA, and Western blot techniques (Grassi et al., 2008). The primary difficulty in the development of antemortem assays for PrPd lies in the inability to detect extremely small amounts of infectious prion protein that does not contain any nucleic acid, and can therefore not be amplified by traditional molecular techniques. In response to this need, Castilla and colleagues (2005) developed a means to amplify prion proteins using a cyclic protein misfolding amplification assay (PMCA). Using this system, PrPd below the sensitivity of current assays has been amplified from rodent, deer, and sheep blood samples, providing an opportunity to develop new and highly sensitive antemortem assays for prionoses. Recent data has indicated that this technique is rather sensitive to changes in amplification conditions, and may also result in nonspecific amplification of PrPd (Thorne & Terry, 2008). Therefore, a number of other techniques are currently in development that focus on identification of low levels of PrPd in lymphoid tissues for antemortem diagnostics. As mentioned above, B-cell follicles of germinal centers as well as those from chronic inflammatory sites acquire and apparently amplify PrPd. In response to this, lymphoid tissues harvested from the third eyelid and rectal mucosa of sheep have been shown to harbor PrPd prior to clinical disease, and provide biopsy sites appropriate for development of antemortem assays (Grassi et al., 2008). Other investigators are attempting to develop in vitro cell culture systems as an ex vivo bioassay for infectivity (Atarashi et al., 2006). These systems show promise, but might not easily be developed into a high-throughput, low-cost diagnostic for prion disease. With these issues in mind, exploratory research into novel detection systems capable of amplifying the relatively small PrPd signal obtained using conventional techniques examines the use of nonantibody molecules to selectively and specifically bind PrPd, PrPd-specific antibodies, ImmunoPCR-based techniques, and hybrid assays capable of differentiating PrPc and PrPd in vitro (Reuter et al., 2009; Safar et al., 2008). While many of these assays have proven viable in a research setting, to date, none have been demonstrated sufficiently robust for widespread commercial use. This work was supported by the NIAID-NIH PO1 AI 77774-01 “Pathogenesis, Transmission and Detection of Zoonotic Prion Diseases,” by funds from the Kansas Bioscience Authority, and by the South Dakota Agricultural Experiment Station. The authors would like to thank Stacy Lindblom-Dreis and Audree Gottlob for assistance in preparation of the manuscript. The authors would like to thank EngDesign for contributions to Figures 2 and 3, and Stacy Lindblom-Dreis and Audree Gottlob for assistance in preparation of the manuscript.

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INNATE IMMUNITY TO MICROBIAL INFECTIONS

III



15 Innate Immunity to Viruses AKIKO IWASAKI

INTRODUCTION

they can gain access to viral nucleic acids. This appears to be a well-suited strategy as most viruses localize to the endosome either during entry and uncoating, or during assembly and budding. The function of these endosomal TLRs require trafficking by the endoplasmic reticulum (ER) membrane protein, UNC-93B (Brinkmann et al., 2007; Tabeta et al., 2006). UNC-93B physically interacts with TLR3, 7, and 9 via the transmembrane domain (Brinkmann et al., 2007), and transports these TLRs from the ER to the endosome (Fig. 1). Mutations in the UNC-93b1 gene was found in two patients with severe HSV encephalitis (Casrouge et al., 2006), indicating the importance of the transport of endosomal TLRs via UNC-93B in antiviral defense.

Viruses are the most abundant infectious agents on earth and the most primitive form of life, predating us by billions of years. Because viruses rely on the host cell machinery to replicate, eukaryotes have developed a variety of means to recognize and destroy viruses within the cells. Due to the fact that viruses utilize host cell machinery to replicate, the host has the daunting task of distinguishing virus infection signatures from self-molecules. This chapter will provide an overview of the mechanisms used to sense virus infection (innate recognition), the cytokine system that evolved to rapidly induce antiviral states in neighboring cells (type I interferons), and the methods used to contain and destroy viruses (effector functions).

TLR3 Expression

PATTERN RECOGNITION RECEPTORS (PRR)

TLR3 is expressed by a variety of cells in humans and mice, including conventional DCs, B cells, fibroblasts, and epithelial cells, but not in plasmacytoid DCs (pDCs).

Toll-Like Receptors (TLRs)

TLRs are members of the PRR family, which recognize evolutionarily conserved molecular patterns associated with microorganisms, termed pathogen-associated molecular patterns (PAMPs) (Janeway, 1989). TLR triggering, following virus detection, catalyzes a complex signaling cascade initiated by the Toll/interleukin-1 receptor (TIR) domain in the cytoplasmic tail of the TLR, which ultimately leads to expression of variety of genes (Akira & Takeda, 2004). TLR stimulation bifurcates into two main pathways, culminating in the synthesis of proinflammatory cytokines (NF-kB-dependent) and antiviral cytokines, type I interferons (IFNs) (IRF-dependent). In addition, TLR signaling results in the activation of the mitogen-activated protein kinases (MAPKs). Type I IFNs, in turn, induce effector molecules that directly suppress viral replication (see below). Other cytokines efficiently recruit immune cells to sites of virus infection. In particular, TLR recognition by dendritic cells (DCs), the most potent antigen presenting cells, enables their activation and antigen-presenting function, effectively linking innate recognition to the induction of adaptive immunity (Iwasaki & Medzhitov, 2004). Viruses are recognized by a group of TLRs that reside in the endosomal membrane—namely, TLR3, 7, 8, and 9—where

Signaling

Upon PAMP recognition, TLR3 recruits TRIF via its TIR domain (Fig. 2). TRIF is responsible for activation of both NF-kB and IRF3, leading to proinflammatory cytokines and type I IFN gene expression, respectively. TRIF binds to TRAF6, which is a RING-domain E3 ligase capable of polyubiquitinylating TRAF6 and NEMO. Ubiquitinylated TRAF6 and NEMO recruit TAK1 complex (consisting of TAK1 and TAB proteins), leading to the activation of MAPK. TRIF also binds to receptor interacting protein (RIP)1 to initiate NF-kB activation. To activate the type I IFN genes, TRIF also recruits TRAF3, which engages TBK1 and subsequent activation of IRF3. IRF-3 translocates to the nucleus and binds to promoters of type I IFN genes.

Pattern Recognition

Double stranded RNA intermediates are produced during the replication cycle of most RNA viruses (except for retroviruses). DNA viruses also produce dsRNA by convergent transcription of their genomes. It has long been known that dsRNA triggers IFN production. The first TLR implicated in viral nucleic acid recognition was TLR3 (Alexopoulou et al., 2001). TLR3 responds to the artificial dsRNA mimic polyinosinic-polycytidylic acid (poly IC) and dsRNA viral

Akiko Iwasaki, Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520.

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FIGURE 1 Intracellular trafficking and processing of endosomal TLRs. TLR3/7/9 require an ER-chaperone UNC93B for transport to the Golgi and the endolysosome. Pro-form TLR9 (fulllength) is cleaved by cathepsins in the endolysosome, which is required for MyD88 recruitment and signaling. Endosomal TLRs recognize viral nucleic acids and dimerize to induce signaling, leading to the transcription of proinflammatory cytokines (via NF-kB) and type I IFNs (via IRFs).

genome (Alexopoulou et al., 2001) when provided extracellularly (Gitlin, 2006; Kato et al., 2006). However, evidence supporting TLR3-mediated recognition of authentic viral replication intermediates remains elusive. TLR3 may be involved in recognizing virus-produced dsRNA in the context of phagocytized apoptotic cells (Pichlmair & Reis e Sousa, 2007). Humans with dominant negative mutations in the tlr3 gene suffer from neonatal herpes infection (Zhang et al., 2007), indicating the importance of this receptor in protection against HSV-1 in the CNS.

TLR7/8 Expression

In humans, TLR7 is expressed in plasmacytoid DCs and B cells, while TLR8 is expressed by myeloid DCs and monocytes. In mice, TLR7 is expressed by pDCs, conventional DCs (except for CD8a1 DCs) and B cells (Iwasaki & Medzhitov, 2004). Mouse TLR8 was considered to be not functional and therefore has not been well-studied.

Signaling

TLR7, 8, and 9 are all expressed in the endosomes and share similar signaling pathways. Upon engagement, these receptors recruit MyD88 via the death domain. MyD88 subsequently interacts with the death domain of several IRAK proteins to induce both NF-kB and IRF7 activation. IRAK4 is required for activation of NF-kB pathway by interacting with TRAF6, which in turn activates the TAK1 complex, leading to activation of NF-kB and MAPK path-

ways (Fig. 2). NF-kB activation follows the recruitment of IRF5 to the MyD88 complex. On the other hand, MyD88 also recruits IRF7, which forms a signaling complex with IRAK4 and TRAF6. TRAF3 also binds MyD88 and IRAK1 to induce IRF7 activation. Unlike TLR3, TLR7 and 9 utilize IRF7 and not IRF3 for activation of type I IFN genes in pDCs. Interestingly, in conventional DCs, TLR7 and 9 utilize MyD88 and IRF1 to induce IFN-b but not IFN-a genes (Pichlmair & Reis e Sousa, 2007).

Pattern Recognition

TLR7 was originally shown to be responsible for mediating the antiviral effects induced following delivery of imidazoquinoline compounds (Hemmi et al., 2002). It is now known that TLR7 and TLR8 recognize single-stranded RNA (ssRNA) and induce innate immune responses to ssRNA-viruses. TLR7 is required for type I IFN and cytokine responses to influenza, Sendai virus (SeV), and vesicular stomatitis virus (VSV). Uridine and ribose, the defining signatures of RNA, are both necessary and sufficient for TLR7 stimulation (Pichlmair & Reis e Sousa, 2007). Furthermore, viral fusion and/or uncoating and endosomal acidification are required for TLR7-dependent recognition. Viral RNA, synthetic polyU RNA, and even nonviral, cellular RNA in the endosome is sufficient to stimulate TLR7-dependent cytokine production (Pichlmair & Reis e Sousa, 2007), indicating that any RNA localized to the endosome is able to trigger TLR7 activation. While influenza virus is recognized upon endocytosis by TLR7, other viruses such as VSV and SeV require replication in the cytosol prior to recondition

15. Innate Immunity to Viruses

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FIGURE 2 Signaling cascade of endosomal TLRs. Upon recognition of dsRNA, TLR3 utilizes TRIF to induce activation of NF-kB, MAPK, and IRF3 pathways. In contrast, TLR7 and 9 recognize viral RNA and DNA and engage MyD88 to induce NF-kB/IRF5, MAPK, and IRF7 activation. These transcription factors enter the nucleus, bind to conserved sequences in the promoter regions, and induce expression of a variety of genes required for innate and adaptive antiviral defense.

by TLR7. The latter type of viruses is recognized by TLR7 in the endosome upon delivery of cytosolic viral replication intermediates through the process of autophagy (Lee et al., 2007) (see below).

TLR9 Expression

TLR9 is expressed in pDCs and B cells in humans. Mice express TLR9 in pDCs, B cells, macrophages, and conventional DCs.

Pattern Recognition

TLR9 is located in the endosome and mediates recognition of viral DNA. Originally, bacterial DNA sequences containing hypomethylated CpG motifs were shown to activate TLR9 (Hemmi et al., 2000). More recent studies showed that TLR9 might recognize the sugar-base-backbone, 2deoxyribose, of phosphodiester DNA irrespective of the CpG

content (Haas et al., 2008). TLR9 is the principal means by which HSV-1 and HSV-2 stimulate type I IFNs in pDCs in vivo (Iwasaki & Medzhitov, 2004). Interestingly, a TLR9 molecule “retargeted” to the plasma membrane was unable to respond to viral nucleic acids, however it did respond to self-DNA, which did not stimulate wild-type TLR9 (Barton et al., 2006). These results suggest that not only is endosomal localization important to trigger TLR-viral nucleic acid interactions, but also that endosomal TLRs may limit access to nonviral nucleic acids via an active sequestering mechanism in the endosome. It has long been known that CpG stimulation depends upon acidification of the endosome. Recent studies shed light on why TLR9 signaling requires acidified endosomal compartment. While full-length and cleaved forms of TLR9 are both capable of binding ligands, only the processed form recruits MyD88 upon activation (Ewald et al., 2008; Park et al., 2008). The ectodomain of TLR9 must first be cleaved in the endolysosomes by cathepsins in order to become competent for signaling

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(Fig. 1). Since the full length TLR9 is unable to signal, this mechanism is also likely to explain how aberrant activation of TLR9 from the cell surface (e.g., upon recognition of selfDNA) is avoided.

Regulators of Endosomal TLR Signaling

To avoid damaging effects of prolonged inflammation, it is essential to terminate TLR signaling once the key innate effector genes are turned on and the process of adaptive immune priming has been initiated. Several mechanisms account for negative regulation of TLR signaling, most of which rely on negative feedback loops. Examples of these include the following (for complete description, see review by Liew et al., 2005). A20 is a deubiquitinase that is required for the termination of TLR-induced NF-kB activation. A20 removes ubiquitin moieties from TRAF6, which is required for TLR signaling. IRAK-M is expressed specifically in the monocyte/macrophage lineage and suppresses both TLR and IL-1R signaling by blocking dissociation of IRAK-4 from MyD88 and formation of IRAK-TRAF6 complexes (Kobayashi et al., 2002). Suppressor of cytokine signaling (SOCS) comprises a family of cytokine-inducible intracellular proteins involved in negative regulation of cytokine signaling (Yoshimura et al., 2007). SOCS-1 is also involved in the negative regulation of TLR signaling

by binding to the p65 subunit of NF-kB and facilitating its ubiquitinylation and degradation (Ryo et al., 2003). Both RIP1 and RIP3 are recruited to TRIF upon dsRNA recognition. While RIP1 is essential in TRIF signaling (Fig. 2), RIP3 negatively regulates the TRIF-RIP1-induced NF-kB pathway by competitively binding to TRIF (Meylan et al., 2004). These and other regulators of TLRs ensure rapid termination of signaling and return to homeostasis.

RIG-I-Like Receptors (RLRs)

RNA viruses are efficiently recognized by the cytoplasmic RNA helicases, retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) (Yoneyama & Fujita, 2009). Unlike TLRs, RLRs are expressed by most cell types. Both of these proteins recognize viral RNA through their helicase domain. Binding to viral RNA catalyzes a conformational rearrangement, which exposes a caspase activation and recruitment domain (CARD) to initiate antiviral signaling (Yoneyama & Fujita, 2009) (Fig. 3). Both RIG-I and MDA5 utilize a common adaptor molecule termed mitochondria antiviral signaling protein (MAVS), which localizes to the outer mitochondrial membrane. MAVS activates two protein kinase complexes, one consisting of FADD and caspases 8/10 and the other containing TANK, NAP1, and SINBAD (Fig. 3). The former

FIGURE 3 Viral recognition and signaling via RLRs. RIG-I recognizes 5 triphosphate RNA and/ or short dsRNA (or a panhandle RNA structure which contains both) whereas MDA5 recognizes long dsRNA. RNA binding to the helicase region enables exposure of the CARD domains of RLRs, which bind to the CARD domain of MAVS located on the outer mitochondrial membrane. MAVS recruits TRAF3 and activates IRF3 and NF-kB pathways. LGP2 also binds to dsRNA but lacks the CARD domain necessary for signaling.


activates the IKKa/IKKb/IKKg complex while the latter activates TBK1/IKKi complex, leading to the nuclear localization of transcription factors NF-kB and IRF3, respectively. An adaptor protein MITA (also known as STING) found on the outer mitochondrial membrane forms a complex with MAVS, RIG-I, and TBK1 (Ishikawa & Barber, 2008; Zhong et al., 2008). Analysis of animals deficient in the RLRs revealed important and distinct roles for these sensing molecules in innate immunity. These molecules cumulatively provide the host with the ability to recognize a large group of viral pathogens in infected cells and mediate a critical aspect of innate antiviral defense. RIG-I and MDA5 are the critical sensors of viral infection in most cell types including fibroblasts and conventional dendritic cells (DCs), while plasmacytoid DCs rely upon the TLR pathway for RNA virus recognition (Kato et al., 2005).

RIG-I

RIG-I consists of two N-terminal tandem caspase activation and recruitment domains (CARDs), a central DExH box RNA helicase/adenosine triphosphatase (ATPase) domain, and a C-terminal regulatory domain (RD) (Fig. 3). RIG-I functions as a critical PRR for a number of viruses including SeV, VSV, flu, hepatitis C virus, and Japanese encephalitis virus (Kato et al., 2006). RIG-I recognizes ssRNA that contain 5 triphosphate end—but not 5 OH or a 5-methylguanosine cap—that is longer than 23 nucleotides and contains uridine- or adenosinerich ribonucleotide sequences (Pichlmair & Reis e Sousa, 2007). RIG-I can also recognize dsRNA in the absence of 5 triphosphate in some cases with a preference for shorter length dsRNA compared to those recognized by MDA-5. Analysis of the central DExH box RNA helicase/ATPase domain of RIG-I revealed an ATP-powered dsRNA translocation activity. The CARD domains suppress translocation in the absence of 5-triphosphate, and the activation by 5-triphosphate triggers RIG-I to translocate preferentially on dsRNA in cis (Myong et al., 2009). This ATPase activity is required for RIG-I signaling, indicating that RIG-I recognizes two distinct features of in viral RNA simultaneously, triphosphate at the 5 end and dsRNA. RIG-I indeed selectively detects blunt short doublestranded 5 triphosphate RNA, which is common in the panhandle region of single-stranded RNA viral genomes (Schlee et al., 2009).

MDA5

MDA5 serves as a sensor for cytosolic synthetic dsRNA and picornaviruses (Gitlin, 2006; Kato et al., 2006). There appears to be some level of redundancy in RIG-I and MDA5 in recognition of certain viruses. Dengue virus and West Nile virus are recognized by both MDA5 and RIG-I, and either of these sensors is sufficient to induce type I IFN production (Loo et al., 2008). The precise viral ligand of MDA-5 is unknown. Comparison of synthetic and viral dsRNA revealed that MDA-5 preferentially recognizes longer dsRNA that is at least 2 kbp in length, while RIG-I is activated by shorter dsRNA (Yoneyama & Fujita, 2009). Therefore, MDA5 likely recognizes large dsRNA structures generated in the cytosol during virus infection.

LGP2

The third member of the RLR family, laboratory of genetics and physiology 2 (LGP2), displays sequence homology to the helicase domains of RIG-I and MDA5; however LGP2 lacks a CARD domain and therefore has been proposed to serve as a negative regulator of both sensors. Overexpression studies indicated that LGP2 does not activate the production of type I IFNs on its own,

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but inhibits RIG-I and MDA5-mediated signaling in a dose-dependent manner (Pichlmair & Reis e Sousa, 2007). Interestingly, studies of lgp2 knockout mice indicated that LGP2 may play both positive and negative regulatory roles in MDA5- and RIG-I mediated innate immunity (Yoneyama & Fujita, 2009). The loss of lgp2 augments type I IFN production in response to transfected poly I:C (MDA-5 agonist) or VSV infection (RIG-I agonist). In contrast, LGP2 is required for efficient antiviral responses following EMCV infection (MDA5 agonist). Therefore, LPG2 can serve as a positive or negative regulator of RLR signaling depending on the type of viral infection. The precise mechanism underlying the opposing functions of LPG2 is still unclear.

Regulators of RLR Signaling

Recent studies shed light on new molecules that are responsible for the regulation of RLR-mediated signaling (Yoneyama & Fujita, 2009). The CARD domains of RIG-I are polyubiquitinylated by tripartite motifcontaining 25 (TRIM25). Ubiquitinylation of CARDs by TRIM25 enables RIG-I to interact with the CARD on MAVS to elicit downstream signaling that leads to IFN and cytokine expression. On the other hand, RIG-I also undergoes ubiquitinylation by the ubiquitin ligase RNF125, which leads to proteasomal degradation. NLRX1 is a member of the NOD-like receptor (NLR) family (see below), which is expressed on the outer membrane of the mitochondria. NLRX1 negatively regulates MAVS signaling by disrupting virus-induced MAVS-RLR interactions. The ubiquitin ligase RNF5 mediates degradation of the adaptor protein MITA, thereby regulating RLR signaling. DUBA is a deubiquitinylating enzyme found to negatively regulate IFN production. DUBA selectively removes the lysine-63-linked polyubiquitin chains on TRAF3, thereby dissociating it from its downstream signaling complexes. siRNA knock-down of DUBA augments TLR and RLR-mediated IFN induction, indicating that DUBA acts downstream of both TLR and RLR receptors through intersecting with TRAF3. Atg5–Atg12 heterodimer, which are key molecules in autophagic pathway, can bind to RIG-I, MDA5, and MAVS to block RLR signaling (Deretic & Levine, 2009). Reactive oxygen species that accumulate on the mitochondria enhance RLR signaling. Constitutive autophagy plays an important role in regulating RLR signaling by clearance of damaged mitochondria (Deretic & Levine, 2009). These and other regulators of RLRs ensure rapid termination of signaling and a return to homeostasis.

NOD-Like Receptors (NLRs)

NLRs comprise a large family of intracellular PRRs that regulate innate immunity in response to recognition of various PAMPs and stress signals (Martinon et al., 2009). The NLR family consists of multidomain proteins that contain C-terminal LRR domain, a central NOD domain, and a N-terminal effector domain. NLR proteins can be subdivided into three subfamilies depending on the N-terminal domains: (i) the CARD-containing subfamily (NOD1, NOD2, CIITA), (ii) the PYD-containing subfamily (NLRPs), (iii) and the BIR domain containing subgroup (NAIPs). Recent studies have revealed the importance of NLRP in antiviral defense. The PYD subfamily of NLRPs consists of 14 members. Although the functions of many of the NLRPs are largely unknown, several NLRPs play a key role in the activation of caspase-1 by forming a multiprotein complex known as the inflammasome (Fig. 4). Caspase-1 is an essential mediator of inflammatory response through its capacity to cleave and generate active forms of IL-1b, IL-18,

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FIGURE 4 Viral recognition and activation of inflammasomes by NLRs. Caspase-1 is present as a pro-form in noninfected cells. Upon viral infection, TLR signaling provides signal 1, which induces transcription of pro-forms of IL-1b, IL-18, and IL-33. In addition, virally infected cells, signal 2 activates NLRP3 to form the inflammasome complex consisting of NLRP3/ASC/caspase-1, whereby pro-caspase-1 is cleaved to form active caspase-1. Another pathway leading to the activation of caspase-1 is via recognition of viral cytosolic dsDNA by AIM2, which forms a inflammasome complex consisting of AIM2/ASC and caspase-1. Release of mature forms of IL-1b, IL-18, and IL-33 requires processing by active caspase-1.

and IL-33. IL-1b and IL-18 are potent proinflammatory cytokines, and IL-33 promotes a response mediated by type 2 helper T cells (Dinarello, 2002). In vitro studies have indicated that the formation and secretion of mature IL-1b, IL-18, and IL-33 require a two-step activation mechanism: first, transcriptional and translational upregulation of the pro-forms of these cytokines are induced by TLR signaling, and a second signal that leads to the proteolytic activation of caspase-1. The latter process is mediated by the inflammasome (Fig. 4). What constitutes these signals in vivo during an infection is not known, since injection of agonists of signal 1 or signal 2 alone are sufficient to trigger IL-1b production in vivo. Inflammasome complexes that are important to antiviral defense are described below.

NLRP3-ASC Inflammasome

NLRP3, also known as NALP3/Cryopyrin/CIAS1/PYPAF1, forms a caspase-1 inflammasome (Fig. 4). The NLRP3 inflammasome can be activated by a variety of stimuli,

including endogenous signals from dying cells (i.e., uric acid), crystals (e.g., asbestos, silica, alum), as well as microbial signals such as whole bacteria, bacterial RNA, extracellular ATP, pore forming toxins, or viral infections (Martinon et al., 2009). It is unclear whether microbial ligands directly activate the NLRP3 inflammasome. Instead, the NLRP3 inflammasomes likely sense cellular stress such as disruption in membrane integrity and extracellular ATP released from stressed or damaged cells. Virus infection also results in the activation of inflammasomes. DNA viruses such as adenovirus stimulate the NLRP3-ASC-caspase-1 inflammasomes in vivo (Muruve et al., 2008). However, inflammasomes were not activated by transfection of RNA, Poly I:C, or infection with reovirus (dsRNA virus) or VSV (ssRNA virus) (Muruve et al., 2008), which indicates that RNA PAMPs are insufficient to trigger inflammasome activation. In vivo, NLRP3 deficiency resulted in increased susceptibility to a high dose flu challenge (Allen et al., 2009; Thomas et al., 2009). In addition, after a physiological sublethal dose of


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influenza infection, ASC-dependent inflammasome activation is required to elicit adaptive protective immunity to influenza virus, indicating the importance of NLRs in linking innate viral recognition to adaptive immunity (Ichinohe et al., 2009).

ANCIENT ANTIVIRAL DEFENSE MECHANISMS

AIM2-ASC Inflammasome

RNA Interference

Not all inflammasomes are activated by NLRPs. Recent studies showed that AIM2 couples dsDNA recognition to ASC-caspase-1 inflammasome (Vilaysane & Muruve, 2009). AIM2 is a HIN200 family of protein that contains a dsDNA binding domain (HIN domain) and a PYD domain, which promotes interaction with the PYD domain of ASC. AIM2 recognizes dsDNA in the cytosol and induces oligomerization of ASC and caspase-1, leading to activation of caspase-1 and cleavage of pro-forms of cytokines including IL-1b, IL-18, and IL-33 (Fig. 4). Vaccinia virus, which has a dsDNA genome, was shown to require AIM2 for recognition and activation of caspase-1 inflammasomes (Vilaysane & Muruve, 2009). It is interesting to note that different classes of dsDNA viruses are recognized by NLRP3 (adenovirus) or AIM2 (vaccinia virus) for inflammasome activation. This likely depends on the intracellular location within which the viral genome is accessible to the innate receptors. While cytosolic dsDNA can bind to AIM2 directly and activate inflammasomes, how and where exactly NLRP3 becomes activated by dsDNA is yet to be determined.

p202

Mouse HIN200 protein, p202, is a negative regulator of the AIM2 inflammasome (Vilaysane & Muruve, 2009). No orthologs of p202 have been found in humans. p202 contains two HIN domains but no PYD domain, and binds specifically to dsDNA. Unlike AIM2, p202 cannot bind ASC and therefore appears to function as a dominant negative inhibitor.

Cytosolic DNA Sensors

The existence of a TLR-independent cytoplasmic DNA sensing molecule leading to type I IFN production was suggested from studies utilizing DNA viruses and bacteria (Ishii et al., 2006; Stetson and Medzhitov, 2006). Immunostimulatory DNA (as defined by 45bp dsDNA in the cytosol) stimulates IFN-b in a sequence- and CpG-independent manner. Interestingly, ISD does not engage NK-kB, MAPK, or MAVS pathways (Stetson & Medzhitov, 2006), activating only IRF3-induced pathways. In contrast, poly(dA-dT) poly(dT-dA) dsDNA triggered type I IFNs via IRF3 and NK-kB, suggesting that this form of DNA triggers a separate sensor from ISD (Ishii & Akira, 2006). The sensor(s) for cytosolic DNA remained elusive until recently. First, a molecule called DAI (also known as DLM-1/ZBP1) was identified as a potential intracellular DNA sensing molecule (Takaoka et al., 2007). IFN induction following poly(dA-dT) poly(dT-dA) DNA treatment was abrogated by a specific interfering RNA directed against DAI, suggesting that DAI is a critical cytoplasmic DNA sensor (Takaoka et al., 2007). However, DAI is not the sole sensor of DNA in the cytosol, as DAI-knockout mice still responded to B-form DNA or plasmid DNA (Ishii et al., 2008). More recent studies showed that poly(dA-dT)poly(dT-dA) DNA is recognized by RIG-I upon transcription by RNA polymerase III (Pol III) (Ablasser et al., 2009; Chiu et al., 2009). Pol III transcribes dsRNA containing 5 triphosphate using dsDNA as a template, generating a RIG-I agonist in the cytosol. Therefore, RIG-I is the sensor for cytosolic poly(dA-dT)poly(dT-dA) dsDNA upon transcription by Pol III. However, the sensor for ISD remains to be identified.

Prior to the evolution of RLR, NLR, TLR, and type I IFNs, unicellular and multicellular eukaryotes used a more primitive form of antiviral defense to protect themselves from virus-induced diseases or death. RNAi was first identified as a potent antiviral defense mechanism in plants and subsequently in fungi, nematodes, and insects. The mechanism of RNAi involves two steps (Fig. 5A). First, viral dsRNA is recognized by Dicer-like endonuclease family, which processes it into siRNA. Second, these siRNA are incorporated into RNA-induced silencing complex (RISC), which guide the RNase enzyme AGO to complementary sequences (viral RNA) for cleavage and degradation of viral RNA. In plants and nematodes, but not in insects, this antiviral response is further amplified through a secondary wave of siRNAs generated by RNA-dependent RNA polymerases (RdRPs), which greatly increases the pool of siRNAs available to RISC. To combat antiviral RNAi responses, many plant and invertebrate viruses have evolved to encode proteins that act as suppressors of RNA silencing. Interestingly, in addition to generating RNAi, Dicer-2 was shown to trigger signaling pathways, resulting in the expression of antiviral genes in Drosophila. The latter function requires the helicase domain of Dicer-2, and given its phylogenic relation to RIG-I, a parallel role of Dicer-2 to mammalian RIG-I was discovered in the induction of antiviral genes (Deddouche et al., 2008). In mammalian cells, with the exception of germline cells, RNAi has not been found in virally infected cells. Further, mammalian cells lack the RdRPs to amplify the siRNA and fail to mount a systemic antiviral RNAi response. Therefore, with the evolution of the potent type I IFN system, the RNAi mechanism may have become obsolete for antiviral defense in mammals.

Xenophagy

Autophagy is an ancient, highly conserved pathway responsible for the lysosomal degradation of cytosolic constituents and organelles that is critical to maintaining cellular homeostasis. Recent studies have illustrated an important interplay between autophagy and the innate immune system. Signaling through innate PRRs can lead to the induction of autophagy. Clearance of intracellular pathogens via autophagy, followed by degradation in the lysosome, is referred to as xenophagy and presents an important mechanism of antiviral defense (Deretic & Levine, 2009). Upon xenophagocytosis, virions in the cytosol are engulfed into autophagosomes and degraded in the lysosome for clearance (Fig. 5B). This pathway of viral clearance was shown to be important in protection of Drosophila from VSV infection. Given that multiple human viruses, including HSV-1, HCMV, HIV-1, and gamma herpesviruses, have evolved evasion mechanisms to escape destruction by xenophagy (Deretic & Levine, 2009), this pathway likely plays an important role in viral sequestration and clearance of viruses in humans.

TYPE I INTERFERONS: KEY ANTIVIRAL CYTOKINES

Antiviral mechanisms in vertebrates are highly dependent on the action of type I IFNs. Type I IFNs are a family of cytokines that act early in the innate immune response and are key cytokines capable of inducing an antiviral state in infected and uninfected neighboring cells. In addition

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FIGURE 5 Ancient antiviral effector mechanisms. (A) In plants and invertebrates, the RNAi pathway plays a major role in the recognition and destruction of viral RNA. Viral dsRNA is recognized by Dicer, which process such RNA into short RNAi (20 bp). The processed RNAi is incorporated into the RISC complex, which serves as the guide strand. The guide strand base pairs with a complementary sequence of a viral RNA molecule and induces its cleavage by AGO, the catalytic component of the RISC complex. (B) From insects to humans, xenophagy is used to limit viral replication in the cytosol. A double membrane structure called phagophore forms around the invading or replicating virus, engulfing it into autophagosome. Encapsulated virions are degraded when autophagosomes fuse with lysosomes.

to this antiviral activity, the interferons have a role in regulating the ensuing adaptive immune response. While type II and type III IFNs also have antiviral activities, in this chapter, we will focus on the role of type I IFNs in innate antiviral defense. In humans, type I IFNs consist of 13 a-genes coding for 12 IFN-a subtypes, one b-gene encoding a single IFN-b subtype, and a single v-gene encoding one IFN-v. Type I IFNs bind to IFN-abR, which is a heterodimer of IFN-aR1 and IFN-aR2 chains. High levels of IFN-a subtypes are rapidly secreted from pDCs upon viral recognition, whereas IFN-b can be induced from most virus-infected cell types.

Type I IFN Induction

As described in detail above, stimulation of various PRRs, including TLRs (i.e., TLR3, 4, 7, 8, and 9), RLRs (i.e., RIG-I and MDA-5) and cytosolic DNA sensor(s) leads to the production of type I IFNs. Most virally infected cells trigger the cell’s intrinsic pathway of type I IFN production through activation of RLRs or DNA sensors. In contrast, pDCs recognize viral genomes within the endosome via TLR7 or TLR9 and induce robust secretion of IFN-a and IFN-b. pDCs are thought to express high constitutive levels of IRF7 and are the only cells capable of coupling TLRs to IRF7 directly, thereby leading to rapid and robust transcription of type I IFN genes.

Amplification of IFN Production by IRF7

In the cytosol of most cells, IRF3 is constitutively expressed while very low levels of IRF7 are found (Honda & Taniguchi, 2006). While IFN-a genes contain ISREs, the IFN-b gene also contains binding sites for NF-kB and activator protein 1 (AP1). IRF3 is a potent activator of the IFN-b and IFN-a4 gene but not the IFN-a genes, whereas IRF7 efficiently activates both IFN-a and IFN-b genes. Thus, upon activation of RLRs or TLR3, IFN-b and IFN-a4 production is immediately triggered by the IRF3 dimer binding to the promoter regions of these genes, while the production of other members of IFN-a occurs at a later point (i.e., upon IFN-abR-induced production of IRF7). In addition, IFN-abR signaling increases PRR expression (TLRs and RLRs), leading to further amplification of antiviral signaling pathways.

IFN-abR Signaling

Upon engagement of Type I IFN receptor complex IFN-aR1 and IFN-aR2 by IFN-a, IFN-b, or IFN-v, these receptor subunits dimerize, resulting in phosphorylation of Tyk2, which is associated with the IFN-aR1, by the Janus kinase (JAK) 2 (Fig. 6). Activated Tyk2 subsequently phosphorylates JAK1, which is coupled to the IFN-aR2 chain. Signal transducer and activator of transcription (STAT) 1 and 2 are prebound to the IFN-aR2 chain. Activated JAK1 binds


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FIGURE 6 IFN-abR signaling. Upon binding of type I IFNs, IFN-aR1, and IFN-aR2 dimerize to form an active type I IFN receptor. This induces phosphorylation of JAK1 and TYK2, resulting in phosphorylation of STAT1 and STAT2. STAT1 can form homodimers with another STAT-1 or can form a trimolecular complex between STAT2 and IRF9 (called ISGF3) and can translocate to the nucleus to induce transcription of ISGs via binding to conserved DNA sequences (GAS and ISRE).

to STAT2 and phosphorylates it, creating a binding site for STAT1, causing their dimerization and transport to the nucleus. The STAT1-2 heterodimer forms a transcription complex, ISGF3, with IRF9 to facilitate transcription of ISGs by binding to sequence motif called interferon simulated response element (ISRE). In addition, STAT1 homodimers bind to the IFN-g-activated site (GAS) sequencing motif and induce a different set of ISGs.

viridae families (Sadler & Williams, 2008). However, the role of OAS and RNase L pathway in defense against DNA viruses remain less clear. Interestingly, the cleavage products of RNase L can serve as ligands for RIG-I and MDA5, leading to amplification of RLR pathway (Malathi et al., 2007). Therefore, OAS and RNase L not only degrade viral RNA, but also provide more ligands for the RLR system, leading to induction of further IFNs and IFN-induced genes.

Antiviral Effectors Induced by IFNs

Protein Kinase R (PKR)

Even though more than 300 ISGs are transcribed as a result of type I IFN receptor signaling, the function of the majority of these genes is unknown. ISG products likely exert antiviral functions targeted at multiple stages of virus infection including attachment, fusion, entry, replication, budding, and release. Of the ones that have been studied extensively, those with defined antiviral effector properties will be discussed below.

2–5 Oligoadenylate Synthetase (OAS) and RNase L

OAS and RNase L act in concert to degrade viral RNA in the cytosol. While basal levels of OAS and RNase L are found constitutively, stimulation through the IFN-abR dramatically increases the protein levels of these molecules. Activated by dsRNA, (2–5) OAS converts ATP into ppp(A2pA)n oligomers, which activate latent RNase L. Activated RNase L degrades viral and cellular ssRNAs, inhibiting protein synthesis and viral growth. Mice deficient in RNase L suffer from increased susceptibility to RNA viruses including Picornaviridae, Reoviridae, Togaviridae, Paramyxoviridae, Orthomyxoviridae, Flaviviridae, and Retro-

PKR is a serine/threonine kinase that phosphorylates the a-subunit of eukaryotic translation initiation factor 2 a (eIF2a). PKR becomes activated through homodimerization upon binding to viral dsRNA structures via its dsRNA binding domains. This results in inhibition of translation and a decrease in total cellular and viral protein synthesis, effectively reducing viral production. In addition to its translational regulatory function, PKR has a role in signal transduction and transcriptional control through the IkB/NF-kB pathway. PKR can also mediate apoptosis, cell growth, and autophagy, all of which serve to curb viral synthesis and viral spreading within the host (Sadler & Williams, 2007). Mice genetically deficient in PKR or who express dominant negative forms of PKR are susceptible to infection with rhabdovirus, orthomyxovirus, and orthobunyavirus.

Orthomyxovirus Resistance Gene (Mx) Proteins

Mx proteins belong to a family of GTPases consisting of MxA and MxB in humans and Mx1 and Mx2 in mice (Haller et al., 2007). The Mx proteins have a large N-terminal GTPase domain, a central interacting domain (CID), and a C-terminal leucine zipper (LZ) domain. Both the CID and the LZ domain

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are required to recognize target viral structures. Viral targets of Mx proteins include orthomyxoviruses, paramyxoviruses, rhabdoviruses, togaviruses, and bunyaviruses. Remarkably, transgenic expression of human MxA in IFN-abR-deficient mice confers full resistance to otherwise fatal infection with Thogoto virus, influenza virus, VSV, LaCrosse virus, or Semliki Forest virus, indicating that this effector molecule is sufficient for protective innate defense against these viruses. The main viral target seems to be viral nucleocapsid-like structures (Haller et al., 2007). Mx proteins are thought to survey exocytic events and mediate vesicle trafficking to trap essential viral components. It is interesting to note that most inbred strains of mice harbor defective Mx1 genes. Therefore, studies using inbred mice must be interpreted with the caveat that they are Mx-1 deficient.

TRIM5a

Members of the tripartite motif (TRIM) protein family are involved in various cellular processes, including cell proliferation, differentiation, development, oncogenesis, and apoptosis (Nisole et al., 2005). There are 66 known members of TRIM proteins in human, and they are characterized by the presence of RING, B-box, and coiled-coil domains. Many of the TRIM proteins display antiviral properties, particularly against retrovirus entry and release. TRIM5a has been extensively studied as a key factor responsible for the antiretroviral activities. TRIM5a is thought to perturb the controlled uncoating of the subviral particle prior to reverse transcription by recognizing and degrading the capsid protein of retroviruses, resulting in a block of viral replication. Interestingly, TRIM5a from Old World monkeys confers potent resistance to HIV-1, but not SIV, while the human ortholog of TRIM5 is unable to specifically target HIV. Strikingly, the differential ability of human and monkey TRIM5a to restrict HIV hinges on a single amino acid, R332 (human) versus P332 (monkey) (Li et al., 2006). It is likely that HIV and SIV have evolved in their natural hosts to evade interaction with TRIM5a.

APOBEC Family

APOBEC (apolipoprotein BmRNA-editing catalytic polypeptide) proteins are a group of cytidine deaminases. Members of the APOBEC family contain either one or two catalytic deaminase domains. APOBEC becomes encapsidated into retroviral virions in producer cells. Upon viral fusion and entry into a new host cell, APOBEC deaminates cytosine residues in nascent retroviral cDNA (Harris & Liddament, 2004). The resulting uracil residues function as a template for the incorporation of adenine, which, in turn, can result in strand-specific C/G to T/A transition mutations that affect virus viability. The antiviral activity of APOBEC3G is strongly inhibited by HIV-1 Vif protein, allowing the virus to replicate virtually unimpaired in APOBEC3G-expressing host cells. Vif induces the ubiquitin-dependent degradation of some of the APOBEC proteins. However, Vif is also able to prevent encapsidation of APOBEC3G and APOBEC3F through degradationindependent mechanisms.

Tetherin and Viperin

Tetherin (also known as BST-2, PDCA-1, CD317) is a GPIanchored protein that is highly expressed upon type I IFN stimulation. Tetherin associates with lipid rafts and inhibits retrovirus particle release in the absence of Vpu (Neil et al., 2008). Vpu utilizes the beta-TrCP E3 ubiquitin ligase complex to induce endosomal trafficking events that remove tetherin from the cell surface, rendering it incapable of restricting the release of enveloped viruses (Mitchell

et al., 2009). Thus far, tetherin has been implicated in restricting the release of members of the retrovirus, filovirus, and arenavirus families. Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon inducible) is another IFN-inducible protein that is known to impair the release of influenza virus. Viperin does so by disrupting lipid rafts via suppression of the activity of farnesyl diphosphate synthase, a key enzyme in isoprenoid biosynthesis (Wang et al., 2007).

ISG15

ISG15 is an ubiquitin-like molecule capable of modifying proteins. The enzyme UBE1L (E1-like ubiquitin-activating enzyme) was shown to be the specific ISG15-activating enzyme (Sadler & Williams, 2008). Two E2 ubiquitin-conjugating enzymes, UBCH6 and UBCH8, serve as ISG15 carriers. Subsequently, two E3 ubiquitin ligases, HERC5 (homologous to the E6-associated protein C terminus [HECT] domain and RCC1-like domain containing protein 5), and TRIM25 conjugate ISG15 to protein substrates. All enzymes identified in the ISGylation pathway are coordinately induced by type I IFNs. There are over 150 targets of ISGylation that have been identified, including those involved in IFN induction (RIG-I, STAT1), and other IFN-induced antiviral effector proteins

CONCLUSIONS

Antiviral defense in vertebrates is mediated through a multipronged approach, initiated by recognition of various viral signatures in both infected cells and in uninfected professional sensors such as pDCs, leading to the secretion of type I IFNs. Type I IFNs stimulate IFN-abR and induce hundreds of genes whose products act on the invading virus to halt its replication and transmission. The host has learned to utilize viral recognition not only for antiviral innate defense, but also for activation of adaptive immunity, which provides long-term protection (covered in detail in Chapter 19 & 20). Not surprisingly, viral pathogens have evolved numerous strategies to evade all aspects of antiviral defense (covered in detail in Chapter 31). Future challenges in the field include identification and understanding of more innate antiviral effector mechanisms and to utilize such information to develop effective antiviral agents. Development of a counterpart to bacterial antibiotics is urgently needed for emerging and reemerging viral pandemics in future years to come.

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16 Natural Killer Cell Response against Viruses JOSEPH C. SUN AND LEWIS L. LANIER

INTRODUCTION TO NATURAL KILLER (NK) CELLS

mouse cytomegalovirus (MCMV) in this setting of immunodeficiency (Bukowski et al., 1985). More recently, specific subsets of NK cells in humans and mice have been shown to undergo prolific expansion during CMV infection and contribute to host protection (Dokun et al., 2001; Guma et al., 2004; Sun et al., 2009a). Over the 3 decades since the discovery of NK cells, numerous studies have conclusively demonstrated the importance of these cells in host immunity and defense against certain viral infections. NK cells have evolved a sophisticated repertoire of germline-encoded activating and inhibitory receptors to regulate their activity and ensure protection of the host against pathogens, yet also prevent deleterious autoimmune responses (Lanier, 2005). Initially following their discovery, NK cells were only considered “nonspecific” in their interactions with target cells. However, NK cells were later found to specifically recognize and kill tumor cells lacking MHC class I, while sparing the same tumors expressing MHC class I (Karre et al., 1986); seminal observations gave rise to the “missing self” hypothesis, which explained the rules by which NK cells function. The subsequent identification and characterization of inhibitory receptors on NK cells provided a mechanism whereby NK cells participate in immunosurveillance against virally infected cells that may have downregulated MHC class I expression to avoid recognition by CD81 cytotoxic T cells (CTL) (Lanier, 2008). Furthermore, many activating receptors on NK cells have been recently characterized (including several that recognize virus-derived components), leading to our understanding that activation of NK cells is stringently regulated by the engagement of inhibitory and activating receptors (Lanier, 2005). Thus, like T cells of the adaptive immune system, NK cells respond with specificity to both self and non-self (foreign) ligands. Viruses that have actively fused with cellular membranes and have injected viral proteins and genetic material into the cytosol of all somatic cells can be detected by intracellular defense mechanisms (cytosolic sensors that will be described below). Soon after viral infection, macrophages and neutrophils (and other granulocytes) provide containment by capture (via phagocytosis) and degradation of the virus particles. Afterwards, NK cells and T cells arrive and provide a more “specific” line of defense against cells that are infected (Fig. 1). In virus-infected cells, the proteosome degrades cytosolic host and viral proteins and processes

During viral infection, cells of the innate immune system such as monocytes, dendritic cells (DCs), natural killer (NK) cells, and polymorphonuclear leukocytes serve several functions: alerting the host to invading pathogens, providing early containment of pathogens, mediating antimicrobial effects against pathogens, directing the nature of the immune response, and participating in wound healing and tissue repair. Although NK cells are not the earliest cell to respond to infection at the site of injury (cells of the myeloid lineage respond within minutes to hours, whereas NK cells respond on the order of hours to days), they represent a crucial early line of defense against pathogen invasion, and are thought to bridge innate and adaptive immunity by keeping certain viruses in check until antigen-specific B and T cells are activated, recruited, and expanded over the course of days to weeks following initial infection. NK cells form a distinct subset of lymphoid cells derived from the bone marrow that possess innate immune characteristics and functions, and may have evolved specifically to provide protection against viruses (Lanier, 2008). NK cells comprise 2% to 5% of circulating leukocytes in the blood of mice, and as much as 20% of lymphocytes within human peripheral blood mononuclear cells, and are found at varying frequencies in nearly all tissues. NK cells recognize viral infection through surface receptors, leading to cell activation and initiation of effector functions (Lanier, 2005). NK cells were first identified in the 1970s for their ability to mediate cytotoxicity against tumor cell lines without prior sensitization (unlike naïve B and T cells, which require “priming”) (Kiessling et al., 1975a, 1975b). Subsequently, NK cells were observed to also mediate enhanced cytotoxicity during viral infection (Welsh, 1978) and against virally infected cells (Ching & Lopez, 1979). The deficiency of NK cells in humans or mice by genetic mutation or antibody-mediated depletion results in susceptibility against several different viruses (Biron et al., 1999; Orange, 2006). Conversely, the adoptive transfer of NK cells into newborn mice protects against a lethal challenge with Joseph C. Sun and Lewis L. Lanier, Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, CA 94143.

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FIGURE 1 Specific recognition of virally infected cells by the immune system. During viral infection of a target cell, NK cells, and CD81 cytotoxic T lymphocytes (CTL) can respond by sensing the presence of virus through various mechanisms. In the cytoplasm of infected cells, viral proteins generated from translation of viral transcripts can be processed by the proteosome into peptides. Viral peptides presented on MHC class I will be recognized by CTL expressing specific T-cell receptors (TCR). Viral transcription can also result in products that are directly expressed on the cell surface as whole proteins. NK cells expressing ligand-specific activating receptors, such as Ly49H, directly recognize these viral molecules. Lastly, viral infection can lead to cellular “stress” and the upregulation of stress-induced molecules such as members of the NKG2D family of ligands. All NK cells and subsets of CTL express the activating NKG2D receptor and will recognize the presence of viral infection via the expressing of NKG2D ligands, unless the virus evolves mechanisms to prevent expression of the NKG2D ligand proteins on the surface of infected cells.

them into peptides for presentation on MHC class I (Fig. 1). Viral peptides bound to MHC class I are recognized by the T-cell antigen receptor on CD81 T cells (Fig. 1), leading to cytotoxicity of the infected cell via perforin and granzymes. Cytosolic recognition of viral components also results in the induction of stress-associated pathways and the expression of stress-associated ligands on the cell surface, such as MIC and ULBP families of proteins in humans, and the Rae-1, H60, and MULT1 family of proteins in mice. These ligands are specifically recognized by NKG2D (Raulet, 2003), an activating receptor that is expressed on NK cells and T cells and leads to their activation upon ligation (Fig. 1). Lastly, in some cases, specific recognition of virally infected cells can occur when transcription and surface expression of viral

molecules occurs leading to recognition by certain activating receptors on NK cells; antigen-specific engagement by the NK cell leads to activation of effector function and lysis of the infected cell (Fig. 1).

DIRECT VIRAL CONTROL BY NK CELLS

NK-cell activation and effector functions are well-documented during CMV infection; however, there are other viral infections in which NK cell-mediated control has been implicated (Lodoen & Lanier, 2006). The documented NK cell responses against different viruses in humans and mice are summarized in Table 1. NK cells appear to be most important in mediating host resistance to members of the herpesvirus family. The first

16. Natural Killer Cell Response against Viruses

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TABLE 1 NK cells in host immunity against virusesa Virus MCMV

HCMV

HSV-1 VZV EBV Vaccinia virus Ectromelia virus

HIV-1 Influenza A virus

Hepatitis B virus Hepatitis C virus LCMV VSV Ebola virus Coxsackie virus Papillomaviruses a

NK-cell response Subsets of NK cells mediate effector function (IFN-g secretion and cytotoxicity) upon specific recognition of MCMV components, conferring resistance in C57BL/6 and MA/My mouse strains. NK cell deficiency results in disseminated HCMV infection. NK cells expressing CD94/NKG2C specifically expand during HCMV infection. NK cell-secreted IFN-g and TNF-a inhibit viral replication. Humans and mice with NK cell defects or lacking NK cells are susceptible to HSV-1 infection and HSV-induced lesions. Humans deficient in NK cells succumb to disseminated VZV infection. Patients with NK cell defects present with chronic active EBV infection. Depletion of NK cells in mice increased viral titer. Vaccinia virus enhances NK cell-mediated cytotoxicity. NK cell depletion in C57BL/6 mice increased viral titers. NK cell-mediated cytotoxicity, secretion of IFN-g, and NKG2D receptor play a role in resistance to infection. NK cell activity increased during viremic HIV infection. KIR3DS1 and Bw4-80I associated with a slow progression to AIDS. NK cells produce IFN-g and mediate cytotoxicity in response to viral infection. NK cell depletion results in poor disease outcome and mortality. NKp46 receptor shown to bind influenza virus hemagglutinin. NK cells play a role in liver disease progression. Blocking NKG2D prevents acute liver disease in mouse models of HBV. The ability to resolve HCV infection correlated with KIR2DL3 and HLA-C group 1 alleles. LCMV infection enhanced NK cell cytotoxicity; however, NK cell depletion does not affect viral titers. NK cell cytotoxicity enhanced during viral infection and replication. NK cells infiltrate the CNS. NK cells exhibited enhanced cytokine secretion and cytotoxicity, and protected against viral infection. NK-cell-deficient mice were susceptible to Ebola virus. NK cell cytotoxicity induced during infection. NK cell depletion results in increased viral replication and severe myocarditis. NK cell deficiency in humans correlated with papillomavirus-induced cervical carcinomas.

Biron et al., 1999; Lodoen & Lanier, 2006; Orange, 2006.

study of a human patient with a selective deficiency in NK cells demonstrated a susceptibility to varicella zoster virus (VZV) and herpes simplex virus (HSV) in addition to HCMV (Biron et al., 1989). Recently, another NK cell-deficient child died of VZV infection, demonstrating the crucial role NK cells play in control of this virus (Etzioni et al., 2005). Although NK cells are activated during HSV-1 infection, whether a direct NKcell-mediated control of replication of HSV-1 in mice exists is controversial. Genetic resistance to HSV-1 has been mapped to a locus on mouse chromosome 6 distinct from that of the gene complex responsible for MCMV resistance (Vidal & Lanier, 2006). During infection with ectromelia (mousepox) and vaccinia virus, NK-cell depletion in the resistant C57BL/6 mice results in immense viremia and mortality as compared to undepleted control mice. With all mouse studies, many factors such as viral dose, route of infection, and genetic background of the host likely play an important role in whether a productive NK cell response is mounted and whether this response confers resistance against a particular virus. NK cells are also activated and may provide protection during infection with HIV-1 and hepatitis viruses. Expression of certain KIR and HLA polymorphisms have been reported in genetic epidemiological studies to significantly influence disease outcome during HIV-1 and hepatitis C virus (HCV) (Kulkarni et al., 2008). HIV-infected individuals that possess

certain alleles of the KIR3DL1 and HLA-B genes have a considerably delayed progression to AIDS. During HCV infection, the ability to clear viral infection was associated with individuals carrying the KIR2DL3 and HLA-C group 1 alleles. Interestingly, both KIR3DL1 and KIR2DL3 are inhibitory receptors, and thus the requirement to engage these receptors for host benefit against viral infection is counterintuitive. Functional studies are required to elucidate the mechanisms behind viral resistance in both infections.

VIRUS DETECTION AND CYTOKINE SECRETION BY ANTIGEN-PRESENTING CELLS

During viral infection, antigen-presenting cells (APCs) sense viral nucleic acids via members of the Toll-like receptor family (TLR3, 7, 8, and 9) and intracellular sensors such as the RNA helicases RIG-I, mda5, and LGP2 (Fig. 2). Within endosomes, TLR3 recognizes double-strand viral RNA, TLR7 and 8 recognize single-strand RNA viruses, and TLR9 recognizes unmethylated CpG motifs found in DNA viruses (Takeda et al., 2003). Whereas TLR3 signaling requires the adapter molecule TRIF, TLR7, TLR8, and TLR9 use MyD88, and signaling downstream of these adaptor molecules leads to activation of IFN regulatory factors (IRF3 and IRF7, respectively), along with NF-kB (Kawai & Akira, 2008).

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FIGURE 2 NK-cell activation during viral infection. During viral infection, viral particles that actively infect or are taken up by DCs will trigger TLRs and intracytosolic sensors, leading to activation and maturation of the DC. Activated DCs produce type I IFNs and proinflammatory cytokines such as IL-12. pDCs sense viral infection primarily through TLR7 and TLR9, and are cells specializing in type I IFN production. Type I IFNs and IL-12 will activate NK cells, which constitutively express receptors for these cytokines. NK cells can also be stimulated when cell surface activating receptors engage viral or virally induced ligands. Activation of the NK cell leads to multiple effector functions such as cytokine secretion and cytotoxicity.

RIG-I, mda5, and LGP2 recognize viral RNA in the cytoplasm, and RIG-I and mda5 signal using the mitochondriaassociated CARD-containing adapter protein MAVS (also known as IPS-1, VISA, or Cardif), leading to downstream activation of IRF3 and NF-kB (Kawai & Akira, 2008). The importance of the antiviral RIG-I pathway was demonstrated by the susceptibility of MAVS-deficient mice infected with the RNA virus VSV (vesicular stomatitis virus), despite a robust induction of a systemic type I IFN response by TLRdependent recognition by plasmacytoid DCs (pDC). In addition to TLR9 recognition of viral DNA, ZBP-1 or DAI (DNA-dependent activator of interferon-regulatory factors) can contribute to host defense against DNA viruses as a cytosolic DNA sensor. More recently, components of the inflammasome (ASC, Caspase 1, NLRP3, and AIM2) have been shown to participate in the detection of RNA (influenza) and DNA (vaccinia) viruses. Altogether, these mammalian sensors of viral nucleic acids provide a cell-intrinsic defense leading to global inflammation early after viral invasion.

In response to virus detection, type 1 interferons (IFNs) and proinflammatory cytokines (such as IL-6, IL-12, and TNF-a) are rapidly induced (Garcia-Sastre & Biron, 2006). Although virtually all nucleated cells can produce type I IFNs, a specialized type I interferon-producing cell, named the plasmacytoid dendritic cell, engages viral nucleic acids specifically via TLR7 or TLR9, and produces large quantities of IFN-a, which act systemically (Fig. 2) (Liu, 2005). The generation of a system-wide inflammatory response results in: (i) neighboring uninfected cells are “alerted” of potential invaders, (ii) vasodilation occurs at the site of inflammation, and increased permeability of the endothelium promotes leukocyte extravasation, and (iii) lymphocytes are activated and mediate effector function against the virus or virally infected cells. Interestingly, different viruses induce varying levels of type 1 IFNs and different proinflammatory cytokines. In two well-characterized viruses commonly used to study immune responses in mice, MCMV induces both type I IFNs and IL-12 (along with TNF-a and IL-6), whereas


LCMV induces exclusively type I IFNs (with undetectable levels of IL-6, IL-12, or TNF-a). Because type I IFNs and IL-12 are potent inducers of NK cell activation (Fig. 2), the following two sections will be primarily devoted to the cellspecific effects of these cytokines and the characterization of downstream events subsequent to triggering of the type I IFN and IL-12 receptors.

INTERFERONS AND INFLAMMATORY CYTOKINES ACTIVATE NK CELLS

For decades, type I IFNs have been known to be a family of cytokines that are important for immune regulation and defense against viruses. The IFN-a/b family contains several IFN-a genes and only one IFN-b gene, located on chromosome 9 in humans and chromosome 4 in mice (Theofilopoulos et al., 2005). Type I IFNs were originally described as a “virus interference” soluble factor, experimentally characterized by their secretion from cells exposed to inactivated viruses that then inhibited subsequent infection with live viruses. Ablating the pleiotropic activity of type I IFNs during viral infection in mice containing a genetic deletion of the type I interferon receptor resulted in higher virus replication and severe pathology or death. In addition to their antiviral effects and activating dendritic cells in an autocrine fashion to promote antigen presentation, type I IFNs act directly on NK cells, resulting in their activation and enhancing their effector mechanisms (Fig. 2). This interferon-derived activation signal is necessary as a complement to NK cell recognition of viral or virally induced (“danger” or “stress”) ligands (Fig. 2). In mouse models of viral infection, NK cells exposed to type I IFNs have been shown to proliferate and efficiently mediate cytotoxicity (Fig. 2). Signaling through the type I IFN receptor activates the induction of hundreds of genes that are responsible for generating an “antiviral state” (van Boxel-Dezaire et al., 2006), a description commonly used, but not fully characterized at the molecular level. Some IFN-a/b-activated gene products have been shown to broadly interfere with the viral life cycle by inhibiting or altering protein synthesis (Stetson & Medzhitov, 2006). Among the IFN-a/binduced antiviral pathways implicated in infected cells are the 2,5-oligoadenylate synthetase (OAS) and RNase L, PKR, the Mx GTPases, and the RNA-specific adenosine deaminase ADAR, and DNA-specific and cytidine deaminase APOBEC3. Proteins such as OAS are activated upon viral dsRNA binding, and these enzymes produce 2–5 oligoadenylates, which activate a latent nuclease RNase L. RNase L subsequently degrades both viral and host RNA transcripts, and results in decreased protein synthesis and inhibited viral replication. Another well-characterized IFNinduced pathway of viral inhibition involves the serinethreonine kinase PKR, which phosphorylates downstream substrates (such as elongation initiation factor eIF-2a) leading to termination of viral and host mRNA translation. Mx and guanylate-binding proteins, which are large GTPases belonging to the dynamin superfamily, also have direct antiviral activity and can inhibit at an early stage of the viral replication cycle (e.g., influenza virus and VSV). The IFN response also induces expression of the adenosine deaminase ADAR and the recently discovered APOBEC3 family of cytidine deaminases, which catalyze the deamination of adenosine to inosine in dsRNA and cytidine to uracil in DNA, respectively, generating deleterious mutations in the viral genome and leading to altered protein synthesis or transcript degradation (Chiu & Greene, 2008). In particular, APOBEC3G (CEM15) was identified by its ability to inhibit HIV-1 infection. The importance of type I IFN

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for host defense is demonstrated in the redundancy found between the pathways that inhibit viral replication. Not surprisingly, many viruses encode a substantial amount of gene products dedicated to interfering with the IFN pathway. For example, the Vif (virion infectivity factor) protein found in primate immunodeficiency viruses such as HIV-1 is required during the late stages of virus production to suppress the antiviral activity of APOBEC3G in human T lymphocytes. Poxviruses such as vaccinia have been shown to secrete a soluble type I IFN receptor analog that sequesters the cytokine so that it cannot bind to host cells and signal to induce an antiviral response. Hepatitis C virus and VSV modulate expression of the type I IFN receptor by hijacking host ER machinery and inducing the unfolded protein responses, which leads to degradation of IFNAR1 and the inhibition of type I IFN signaling, allowing the virus to avoid triggering innate immune defenses. Other viral mechanisms to inhibit the type I IFN pathway have been found in different virus species (Garcia-Sastre & Biron, 2006), and many more will be elucidated in the days to come. Whether the virus targets cytokine induction, receptor ligation, signaling, or induced antiviral gene products, the disruption of the type I IFN pathway represents a mechanism by which the virus evades innate immune defenses, propagates within the host, and disseminates among the population. IL-12, like type I IFNs, is a proinflammatory cytokine produced early after infection with many viruses and has potent effects on NK cells (Fig. 2). Originally identified as NKSF or natural killer cell stimulatory factor, IL-12 is produced by monocytes, macrophages, DCs, and neutrophils in response to triggering through TLRs and other receptors (Trinchieri, 2003). IL-12 induces the NK cell to produce IFN-g, a powerful activator of antimicrobial properties in macrophages and other phagocytes, and drives a strong TH1 response. NK cells and activated/memory T cells are thought to be major producers of IFN-g early on during viral or bacterial infection as a “bystander” response to an inflammatory environment. Interestingly, whereas IL-12 positively influences IFN-g production, high concentrations of type I IFNs downregulate IL-12 expression both in humans and in mice. Because of the potency of IFN-g and the inflammatory response during viral infection, regulatory mechanisms exist to limit the effects of IL-12. The antiviral properties of NK-cell-secreted IFN-g during infection is discussed below. Many other inflammatory cytokines are produced by monocytes and DCs during viral triggering of TLRs including TNF-a (tumor necrosis factor-a), IL-1, IL-6, IL-15, and IL-18. During different viral infections, differential expression of these cytokines can individually contribute either directly or indirectly to the antiviral response (Guidotti & Chisari, 2001). TNF-a plays an important role in the recruitment of immune cells to the site of infection, thereby inhibiting the spread of the viral infection. This cytokine directly mediates cytotoxicity and necrosis, and was originally identified as a key player during inflammatory responses. TNF-a interferes with viral replication in several ways, but is thought to synergize with IFN-g to enhance inflammation, upregulate expression of MHC class I, and induce the production of reactive nitrogen and oxygen species. Antagonizing the biological activity of TNF-a during treatment of immune-mediated inflammatory diseases in some circumstances leads to enhanced viral replication and reactivation of hepatitis and other viruses. As with type I IFNs, certain viruses have evolved mechanisms to thwart the TNF pathway and evade immune responses to favor viral dissemination. IL-1 and IL-18 are members of a proinflammatory cytokine family produced soon after

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viral infection and exist in an inactive form that requires processing by caspase-1 to a biologically active mature form (Dinarello, 2009). Both IL-1a and IL-1b act as costimulators of T cells (along with antigen), and also drive production of IL-6, another inflammation-inducing cytokine. Abrogating IL-1 function in systemic autoimmune diseases reduces IL-6 levels and inflammation. IL-1 is released from dying cells, acting as a “danger” signal to alert surrounding cells to host damage in response to pathogens. IL-18 has been implicated in the priming of NK cell responses against MCMV and herpes simplex virus (HSV)-1. Lastly, IL-15 is another critical cytokine produced in response to viral infection. While IL-15 is indispensable during NK cell development and peripheral homeostasis and is essential for the generation of memory T cells (Ma et al., 2006), our recent studies have suggested that IL-15 is not essential for an NK cell response against MCMV (Sun et al., 2009b), although

it might be involved in NK-cell-mediated responses to other viruses. Although the effects of certain individual cytokines on NK cells remain to be characterized, the early global inflammatory response is clearly critical in the activation and recruitment of NK cells in the host immune defense.

SIGNALING EVENTS DOWNSTREAM OF CYTOKINE RECEPTORS

The type I IFNs bind a shared heterodimeric receptor consisting of the IFNAR1 and IFNAR2 chains, which associate via their intracellular tail with tyrosine kinases TYK2 (tyrosine kinase 2) and JAK1 (Janus activated kinase 1), respectively (Platanias, 2005) (Fig. 3). Binding of type I IFNs to their receptor results in ligand-dependent rearrangement and dimerization of the receptor subunits, followed by activation of the classical JAK-STAT signaling

FIGURE 3 Type I IFN and IL-12 signaling in NK cells augments effector functions. During many viral infections, type I IFN (IFN-a/b) and IL-12 are produced by infected cells. NK cells possess the cytokine receptors to sense inflammatory cytokines. Both type I IFNs and IL-12 bind heterodimeric receptors that signal through the JAK-STAT pathway. The type I IFN receptor induces phosphorylation of JAK1 and TYK2, resulting in phosphorylation and dimerization of STAT1 and STAT2. Along with IRF9, STAT1-STAT2 form the ISGF3 complex, and following nuclear translocation activates transcription of IFN-induced gene targets. Similarly, the IL-12 receptor consists of two chains that phosphorylate JAK2 and TYK2 during signal transduction, leading to phosphorylation and activation of various STATs including STAT4. STAT4 homodimers translocate into the nucleus and promote the transcription of target genes. Type I IFN and IL-12 signaling in NK cells can result in the induction of an “antiviral state,” the production of effector cytokines, cytotoxicity of virally infected cells, and proliferation.


pathways (Fig. 3). Tyrosine phosphorylation of STAT1 (signal transducer and activator of transcription 1) and STAT2 leads to the formation of the STAT1/STAT2/IRF9 complex, which translocates to the nucleus, binds target genes, and initiates transcription of IFN-stimulated response elements (Fig. 3). In addition to the cell-intrinsic antiviral effects of type I IFN signaling, NK cells exposed to type I IFNs become activated to mediate killing of virally infected target cells through their directional release of cytolytic granules into the infected cells and by the increased expression of TRAIL (TNF-related apoptosis inducing ligand). Although type I IFNs have been shown to induce blastogenesis of NK cells, whether a direct role exists in their ability to drive NK cell proliferation in vivo during viral infection remains to be determined. IL-12 is a heterodimeric cytokine consisting of a covalently linked 35 kDa (p35) subunit, (located on chromosome 3 in mice and humans) and a 40 kDa (p40) subunit (located on chromosome 11 in mice and chromosome 5 in humans) (Trinchieri, 2003). The IL-12 p40 subunit associates with another cytokine family member, p19, to form IL-23), and can be secreted as a monomer or homodimer. The IL-12 receptor is composed of two chains, IL-12Rb1 and IL-12Rb2, which associate with TYK2 and JAK2, respectively (Fig. 3). Phosphorylation of TYK2 and JAK2 upon binding of IL-12 to the IL-12 receptor results in signal transduction via the JAK-STAT pathway, with tyrosine phosphorylation and translocation of STAT4, which is responsible for the downstream effects of IL-12 signaling (Fig. 3). NK cells and certain T cell subsets exposed to IL-12 rapidly produce IFN-g, and this response occurs independently of antigen-specific receptor ligation.

DIRECT RECOGNITION OF VIRUSES BY NK CELLS

NK cells possess many activating and inhibitory receptors, and specific ligation of these receptors determines whether NK cells become activated (Lanier, 2005). Whereas inhibitory NK cell receptors generally engage MHC class I, activating receptors are thought to recognize pathogen components, but have not been well-characterized. NK cells also possess the activating NKG2D receptor, which recognizes hostencoded proteins (in humans the MICA, MICB, and ULBP family and in mice the Rae-1, H60, and MULT1 family) that are expressed on “stressed” cells, including virus-infected and transformed cells (Lanier, 2005; Raulet, 2003). Whereas B cells recognize the intact viral proteins and T cells recognize processed viral peptides presented on MHC complexes, the mechanisms by which NK cells directly recognize viral components have not been entirely elucidated. In humans and mice, convergent evolution has resulted in the KIR and Ly49 receptor families, respectively, both containing activating members that have been implicated in detection of virusinfected cells (Lanier, 2005). The best example of direct viral recognition by specific NK cell subsets is found in mouse and human cytomegalovirus (MCMV and HCMV) infections. Certain strains of mice were shown to be resistant to MCMV, and the resistance genes were genetically mapped to the “NK complex” locus located on chromosome 6 (Vidal & Lanier, 2006). In C57BL/6 mice, NK cells bearing the activating Ly49H receptor confer resistance against MCMV infection; conversely, mice strains and genetic knockout mice lacking the Ly49H receptor are susceptible to viral infection. The viral ligand of Ly49H is the m157 glycoprotein, a MHC class I homolog encoded by MCMV, likely acquired during virus evolution to engage inhibitory receptors and allow for degradation of host MHC class I to circumvent T cell recognition (Fig. 4). When Ly49H1 NK cells

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encounter MCMV-infected cells, the Ly49H receptor signals via the ITAM-containing adapter molecule DAP12, leading to NK cell activation, secretion of cytokines, killing of the infected target cells, and resistance against MCMV infection. Recently, another activating Ly49 receptor has been identified in the MA/My mouse strain. Ly49P specifically recognizes MCMV infected cells that express the MCMV-encoded m04 protein in conjunction with a host MHC class I, H-2Dk (Kielczewska et al., 2009). Although the precise nature of the ligand requires further elucidation, this case is interesting because the recognition of MCMV infection by the Ly49P receptor is MHC-restricted, similar to MHC-restricted recognition by T cells. In humans, several groups have described the expansion of a specific subset of NK cells during infection with HCMV (Guma et al., 2004; Kuijpers et al., 2008) or in cultures of NK cells with HCMV-infected fibroblasts (Guma et al., 2006). In the patient studies, a substantial increase in the percentage of NK cells bearing the CD94-NKG2CDAP12 receptor complex was observed in HCMV-infected individuals compared to the same individuals preinfection or in HCMV-seronegative adult blood donors (Guma et al., 2004; Kuijpers et al., 2008). The identity of the ligand recognized by NKG2C1 NK cells awaits elucidation. Altogether, these studies indicate an important role for direct and specific recognition of infected cells by NK cells during the immune response against viral infection. Immense selective pressures are placed on viruses by NK cells and other cells of the immune system. In order to avoid immune detection, certain viruses have adopted many immune evasion strategies that include blocking expression of molecules (MHC class I and NKG2D ligands) that reveal infection or stress (Fig. 4) and some of these viruses carry in their genomes decoy molecules (MHC class I analogs) that hinder NK cell activation (Fig. 4) (Jonjic et al., 2008; Orange et al., 2002; Tortorella et al., 2000). As discussed above, some viruses encode analogs of cytokine and chemokine receptors that, when secreted, sequester soluble factors that serve to activate or attract NK cells (Fig. 4) (Alcami, 2003). Viruses that evade or delay the immune response have a better chance at ensuring their propagation and survival. Viruses that employ some of the NK cell evasion strategies are found in Table 2.

EFFECTOR FUNCTIONS OF NK CELLS

Although NK cells have been implicated in host defense against numerous viruses, including herpesviruses, poxviruses, HIV, and even Ebola virus, the mechanism of NK cell effector function generally remains the same. NK cells respond early and in a nonantigen-specific fashion by secreting cytokines (such as IFN-g and TNF-a) and chemokines (MIP-1a). Following activation, NK cells in some cases can kill virally infected cells through a variety of cytolytic mechanisms. Lastly, NK cells may proliferate and traffic to different peripheral organs to mediate antiviral effects. The early secretion of IFN-g by NK cells after viral infection is driven by inflammatory cytokines produced by APCs, but also feed back to stimulate the APC. In IFN-g reporter mice (where yellow fluorescent protein reports cytokine transcription) NK cells in the spleen and liver were found to contain abundant amounts of IFN-g transcript even in the absence of infection or stimulation (Stetson et al., 2003). The pre-existing pool of IFN-g mRNA in resting NK cells explains how NK cells can produce this cytokine so rapidly after activation. The effects of NK cell-secreted IFN-g are broad, and over 200 genes are regulated by this cytokine (Boehm et al., 1997). In contrast to the many type I IFNs, IFN-g is the only type II IFN, encoded on chromosome 12 in humans and chromosome 10 in mice. IFN-g binds the

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FIGURE 4 General immune evasion strategies used by viruses. Many viruses encode immune evasion products that target MHC class I presentation or expression of NKG2D ligands, preventing the detection of viral infection by CTL through their antigen-specific TCR and NK cells via the activating NKG2D receptor. Because down-modulation of MHC class I on the infected cell might result in NK-cell-mediated killing through missing-self recognition, some viruses encode MHC class I decoy ligands, which engage inhibitory receptors on NK cells. Cytokines and chemokines play a crucial role in the activation and recruitment of NK cells and CTL, thus viruses have acquired mechanisms to thwart the production of cytokines such as type I IFNs. Viruses can either directly block the synthesis of inflammatory cytokines or secrete virally encoded cytokine and chemokine receptor analogs that sequester and neutralize the soluble factors so that responding cells cannot receive growth and differentiation signals. Viruses also encode products that directly interfere with the host intracellular sensors that activate transcription of antiviral effector gene products. NK cells and CTL can kill virally infected cells by activating apoptotic pathways in their targets via death receptor ligation and signaling; therefore, some viruses have developed strategies to block apoptosis in the infected until they can complete their replication and new virions are produced.

type II IFN receptor, consisting of the IFNGR1 and IFNGR2 chains (Platanias, 2005). Signaling of the IFN-g receptor on macrophages and DCs leads to pathways of antiviral defense, including the induction of nitric oxide (NO) synthase and NO production, which broadly inhibits replication of ectromelia virus, vaccinia virus, and HSV-1, among other viruses. During the NK cell-DC crosstalk that occurs early after MCMV infection, IFN-g synergizes with TNF-a (also produced by activated NK cells), leading to inhibition of viral replication. Activated NK cells secrete many other growth factors and chemokines, including MIP-1a. Production of MIP-1a by NK cells has a direct antiviral role during HIV infection, as it competes with viral particles for binding to the chemokine receptor CCR5, thus blocking viral entry.

To counter the antiviral role of chemokines, large DNA viruses such as the poxviruses and herpesviruses, have adapted strategies to block chemokines generated by immune cells during the antiviral response (Fig. 4). NK cells were originally described as cells that could rapidly kill tumor targets without prior immunization, and the general mechanism for NK-cell-mediated cytotoxicity occurs through directional secretion of perforin and granzymes into targets. Perforin-dependent pathways of CTL and NKcell-mediated cytotoxicity in humans and mice were shown to be important in studies demonstrating that certain NK cell functional deficiencies resulted in susceptibility to viral infection (Orange, 2006). The ability of resting mouse NK cells to rapidly acquire cytolytic function upon activation


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TABLE 2 Virus evasion of the NK-cell responsea Virus MCMV

HCMV

HSV-1 VZV

EBV Vaccinia virus

Ectromelia virus

Cowpox virus

HIV-1

Hepatitis C virus

a

Evasion strategies Virally encoded m04, m06, and m152 inhibit MHC class I expression. m138, m145, m152, and m155 inhibit expression of NKG2D ligands. m131/129 encodes a chemokine homolog. Virally encoded US2, US3, US10, and US11 sequester MHC class I leading to degradation. UL83 and US6 interfere with proteosomal processing and TAP transport, respectively. UL16 and UL142 prevent NKG2D ligand expression. UL141 targets CD155. UL111A is a homolog of IL-10 and inhibits NK cell activation. UL146 encodes a chemokine homolog and UL144 encodes a TNF receptor homolog. Virally encoded ICP0, US11, and g34.5 interfere with different signaling and effector molecules in the type I IFN response. Viral infection downregulates MHC class I and II expression, and infected cells are desensitized to IFN-g. Virulence factors may interfere with type I IFN and TLR signaling. Virally encoded BNLF2a and BGLF5 disrupt antigen processing and presentation to block MHC class I and II expression. Virally encoded vCKBP and A41L are chemokine-binding proteins thought to interfere with NK cell trafficking. Virus also contains a type I IFN receptor analog that inhibits cytokine activity. Virally encoded serpin-related proteins inhibit Fas-mediated apoptosis. Ectromelia also encodes soluble cytokine receptor homologs that bind and neutralize secreted IL-1 and IL-18. Virus encodes CrmE and other TNF receptor homologs proteins that bind cytokines (TNF) and block their activity. Virus also encodes a competitive antagonist of the activating NKG2D receptor. Virally encoded Nef preferentially downregulates expression of certain HLA and NKG2D ligands. Vif targets the type I IFN-induced APOBEC3G to block antiviral activity. The HCV envelope protein E2 binds CD81 to suppress human NK cell cytokine secretion, cytotoxicity, and proliferation. HCV also inhibits type I IFN signaling by causing degradation of IFNAR1.

Alcami, 2003; Jonjic et al., 2008; Lanier, 2008; Orange et al., 2002; Tortorella et al., 2000.

occurs because NK cells possess an abundant pool of preexisting perforin and granzyme A/B mRNA that can be rapidly translated upon activation. Perforin forms pores in the membrane of infected cells, allowing granzymes entry to trigger pathways leading to programmed cell death (Chowdhury & Lieberman, 2008). The many redundant pathways and activities of granzyme family members likely reflect the numerous mechanisms viruses employ to subvert cell death and also allow for viral replication and propagation (Chowdhury & Lieberman, 2008). Mice deficient in either perforin or granzymes A/B showed higher viral titers and pathology following MCMV, ectromelia, and West Nile virus infection; however, whether the disease outcomes were directly linked to defects in CTL or NK cells was not determined. Other mechanisms of NK-cell-mediated cytotoxicity include the induction of programmed cell death or apoptosis through members of the TNF receptor superfamily, such as Fas ligand (FasL) and TRAIL. These death receptors mediate the apoptosis of virally infected cells through the recruitment and activation of adapter molecules and caspases. IFN-g production by NK cells and CD81 T cells induces Fas (or CD95) expression on target cells, rendering them increasingly susceptible to apoptosis via the Fas pathway. NK cells store newly synthesized FasL on the inner surface of lytic granules, which also contain perforin and granzymes. Thus, simultaneously delivery of FasL and perforin to the area of contact between the NK cell and virally infected cell results in apoptosis of the target in a polarized fashion. Perforin- and granzyme B-deficient NK cells have been

shown to use FasL to mediate apoptosis of their targets. As with other antiviral mechanisms employed by the immune system, certain viruses such as those of the poxvirus family (ectromelia and vaccinia virus) encode proteins related to the serpin family of proteinase inhibitors that inhibit Fasmediated apoptosis (Fig. 4). There is also evidence that cellular cytotoxicity mediated via the death receptor pathway might be responsible for disease progression in certain viral infections, as NK cells expressing TRAIL have recently been shown to contribute to the immune-mediated liver damage during chronic hepatitis B virus infection in humans. Lastly, in both humans and mice, NK cells have been shown to undergo prolific antigen-specific expansion during infection with CMV (Dokun et al., 2001; Guma et al., 2004; Sun et al., 2009a). This clonal-like proliferation, found in Ly49H1 NK cells during MCMV infection, has been measured to be as much as 100-fold in the spleen and 1,000-fold in the liver from a small number of precursors (Sun et al., 2009a). As discussed above, the kinetics of the NK cell response falls between that of traditional innate and adaptive immune responses, and the specific expansion in NK cells shows similar kinetics to antigen-specific T cell responses (peaking at 7–8 days) to viral infection (Sun et al., 2009a). Consequently, NK cells, along with other innatelike immune cells, such as NK T cells, B1 cells, marginal zone B cells, gd T cells, and CD8aa T cells, can serve as a functional and temporal “bridge” from the early responders to conventional T cells and B cells. Because T and B cell responses require days to weeks to become fully effectual,

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the early recruitment and activation of NK cells during viral infection can be crucial. Interestingly, NK cells may also have arisen as an evolutionary “bridge” between the cells of innate and adaptive immunity. It is clear that NK cells possess characteristics attributed to innate immunity; however, recently, several groups have shown that NK cells possess features of adaptive immunity as well (O’Leary et al., 2006; Sun et al., 2009a), including the ability to form long-lived memory cells capable of secondary expansion against virus challenge (Sun et al., 2009a). Whether these newly discovered traits in NK cells can be harnessed towards a new vaccination strategy against viruses remains to be determined.

REGULATION OF THE NK CELL RESPONSE

The innate immune responses during viral infection serve to initiate and direct ensuing adaptive immune responses; however, once T and B cell responses are activated, mechanisms need to be in place to turn off NK cell activity. Because NK cells possess features that allow them to respond rapidly and powerfully, tight regulation of this immune cell subset during development and in the periphery is important to limit collateral damage and to prevent autoimmunity. During their development, NK cells stochastically express inhibitory receptors which, upon ligation with their cognate MHC class I ligands, allows them to become fully functional; developing NK cells possessing inhibitory receptors that do not engage MHC class I are hyporesponsive (Fernandez et al., 2005; Kim et al., 2005). Similarly, in experimental models, when NK cells bore activating receptors that engage their cognate viral ligand during development, these NK cells are deleted or rendered unresponsive in the periphery, safe-guarding against the generation of functional autoreactive cells (Sun & Lanier, 2008; Tripathy et al., 2008). In the periphery, the activity of mature NK cells must also be stringently regulated. NK cell activation during viral infection results in the accumulation of the inhibitory KLRG1 receptor on the NK cells, which binds to members of the cadherin family. Engagement of KLRG1 on NK cells limits both cytokine production and cytotoxicity of target cells, implicating a role for this receptor in tuning down NK cell responses after activation. Additionally, NK cells express a plethora of inhibitory receptors for non-MHC class I ligands that likely serve to regulate their responses. For example, LAIR-1 is an inhibitory receptor expressed by mouse and human NK cells that recognizes certain isoforms of collagen, potentially dampening NK cells responses in tissues. Moreover, mouse and human NK cells express members of the NKR-P1 family that possess inhibitory function and recognize ligands broadly expressed on many normal health tissues. Collectively, such inhibitory receptors that recognize host-encoded ligands may serve to protect healthily normal tissues from NK cell attack during inflammation or viral infection. The cytokines IL-10 and TGF-b also play a role in restraining the NK cell responses elicited by viral infection (Biron et al., 1999). Although a direct regulatory role for IL-10 on NK cells remains unclear during viral infection, IL-10 globally inhibits the production of IL-12 by myeloid cells, thus indirectly limiting NK cell activation. Interestingly, NK cells themselves have recently been shown to be producers of IL-10 during sustained activation, which occurs when an infection persists (De Maria et al., 2007; Maroof et al., 2008). TGF-b is a potent inhibitor of NK cell activity, specifically impeding the proliferation and cytotoxic ability of NK cells. In addition, TGF-b prevents the induction of IL-12, leading to a diminished IFN-g response from NK cells during viral infection. Because viral infections elicit such robust NK cell responses, multiple mechanisms for

regulating NK cells exist, demonstrating the need for the host to maintain tight control of these cytotoxic cells under conditions of global inflammation.

CONCLUSIONS

Decades of studies in both humans and mice have shown that NK cell activation during viral infection contributes to a vigorous immune effector response. The potent antiviral responses of NK cells that directly clear certain viral infections to promote host survival unmistakably demonstrate how crucial these cytotoxic cells are; at the same time, the importance of NK cell activity is equally demonstrated by the discovery of the many strategies different viruses employ to specifically evade detection by NK cells. Although viruses evolve because of the pressures exerted by cells of the immune system, there is evidence that the mammalian immune system also adapts in response to viral evasion mechanisms. As more viral ligands of activating NK cell receptors are uncovered, we will have a greater appreciation of the evolution of mammalian receptors on NK cells at the population level driven by viruses. We thank Carrie Sun for generating the figures. J.C.S. is an Irvington Postdoctoral Fellow of the Cancer Research Institute and L.L.L. is an American Cancer Society Professor and is supported by NIH grants AI066897, AI068129, CA095137, and AI64520.

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17 Innate Immunity against Bacteria THOMAS ARESCHOUG, ANNETTE PLÜDDEMANN, AND SIAMON GORDON

INTRODUCTION

CELLULAR BASIS OF INNATE IMMUNITY TO BACTERIA

The innate immune response to bacteria has been a major determinant of natural selection and evolution throughout the plant and animal kingdoms. The association between host and microbe can be symbiotic or can give rise to devastating epidemics of infectious disease. Since the 19th century heroic age of bacteriology (Pasteur, Koch, and their colleagues), the founding of cellular pathology (Virchow), the study of cellular immunity (Metchnikoff and Ehrlich), and humoral immunity (Von Behring and Ehrlich), among others, these interlinked disciplines have been at the forefront of research, prevention, and treatment of infectious disease. The advent of antisepsis and antibiotics has changed life expectancy of infants and adults, but antibiotic resistance, the emergence of new bacteria, and the persistence of known bacterial infections continue to pose a major threat. The innate mechanisms of resistance to bacteria and other organisms remain a priority for investigation, as does their connection to adaptive immunity, vaccine development, and autoimmunity. Many milestones were achieved in the 20th century, such as delineation of leukocyte differentiation and activation; the description of surface receptors for opsonic and nonopsonic recognition of microbes, especially the Tolllike receptors (TLR) and signaling pathways; molecular characterization of cytokines such as TNF and interferon-g (IFN-g) and other mediators of cellular interaction, bacterial killing, and inflammation. Progress proceeds apace in genetic analysis, global transcriptional responses, and cellular biology of host-pathogen interactions. In this review, we summarize cellular and humoral mechanisms of innate immunity to bacteria in mammals. We consider microbial ligands that serve as recognition structures for cellular receptors, secreted bactericidal molecules, and humoral proteins, such as complement components and pentraxins. Reviews of relevant topics are found in selected recent publications (Gordon, 2008b; Russell & Gordon, 2009; Talks, 2009).

The Innate Immune Cell: General Aspects

Diverse myeloid cells are specialized to perform a range of homeostatic and defense functions against bacteria and their products. Host-microbial interactions induce trophic, as well as injurious and repair processes ranging from commensals to virulent pathogens. In mammals, differentiated myeloid cells are adapted to recognize and respond to microbial and altered host ligands, expressing common as well as distinctive features of their distribution, activation, and turnover. Cells are able to react to bacteria directly, through a range of receptors, as well as indirectly, through intercellular and humoral interactions, locally and systemically, that underlie innate and adaptive immunity. In addition, innate lymphoid cells provide another bridge to acquired immunity. Nonhemopoietic cells, especially epithelia (including Paneth cells), also contribute to innate immunity as barriers and through biosynthetic responses. Table 1 provides a summary of the different cell types and their interactions with bacteria. Further details of receptors and responses will be given below. The production, differentiation, and activation of hemopoietic cells are described in many texts. These cells are distributed in different tissue compartments to provide a widely dispersed defense system in lympho-hemopoietic and other organs. Their mobilization, recruitment, and entry to tissues have been studied extensively, depending on selective chemotactic signals and receptors, and migratory and adhesive interactions. Once within tissues, they interact with matrix and neighboring cells and display heterogeneous phenotypes as they adapt to their local microenvironment. They vary in life span, they may die or emigrate, and ingest, kill, or coexist with bacteria through preformed or induced products. Although bacterial models of disease and naturally occurring infections have taught us a great deal about particular cellular responses of the host (described below), we raise some questions of general interest. The specific nature of the host response to different infectious agents depends, in part, on particular microbial ligands, but discrimination of foreign from normal and abnormal host constituents is not entirely understood. Different leukocytes must have evolved, in large measure, to coexist with pathogenic microbes, as well as to deal with infection, yet we know little about the selective pressures concerned. Conversely, bacterial adaptation, evasion, and selection have taught us a great deal

Thomas Areschoug, Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sölvegatan 23, 22362 Lund, Sweden. Annette Plüddemann, Department of Primary Health Care, University of Oxford Old Road Campus, Oxford, OX3 7LF, UK. Siamon Gordon, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom.

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TABLE 1

Selected properties of innate leukocytes, with reference to bacterial infectiona

Cell type

Characteristics

PMN(neutrophils)

Abundant in bone marrow, blood Short lived in tissues Chemotaxis Preformed products in distinct granules and new synthesis Opsonic receptors, TLRs, TREMs Assembly phagocyte NADPH oxidase

Basophils

Minor, circulate in blood Depend on IL-3, IL-33 Preformed granules, contents and new synthesis Distinct receptors (IgE), TLR Very rapid production of IL4, IL-13 Heterogeneous, mucosal, tissue Variable lifespan Receptors: IgE,C3a,C5a,TLR Preformed granules Histamine, proteinases Prostanoids, leukotrienes Minor except parasitic infections, allergy syndromes Short lived Activation dependent Can be phagocytic Preformed granules (MBP, cationic protein, peroxidase, neurotoxin) Can synthesize cytokines Low, in blood, long lived Subpopulations Migratory, chemotaxis Adhesion Phagocytic (FcR, CR, TLR, other) Active biosynthesis: mRNA, protein Secretory, respiratory burst Cytokines, leukotrienes Tissue populations Long lived, adherent Constitutive, induced Heterogeneous, resident Activated (innate, alternative, classical, deactivated) Phagocytic opsonic FcR, CR Nonopsonic (TLR, SR, lectin-like) Other receptors (e.g., GPCR) Regulatory (e.g., TREMs, CD200) Biosynthetically active, cytokines (e.g., IL-1, TNF, IL-6, IL-10) Low mw metabolites Inducible MHCII

Mast cells

Eosinophils

Monocytes

Macrophages

Antibacterial response (direct, indirect)

Comment

Pyogenic Granulocytosis Acute inflammation Diapedesis Degranulation Phagocytosis Killing Respiratory burst Apoptosis AMPs PGLYRP-1 Transient recruitment Innate Th2 response Degranulation Direct and indirect, by IL-3, IL-18

Infection Neutropenia LAD Septic shock Elastase, MPO Radicals, CathG Tissue injury CGD, immune complex disease

Direct, indirect Degranulation Mediator release

Allergy, Inflammation Interactions: smooth muscle epithelium, other innate leukocytes

Granuloma (e.g., M. tuberculosis) Cytotoxic ECP

Allergy, inflammation Broader interactions Leukocytes Tissue injury

Source resident, recruited Tissue Mw, osteoclasts, cDC Diapedesis Killing AMPs, sPLA2 Reactive oxygen intermediates

Chronic infection Inflammation Granuloma

Phagocytosis bacteria, apoptotic, necrotic cells Killing Digestion Clearance antigens AMPs, sPLA2 Mediators Reactive intermediates, oxygen, nitrogen, prostanoids Activation primed T cells Suppression

Trophic Cytotoxic Inflammation Acute, chronic granuloma Repair Fibrosis Tissue injury Systemic fever, wasting Source, response cytokines

Allergy Parasitic Inflammation Effector, regulator Eos, PMN Proteinase allergens


17. Innate Immunity against Bacteria TABLE 1

(Continued)

Cell type Dendritic cells, cDC

Plasmacytoid dendritic cells (pDC)

NK cells

NKT cells

Innate B lymphocytes

Paneth cells

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Antibacterial response (direct, indirect)

Characteristics Minor, blood, lymph, tissues Short to medium lived Motile Chemokine receptors (e.g , CCR7) Adhesion Maturation/activation Constitutive MHCII, DM association Limited digestion, Multivesicular bodies Phagocytosis (immature) Opsonic receptors (FcR, CR) Nonopsonic TLR, SR, lectin CD1d Cytokine production (e.g., IL-12, IL-18) Exosome formation Minor, blood and tissue Short-lived mixed phenotype CD123, IL-3R, TLR synthesis Type I interferon Blood, tissues Activatory/inhibitory receptors MHC I recognition Antigen markers Products, granzyme, interferon gamma Adaptive? Mixed phenotype Restricted TcR repertoire, especially CD1d family Produces interferon-gamma and other cytokines Different location, surface, antibody repertoire, growth (e.g., B1 versus B2, marginal zone B1) Semi-invariant BcR, 1/2 CD11b Small intestinal crypt Metaplasia of large intestine Epithelial surface markers Phagocytic Preformed granules, rich in lysozyme (P), also sPLA2, AMPs, TNF

Adaptive immunity Tolerance Migration Induced by LPS, other ligands Antigen capture, processing, presentation and activation of naive T and B cells Interactions with other innate cells Binding mycobacterial lipids

Comment Bacteria regulate APC Direct and cross

Although mainly viral recognition, also bacterial responses

Early studies Listeria monocytogenes infection

“Missing self” Viral infected targets Tumors

Bacterial lipid recognition Activation of other innate cells, including APC, NK cells

Asialoganglioside Host ligands

Rapid production of antibody (IgM)

High self-reactivity

Bacteria stimulate degranulation

a Auffray et al., 2009; Cohen et al., 2009; Gessner et al., 2005; Gordon, 2008a; Lopes-Carvalho et al., 2005; Martinez et al., 2008; Merad et al., 2008; Min & Paul, 2008; Russell & Gordon, 2009; Schroeder, 2009.

about normal host functions as well as about the diseases associated with innate and acquired immunity. Furthermore, the interplay between different innate cells, such as dendritic and NK cells, or between neutrophils and macrophages, is only beginning to be studied. The host-pathogen interactome provides a powerful field for discovery.

Bacteria: General Aspects

The bacterial microbiome is diverse and dynamic, varying from commensals in the gut, skin, and lung, and symbiotic

with the host, to virulent bacteria, able to produce endoor exotoxins and other secreted molecules. The organisms associated with mammalian hosts include aerobes and anaerobes, extra- and intracellular pathogens, gram-negative or gram-positive, motile or nonmotile bacteria. They can differ in wall composition, appendages, metabolism, ability to take up DNA, become latent, or form spores. They are able to expose or mask surface molecules, release enzymes and metabolites—which can be recognized by the innate immune system (as shown in Table 2)—and include

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TABLE 2

Selected bacterial ligands recognized by the innate immune systema

Structure

Cellular receptors (Examples)

Source (Examples)

Comments

Lipopolysaccharide (LPS)

E. coli

TLR4, CD14, SR-A

Inflammatory response, phagocytosis Inflammatory response, phagocytosis

Lipoteichoic acid (LTA)

S. aureus

CD36, TLR2/6?, SR-A

S. aureus Gram-negative bacteria, Bacillus spp Gram-positive cocci

TLR2, CD14 NOD1 NOD2

Inflammatory response Inflammatory response Inflammatory response

S. aureus, group B Streptococcus (GBS) Gram-positive bacteria

CD36, TLR2/6 CD14, TLR1/2

Inflammatory response Inflammatory response

Trehalose dimycolate Lipoarabinomannan (LAM)

M. tuberculosis M. tuberculosis

MARCO, TLR2, CD14 DC-SIGN, CD14, CD1

Mycolic acid a-galactosyl diacylglycerol

M. tuberculosis Borrelia burgdorferi

CD1 CD1

Inflammatory response Phagocytosis, inflammatory response, antigen presentation Antigen presentation Antigen presentation

Sialic acids

N. meningitidis, GBS

Siglecs

Terminal mannose, fucose or GlcNac

S. pneumoniae, K. pneumoniae

MR, DC-SIGN

Phagocytosis? Down regulation of inflammatory response Phagocytosis

Gram-positive and gram-negative bacteria GBS

TLR9

Inflammatory response

TLR7

Type I IFN response

TLR5, NAIP5, IPAF

Inflammatory response, activation of caspase-1 and IL-1b production Phagocytosis, inflammatory response Phagocytosis

Peptidoglycan Polymeric peptidoglycan meso-DAP Muramyl di-peptide (MDP)

Lipoproteins Diacylated lipoproteins Triacylated lipoproteins

Lipids

Carbohydrates

Nucleic acids DNA RNA

Proteins Flagellin

OmpA

Salmonella enterica serovar Typhimurium, Shigella flexneri, Legionella pneumophila Enterobacteriaceae

LOX-1, SREC-I, TLR2

Various surface proteins

N. meningitidis, GBS

SR-A, MARCO

a

Akira, 2009; Areschoug & Gordon, 2008; Cohen et al., 2009; Plüddemann et al., 2006; Taylor et al., 2005; van Kooyk & Rabinovich, 2008.

carbohydrates, proteins, lipids, and nucleic acids, recognized in the intact bacterium or after breakdown by a wide range of cellular and humoral receptors. A great deal of progress has been made in characterizing bacterial ligands, such as lipopolysaccharide, peptidoglycans, di- and triacylated lipoproteins, and their breakdown products.

Recognition by Innate Immune Cells: General Features

Janeway introduced the concept of “pattern recognition” of nonself (i.e., foreign structures on molecules) by receptors of antigen presenting cells (APC), which are germ-line encoded, in contrast with the somatic rearrangement of T- and B-lymphocyte-specific receptors responsible for adaptive immunity. As more receptors and their ligands were identified, it became clear that the receptors do not distinguish “pathogens” per se, but rather microbial structures in general (Gordon, 2008a).

Nor is it clear that it is “pattern recognition” by these receptors, rather than recognition of specific molecules. It has also become apparent that the problem of “foreign” versus “self” discrimination is not determined by the receptors alone, since many have dual recognition properties, including “modified self” and even “normal self.” Different signaling by concomitant TLR stimulation, for example by microbes versus apoptotic cells, may be responsible for immune activation rather than inhibition (Savill et al., 2002). The outcome of foreign recognition, with resultant activation of proinflammatory/ immunogenic responses, versus nonactivation or tolerance, cannot yet be explained. Innate membrane recognition receptors can be classified as opsonic, especially Fc and complement receptors, and nonopsonic, as TLR and non-TLR, including lectin-like receptors and scavenger receptors (SR). Receptors can be expressed at the cell surface, or within vacuoles/endocytic

17. Innate Immunity against Bacteria

vesicles. Distinct families of cytosolic recognition molecules include Nod-like (NLR) and Rig I-like (RLR) receptors. Different myeloid cells vary in the expression and regulation of these receptors, representative structures of which are illustrated in Fig. 1. The role of selected receptors in innate recognition of bacteria is summarized in Table 3, to illustrate their diversity, as well as common properties.

Phagocytosis, Interaction with Intracellular Bacteria: General Features

Understanding the phagocytic process is mainly based on studies with opsonic receptors in macrophages. Figure 2 shows a schematic representation of nonopsonic uptake of a range of intracellular pathogenic bacteria, to illustrate their strategy for infection and survival in macrophages. The resultant host-microbe interaction determines the outcome of infection. The phagocytic vacuole is mainly formed from the plasma membrane, but a role for the endoplasmic reticulum has also been proposed. The macrophage response is dynamic, involving fusion and fission, coupled to the actin-myosin contractile apparatus, acidification, and killing, followed by digestion, antigen processing, and presentation, especially in the case of the specialized APC, the conventional DC (cDC) (Akira, 2009; Gordon & Trinchieri, 2009). Conversely, different pathogenic bacteria counteract this process to arrest phagosome maturation and acidification by vacuolar ATPase or phagosome-lysosome fusion, as in the case of Mycobacterium tuberculosis. Other bacteria, such as Listeria monocytogenes, lyse the phagolysosomal membrane to escape into the cytosol, where they nucleate actin to spread within and between cells. Yet another strategy is employed by Legionella pneumophila, which can enter the cell via a coiling mechanism, its porin inducing a novel vacuolar membrane, whereas Brucella seems to interact directly with the endoplasmic reticulum/Golgi network. On the other hand, extracellular bacteria that do not replicate intracellularly are believed to remain in the phagosomes/phagolysosomes and are mostly efficiently killed. However, some species, such as Staphylococcus aureus have been suggested to remain viable inside macrophages for extended periods of time, using these cells as a reservoir. However, in most cases, the mechanisms for such increased intracellular survival remain unclear.

Responses to Phagocytosis

The downstream signaling, altered gene expression and biosynthetic response have been studied intensively, especially in the case of TLR-mediated sensing of bacterial ligands. Diverse TLR associate with adaptor molecules, such as MyD88, and activate transduction pathways, such as NF-kB, inducing a complex range of transcription changes after translocation of transcription factors to the nucleus. Translation and posttranslational modifications of effector molecules offer targets for evasion by pathogenic bacteria. A major response of granular leukocytes is the process of degranulation, vividly illustrated by time-lapse microscopy in the pioneering studies of James Hirsch and Zanvil Cohn, with neutrophils and eosinophils (Steinman & Moberg, 1994). Mast cells also provide an important model to study the cell biology of granule release. In both instances, opsonins (IgG, complement, cytophilic IgE) have been important in initiating degranulation, whereas this is less well characterized under nonopsonic conditions. A major outcome after microbial binding and uptake by innate phagocytes, especially macrophages, is the elaboration and release of low molecular weight products (e.g., reactive oxygen and nitrogen metabolites, arachidonates), and a range of cytokines, both pro-and anti-inflammatory. The antibacterial lytic and static activities secreted vary with the

213

cell type, and may include antimicrobial peptides (AMPs), secreted phospholipase A2 (sPLA2), peptidoglycan recognition proteins (PGRPs), chemokines, RNases, among others. In the case of neutrophils, the granules contain lysozyme, myeloperoxidase, and potent neutral proteinases (e.g., elastase, collagenase, Cathepsin G), as well as defensins, lactoferrin, and vitamin B12-binding protein. Monocytes contain only rudimentary granules, with lysozyme and myeloperoxidase, but also have an active respiratory burst and arachidonate metabolism. Mature macrophages are able to secrete a wide range of products depending on the activation state of the cell (Gordon, 2008b). TLR and Dectin-1, for example, initiate production of a respiratory burst, depending on priming of the cell by IFN-g, which is released by NK cells, or by interleukin 4/13 (IL-4/13), which is released by basophils. The killing and bacteriostatic mechanisms of activated macrophages are mainly due to the combination of reactive oxygen and nitrogen metabolites generated by the NADPH oxidase and inducible nitric oxide synthase in the plasma membrane, whereas the lysosomal acid hydrolases degrade dead organisms. The breakdown products of peptidoglycan, such as meso-DAP and muramyl di-peptide (MDP), can enter the cytosol for recognition by intracellular sensors, such as NOD1 and NOD2. These and other CARD-domain sensors, some of which are illustrated in Fig. 3, result in inflammasome activation, caspase cleavage, and IL-1 release. Human genetic defects in regulation of this important cytosolic recognition system can cause hyperinflammatory syndromes, even in the absence of obvious pathogens (Areschoug & Gordon, 2008). The role of type I interferon pathways in bacterial resistance is complex and may be deleterious, compared with its role in antiviral resistance. It is also not clear whether the cytoplasmic RIGI-like sensors, so important in viral defense, are implicated in intracellular bacterial infection. However, recent findings indicate that type I Interferons are important in the defense against extracellular bacteria, where TLR7 has been suggested to play an important role (Mancuso et al., 2009). Pathogens such as M. tuberculosis also respond in turn to the stress induced by a hostile intracellular environment, and resist oxidative killing mechanisms. The interplay between host cell and microbe in latent infection has been difficult to study. In selected cases, intracellular bacteria can induce cell death; apoptosis may be a host defense mechanism, as is autophagy induced by sublethal injury to cytoplasmic organelles. Apart from the above responses by both host cells and pathogens, the maturation of DCs by bacterial products serves to activate the adaptive immune response. This involves capture of peptide or lipid antigens, by MHC or CD1 molecules, DC maturation, restricted processing by proteases such as cathepsin S and antigen presentation, either directly or by cross-presentation. This process depends on the interaction of ligands with MHC Class II, CD1, or MHC Class I molecules, and activation of different subclasses of T lymphocytes. The inflammatory response, mediated by myeloid as well as lymphoid cells, can have profound systemic effects (e.g., fever, hypotension, steroid release, altered metabolism) as well as promoting tissue injury and repair. Persistent bacterial infection can induce chronic inflammation, granuloma, and macrophage giant cell formation and fibrosis, to which all innate leukocytes can contribute.

Modulation of Innate Immunity to Bacteria

The outcome of host-microbial interaction also depends on additional factors to those mentioned, operating on either partner. Apart from extrinsic immunomodulators, which can prime or down regulate host cell functions, age (both neonatal and the elderly) and genetic or acquired loss of

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FIGURE 1 Innate immune receptors. Schematic structures of selected surface glycoproteins expressed by myeloid leukocytes. (A) Recognition receptors, to illustrate the variety of nonopsonic receptors (CD11b/CD18 is also an opsonic receptor for complement). N and C indicate orientation. (B) Regulatory molecules and paired receptors. See text and references for details (Barclay et al., 2002; Lanier, 2009; N’Diaye et al., 2009; Taylor et al., 2005; van Kooyk & Rabinovich, 2008; Yona et al., 2008).

function influence the outcome. Mutations in the IFN-g/ IL-12 axis can cause primary innate immunodeficiency, as do mutations in the IRAK 4 signaling pathway. What is not clear is why enhanced susceptibility is restricted to only selected organisms, possibly because of redundant innate defenses. On the microbial side, there is the interplay between viral and subsequent bacterial infection. Metabolic disorders such as diabetes also affect the innate response, although mechanisms are not clear. Finally, adjuvants used to boost adaptive responses can prime innate responses with deleterious effects on the host. Ultimately, it may be possible to enhance innate resistance to bacterial infection, in combination with antibiotic therapy, subject to a better understanding of the cellular and molecular mechanisms involved.

THE ROLE OF INNATE RECOGNITION RECEPTORS IN BACTERIAL INFECTION

Figure 1 and Tables 3 and 4 summarize the properties of membrane molecules mainly expressed by myeloid cells, which contribute to recognition and regulation of innate immunity to bacteria. In this section we highlight the role of selected receptors in antibacterial responses (Areschoug & Gordon, 2008; Taylor et al., 2005).

CR3

CR3, a b2 integrin, plays a dual role in direct and complement-mediated (opsonic) recognition of bacteria, as well as in myeloid cell recruitment to infection by both extra- and intracellular pathogens. It is vital in innate resistance to fast-growing bacteria such as L. monocytogenes, and is one of several receptors utilized by M. tuberculosis to enter macrophages, possibly because of a poorly understood dampened signaling response. The CR3 molecule shares with other nonopsonic receptors apparent promiscuity in its range of microbial and host-derived ligands. It contributes to the leukocyte adhesion deficiency (LAD) syndrome, a human inborn error, due to the role of the common b2 chain in expression of CR3, LFA-1, and CD11c by myeloid leukocytes.

TLR and Associated Molecules

Recognition of bacterial ligands such as LPS by TLR4 has served as a paradigm in understanding mechanisms by which these leucine-rich receptors sense bacteria. By itself, TLR4 does not bind LPS, but rather depends on formation of a recognition complex consisting of TLR4, MD2, and LBP, an LPS-binding protein in plasma that facilitates the transfer of hydrophobic lipid structures to CD14. The TLR2/6 heterodimer is important for sensing gram-positive bacteria, initially thought to be due to recognition of lipoteichoic acid


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FIGURE 1 (Continued)

(LTA), but more recently it has been shown to primarily be due to sensing of the lipid anchor of bacterial surface lipoproteins. However, binding of gram-positive lipoproteins/ lipopeptides depends largely on the class B scavenger receptor CD36, which associates with TLR2/6 in lipid rafts at the cellular surface (Areschoug & Gordon, 2009). TLR5 recognizes flagellin from both gram-negative and grampositive bacteria. Recent studies suggest that TLR7 may recognize bacterial RNA and is of importance for the defense against extracellular bacterial pathogens (Mancuso et al., 2009). Structural studies have provided insights into TLR function and the effects of polymorphisms and mutations, some of which are implicated in altered resistance to infection. Proteolytic cleavage has been described in activation of intravacuolar TLR9, which recognizes bacterial CpG DNA. Several intracellular bacteria have evolved mechanisms to evade TLR responses through interference with various stages in the TLR signaling pathways. Ongoing studies on the adaptor molecules, such as MyD88 and TRIF, and kinases such as IRAK-4, have contributed to knowledge of human and experimental infection and possible therapeutic manipulation, including development of immune adjuvants based on oligodeoxy nucleotides.

Scavenger Receptors (SR)

Although initially described as an endocytic receptor implicated in macrophage foam cell formation resulting from uptake of modified low density lipoprotein, and therefore important in atherogenesis, the class A scavenger receptor (SR-A) has been shown to be important in innate immu-

nity to bacterial challenge by S. aureus, L. monocytogenes, Streptococcus pneumoniae, and more recently, Neisseria meningitidis (NM). NM express not only lipid A, a known ligand, but also several novel surface protein ligands for SR-A. In a bacteremia/septicemia model of infection by NM, it was shown that SR-A is host protective against ligand-bearing bacteria (Plüddemann et al., 2009). Masking of bacterial ligands for SR-A in Streptococcus pyogenes by M protein, an important virulence factor, has been demonstrated as a bacterial evasion strategy. The structurally related class A SR MARCO (macrophage receptor with collagenous structure) is more restricted in expression in resident macrophages, but is readily induced in macrophages by TLR stimulation. It has overlapping but distinct specificity for polyanionic ligands compared with SR-A, also binding and ingesting NM, as well as contributing to innate resistance to S. pneumoniae. MARCO has recently been shown to contribute to M. tuberculosis signaling and activation by trehalose dimycolate (cord factor), together with TLR2 and its associated recognition complex. Other SR, such as LOX-1, play a role in binding OmpA, a conserved outer membrane protein in the Enterobacteriaceae family. The Class B SR, CD36, contributes to mycobacterial entry, as shown by ENU mutagenesis, as well as to the clearance of apoptotic cells and Th-2 cytokine-induced macrophage giant cell formation (Plüddemann et al., 2006).

CD1

In contrast to the well-known role of MHC I and II in peptide antigen presentation by APC, the special role of various CD1

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TABLE 3

Properties of selected innate recognition membrane receptorsa

Receptor

Structure

Expression (predominant)

Ligands

Comments

Opsonic FcR gamma

IgSF

PMN, monocytes, Mw, cDC, NK, B cells

IgG Fc

ITAM, activatory ITIM, inhibitory Antibodies to allergen

FcR epsilon

IgSF

IgE Fc

CR3 (CD11b/18)

b2 integrin GPCR GPI anchor

Direct and opsonic (iC3b) C5a fragment LPS binding Protein

Phagocytosis, adhesion

C5aR CD14

Basophils, mast cells, eosinophils, Mw PMN, monocytes, Mw, cDC PMN, monocytes Monocytes, PMN, Mw

PMN, monocytes, Mw, cDC, basophils, inducible eosinophils Constitutive, all Constitutive cDC, B cells, inducible monocytes, Mw cDC

Range microbial and endogenous

TLR 3, 7, 8, 9 vacuole others surface

Peptides Peptides

CD8 T cells CD4 T cells

Microbial lipids (Mycobacteria, gramnegative bacteria) Also self lipids

Trafficking in APC, NKT-restricted (CD1d)

Beta glucan, non CHO? Mannose

Hemi-ITAM, cell activation Signals via common FcR gamma chain Broad range microbial, endogenous cell interactions Endocytic, double lectin, targeting

Myeloid cell activation Complex TLR4/MD2

Nonopsonic TLR(1–11)

MHC I MHC II

Leucine-rich repeat TIR domain (homo/ heterodimer) IgSF IgSF

CD1

IgSF

Lectins Dectin-1

C-type lectin-like

Dectin-2

C-type lectin-like

Mw, PMN, cDC, selected lymphoid subpopulations myeloid

DC-SIGN/SIGNR1

C-type lectin

APC, marginal zone

Mannose, fucose, GlcNAc

Mannose R

Mw, cDC, select endothelia, mesangial cells

Siglec-1

C-type multilectin, Cysteine-rich domain IgSF

Siglec H

IgSF

Branched mannose, fucose, GlcNAc Sulfated gal Sialic acid (e.g., Neisseria Cell-cell interaction meningitidis [NM]) and host glyco-conjugates Antigen marker

selected Mw (marginal metallophils, subcapsular sinus Mw) pDC

Scavenger receptors SRAI/II (Class A)

MARCO (Class A) CD36 (Class B)

Collagenous, transmembrane 1/2 cysteine-rich domain (SRCR) Collagenous, transmembrane, SRCR 2-span transmembrane

Phagocytosis (bacteria, Mw, cDC, select endothelia Polyanions (e.g., LPS, apoptotic cells) LTA, NM proteins, endocytosis, adhesion modified host proteins) Selected Mw, cDC (outer NM proteins, selected Induced by TLR, marginal zone Mw) host proteins coreceptor TB APC, platelets, EC, retinal Mycobacteria, Apoptotic Adhesion, phagocytosis, pigment epithelium cells, Oxidized LDL Mw fusion

a NB myeloid cells also express a wide range of receptors for host ligands-cytokines, growth factors chemokines. Cohen et al., 2009; Taylor et al., 2005; van Kooyk & Rabinovich, 2008.

isoforms in the binding and presentation of complex lipid antigens, mainly by cDC, has only been described relatively recently. The hydrophobic pockets of CD1 are able to accommodate a range of mycobacterial lipids, and the endocytosis and intracellular trafficking of CD1 has received a great deal of attention, especially for its role in activation of NK T cells, which express a highly restricted range of TcR. A marine sphingoglycolipid has been widely used experimentally

to activate these cells in vivo. Endogenous host ligands have also been characterized (Cohen et al., 2009).

Lectin-Like Receptors

Glycoconjugates can be recognized by a range of cellular receptors (e.g., DC-SIGN, MR, Dectin-2, Langerin) with broadly similar specificity for terminal mannose, fucose, and N-acetyl glucosamine structures; affinity and optimal


FIGURE 2 Selected bacterial pathogens evade distinct phagocytic mechanisms. Pathogenic bacteria have developed several mechanisms to enter and survive inside macrophages. Following an LPS-dependent, lipid raft-mediated entry, Brucella abortus is found in an early Brucella-containing vacuole (BCV) that acquires early endosome markers Rab5 and EEA-1. BCVs then mature into acidic intermediate vacuoles that accumulate LAMP-1, but not Rab7, avoiding interactions with late endosomes and fusion with lysosomes. BCVs then interact with ER exit sites (via VirB type IV secretion system) leading to fusion with the ER, generating an ER-derived organelle permissive for bacterial replication. Replicative BCVs exclude LAMP-1 and acquire various ER markers as a result of membrane exchange with the ER. Bacterial replication is thought to occur through fission of the BCV into two daughter BCVs via further accretion of ER membranes. Legionella pneumophila resides and multiplies in a vacuole studded with ribosomes due to interaction with the rough endoplasmic reticulum (RER). The organism inhibits acidification of its phagosome and secretes effector molecules via its type IV secretion system into the cell, which inhibit phagosome/lysosome fusion. The Francisella tularensis phagosome acquires the early endosome markers EEA1 and Rab5 and then matures into a late endosome defined by the presence of the markers Lamp1, Lamp2, and Rab7. The late endosome does not acidify and the phagosomal membrane is disrupted, releasing the bacteria into the cytosol. The Mycobacterium tuberculosis phagosome acquires the early endosome marker Rab5 but excludes the late endosomal Lamps and Rab7. Mycobacteria have characteristic thick cell wall, which is hydrophobic, waxy, and rich in mycolic acids/mycolates and allows them to survive inside phagosomes. This organism also inhibits acidification of the phagosome and produces molecules that block fusion with the lysosome, allowing it to reside and replicate in this modified phagosome. Acidification of the Listeria monocytogenes phagosome is essential for the perforation of the phagosomal membrane and escape of the bacteria into the cytosol. Here they mobilize the cell’s own actin polymerization machinery (via the bacterial ActA protein) to move within the cell and then from cell to cell (Celli, 2006; Clemens & Horwitz, 2007; Flannagan et al., 2009).

217

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FIGURE 3 Macrophage sensing of intracellular Salmonella enterica serovar Typhimurium is mediated by detection of monomeric flagellin, which is secreted by the bacterial type III secretion system and is dependent on the S. Typhimurium SipB protein. Upon stimulation, inactive monomeric Nlrc4 (formerly IPAF, ICE-protease activating factor) oligomerizes to form active NLRC4 inflammasome. The NLRC4 inflammasome then recruits ASC (apoptosis-associated speck-like protein containing a CARD system), which, in turn, recruits procaspase-1. Proteolytic cleavage activates caspase-1, which induces release of interleukin-1b, interleukin-18, and macrophage cell death. NLRC4 is also involved in sensing of Shigella flexneri and Legionella pneumophila. Sensing of S. flexneri is mediated by the bacterial type III secretion system protein lpaB. L. pneumophila sensing is mediated by Naip5 (neuronal apoptosis inhibitor protein 5) detection of monomeric flagellin secreted by the type IV secretion system, which induces caspase-1 in conjunction with NLRC4. Listeria monocytogenes and Staphylococcus aureus produce microbial toxins (e.g., maitotoxin, aerolysin, nigericin) and activate other signals (ATP and uric acid), which activate the NLPR3 inflammasome. This results in activation of caspase-1, and active caspase-1 can then process pro-IL-1b and pro-IL-18 and induce macrophage cell death. The specific NOD-like receptor (NLR) protein that detects intracellular Francisella tularensis remains to be identified; however, ASC is essential in the immune response against F. tularensis. CARD, caspase-recruitment domain; LRR, leucine-rich repeat; NACHT, domain present in NAIP, CIITA, HET E and TP1. (For further details see Brodsky & Monack, 2009; Mariathasan & Monack, 2007).

ligand structure vary, although there is overlap (van Kooyk & Rabinovich, 2008). DC-SIGN has been implicated in a range of microbial infections, including bacteria, viruses, and fungi, as well as in endogenous host ligand recognition, and its isoforms/variants differ in humans and mice. The multilectin MR (mannose receptor) is a more complex endocytic receptor, implicated in clearance of endogenous ligands such as lysosomal hydrolases, as well as in antigen capture and targeting, as described above. Its phagocytic function is more problematic and studies with Candida albicans indicate that the mainly intracellular MR is recruited to newly formed phagosomes only after internalization. The MR has been implicated in mycobacterial as well as other bacterial, viral, fungal, and parasitic infections and offers a possible therapeutic target for intracellular drug delivery.

Studies on MR knockout mice have shown divergent effects in models of inflammation. Mannose recognition has recently been implicated in Th17 lymphocyte activation via Dectin-2, and possibly the MR itself. The Dec-205 antigen of cDC has been utilized to target antigens efficiently to cDC in vivo. Although it belongs to the MR family, it remains an orphan receptor since no carbohydrate or other ligand has been discovered. Dectin-1, an important receptor for fungal b-glucans, contains a cytoplasmic hemi-ITAM implicated in signaling in APC. There have been reports of uncharacterized ligands in bacteria, as well as unidentified non carbohydrate ligands in subpopulations of lymphocytes. Its activatory functions have been well studied in macrophages and cDC, and involve syk, CARD 9, bcl10, and malt1.

17. Innate Immunity against Bacteria TABLE 4

Properties of selected membrane molecules that regulate myeloid cell innate responsesa

Molecule

Structure

Expression

Ligands

Function

EMR2

EGF-TM7

PMN, monocytes, Mw, cDC

Chondroitin sulfate B, selected E. coli

TREM1

IgSF

CD200

IgSF

PMN, monocytes, Mw, cDC Broad hemopoietic, inducible Mw by innate stimuli

Potentiate PMN, respiratory burst, degranulation cDC migration, maturation Potentiation PMN

CD200R

Inhibitory pair controls Mw activation

SIRPa

IgSF

CD47

DAP12

GPI-anchor NK, Mw

Pairs various recognition receptors (e.g., TREM2, CD200)

Inhibitory pair regulation phagocytosis ITAM, activation induces fusogenic program in Mw signals through common FcR gamma chain

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a NB other receptors and costimulatory antigens regulate T-cell activation. Barclay et al., 2002; Lanier, 2009; N’Diaye et al., 2009; Yona et al., 2008

Sialic acid recognition of host and bacterial ligands, for example on N. meningitidis, can be mediated by Siglec-1 (sialoadhesin), the member of a family of receptors specific for sialyl structures. Siglec H, whose binding properties and functions are less well defined, is highly expressed on plasmacytoid DC. It has been suggested that the siglecs promote phagocytosis of sialic-acid-expressing pathogens, but recent studies indicate that bacteria utilize the inhibitory function of siglecs to down regulate cellular activation, phagocytosis, and inflammatory responses in macrophages/neutrophils (Carlin et al., 2009a, 2009b).

NOD-Like Receptors (NLR)

Internalized bacteria and their products can also interact with cytosolic NOD-like receptors (NLRs). This family of receptors is structurally related and consists of several members, including NOD (nucleotide-binding oligomerization domain), NAIP (neuronal apoptosis inhibitor), NLRP3 (formerly NALP; NACHT, LRR, and PYD domains), and NLRC4 (formerly IPAF; ICE-protease activating factor). NLR proteins have three structural domains, consisting of (i) a variable N-terminal protein–protein interaction domain, defined by the caspase recruitment domain (CARD), pyrin domain (PYD), or the baculovirus inhibitor domain (BIR); (ii) a centrally located NOD domain that facilitates self-oligomerization during activation; and (iii) a C-terminal leucine-rich repeat (LRR) responsible for binding/detecting pathogen-associated molecular patterns (PAMPs). The PYD or CARD domain of NLR is the link to downstream adaptors (such as apoptosis-associated speck-like protein containing a CARD [ASC]) or effectors (such as caspase-1). The BIR domain is proposed to act as caspase inhibitor. NOD1 and NOD2 sense bacterial peptidoglycan and trigger nuclear factor-kB (NF-kB) signaling via their aminoterminal CARD. NOD2 is necessary for the activation of caspase-1 in response to both MDP and anthrax lethal toxin. Nlrp3 interacts through PYD–PYD homotypic interactions with ASC, which then mediate caspase-1 activation through the CARD domain. Nlrc4 can sense PAMPs on its own and possesses a CARD domain at the N-terminal, and thus may directly activate caspase-1 without ASC recruitment. Nlrc4 senses flagellin delivered to the cell cytoplasm by secretion systems of bacteria such as Salmonella enterica serovar Typhimurium, Legionella pneumophila, and Pseudomonas aeruginosa, although in the case of

Legionella the protein Naip5 is also required. Shigella flexneri infection is also sensed by Nlrc4, however this bacterium does not express flagellin, and this signal remains to be elucidated. Caspase-1 activation then results in the release of IL-1b, IL-18, and macrophage cell death (Fig. 3) (Brodsky & Monack, 2009; Mariathasan & Monack, 2007).

Rig I-Like (RLR) Receptors

The RIG I-like (RLR) receptors are mainly involved in antiviral responses, recognizing double-stranded RNA; however, a recent publication showed the involvement of RIG-I in TLR-stimulated phagocytosis. LPS stimulation of macrophages induced RIG-I expression and RIG-I-deficient macrophages exhibited impaired phagocytosis, while RIG-I-deficient mice were more susceptible to Escherichia coli infection. Therefore the regulatory functions of RIG-I may also extend to bacterial responses (Kong et al., 2009).

SOLUBLE/SECRETED ANTIBACTERIAL HOST DEFENSE MOLECULES

As described above, initial recognition, sensing, and phagocytosis of bacteria and their products by host cells leads to an inflammatory response and release of various antibacterial molecules, either preformed or induced, that are released intracellularly, locally, or secreted into various body fluids. Many of these molecules are able to directly kill bacteria by various mechanisms and some are specialized for protection against bacterial pathogens, recognizing structures unique to bacteria. Moreover, many antibacterial molecules seem to act synergistically. Below we summarize selected soluble/secreted host molecules in the innate defense against bacterial pathogens.

Neutrophil Serine Proteases and Other Granular Proteins

The granules of neutrophils contain several different serine proteases, including elastase, cathepsin G, and protease 3 that effectively kill engulfed bacteria (Pham, 2006). These enzymes are normally 30 to 35 kDa in size, and contain a serine residue in the active site. The generation of mice deficient in elastase or cathepsin G has demonstrated that these molecules are crucial for resistance against infection with selected gram-positive or gram-negative pathogens. However, the precise mechanisms by which these proteases

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kill bacteria are unknown, and may be unrelated to their enzymatic activity. The serine proteases are also released into the extracellular environment where they may participate in regulation of inflammation by various mechanisms (e.g., proteolytic modification of cytokines and chemokines and activation of cellular receptors such as TLR4 and the formyl peptide receptor [FPR]). Lactoferrin is found in secondary neutrophil granules and has been shown to have direct antibacterial activity by at least two distinct properties (Ward et al., 2005). First, lactoferrin has strong iron-binding properties, which allows the protein to sequester free iron and maintain an environment refractory to bacterial growth. Second, lactoferrin has also been shown to have direct bactericidal activity by a mechanism independent of its iron sequestration function; it binds directly to gram-negative bacteria, causing release of LPS and increased membrane permeability. Myeloperoxidase is crucial for the conversion of hydrogen peroxide to hypochlorous acid, a highly toxic and microbicidal agent.

Antimicrobial Peptides

The antimicrobial peptides (AMPs) were initially discovered as important effectors of innate immunity in insects and were shortly thereafter described in mammals (Brown & Hancock, 2006). Some AMPs are expressed constitutively by epithelial cells, but most AMPs are induced in epithelial cells as well as monocytes/macrophages and neutrophils during inflammation in a TLR-dependent manner. The AMPs represent an extremely diverse group of peptides but virtually all AMPs share the overall feature in being cationic and hydrophobic. In mammals, there are two major families of AMPs: the cathelicidins and the defensins. The cathelicidins are a-helical peptides with a conserved 14 kDa N-terminal “cathelin” (cathepsin L inhibitor)-like domain and a variable C-terminal region. Both humans and mice have only a single cathelicidin gene that encodes the proteins LL-37/hCAP18 and CRAMP (cathelin-related antimicrobial peptide), respectively. The defensins are small peptides that range from 2 to 6 kDa in size with a conserved three-dimensional structure. On the basis of the size and arrangement of the disulfide bridges, the defensins are subdivided into three major groups, a, b, and u. Direct evidence for a role of AMPs in the innate immune defense against bacterial infection has been provided in several studies using knockout mice deficient in specific AMPs, showing that these mice were more sensitive to bacterial infection and colonization compared to their WT counterparts (Brown & Hancock, 2006). Moreover, human individuals with AMP deficiencies have an increased risk of suffering from various bacterial infections. However, the primary role of AMPs in innate host defense is not entirely clear. Under certain in vitro conditions, AMPs exhibit direct antimicrobial activity against both gram-positive and gram-negative bacteria. The mechanism of action is believed to be similar for the cathelicidins and the defensins; the cationic charge of the AMPs attracts them to negatively charged phospholipid groups in the outer leaflet of the bacterial membrane. The hydrophobic properties enable the AMPs to insert into the membrane, forming transient pores that eventually cause osmotic lysis of the bacterium. However, the direct antimicrobial action of many AMPs is antagonized by physiological salt conditions, monovalent and divalent ions, and serum, which indicate that the direct antimicrobial activity of most AMPs may be limited in vivo (Bowdish et al., 2005). In recent years, several additional roles have been described for AMPs in immune defense. Both the cathelicidins and the defensins can act as chemotactic factors for various effector cells. Moreover, in addition to direct

chemotactic activity, several AMPs can also induce release of multiple cytokines from host cells, which in turn leads to an increased inflammatory response and chemotaxis. However, the cathelicidins have also been shown to have antiinflammatory properties as they down regulate TLR4- and TLR2-dependent cytokine release from DCs and prevent LPS-induced sepsis in animal models. Cathelicidins exert their immune-modulating effects by several mechanisms, including direct binding and neutralization of LPS and interactions with host cell receptors or intracellular signaling molecules (Brown & Hancock, 2006).

Enzymatically Active Antibacterial Molecules

An important group of bactericidal molecules are enzymes that kill bacteria by enzymatic attack on microbial cell walls or phospholipid membranes, including lysozyme, secreted phospholipase A2 (sPLA2), and the peptidoglycan recognition proteins (PGRPs). Lysozyme is produced by a variety of cells and is present in large concentrations in a number of secretions, including tears and saliva. It is a glycosidase that hydrolyzes the 1,4-b-glycosidic linkages between the N-acetylglucosamine and N-acetylmuramic acid moieties in the peptidoglycan structure. Although lysozyme itself is bactericidal only against a limited number of gram-positive bacteria, it has been shown to act synergistically with complement and some AMPs, enhancing their antibacterial effect (Dziarski & Gupta, 2005). The secreted phospholipases A2 (sPLA2) are small (14 to 18 kDa) basic proteins that hydrolyze the sn-2 bond of phospholipids, resulting in the release of a fatty acid and a lysophospholipid (Nevalainen et al., 2008). Today more than 10 isoforms have been identified; with regard to a role in innate immune defense, the most extensively studied sPLA2 isoform is GIIA. The main source of GIIA sPLA2 is believed to be liver hepatocytes, but also macrophages, blood platelets, and Paneth cells. It is unclear whether GIIA sPLA2 is synthesized by neutrophils. Expression of GIIA sPLA2 is induced during inflammation by proinflammatory cytokines, or bacterial products such as LPS, resulting in release of GIIA sPLA2 into the circulation. GIIA sPLA2 has been shown to have direct bactericidal activity in vitro against a wide range of gramnegative and gram-positive bacterial pathogens, including E. coli, S. enterica serovar Typhimurium, Helicobacter pylori, L. monocytogenes, S. aureus, and Bacillus anthracis. Using an animal model employing transgenic mice overexpressing human GIIA sPLA2 in a C57B/6J background (that lacks functional murine GIIA sPLA2), it has been shown that human GIIA sPLA2 provides significant protection against infection with several bacterial pathogens in vivo, including B. anthracis. GIIA sPLA2 is highly basic, which allows it to penetrate the bacterial cell wall and gain access to the bacterial membrane, where it hydrolyzes bacterial phospholipids in a Ca212dependent manner (Nevalainen et al., 2008). The peptidoglycan recognition proteins (PGRPs) are present in most invertebrate and vertebrate animals (Royet & Dziarski, 2007). As the name indicates, these molecules bind to bacterial cell wall peptidoglycan, but the main role of these interactions in innate immune defense varies among different PGRP members. In insects, the PGRPs mainly function as PRRs that sense microorganisms, resulting in generation of AMPs; in mammals, secreted PGRPs function mainly by direct bactericidal activity. All PGRPs contain at least one C-terminal PGRP domain that is homologous to bacteriophage and bacterial type 2 amidases. In mammals, four PGRPs have been identified, designated PGLYRP-1, PGLYRP-2, PGLYRP-3, and PGLYRP-4. PGLYRP-2 is constitutively expressed in the liver and secreted into the bloodstream, but it can also be induced locally in epithelial cells by bacteria and cytokines. PGLYRP-2 acts as an amidase,


hydrolyzing the amide bond between N-acetylmuramic acid and l-alanine in peptidoglycan, resulting in removal of the stem peptides from the glycan chain. The other three human PGRPs are also bactericidal, but it is unclear whether they have enzymatic activity. PGLYRP-1 is expressed primarily in neutrophil granules, while PGLYRP-3, and PGLYRP-4 are expressed in the skin, eyes, oral cavity, and intestinal tract. Interestingly, human PGRPs kill both gram-positive and gram-negative bacteria synergistically with other classes of antibacterial molecules, such as sPLA2, lysozyme, and a- and b-defensins when compounds are present in sub-bactericidal concentrations (Royet & Dziarski, 2007).

Other Antibacterial Host Molecules

A hallmark of the inflammatory response is the production of chemokines, small peptides ranging from 70 to 130 amino acids that induce chemotaxis in target cells expressing the appropriate chemokine receptors. Although the main functions of chemokines are related to homing and inflammation, recent data indicate that many chemokines have antibacterial activity, but the mechanism is not understood. However, many antibacterial chemokines have structural properties similar to those of AMPs, making it a possibility that the chemokines act by disrupting bacterial membranes (Eliasson & Egesten, 2008). Another group of bactericidal molecules are members of the RNase A superfamily, which are characterized by homology with bovine RNase A (Rosenberg, 2008). Eight human members (RNase 1–8) of the RNase A superfamily have been described, and five additional genes in the human genome that are related to the RNase A ribonucleases have been identified (RNases 9–13). They are all cationic, share a unique three-dimensional disulfide bonded structure, and the ability to hydrolyze polymeric RNA, but the bactericidal properties of the RNases are, in most cases, unrelated to their enzymatic activity. RNases are expressed by a variety of cells, but some of the best characterized members with regard to bactericidal properties are expressed by eosinophils and Paneth cells. For example, the eosinophil granule protein (ECP) is strongly bactericidal against a number of bacterial species, and Angiogenin-4 (Ang-4), expressed by Paneth cells, exhibits broad spectrum bactericidal activity (Rosenberg, 2008).

HUMORAL INNATE IMMUNITY AGAINST BACTERIA The Complement System

The complement system, which consists of about 35 serum or membrane bound proteins, is an important part of the humoral branch of the innate immune system and is of central importance in the defense against bacterial pathogens (Walport, 2001a, 2001b). Complement proteins are mainly synthesized by liver hepatocytes, but significant amounts are also produced by monocytes, tissue macrophages, and epithelial cells lining the gastrointestinal and genitourinary tracts. Several complement proteins circulate in serum as inactive proenzymes, which are activated by specific cleavage of the molecule. Activation of complement triggers a tightly regulated self-amplifying sequential enzyme cascade, resulting in several important physiological activities, including: (i) opsonization and phagocytosis of invading microorganisms, (ii) chemotaxis and activation of leukocytes, (iii) direct lysis of microorganisms, (iv) clearance of immune complexes, and (v) induction of antibody responses. The initial recognition of pathogenic bacteria is mediated by three different molecules, C1q, mannose-binding lectin (MBL), and C3b, which trigger activation of the classical,

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lectin, and alternative pathways, respectively. The classical pathway is initiated through the interaction between C1q, which is a part of the C1-complex and the Fc part of antibodies (IgG or IgM) bound to antigens on the bacterial surface. Moreover, C1q can also bind directly to bacteria by interacting with LPS, porins, or various capsular polysaccharides. The lectin pathway is initiated through the binding of MBL to mannose, N-acetyl glucosamine residues or fucose on the surface of bacteria. Ficolins, members of the collectin family, also activate the lectin pathway through recognition of carbohydrates at the bacterial surface. The alternative pathway, which is mainly considered to act as an amplification pathway, is initiated by surface deposition of C3b. Importantly, activation of complement leads to the formation of either of two different C3-convertases, which catalyze the key reaction in the complement system: the conversion of C3 to C3b. The C3b molecule is rather unstable and is rapidly inactivated by factor I and its cofactors to C3bi at the bacterial surface. Both C3b and C3bi promote phagocytosis through the interaction with complement receptors CD35 (CR1) and CD11/CD18b (CR3) expressed on macrophages and neutrophils. Further activation results in formation of either of two C5-convertases, which initiate the formation of the terminal membrane attack complex (MAC) C5b-9 by catalyzing the conversion of C5 to C5b. MAC is inserted into the membrane of some gram-negative bacteria, causing osmolysis. Gram-positive bacteria, however, are resistant to MAC due to their thick peptidoglycan layer. Importantly, cleavage of C3 and C5 by the two convertases results in formation of the C3a and C5a molecules, which are important chemoattractants for phagocytic cells. Complement also plays a role in the induction of antibody responses, thus providing a link between innate and adaptive immunity. One of the key players in this mechanism may be natural IgM, which, upon binding to bacterial antigens, induces complement deposition at the bacterial surface. One of the deposited molecules, the C3b degradation product C3d, interacts with complement receptor 2 (CR2) on B cells, acting as a coreceptor. Simultaneous binding of CR2 and membrane-bound antibody on B cells to antigen is believed to lower the threshold for B-cell activation and uptake of antigen, resulting in enhancement of B-cell efficiency in inducing specific antibody responses and induction B-cell memory (Walport, 2001a, 2001b). The major importance of complement in innate immune defense against bacterial pathogens is underlined by studies on families with various deficiencies in complement proteins (Tedesco, 2008). C3-deficient individuals suffer from repeated life-threatening infections with encapsulated bacteria such as S. pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, which can be explained by the important role of this complement protein in promoting phagocytosis and bactericidal activity. Recurrent otitis media, meningitis, and pneumonia are the most characteristic infections among these bacteria. C2 and MBL-deficiency has been shown to be associated with an increased risk for septicemia and pneumonia caused by S. pneumoniae. Deficiencies in the proteins forming MAC are almost exclusively associated with an increased risk of infections caused by Neisseria meningitidis, indicating that MAC primarily is of importance in the defense against this particular gram-negative bacterial pathogen (Walport, 2001a, 2001b). Another part of the humoral branch of the innate immune system are members of the pentraxin superfamily, the short pentraxins C-reactive protein (CRP) and serum amyloid P-component (SAP), and the long pentraxin PTX3 (Mantovani et al., 2008). CRP is rapidly produced by hepatocytes in response to inflammatory signals such as IL-6, and subsequently secreted into the circulating blood. SAP is also produced by hepatocytes, but the concentration of SAP is relatively stable. PTX3 is produced by both innate and

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resident cells in peripheral tissues in response to inflammation and TLR activation. The pentraxins have been shown to bind a wide range of pathogenic bacteria, including S. aureus, P. aeruginosa, Klebsiella pneumoniae, and S. pneumoniae. A bacterial ligand identified for CRP is the C-polysaccharide on S. pneumoniae, an interaction dependent on Ca21 ions, and it is likely that CRP binds to similar polysaccharide structures on other bacteria. The main function of surface bound CRP is believed to be activation of the classical pathway of complement through interaction between CRP and C1q. SAP also binds C1q, but its role in complement activation is less well defined. For PTX3 it has been demonstrated that an important bacterial ligand is the conserved Enterobacteriaceae OmpA protein, an interaction independent of Ca21. The OmpA protein binds to and is internalized by DC and macrophages, activating these cells in a TLR2 dependent manner. The innate immune response to OmpA involves recognition by the scavenger receptors LOX-1 and SREC-I, which results in production of PTX3, which in turn binds OmpA, thus amplifying the inflammatory response (Mantovani et al., 2008).

CONCLUSIONS

The human body possesses diverse innate weaponry against bacterial pathogens. Various cell types, both phagocytic and nonphagocytic, express different combinations of innate receptors, both extra- and intracellular, recognizing a wide range of bacterial ligands, including LPS, peptidoglycan, lipoproteins, carbohydrates, nucleic acids, and proteins. The cells respond by secreting pro- and anti-inflammatory cytokines and various bacteriostatic/bactericidal effector molecules, many of which act in synergy to achieve efficient killing of the target bacterium. The humoral arm of the innate immune system (in particular, complement) is essential for protection against both gram-positive and gram-negative bacteria through the important function of these host proteins to act as opsonins and chemoattractants. Importantly, several innate immune receptors/ effector proteins enhance the adaptive immune response by various mechanisms, providing significant links between innate and acquired immunity. Many bacterial pathogens, however, have evolved mechanisms to evade or manipulate the innate immune defenses, or sometimes even utilize innate receptors for their own benefit. The interplay between the innate immune system and different virulence factors expressed by bacterial pathogens will remain an important field of study to understand the molecular pathogenesis of important infectious diseases and the possibilities to develop novel therapies against important bacterial pathogens.

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18 Innate Immunity to Parasitic Infections CHRISTOPHER A. HUNTER AND ALAN SHER

INTRODUCTION

on host and microorganism. This is evident from the numerous strategies employed by protozoa and helminths to evade these antimicrobial mechanisms and underscores the importance of studying innate immunity in order to understand the host/parasite adaptation. One question that has preoccupied this field for almost 20 years is how the innate immune system can distinguish different classes of pathogen and direct appropriate T-cell dependent effector functions. Thus, while the innate immune system is the first to be triggered in response to invading microbes, it is now appreciated that these events can determine the class of the subsequent adaptive immune response. This is exemplified by the classification of CD41 T cell responses into the Th1 and Th2 subsets associated with resistance to different classes of parasite. For example, while helminths are an evolutionarily diverse group of organisms, they are unique among infectious agents in that they are multicellular and, with few exceptions, fail to replicate in their definitive hosts. Resistance to these worms is typically dependent on a highly polarized Th2-type cytokine response pattern, characterized by the production of IL-4, that is reminiscent of the patterns seen in allergy. In contrast, protozoa are unicellular and occupy intracellular and/or extracellular host niches. They normally elicit strong Th1-type responses dominated by the production of IFN-g (interferon-g), which in turn activates cell-mediated immunity associated with protection against this class of organism. The innate capacity of dendritic cells and macrophages to produce the cytokine IL-12, which promotes the ability of NK (natural killer) and T cells to make IFN-g, placed these phagocytes at the core of this process. In contrast, much less was known about how dendritic cells influence the development of Th2 responses and many groups have focused on understanding the events during infection that led to the early production of IL-4. Recent studies have gone a long way to understanding this process and revealed complex networks in which eosinophils, basophils, and epithelial cells contribute to the programming of appropriate T-cell activities during helminth infections. Nevertheless, with the continuing identification of additional T-cell subsets (Treg, TFH, Th17), their relevance to the control of parasitic infections and how the innate immune system promotes their development remains uncertain. There are also several practical aspects to understanding how these systems operate and defining the innate pathways through which pathogens polarize adaptive

Although parasitism implies mutual coexistence of host and infectious agent, the immune response plays a critical role in the establishment and maintenance of this balance. Traditionally, the control of parasitic infections was thought to be the exclusive domain of the acquired immune system and typical innate functions, such as the ability of phagocytes to engulf and destroy invading microorganisms or complement components to kill extracellular parasites, were primitive effector mechanisms. Since the start of the 1990s, it has been recognized that the early interactions between the host innate system and pathogens shape subsequent adaptive responses and the outcome of infection (resolution, latency, disease). Consequently, there is now an appreciation that the innate events that underlie these functions are complex and a number of receptors, including Toll-like receptors (TLRs), have been implicated in the ability to recognize parasites specifically or which act as part of a surveillance system that distinguishes non-self from self. How these pattern recognition receptors distinguish diverse parasites and influence adaptive responses, as well as the ability of pathogens to evade these antimicrobial effector functions, are major themes of this chapter. As noted previously in this volume, parasitic organisms are diverse in their biology and host habitats, and consequently there is the need to tailor the protective response to each individual microorganism. The highly divergent lifestyles and patterns of adaptive immune responses associated with control of helminth and protozoan parasites are also reflected in the distinct innate responses they trigger and in their consequences for the pathogen. In the case of protozoa, innate immunity has an important role in limiting early parasite replication until cognitive T- and B-lymphocyte responses can dominate. Alternatively, the innate recognition of helminths may function to limit the number of invading larvae and perhaps slow their development prior to the onset of adaptive immunity. Consequently, in order to be successful, parasites have to survive the innate response and this hard-wired system provides a powerful selective pressure Christopher A. Hunter, Department of Pathobiology School of Veterinary Medicine, University of Pennsylvania, Rm 313, Hill Pavilion, 380 South University Avenue, Philadelphia, PA 19104-4539. Alan Sher, Laboratory of Parasitic Diseases NIAID, NIH Building 50, Room 6140, 50 South Drive, MSC-8003, Bethesda, MD 20892-8003.

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functions may lead to new approaches for improving the efficacy of vaccination and directing the development of appropriate adaptive responses that are pertinent to immunoregulation in general. Moreover, since, by definition, the protective mechanisms in question do not depend on conventional T- and B-cell recognition, they may be of particular use for the design of strategies to limit infection in immunocompromised individuals.

HUMORAL MECHANISMS OF INNATE IMMUNITY Activation and Evasion of Complement

Perhaps the simplest forms of innate immunity are represented by the presence of preexisting, soluble factors that can recognize and destroy invading parasites. While the classical pathway of complement activation is typically associated with antibody responses, the alternative pathway represents one of the oldest, evolutionarily conserved, defense mechanisms that provide initial protection against invading microorganisms. This recognition system, detailed in chapter 6, is based on the continuous turnover of the C3b molecule on all surfaces, but which is normally inactivated by different complement regulatory factors such as Factors H and I. The surfaces of most parasites lack these regulatory proteins and this can result in the accumulation of C3b, the initiation of the complement cascade and formation of the membrane attack complex (MAC), and pore formation in the surface membranes. There is also a lectin-mediated pathway in which mannose residues on parasite surfaces are recognized by a mannan-binding lectin (MBL), which initiates complement activation. Irrespective of how the complement system becomes activated, the formation of the MAC can lead to direct parasite lysis or the deposition of complement components, which will opsonize parasites for phagocytosis. These events are also relevant to other processes that act to coordinate and regulate immunity. Thus, complement activation components are chemotactic and attract immune cells to the site of infection and have additional properties that can also amplify the adaptive arm of the immune system. Because the complement system represents such an important first line of defense in resistance to pathogens, many parasites have developed a variety of developmentally regulated strategies to subvert complement-mediated attack. Some of the best characterized instances for trypanosomatids show that the preinfective forms of parasites found in insect vectors are susceptible to lysis via the alternative pathway of complement activation, whereas the infective forms can evade this defense mechanism. It is notable that for some apicomplexans such as Plasmodium, Theileria, and Babesia, while the ability of the infective sporozoite stages to avoid complement activation is essential for these pathogens to establish infection, the basis for resistance is not well understood. Perhaps the best illustration of the importance of evading complement is provided by different stages of the African trypanosome, Trypanosoma brucei. For this hemoparasite, the procyclics found in the midgut of the tsetse fly, are highly susceptible to the alternative pathway of complement activation (Ferrante & Allison, 1983). However, as these forms develop into the infective metacyclic stage in the salivary glands, they express a thick surface glycoprotein that provides a physical barrier that protects against the MAC. While it is likely that this coat first developed as a means to evade complement-mediated lysis, the ability to sequentially express antigenically distinct forms of this

surface glycoprotein, forms the basis for antigenic variation, which allows this parasite to persist in the face of an adaptive immune response (see chapter 36). The South American trypanosome, T. cruzi, has also evolved ways to avoid the complement system. Similar to the African trypanosomes, the epimastigote stage of T. cruzi, found in the gut of the reduvid vector, is susceptible to lysis by the alternative pathway of complement, but, as it matures into the infective trypomastigote, it develops several mechanisms that either prevent efficient complement activation or lead to its shedding from the surface. The identification of the trypomastigote gp160 antigen as a homolog of the host complement regulatory protein, decay-accelerating factor (DAF) was an important step in understanding how these parasites evade complement. Like DAF, gp160 can bind to C3b and C4b (Norris & Schrimpf, 1994) and inhibit the uptake of subsequent members of this complement cascade, thus preventing convertase formation and lysis of the parasite. In addition, the binding of C3b to gp160 allows a parasite protease to cleave this complex, which may represent a mechanism to avoid lysis as well as complement-mediated opsonization. Importantly, whereas complement-sensitive epimastigotes fail to express gp160, epimastigotes transfected with gp160 are resistant to complement-mediated lysis (Norris, 1998). A similar set of challenges are also relevant to other trypanosomatids and the extracellular promastigote stage of Leishmania sp. is found in the midgut of the sand fly vector and can activate the alternative lectin-mediated pathways of complement. As promastigotes develop into the infective metacyclic stage present in the proboscis, their membrane is altered to prevent the insertion of the lytic C5b-C9 complex. This change correlates with their ability to express a modified form of a surface lipophosphoglycan (LPG), which is approximately twice as long as the form on promastigotes and which may inhibit the insertion of the MAC into the surface membrane of the parasite (McConville et al., 1992). However, the importance of LPG in the virulence of different Leishmania species is variable, as gene-targeting studies indicate that LPG deficient promastigotes of L. mexicana are still infective (Ilg, 2000), while its absence in L. major impacts on virulence and infectivity (Spath et al., 2000). Another developmental change that occurs during generation of metacyclics is the increased expression of the surface proteinase gp63, which can cleave C3b to the inactive iC3b form and so prevent deposition of the C5b-C9 complex (Brittingham, 1995). Parasites associated with mucosa are also susceptible to the effects of complement, which is an important factor that limits invasion across these barrier surfaces. Several studies have implicated the MBL in resistance to Cryptosporidium parvum and a patient with a primary genetic defect in the MBP allele that lacked serum levels of this lectin had persistent C. parvum infection (Summerfield et al., 1995). Consistent with this finding, subsequent work revealed that MBP can bind to sporozoites of C. parvum and was capable of activating C4. Moreover, in a small cross-sectional study, these authors reported that in patients with AIDS, the risk of cryptosporidiosis was increased if they had mutations in MBL (Kelly et al., 2000). The protozoan Entamoeba histolytica normally produces a nonpathogenic infection in the gut, but can cause local disease at this site and in severe cases can result in liver abscesses. Once this extracellular pathogen has invaded tissues, it is exposed to soluble host factors and in experimental animal models the depletion of complement results in elevated disease (Capin et al., 1980). Although there are contradictory reports on the capacity of pathogenic

18. Innate Immunity to Parasitic Infections

versus nonpathogenic forms to activate the alternative complement pathway and their susceptibility to lysis, these amoebae have developed the ability to prevent complement-mediated lysis. Understanding the mechanistic basis for this differential sensitivity was aided by the identification of a parasite-derived galactose-specific adhesin, which confers resistance to complement sensitive amoebae (Braga, 1992). This parasite-derived product appears similar to the host molecule CD59, a known inhibitor of the assembly of the MAC. In addition, it has been proposed that the ability of these amoebae to incorporate host complement regulatory proteins into their membranes is one mechanism that prevents the insertion of the MAC (Gutierrez-Kobeh et al., 1997). Moreover, the extracellular cysteine proteinase of E. histolytica can specifically cleave C3 and while this leads to activation of the alternative pathway, it may also inactivate C3a and C5a and so prevent their immunoregulatory and chemotactic effects (Reed et al., 1989). Thus, there are multiple possible ways for this amoeba to evade complement activation, but understanding the relationship between these different mechanisms, relative susceptibility to complement and virulence remains a major challenge. For helminths, there are numerous reports that have described the deposition of complement on the surfaces of these organisms but the relative importance of such observations was unclear. Several studies have begun to address this issue and the markedly enhanced survival of microfilaria in MBLdeficient mice indicates the significance of the ability of MBL to bind to the surface of Brugia malayi microfilaria and activate C3. However, whether MBL promotes resistance to B. malayi through direct complement-mediated killing or through its ability to promote antibody responses is uncertain (Carter et al., 2007). Regardless, other reports have provided evidence that the alternative pathway that leads to C3 activation is required for resistance to larval strongyloides (Kerepesi et al., 2006). The basis for this protective effect is unclear but the activation of C3 and C5 promotes eosinophil extravasation and their production of IL-4 and IL-13 and this may provide the link between the role of the alternative pathway for the recognition of Nippostrongylus brasiliensis and recruitment of eosinophils required to destroy this nematode (Giacomin et al., 2008). Several helminths have also evolved the ability to antagonize complement activation. Larvae of the nematode Anisakis express a secreted product that inhibits the early cascade that leads to complement activation (Garcia-Hernandez et al., 2009). In contrast, other worms can use host complement regulatory proteins to limit local activation. For example, the wall of the hydatid cyst of Echinococcus granulosus contains the host complement regulatory protein, factor H (Diaz et al., 1997), while larvae and adults of Schistosoma mansoni express host decay accelerating factor on their tegumental surfaces (Fishelson, 1995). These examples demonstrate that the ability of parasites to mimic complement-regulatory molecules, or to acquire them from the host, represents a central strategy for evading complement-mediated damage by the innate immune system.

Trypanolytic Factor

In addition to complement, there are other soluble factors that mediate innate immunity to parasitic infections. In particular, the resistance of humans to T. brucei is determined in part by a primate specific, innate cytolytic defense mechanism that restricts the host range of African trypanosomes. With the recognition that human blood contained a factor that would lyse certain species of trypanosomes, fractionation of serum led to the characterization of different complexes that contained high-density lipoproteins that mediate this

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cytolytic effect. Subsequent studies demonstrated that this trypanosome lysis factor (TLF1), is composed of several common apolipoproteins, as well as a haptoglobin related protein (Hpr) (Smith et al., 1995). A second cytolytic complex, TLF2, has also been identified, which shares many of the components of TLF1 but contains a unique IgM component, and has a lower lipid content (Raper et al., 1999). The finding that TLF has to undergo receptor-mediated uptake and enter an intracellular acidic compartment to mediate cytotoxicity raised questions about how this factor actually killed parasites (Hager & Hajduk, 1997). It has now been proposed that ApoL1 has structural similarity to a family of pore forming molecules that includes Bcl2, and that the ability to form anionic pores in parasite membranes provides the basis for its cytolytic effects (Perez-Morga et al., 2005). Interestingly, the antimicrobial activities of these lipoprotein complexes does not appear to be restricted to African trypanosomes but are also relevant to Leishmania and Plasmodium sp. (Imrie et al., 2004; Samanovic et al., 2009). This latter observation may explain the long-standing difficulty associated with adapting malaria parasites to in vitro culture in media containing human serum. Although TLF provides a potent antimicrobial mechanism in humans capable of killing T. brucei, the species that infect humans (T. b. gambiense and T. b. rhodesiense) are not susceptible to TLF-mediated cytolysis. Possible mechanisms that underlie this resistance include the inability of these parasites to internalize TLF and the expression of higher levels of antioxidants that protect from TLF-mediated peroxidase activity (Smith et al., 1995). A molecular basis for parasite resistance to TLF is provided by studies that have correlated resistance to TLF to the expression of a serum resistance associated (SRA) gene, which is homologous to the variant surface glycoprotein. Indeed, transfection of SRA into T. brucei confers resistance to human serum, identifying this gene as being critical for the adaptation of T. b. rhodesiense to survive in humans (Xong et al., 1998). A better understanding of these processes may lead to therapeutic approaches to render T. b. gambiense and T. b. rhodesiense susceptible to TLF. In this regard, transgenic mice expressing different human TLF components have recently been developed that display trypanolytic activity in vivo (Molina-Portela et al., 2008). These studies provide a proof of principle that similar approaches may be useful in the generation of livestock that are resistant to multiple species of trypanosomes.

CELLULAR MECHANISMS OF INNATE IMMUNITY TO PROTOZOA Innate Defenses Mediated by Phagocytes

Perhaps the most basic cellular mechanism associated with resistance to infection involves the ability of phagocytes to engulf invading microorganisms. This can lead to the isolation of pathogens within a hostile lysosomal compartment accompanied by a respiratory burst that represents a first line of defense that results in death of many parasites. This is illustrated by the ability of mononuclear phagocytes to kill many parasites in vitro and monocytes appear to have a critical role in limiting early parasite replication. Given this barrier, it is not surprising that many pathogens have developed ways to evade these responses and some can even survive inside phagocytes, which, in turn, protects them from humoral effector molecules. For Leishmania species, these organisms have evolved to proliferate within the macrophage lysosomal system and a number of strategies have been proposed that allows these parasites to survive

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in this hostile environment. This includes the ability of newly phagocytosed promastigote to modify the host cell cytoskeleton to prevent phagolysosome maturation (Lodge & Descoteaux, 2005), and so allow differentiation to the amastigote stage, which is resistant to hydrolases (VannierSantos et al., 2002). In contrast, the intracellular survival of T. cruzi is dependent on the ability of the parasite to escape from the phagolysosome. This process may be facilitated by this protozoan’s expression of a homologue of C9 that can disrupt the phagosome membrane allowing escape of the parasite into the cytoplasm (Andrews et al., 1990). Importantly, it appears that exposure to the lysosomal environment provides a developmental signal that prevents the motile trypomastigote stage from exiting the cell and allows completion of the intracellular phase of the infection (Andrade & Andrews, 2004). A third strategy to allow intracellular survival is illustrated by the ability of T. gondii to actively invade cells and form a parasitophorous vacuole, which does not undergo acidification. If instead the parasite is forced to enter the cell by a phagocytic pathway, it is exposed to the normal phagolysosomal system and killed (Joiner et al., 1990). While these direct antimicrobial activities of professional phagocytes are implicitly linked to resistance to infection, some parasites have co-opted these events to promote their survival and perhaps even dissemination. The opsonization of Leishmania sp. with iC3b leads to their phagocytosis via the CR1 and CR3 receptors, which fail to trigger the respiratory burst and targets the parasites to the macrophage (their host cell of choice) and is also important for the intracellular survival of the parasite. This perspective has been modified in recent years by studies implicating neutrophils as the initial cell type that phagocytoses L. major (Laskay et al., 2008). Real time imaging has demonstrated rapid swarms of neutrophils within hours at the site of sand fly bites and that depletion of neutrophils prior to vector feeding leads to reduced rates of infection (Peters et al., 2008). Experimental data have provided evidence for a model in which the death of infected neutrophils by apoptosis leads to their phagocytosis and creates an anti-inflammatory environment. One other consequence of these events is that they would also deliver the newly transformed amastigotes directly into the macrophage, thus making neutrophils “Trojan horses” for the parasite (Laskay et al., 2008). This provocative idea is also proposed for T. gondii based on the evidence that dendritic cells and macrophages infected with this organism are able to migrate from sites of infection and carry the parasite to other sites of the body (Lambert et al., 2006). Various imaging studies have also provided evidence that the lysis of infected DC promotes the ability of tachyzoites to infect CD81 T cells or NK cells (Lambert & Barragan, 2009). The topic of parasite dissemination and the extent to which host cells are utilized is clearly relevant to many organisms and understanding these events could provide new insights into how intracellular pathogens pirate host cell machinery. One theme that should be apparent from the studies described above is that many parasites are specialized to survive and replicate in macrophages and other host cells. This, in turn, would eventually lead to cytolysis of infected cells and so, many of these organisms have the potential to cause significant disease if they are not appropriately controlled. One strategy employed by infected hosts is to use cytokines to instruct macrophages to increase their phagocytic and metabolic activity and augment their capacity to effectively deal with intracellular organisms. Numerous in vitro studies have shown that while resting macrophages can support the replication of many intracellular parasites, if these cells are preactivated with IFN-g, alone or in combination with

other stimuli such as TNF-a (tumor necrosis factor-a), then they will destroy these parasites. Subsequently, it was recognized that the ability of IFN-g to upregulate enzymes such as iNOS and the production of various oxygen free radicals is an important component required to limit intracellular replication. However, several in vivo studies suggested that IFN-g also had other protective effects and raised new questions about the actual mechanisms employed by macrophages to limit parasite replication. Particular attention has focused on a family of IFN-g-induced GTPases in these events (Taylor et al., 2007). To date, their mechanisms of action are unknown, although in the case of Toxoplasma, there is evidence that the GTPase IGTP (Irgm3) regulates autophagy-mediated killing of tachyzoites and that the ability to evade this mechanism is linked to parasite virulence (Zhao et al., 2009a, 2009b). Nevertheless, it is not clear yet whether similar mechanisms explain the role of other p47GTPases in IFN-g dependent killing of different intracellular protozoa and bacteria (Taylor et al., 2007).

Innate Immunity and the Development of IFN-g Mediated Control of Intracellular Protozoa

The section above highlighted the role of IFN-g in activating macrophages to kill intracellular parasites, and while this phenomenon was first described in the early 1980s, it was not appreciated at the time that the innate immune system can serve as a source of IFN-g and by this mechanism contribute to early pathogen control. Historically, it was recognized that infection of mice with different parasitic organisms or challenge with parasite antigens results in a transient increase in NK cell activity (Scott & Trinchieri, 1995). These large, granular lymphocytes share a common developmental pathway with T cells, but lack a T-cell receptor and cannot directly respond to foreign antigens. Nevertheless, like T cells, they can recognize class I molecules, have cytotoxic functions, and produce a number of cytokines, in particular IFN-g. There is experimental evidence that these cells can lyse cells infected with parasites but it seems that the early production of IFN-g by NK cells is the most critical mechanism that prevents parasites from overwhelming the host prior to the development of adaptive responses to many other intracellular pathogens (Scott & Trinchieri, 1995). The discovery of IL-12 and the early description of its role in activating NK cells to produce IFN-g provided a framework to understand how different cell populations could interact to provide sophisticated forms of innate immunity. It was quickly recognized that the ability of many intracellular parasites to induce IL-12 has a critical role in the activation of NK cells to produce IFN-g and so provide a T-cell independent mechanism of resistance to infection (Scott & Trinchieri, 1995). This model raised new questions about the cellular sources of IL-12 during these infections and initial reports identified inflammatory macrophages and neutrophils as contributing to the production of IL-12 during multiple infections. Further studies using soluble products of T. gondii tachyzoites showed that CD8a1 dendritic cells can respond to this stimulus and produce IL-12 as well as other proinflammatory cytokines, especially during the initial stages of Toxoplasma infection (Reis e Sousa et al., 1997). Genetic approaches to delete DC confirmed their critical role as a source of IL-12 during toxoplasmosis (Liu et al., 2006). It is now recognized that murine dendritic cells have the capacity to make IL-12 in response to Leishmania amastigotes and promastigotes and thus may provide the initial source of the cytokine during Leishmania infection (Scott & Hunter, 2002). It should be noted that there are multiple DC subsets and plasmacytoid DC also have recently been


implicated as a source of IL-12 during toxoplasmosis (Pepper et al., 2008). Distinguishing the role of these DC subsets in different phases of the immune response is an ongoing area of investigation in multiple parasitic diseases. Although IL-12 is a critical cytokine required to stimulate NK cell production during parasitic infections, its ability to stimulate NK cells to produce high levels of IFN-g is dependent on other soluble and cell bound ligands. Several studies have identified TNF-a, IL-1, and IL-18 as being important cofactors for IL-12-induced NK cell production of IFN-g in response to many parasites (Korbel et al., 2004). Costimulation also has a role in the NK-cell-mediated response to intracellular parasites, and many cells infected with intracellular parasites have been shown to upregulate expression of B7, the ligand for the costimulatory molecule CD28. This molecule is typically associated with providing the second signal required for optimal T-cell responses but activated NK cells also express CD28 and stimulation through this interaction enhances IL-12-induced, NK cell production of IFN-g and innate resistance to T. gondii (Hunter et al., 1997). These observations are likely relevant to other parasites and the ability of L. donovani to prevent infected macrophages from up regulating the expression of costimulatory molecules (Kaye et al., 1994) is consistent with subversion of optimal innate responses and correlates with the ability of L. donovani to bypass NK cell production of IFN-g. Although many of the studies on NK cells have been performed using murine models, there is evidence for similar pathways in humans. Thus, stimulation of human PBMCs with subcellular components of T. gondii will activate human NK-cell cytotoxicity (Sharma et al., 1984). Similarly, P. falciparum infected erythrocytes added to PBMCs induces an IL-12 dependent production of IFN-g (Artavanis-Tsakonas, 2002), but the function of NK cells during malaria appears more complex than just the production of cytokines. While early reports demonstrated that NK cells can lyse erythrocytes infected with P. falciparum, subsequent work has shown that stimulation of PBMC with infected erythrocytes results in stable conjugates between NK cells and infected cells and that responding NK cells increased expression of the activating molecules CD94 and NKG2A (Artavanis-Tsakonas, 2003). Other ligands are also likely to be involved and NK cells express a wide array of activating and inhibitory receptors that are just being explored in this context. Understanding how infection affects the repertoire of these diverse receptors is an area that needs to be explored. While this section has focused on NK cell production of IFN-g, these lymphocytes also produce other cytokines, such as IL-10 and TGF-b, that are relevant to cell mediated immunity. NK cells are a prominent source of IL-10 in L. donovani infection (Maroof et al., 2008) and IL-10 is a potent antagonist of accessory cell functions required to activate NK and T cells. Similarly, TGF-b (a product of many cell types including macrophages and NK cells) has been shown to inhibit NK cell production of IFN-g and resistance to T. gondii and L. major (Hunter et al., 1995; Laouar et al., 2005). In addition, the production of IL-10 and TGF-b during parasitic infection can antagonize macrophage effector functions and, by so doing, enhance the survival of intracellular parasites. It is unclear whether the production of these inhibitory cytokines represents a parasite strategy to inhibit protective host responses or simply reflects a balanced host response required to prevent the development of immunopathology. It is also becoming apparent that NK cells have other regulatory activities that are likely relevant to parasitic infections. They are a source of IL-17 during toxoplasmosis (Passos et al., 2010), a cytokine that is linked to mobilization of neutrophil responses. Other reports in

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noninfectious models have also suggested that NK cells are a potent source of IL-22, a member of the IL-10 family, and further studies are needed to investigate the respective roles of IL-17 and IL-22 in promoting either inflammation or barrier function during parasitic infection.

Innate Cellular Responses Associated with Resistance to Helminth Infection

Although antibody responses are characteristic of resistance to helminths, there are several innate interactions of these parasites with granulocyte populations associated with Th2 type responses. Thus, eosinophilia or mastocytosis are characteristic features associated with the tissue migratory phase of multiple helminths but reductions in mast cells numbers do not affect the expulsion of T. muris (Koyama & Ito, 2000). Cercariae of S. mansoni are capable of activating the release of histamine from mast cells in vitro (Catto et al., 1980) and this could be important in attracting inflammatory cells into areas where these parasites are migrating. Moreover, mobilization of mast cell precursors is observed within hours after infection with Trichinella spiralis and these cells are required for optimal expulsion of this worm through their ability to disrupt epithelial cell barriers and promote intestinal permeability (McDermott et al., 2003). Such barriers are also important for control of T. muris and increased epithelial turnover in Trichuris and death of these cells may play a role in worm expulsion (Cliffe et al., 2005). In addition, compromising innate signals mediated through the NF-kB system in epithelial cells appears to limit protective immunity to this helminth (Zaph et al., 2007). Whether this is a consequence of deficient innate recognition, altered immune regulation, or due to differences in epithelial cell survival remains to be resolved. The observation that eosinophils kill opsonized schistosomula of S. mansoni in vitro and the histological association of these cells with dead or dying larval stages of various helminths suggests an important role for this cell population in resistance to the tissue migratory, larval stages of various helminths. However, that many of these events were dependent on the opsonic effects of antibody and eosinophilia is thought to be largely dependent on the production of IL-5 by T cells and thus appears to represent an adaptive immune response. Nevertheless, there is also a T-cell independent eosinophilia in mice infected with Toxocara canis which is dependent on IL-5 (Takamoto et al., 1997). Since NK cells can produce IL-5, this may represent a mechanism whereby NK cells can influence the development of effector mechanisms required for resistance to worms. In support of the latter hypothesis, the production of IL-5 by NK cells may contribute to the regulation of eosinophilia in response to Heligmosomoides polygyrus (Svetic et al., 1993). In direct contrast, it has been reported that the growth of the filarial worm B. malayi is dependent on host NK-cell function. Thus, comparisons of worm survival and development in different strains of mice with varying levels of NK-cell activity revealed that mice with reduced NK-cell activity are nonpermissive to growth of B. malayi, whereas SCID mice with normal NKcell activity are susceptible and depletion of NK cells in SCID mice prevents growth of this parasite (Babu, 1998). NK-cell production of IL-13 during Trichinella infections is also responsible for the disruption of intestinal tissue architecture and induction of goblet cell hyperplasia, and these cells also appear to play an important role in the remodeling of intestinal tissues (McDermott et al., 2005). The above findings illustrate the complex interplay between NK cells and the various granulocytic populations associated with protection against helminths.

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THE MOLECULAR BASIS FOR INNATE RECOGNITION OF PARASITES

With the realization that a variety of cell populations are able to provide innate recognition of distinct classes of parasites, it was implicit that they would express receptors that allow specific recognition of these organisms based on some unique motif or pattern that leads to the production of IL-12 and other inflammatory mediators. Perhaps the best characterized pattern recognition receptors (PRR) are the evolutionarily conserved TLRs that were identified based on genetic approaches that identified human homologs of Toll-like receptors (TLRs) from Drosophila and the ability of one of these homologs (TLR4) to recognize LPS. Since then, a family of 12 different TLRs has been identified and their specific ligands characterized. TLR ligands have now been identified in nearly every class of microbe and, in the case of parasites, in both helminths and protozoa, although their functional importance is more firmly established in the latter group of pathogens. Interestingly, many of the TLRs appear to be specific for multiple ligands shared by different parasites such as TLR4 and TLR2, which recognize different GPI anchors and TLR9, which can identify unmethylated cytosine phosphate guanosine (CpG) motifs in the DNA of many protozoa (Shoda et al., 2001). Because many TLRs signal through an adapter molecule called MyD88, MyD882/2 mice have become an important tool for testing the general role of TLRs in resistance, and MyD88-dependent mechanisms of resistance to many protozoan parasites have been identified (Gazzinelli & Denkers, 2006). However, the interpretation of these findings as showing a function for innate TLR-mediated recognition has to be tempered by the role of MyD88 in unrelated signaling pathways, such as those mediated through the IL-1R family, or in effector lymphocytes functioning in the adaptive immune system. Some of the major recent findings on the role of TLRs in the innate immune response to parasites are summarized below.

Recognition of Protozoa

The glycosylphosphatidylinositols (GPI) lipid anchors of many parasite surface proteins are capable of activating host protein kinases and stimulating the production of proinflammatory cytokines. Thus, GPIs of phylogenetically diverse parasites can directly simulate macrophages to produce a number of proinflammatory cytokines. Interestingly, the GPI of T. cruzi trigger cytokine production via TLR2, while those derived from epimastigotes preferentially stimulate TLR4. This difference is associated with additional galactose residues and unsaturated fatty acids present in the GPI anchors isolated from trypomastigotes (Campos et al., 2001). In addition, DNA from T. cruzi stimulates cytokine production in infected DC and macrophages through TLR9, and recognition by TLR2 and TLR9 together appear to explain MyD88 dependent innate resistance to the parasite in vivo (Bafica et al., 2006). In addition, a role for the alternative TLR adaptor TRIF has been documented in T. cruzi infection and linked to the production of Type-1 IFN (Koga et al., 2006), although the specific TLR mediating this effect has not been identified. The above findings may be relevant to susceptibility to the human disease, since patients with genetic polymorphisms in the adaptor MAL/ TIRAP, that result in a reduced capacity to signal through TL2/4, have less risk of developing Chagas’ cardiomyopathy (Ramasawmy et al., 2009). In contrast to the activating effects of many parasite GPIs, the LPG and GIPLS of Leishmania have been shown to have a number of down-modulatory effects on macrophage function, including inhibition of IL-12 production as well as reduced expression of iNOS and TNF-R.

An insight into the molecular basis for these events is provided by studies in which it was shown that LPG can interfere with signaling in infected cells and is unable to activate protein kinase C (Olivier et al., 2005). Thus, it appears that variation in structure of parasite GPIs imparts different properties of signal transduction upon this class of glycolipid. In this regard, while TLR2 recognition of LPG has been implicated in MyD88 dependent resistance to L. major (de Veer et al., 2003), the role of TLR2 in L. brasiliensis is paradoxically associated with inhibition of DC responses (Vargas-Inchaustegui et al., 2009). In addition to TLR2, TLR9 has now been linked to the regulation of innate immunity to Leishmania presumably through the recognition of CpG motifs in DNA by DC and subsequent activation of NK cells (Schleicher et al., 2007). Toxoplasma contains GPI anchors, which, like their counterparts in T. cruzi, stimulate cytokine production by macrophages through TLR2/4 and in the case of TLR2, this response has been linked in some, but not all, in vivo studies to host resistance (Gazzinelli & Denkers, 2006). IL-12 production by T. gondii exposed dendritic cells has been shown to be due to recognition by TLR11 of profilin, an actin associated protein that is also required for parasite invasion (Plattner et al., 2008). These PRR may also have other roles in the overall pathogenesis of toxoplasmosis, and following oral infection with T. gondii, a breakdown of intestinal barrier function may allow signals from gut microflora, through TLR2, 4, and 9, to contribute to IL-12 production and Th1 priming (Benson et al., 2009). While apparently not important for control of bloodstage infection, TLR2 recognition of GPI in Plasmodium has been postulated to play a role in cerebral malaria and the GPIs themselves to serve as the “malaria toxins” previously proposed in earlier studies. More recently, hemozoin, the pigment that forms in erythrocytic stages of malaria as a result of hemoglobin degradation, has been shown to stimulate TLR9 (Coban et al., 2005). However, an alternative model has been proposed in which the AT rich parasite DNA motifs form complexes with hemozoin, which are then endocytosed, thereby triggering TLR9 signaling (Parroche et al., 2007). There is evidence in murine models that supports a role for TLR9 signaling in malaria immunopathology but, in one human genetic study, no association was found between a TLR9 polymorphism and severe malaria (Campino et al., 2009). Recently, a different innate recognition pathway has been implicated in the induction of malaria immunopathology. Parasite-derived hemozoin crystals were shown to trigger tissue inflammation by a mechanism involving the NALP3 inflammasome (Dostert et al., 2009; Tiemi Shio et al., 2009). The latter pathway is involved in triggering the cleavage of pro-IL-1b into the mature, bioactive cytokine and involves the NLR (Nod-like receptor) family of pattern recognition receptors. The involvement of non-TLR pattern recognition pathways has also been implicated in the IRF3 dependent production of Type-1 IFN during early T. cruzi infection (Chessler et al., 2008) and in the NF-AT dependent activation of DC and Th1 cells in the murine response to the same protozoan pathogen (Kayama et al., 2009). Indeed, as is the case with other types of pathogens, it is likely that a variety of different pattern recognition families participate in the recognition of protozoa and the identification of these interactions is a fertile ground for future research.

Recognition of Helminths

As noted above, while parasitic helminths clearly possess TLR ligands, TLR signaling appears to be largely suppressed rather than triggered during helminth infection. In addition, when it is triggered by helminth products TLR signaling has


an immunosuppressive rather than immunostimulatory outcome, as exemplified by the phosphorylcholine-containing filarial protein ES62 and its inhibitory interaction with TLR4 (Goodridge et al., 2007). Reduced TLR expression has been described during human lymphatic filariasis and proposed as a means of immune dysregulation (Babu, 2005). In contrast, TLR stimulation in filarial infection is primarily associated with immunopathology and, in this situation, is mediated not by the worms themselves but by their bacterial Wolbachia endosymbionts, which possess TLR agonists (Hise et al., 2007). It has been proposed that continuous exposure to these bacterial TLR ligands may induce a state of macrophage tolerance that would nonspecifically contribute to the generalized depressed responsiveness seen in chronic filarial infection (Turner et al., 2006). In the case of schistosome infection, in vitro studies with newly transformed larvae have suggested that TLR4 signaling may play a role in setting up the early Th1 response to the parasite through triggering of IL-12 production by DC. However, once the adult worms start laying eggs and the Th2 response evolves, TLR signaling appears to be suppressed and eggs and soluble egg extract have actually been shown to inhibit LPS-induced TLR4 signaling in DC (Kane et al., 2004). Moreover, the egg-induced Th2 response is itself MyD88 and TRIF independent and appears to depend instead on an as yet to be identified recognition pathway involving the secreted glycoprotein omega-1 (Kane et al., 2008; Marshall, 2008; Steinfelder et al., 2009). Although eggs have been described to contain a dsRNA species that stimulates TLR3 dependent Type-1 IFN production by DC, the role of this activity in the host response to the ova is currently unclear. Interestingly, a recent report studying the inhibitory effects of a schistosome larval protein (Sm16) on LPS and PolyI:C induced cytokine responses in human PBMC concluded that the target of this suppression is downstream of the TLR complex itself (Brannstrom et al., 2009). Thus, in general, it appears that helminth products suppress rather than stimulate TLR signaling, although the mechanisms involved are still poorly defined.

THE ROLE OF THE INNATE RESPONSE TO PARASITES IN DETERMINING THE NATURE OF ADAPTIVE IMMUNITY

Although innate immunity has an important role in resistance to acute infections, it is the adaptive response, characterized by the selective expansion of T and B cells, that is required to provide long-term immunological control of many chronic parasitic infections. The last decade has seen a major effort to investigate the link between innate and adaptive immunity, and, in particular, to define the role played by innate signals in accessory cells that regulate antigen processing and presentation, provide costimulation, and produce cytokines that affect the onset, duration, magnitude, and class of the adaptive immune response. While it is clear that in the responses to certain parasite products, TLR signaling can explain this link, it is unlikely to be the sole mechanism. Protozoan and helminth infections, which involve sharply divergent T-cell effector outcomes, provide powerful models for studying the interplay between the innate and adaptive arms of the immune system and for identifying these additional pathways.

Initiation of Th1 Responses

While innate production of IL-12 is important for NK-cell activation, this cytokine is also critical for the development of protective T-cell responses required for long-term control

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of many intracellular parasites. The ability of IL-12, which is often produced as a consequence of TLR stimulation of DC and other accessory cells, to drive development of Th1-type responses is a function of the direct and indirect activities of this cytokine. IL-12 has direct effects on the development of Th1 responses, but its ability to stimulate the production of IFN-g by NK cells early in the course of infection can also influence the development of subsequent antigen specific Th1 CD41 T cell required for resistance to L. major (Scharton-Kersten et al., 1995). The mechanism whereby IFN-g affects the development of Th1 responses is not clear, but may be due to its ability to directly trigger T cells and prevent their ability to develop into Th2 cells. The identification of a key role for IL-12 in the activation of NK cells and development of Th1 type responses has led to its use in experimental models to enhance host resistance to many parasitic infections. In addition, IL-12 has been used to modulate vaccination responses to enhance immunity to intracellular parasites and for the development of an antipathology vaccine during schistosomiasis (Afonso, 1994; Wynn et al., 1995). With the discovery that TLR ligands are themselves potent inducers of accessory cell IL-12 production, these agonists have replaced the cytokine itself as adjuvants for vaccination. From the parasite’s point of view, delaying the initial induction of IL-12 and other innate responses involved in T-cell response initiation and polarization may represent a strategy for immune evasion. In this regard, it has been proposed that in the initial stages of T. cruzi infection, the parasite temporarily escapes early TLR recognition, which results in a delay in the induction of protective CD81T cell responses (Padilla et al., 2009). Similarly, virulence in T. gondii strains has been shown to correlate with reduced DC responses and the delayed induction of parasite specific CD81 T cells (Tait et al., 2010). The ability of protozoan pathogens to impair DC function is discussed in chapter 35.

Initiation of Th2 Responses

From some of the earliest studies it became clear that IL-4 has an important role in the development of Th2 type responses, which are generally associated with resistance to helminths and susceptibility to protozoan parasites. For example, the early burst of IL-4 produced in BALB/c mice infected with L. major has been linked with the downregulation of the IL-12 receptor b2-chain expression on CD41 T cells and the inhibited development of protective Th1 responses (Himmelrich et al., 1998). Nevertheless, while Th2 responses are clearly enhanced in the presence of IL-4, there is increasing evidence that this cytokine is not absolutely required for Th2 response initiation. Regardless, the exact role played by IL-4 (or other factors produced by the innate immune system) in directing Th2 development remains an important question. The recent generation of cytokine reporter systems to follow the production of IL-4 in vivo has provided unexpected insights into the source of the cytokine during Th2 polarizing helminth infections. Several innate cell types were found to be associated with IL-4 production, including mast cells, basophils, and eosinophils. The Th2 cytokine IL-13, which shares elements of the IL-4 signaling pathway, also plays a role in determining susceptibility to L. major, as well as resistance to N. brasiliensis, and other nematodes (Finkelman et al., 2004; McKenzie, 2000). As indicated previously, many cell types, including mast cells, basophils, and NK cells, can secrete this cytokine. Whether innate production of IL-13, in addition to promoting effector functions in parasitic infection, is also involved in directing development of Th2 responses remains an open question.

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In addition to serving as an innate source of Th2 polarizing cytokines, innate cells such as eosinophils and basophils have been shown to express MHC class II molecules and to be capable of antigen presentation in vitro and in vivo. In the case of basophils, evidence from recent studies has pointed to the idea that because they possess the dual functions of IL-4 production and antigen presentation, these cells may serve as professional APCs (antigen-presenting cells) for the selective induction of Th2 responses during helminth infection (Padigel et al., 2007; Perrigoue et al., 2009), replacing the role traditionally thought to be held by DCs, which do not appear to express biologically significant levels of IL-4. Nevertheless, as discussed above, IL-4 is not required for Th2 polarization in a number of different helminth infections, and IL-4-deficient DCs exposed to helminth products such as SEA are fully able to polarize Th2 responses in vitro and upon transfer in vivo (MacDonald & Pearce, 2002). While the IL-4 independent mechanisms by which DCs trigger Th2 polarization are poorly understood, a recent study suggests that in the case of schistosome egg products this may involve an effect of the parasite T2 ribonuclease omega-1 on the DC cytoskeleton. This, in turn, is reflected in reduced DC contact with T lymphocytes and lowers the overall strength of the activation signal that DCs deliver to these cells (Steinfelder et al., 2009). As exemplified by the current debate concerning the role of DCs versus other accessory cells in Th2 polarization, the study of the innate signals by which helminths trigger Th2 responses is a fascinating area for future research and may yield important lessons that can be extended to the field of allergic inflammation.

Regulatory Responses and T Cells During Infection,

The study of how the immune system interacts with parasites has generally focused on the events that promote resistance. There is now an appreciation that there needs to be a balanced immune response and that there are a number of mechanisms that prevent the protective responses from becoming pathological (see chapters 8 and 35). For many years, the role of IL-10 in limiting infection-induced inflammation has been recognized, and now other cytokines, such as IL-27, and Tregs have emerged as having critical roles in regulating the outcome of parasitic challenge. Clearly, there are multiple innate sources of IL-10 and IL-27 and cross talk between these cytokines, but there are many questions about how the innate system influences their production. Both IL-10 and IL-27 influence innate functions in the setting of Th1 and Th2 mediated responses (Couper et al., 2008; Stumhofer & Hunter, 2008). It is therefore an attractive concept that parasites might co-opt these regulatory activities to promote their own survival and instances are emerging where this might be relevant. For example, the recent report that NK-cell production of IL-10 inhibits protective immunity to L. donovani (Maroof et al., 2008) highlights the multiple roles of NK cells in innate response to parasites. Accessory cells may also be targeted and glycans of cestodes can trigger innate activation of myeloid suppressor cells, which presumably dampen antiparasite responses (GomezGarcia et al., 2005). Similarly, polar lipids from S. mansoni use TLR2 to prime DCs to induce regulatory T cells (van der Kleij et al., 2002) and in infected TLR22/2 mice, this correlated with reduced Treg responses, decreased egg burdens, and more severe immune pathology (Layland et al., 2007).

CONCLUSIONS

Historically, the concept that innate immunity is important in resistance to parasites was based on the identification of cells with nonspecific effector functions against these pathogens. The field has now advanced to identifying the

cytokines and costimulatory molecules that regulate these innate effector functions and has made considerable progress in defining the molecular signaling pathways involved. However, many questions remain concerning the interactions between the innate immune system and parasites. In particular, we still have a limited understanding of the host receptors involved in early parasite recognition and it is possible that novel mechanisms, not previously identified in the response to other pathogens, may be involved. This is particularly true for parasitic helminths, which represent a phylogenetically distinct and unique group of eukaryotic pathogens. The fact that parasites have evolved strategies to interfere with intracellular pathways such as NF-kB and JAK/STAT signaling associated with innate immunity (see chapter 36) provides a compelling argument for the importance of these pathways in host defense. The ability to apply forward and reverse genetics to define the parasite molecules involved represents an exciting opportunity to finally dissect the molecular basis for these events. This is perhaps best illustrated by recent studies that led to the identification of Toxoplasma rhoptry proteins as inhibitors of STAT signaling (Sibley et al., 2009). Further development and application of these approaches to multiple parasitic systems has the potential to provide major insights into the mechanistic basis for how parasites interfere with innate signaling and, in turn, may reveal novel innate recognition mechanisms. From a practical standpoint, how might this information be used? At present, there are no effective and practical vaccines for protecting against parasitic diseases, and understanding how innate immunity initiates the development of long lived, protective responses to these pathogens may yield new immunization strategies that are essential to move forward in addressing this important public health priority. A.S. was supported by the Intramural Research Program of the National Institutes of Allergy and Infectious Diseases. NIH CAH was supported by NIAID and the State of Pennsylvania.

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ACquired iMMuNiTY TO MiCrOBiAL iNFeCTiONS

IV



19 Acquired immunity against Virus infections EVA SZOMOLANYI-TSUDA, MICHAEL A. BREHM, AND RAYMOND M. WELSH

iNTrOduCTiON

acquired immune system and actively replicate in certain cell types, they may generate many signals that act as costimulatory or “danger” signals and contribute to the efficient activation of acquired immune responses (Matzinger, 2002). In addition, the repetitive surface structure of many viruses may enable them to cross-link B cell receptors, and enhance their efficiency to induce antibody responses (Bachmann et al., 1993; Szomolanyi-Tsuda et al., 2002). In this chapter, we describe the dynamics of T- and B-cell responses elicited by virus infections (see Fig. 1 and 2) and illustrate the functions and importance of these cells with examples from well-studied viral models. This chapter is an updated revision of a chapter with the same name published in 2002 (Szomolanyi-Tsuda et al., 2002). To comply with citation space requirements this previous chapter will be cited for the primary references originally found within that chapter. Copies of the original chapter containing these primary references can be obtained from the authors.

Virus infections activate both the humoral and cellular arms of the antigen-specific acquired immune system, and the combined action of B and T lymphocytes acts to clear the virus and to provide protective immunity against subsequent infections. The importance of acquired immunity against viruses is demonstrated by fatal or persistent infections of viruses in severe combined immunodeficient (SCID) mice, which lack functional T and B cells, under conditions where normal immunocompetent mice would clear the infection without apparent disease or mortality. The relative contributions of the cellular and humoral arms to viral clearance vary with the particular virus. Viruses have evolved by interacting with components of the innate and adaptive immune system and, as a consequence, have developed diverse strategies for replication, spread, and survival in the host. To counter these strategies, the vertebrate immune system has a wide range of effector mechanisms employed by B and T lymphocytes to control virus infections. The humoral and cellular arms collaborate with each other, enhancing or modulating these effector mechanisms. The multiple ways to clear virus provide redundant systems in the case of partial immunodeficiency. Most of our present understanding of antiviral immune mechanisms comes from experiments performed with inbred laboratory mice, because different components of the immune system can be easily manipulated in this animal model. The data obtained in mice, however, have to be carefully interpreted and compared with clinical observations in order to understand human immune functions operating in virus-infected patients. Viruses encode immunogenic protein antigens, but the immune responses to live viruses and inert proteins are fundamentally different. Most inert proteins are poorly immunogenic and have to be administered with adjuvants to generate detectable responses. These adjuvants include Tolllike receptor (TLR) agonists and other agents that stimulate innate immunity-associated cytokines and chemokines. In contrast, live viruses elicit strong immune responses. Because viruses interact with components of the innate and

iNNATe iMMuNiTY AuGMeNTS ACquired iMMuNiTY

Innate immune signals influence both the beginning (Fig. 1) and later stages of acquired T- and B-cell responses. Studies in the past decade have revealed that many viruses are recognized by one or more pattern recognition receptors (PRR), which include several members of the TLR family localized at either the cell surface (e.g., TLR2, TLR4, TLR6), or in the endosomal compartment (e.g., TLR3, TLR7/8, TLR9) (Thompson & Locarnini, 2007). Viral proteins encoded by herpes simplex virus-1 (HSV-1), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (CMV), vaccinia virus (VV), hepatitis C virus (HCV), measles virus, and hepatitis B virus (HBV) can serve as ligands for TLR2, and proteins encoded by respiratory syncytial virus (RSV), measles virus, human T-cell leukemia virus (HTLV), and HBV serve as ligands for TLR4. TLR3 is activated by dsRNA, which is generated during most virus infections and has been shown to be triggered by infections with reovirus, HCV, West Nile virus (WNV), influenza A virus (IAV), RSV, and vesicular stomatitis virus (VSV). TLR7 is a sensor for certain ssRNA structures and it is activated by infections with RNA viruses, such as IAV, Sendai virus, and VSV. TLR9 is activated by viral DNA from DNA viruses such as HSV-1, HSV-2,

Eva Szomolanyi-Tsuda, Michael A. Brehm, and Raymond M. Welsh, Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655.

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FIGURE 1 Kinetics of acquired antiviral immune responses. This shows time kinetics of the antiviral cellular (T and B cell) (Top) and serum antibody (Bottom) responses to a viral infection, progressing from a primary infection to the memory state, followed by a rechallenge with the virus. Innate cytokines include the antiviral IFNs and immunoregulatory and inflammatory cytokines produced by innate immune system cells.

CMV, adenovirus, and adeno-associated virus (AAV). Viral nucleic acids in the cytoplasm and outside of the endosomes can be detected by the cytoplasmic RNA sensors RIG-I and MDA and by recently discovered DNA sensors DAI and AIM2 (Hornung et al., 2009; Takaoka et al., 2007). Activation of one or a combination of several PRRs during virus infection stimulates the synthesis of interferons (IFN) and immunostimulatory and inflammatory cytokines, and plays a major role in orchestrating the adaptive T- and B-cell responses appropriate for the virus.

T-CeLL reSPONSeS

Virus-specific T lymphocytes expressing ab T-cell receptors (TCR) are important for the control of viral infections. Studies with numerous viruses in murine systems have laid the foundation for our current understanding of the functionality of T lymphocytes, including MHC-restriction, the nature of T-cell epitopes, and the hierarchies of epitope recognition (Szomolanyi-Tsuda et al., 2002). The T-lymphocyte response elicited by an antigenic challenge is generally comprised of two cell types: the MHC class II-restricted CD4 T cells and the MHC class I-restricted CD8 T cells (Fig. 2). T lymphocytes recognize antigen derived from either exogenous or endogenous protein

antigens (Fig. 3). This duality enables T lymphocytes to recognize antigens derived from viral proteins that have been internalized by professional antigen presenting cells (APC) and to detect other types of virus-infected cells throughout the body (Rock et al., 2004). In addition to the differences in target recognition, the two T-cell subsets have some preferred functions during the response against the infection. The CD4 T cells serve as regulators of the antiviral immune response by providing help to both CD81 T cells and to B cells by secreting cytokines and by making direct contact via costimulatory molecules in the case of B cells (Fig. 2). In contrast, a main role of CD8 T cells is to eliminate virus-infected cells through cytotoxic mechanisms, and so they are generally referred to as cytotoxic T lymphocytes (CTL). These distinctions are not absolute, as class II-restricted cytotoxic CD4 T cells have been isolated from the peripheral blood of humans infected with viruses such as measles, Epstein-Barr virus (EBV), and human immunodeficiency virus (HIV), and have been shown to lyse viral antigen-expressing B cells in mouse models (Jellison et al., 2005). CD81 T cells have now been demonstrated to synthesize a wide range of cytokines, including IL-2, IFNg, TNF (tumor necrosis factor), and MIP1b, all of which can exert immunoregulatory functions (Callan et al., 2000). Much emphasis has been placed on ascertaining the relative

19. Acquired Immunity against Virus Infections

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FIGURE 2 Cell interactions during acquired immunity. Infecting viruses encounter DCs, which present processed viral antigens on class I MHC to CD8 T cells and on class II MHC to CD4 T cells. B cells capture unprocessed antigens with their B-cell receptor (surface antibody), internalize them, and present processed antigen on their class II MHC to helper CD4 T cells. CD4 and CD8 T cells proliferate and differentiate into effector cells capable of secreting cytokines, providing help to B cells, or becoming CTL. Others become long-lived memory cells. B cells differentiate into shortlived antibody-secreting plasma cells (PCs) or else enter germinal centers, undergo isotype switching and affinity maturation (somatic hypermutation and selection) and become long-lived PCs or memory B cells. fDC 5 follicular DC.

contributions of either T cell subset to the resolution of viral infections, but the optimal control of infection in vivo usually occurs with cooperation between both sets. More recent studies have revealed additional layers of complexity in CD4 T-cell responses (Reiner, 2009). Depending on the cytokine milieu during their initial stages of antigen recognition, CD4 T cells can differentiate into at least four different subsets: Th1 cells, Th2 cells, T regulatory (Treg) cells, and Th17 cells. Th1 cells make high levels of IL-2 and IFNg and are driven through the transcription factor Tbet that responds to and stimulates the synthesis of IFNg. Th2 cells produce IL-4, IL-5, IL-6, and IL-13 and are driven through the transcription factor GATA, which is induced by and stimulates the synthesis of IL-4. Treg cells differentiate in the presence of TGFb and express the transcription factor FoxP3; these cells make immunosuppressive cytokines TGFb and IL-10 and suppress immune responses (Feuerer et al., 2009). Th17 cells are driven by TGFb, IL-6,

and IL-21, express the transcription factor RORgt, and produce the cytokines IL-21 and IL-17, which is proinflammatory and attracts granulocytes into areas of inflammation. The study of Th17 cells in viral infections is still in its infancy. Recent studies of Treg cells have revealed their importance in certain persistent infections, such as with retroviruses, and in discrete viral pathologies, such as with HSV-1 (Rouse et al., 2006; Zelinskyy et al., 2006). Another regulatory CD4 T cell that is considered part of the innate immune system is the NK (natural killer) T cell, which expresses an NK cell receptor and an invariant CD1d MHCrestricted ab TCR and which secretes either IL-4 or IFNg (Bendelac et al., 1997). Conventional NK cells lacking TCR and T cells expressing gd T-cell receptors can play important roles in acute responses to viral infections, but they are not considered part of the acquired immune response, and this chapter will focus more on CD8 T cells and Th1 and Th2 CD4 T cells expressing ab TCR (Fig. 2).

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FIGURE 3 Presentation of viral antigens. (A) Processing and presentation of MHC class I restricted peptides. (1a) Following virus infection viral gene products are synthesized. (2a) Viral proteins are degraded via cellular proteasomes to produce short peptide fragments. (3a) The viral peptides are transported into the endoplasmic reticulum by the transporters associated with antigen processing. Within the ER, peptides associate with class I molecules that are complexed with the beta-2 microglobulin. (4a) This newly formed tripartite complex is then shuttled to the cell surface for recognition by virus-specific CTL. (B) Processing and presentation of MHC class II-restricted peptides. (1b) Newly synthesized MHC class II molecules are localized within the ER and are complexed with the invariant chain (li), which obstructs the peptide-binding site. The invariant chain also participates in the folding of class II molecules and in their transport to the endocytic pathway. The class II complex is shuttled out of the ER into an endosomal compartment (MIIC), where the invariant chain is degraded by proteases, revealing the peptide-binding site. (2b) APCs internalize exogenous viral proteins by endocytosis. The proteins are localized to the endocytic pathway, where they are degraded by proteases into peptides. (3b) As peptide containing endosomes enter the MIIC, the viral-derived peptides bind to class II molecules. (4b) The class II-peptide complexes migrate to the cell surface for presentation.

Virus-Specific T-Cell epitopes

T-cell recognition of antigen is mediated by the TCR, which is structurally similar to immunoglobulin (Wu et al., 2002). As depicted in Fig. 3, CD8 T cells recognize short peptides presented by MHC class I molecules. Proteins contain numerous peptide sequences that conform to binding motifs for MHC class I molecules, but relatively few peptides elicit CTL responses. For the virus-derived peptides that serve as epitopes, distinct hierarchies for their recognition by CD8 T cells have been demonstrated in numerous viral models (Szomolanyi-Tsuda et al., 2002). Epitopes can be arbitrarily categorized as either immunodominant or subdominant, and this classification is dictated by the immunogenicity of a given epitope. Immunodominant epitopes are highly immunogenic and therefore elicit T-cell responses that are easily detectable, whereas T cells specific for weakly immunogenic, subdominant epitopes are often difficult to detect. A number of factors influence the hierarchy of T-cell epitopes, including peptide affinity for the presenting class I molecule, the efficiency of peptide processing and presentation, the availability of reactive T cells within the repertoire (Obar et al., 2008), and “immunodomination,” which is the ability of immunodominant epitopes to negatively influence the induction of T cells specific for weaker epitopes (Szomolanyi-Tsuda et al., 2002;Yewdell & Bennink, 1999). While earlier studies reported that T-cell responses focus on a relatively small number of virus-encoded peptides, more rigorous analyses are now indicating that the number of peptide epitopes recognized can be quite high. For example, T cells from

C57BL/6 mice can recognize at least 20 peptides encoded by the very small virus, lymphocytic choriomeningitis virus (LCMV) (Kotturi et al., 2007) and over 40 peptides from the very large virus, VV (Moutaftsi et al., 2006). Mutation of immunodominant epitopes can sometimes reveal responses to weaker “cryptic” epitopes that would not otherwise be easily detected (Szomolanyi-Tsuda et al., 2002). The early processing and presentation of epitopes allows for CD81 CTL to contribute to the elimination of infected cells prior to the rampant spread of infectious particles. CD41 T cell class-II-restricted peptide epitopes are sometimes longer than class I-restricted epitopes, and the binding motifs for MHC class II-restricted epitopes have not been as completely delineated. To date there have been more examinations of CD8 than of CD4 epitopes in murine viral infections, but over 10 CD4 epitopes have recently been reported for VV infections in C57BL/6 mice (Moutaftsi et al., 2007).

Antigen Presentation and Antigen-Presenting Cells (APC)

Virus-infected cells synthesize large quantities of viral proteins at the peripheral infection site, but the T-cell response does not develop there. Draining lymphoid tissue is the preferred site for T-cell activation (Fig. 4) (Sprent & Surh, 2002; Welsh et al., 2004), so viral antigens should be available within the lymphoid organs draining the site of infection for optimal T-cell stimulation. Viruses with the ability to replicate in many different tissues may reach lymphoid


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FIGURE 4 Progression of virus-induced T cell responses. The T-lymphocyte response to virus infection can be divided into three segments: activation, effector phase, and contraction. After a virus infection at a peripheral site, viral antigens accumulate within draining lymphoid tissue, where they are processed and presented to virus-specific T cells. Following the activation and proliferation of the T-cell population, the activated lymphocytes migrate to the site(s) of infection to eliminate virus-infected cells. Once the host is cleared of viral antigens, the immune system restores homeostasis by deleting a large portion of the T cells. The remaining virus-specific T cells can acquire a memory phenotype.

organs during the normal course of infection by the spread of replicating virus. Viruses may also have the ability to infect professional APCs in the periphery and then be transported by these cells into lymph nodes. Viruses may initially infect the host through respiratory, gut, or genital mucosal surfaces, though they sometimes directly enter the blood stream by way of arthropod vectors, animal bites, or inoculations by syringe (Welsh et al., 2004). Direct entry into the blood stream is likely to quickly deliver the virus into the spleen, a rich environment for T cells, B cells, and APCs. During the more common peripheral routes of infection, viruses will often encounter dendritic cells (DC), which emanate from the bone marrow and lie in wait in peripheral tissue until they encounter “danger” signals associated with infection or tissue damage (Matzinger, 2002; Szomolanyi-Tsuda et al., 2002). In the case of viral infection, the signals may come from direct infection of the DCs or by the DCs sensing damage done to cells in the local environment. Many viruses encode products that activate TLRs and strongly induce type 1 interferon (IFNab) and proinflammatory cytokines (Thompson & Locarnini, 2007). These activated DCs, which may either be infected with virus or else bearing internalized viral antigen, will up regulate expression of the homing chemokine receptor CCR7 and then migrate into the draining lymph nodes where they can initiate immune responses (Sallusto et al., 2000). DCs are thought to be the primary APC for the stimulation of virus-specific T lymphocytes, though macrophages may be an alternate source of antigen presentation (Pozzi et al., 2005). Both types of APCs can process and present antigen and provide costimulation factors that augment T-cell responses. B cells can also serve as APCs, in part by their ability to capture and internalize viral antigen by their surface immunoglobulin, and then present processed viral peptides on their MHCs (Parker, 1993). Generally, DCs in the periphery are in an immature state characterized by the low expression of MHC and costimulatory molecules and by an increased phagocytic activity. Inflammatory cytokines and the process of internalizing antigen can both provide the signals that initiate the transition of DCs to maturity. Mature DCs are the prototypical professional APCs with

high expression of MHC and costimulatory molecules, a decreased ability to capture antigen, and the ability to synthesize cytokines and chemokines. The pathways of viral antigen presentation to T cells vary for different viruses. LCMV can either infect DCs and be transported to the T-cell-rich lymphoid tissue or else can reach these sites during the normal spread of infection. Within the lymphoid tissue LCMV can replicate to high levels, providing ample antigen to stimulate the massive T-cell proliferation observed during infection. IAV also infects DCs, but in contrast to LCMV, the infected DCs do not produce infectious viral particles. Blocking IAV replication prevents the death of DCs, allowing infected DCs to continue presenting viral antigens to T cells. HSV-1 also infects DCs, but the infection blocks the maturation of human DCs, as evident by the lack of costimulatory molecule up regulation, decreased cytokine expression, and a diminished capacity to stimulate T cells in vitro. This should help HSV-1 escape immune surveillance, yet HSV-1 is nevertheless cleared during the acute response. This result suggests that in addition to the classical antigen presentation pathway, the peptides stimulating virus-specific T cells may also be derived from alternative pathways. Studies with poliovirus have indicated that virus-specific CD8 T cells can recognize APC class I MHC-presented peptides derived from exogenous proteins (Szomolanyi-Tsuda et al., 2002).

APCs and Costimulatory Molecules (Signal 2)

Costimulatory molecules are vital for the efficient induction of T-cell responses to many antigens. These receptor/ ligand pairs (signal 2) enhance the TCR-dependent signaling between antigen-specific T cells and APC (signal 1) and are particularly important for the activation of T cells bearing lower affinity TCR to antigen. Examples of such pairs (APC-T cell) are B7.1 or B7.2-CD28, CD27-CD70, 4-1BB(CD137)-4-1BBL, OX40(CD134)-OX40L, HVEMLIGHT, CD30-CD30L, GITRL- and GITR (Greenwald et al., 2005). The significance of different costimulatory pairs varies with the virus. For example, OX40-OX40L interactions are important for VV-specific T-cell responses but not for LCMV-specific responses (Salek-Ardakani et al., 2008).

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Two interactions that have been extensively examined for their contribution to the stimulation of T cells are B7.1 or 2-CD28 and CD40-CD40L. The interactions between B7.1/2 molecules and CD28 deliver a direct activation stimulus for T cells, whereas CD40 and CD40L provide indirect signals mediated through the up-regulation of costimulatory molecules including B7.1 and 2 on the surface of APCs. The engagement of CD28 on T cells with B7 molecules on the surface of the activated APCs, coupled with TCR signaling, enhances T-cell activation by stimulating the synthesis of cytokines (including IL-2) and antiapoptotic proteins (including Bcl-xL), and by up regulating the expression of the a and b chains of the IL-2 receptor (Greenwald et al., 2005). The B7-CD28 interaction is crucial for the activation of CD4 Th cell responses, but its importance for CD8 CTL stimulation appears to be dependent on the characteristics of the immunizing antigen. The activation of CD8 T cells that have a high affinity for their specific peptide is less dependent on CD28 signaling than it is the activation of T cells with lower affinity. Sustained or repeated antigenic stimulation may also alleviate the need for CD28 signaling in CTL induction. The importance of the duration and/or intensity of antigenic stimulation on CD8 T-cell activation was suggested by studies that compared the induction of CTL following infection with two VV strains differing only in the expression of the virulence gene thymidine kinase (tk), which prolongs replication and enhances virus spread. Whereas both viruses elicited comparable VV-specific CTL in wild-type mice, the less virulent, tk2 strain did not elicit CTL in CD28 KO animals. Similarly, the induction of virus-specific CTL by infection with viruses that replicate to high levels in mice, such as LCMV, does not require CD28 signaling, whereas viruses with limited replication capacity, such as VSV and IAV, do require CD28 signaling for CD8 T-cell activation. Thus, costimulatory signals decrease the antigen levels necessary to activate T lymphocytes, thereby making the immune system much more sensitive to low pathogen loads. CD40L expressed on activated CD4 T cells interacts with CD40 expressed on a variety of APCs, including B cells, activated macrophages, and DCs (Greenwald et al., 2005). Whereas CD40-CD40L engagement plays a direct role in the induction of B-cell responses, this interaction has an indirect influence on developing T-cell responses, as it contributes to the completion of the maturation of antigen-bearing DCs. This maturation is manifested by enhanced antigen presentation, up regulation of costimulatory molecules, such as B7 and CD40, and the induction of cytokine secretion. Studies with CD40 KO and CD40L KO mice infected with LCMV and other viruses demonstrated that CD40-CD40L interaction is essential for the induction of CD4 Th cells and CD4 T-cell-dependent antibody responses. The involvement of CD40/CD40L in the activation of CD8 T cells is more subtle; the generation of CD8 CTL can appear normal in the absence of CD40/CD40L signaling, but the CD8 T cells undergo an aberrant differentiation pathway rendering them deficient in recall responses in the memory state. This aberrant differentiation may, in fact, be secondary to the effects of CD40/CD40L on CD4 T cells, which may fail to produce the IL-2 needed for the proper differentiation of CD8 T cells (Williams et al., 2006). In the LCMV model, the induction of primary LCMV-specific CTL was only marginally reduced in CD40L knockout mice, but the CD4 T-cell response was dramatically diminished. These CD40L knockout mice were unable to control infections with high doses of highly virulent LCMV strains.

APC and Signal 3 Cytokines

T-cell activation not only involves signals through the TCR (signal 1) and costimulatory molecules (signal 2) but is also strongly influenced by a variety of cytokines (signal 3). In addition to the well characterized “T-cell growth factor,” otherwise known as IL-2, T-cell responses to infection can be influenced by other cytokines, including IL-12, IFNab, IFNg, and TNF (Kolumam et al., 2005; Whitmire et al., 2007; Williams et al., 2006). IL-12, produced by activated DCs, is important for driving CD4 T cells down a Th1, IFNg-secreting pathway and also can augment the differentiation and activation of CD8 T cells. Studies in IL-12 knockout mice and with IL-12 receptor-deficient transgenic T cells have shown that IL-12 is particularly important for the development of the CD8 Tcell response to VV, but, interestingly, not to LCMV (Xiao et al., 2009). On the other hand, IFNab augments the CD8 T-cell response to LCMV, but less so to VV (Kolumam et al., 2005). This may be because VV is a good inducer of IL-12 and LCMV is a good inducer of IFNab, whereas the converse is not true. Thus, T-cell responses to viruses may prefer to use the cytokines that they strongly induce as signal 3 stimulation factors. IFNg, which induces many of the same signals as IFNab, also can serve as a signal 3 for LCMV-specific T cells (Whitmire et al., 2007). TNF, on the other hand, seems to reduce acute and memory CD8 T-cell responses by mechanisms which may in part involve T-cell-produced TNF limiting the viability of APCs (Brehm et al., 2005; Singh & Suresh, 2007). In general, inflammatory cytokines and IFNg can both influence the induction of T-cell response as well as their contraction after the pathogen is cleared, as it seems that a higher proportion of activated T cells survive contraction if inflammation is limited (Badovinac et al., 2004).

Kinetics of the T-Cell response

MHC-avidin-linked tetramers presenting viral peptides have been useful for quantifying the magnitude of the virus-specific T-cell response (Murali-Krishna et al., 1998) and more recently for quantifying the number of naïve viral peptidespecific precursors, which range from about 15 to 600 per epitope (Obar et al., 2008). These variations in initial starting frequencies likely play some role in determining immunodominance hierarchies but are clearly not the only factors (Obar et al., 2008; Szomolanyi-Tsuda et al., 2002). Using an in vivo limiting dilution method to calculate precursors for the epitome of whole viruses, we find frequencies of about 1 in 3,000 CD8 T cells for LCMV (6400/mouse) and about 1 in 1,400 for VV (12,400/mouse) (Seedhom et al., 2009). These initial starting frequencies are insufficient for control of a viral infection, but T cells are capable of extensive division where ultimately millions of T cells specific to an epitope may be generated. Naïve T cells not specific to the virus do not proliferate, but there may be some low level division of memory T cells specific for other antigens and responding to proliferative cytokines like IL-15. A generalized schematic depicting the kinetics of the T-cell response to a viral infection is shown in Fig. 1. The response is divided into three segments, beginning with the activation and proliferation of the T lymphocytes, followed by the effector phase, and ending with the contraction of the response (Fig. 4). At a later stage, a recall response may occur when the host is reexposed to viral antigen. This recall may lead to a faster and quantitatively larger response, with a deposition of even higher frequencies of memory cells after the clearance of antigen.

Activation of Virus-Specific T Cells

Naïve T cells circulate throughout the body and enter the lymph nodes through high endothelia venules, which express the chemokine, SLC, and the addressin PNAd,


which are the respective ligands for the homing receptors CCR7 and CD62L on naïve T cells (Butcher & Picker, 1996; Sprent & Surh, 2002). Therein, the T cells encounter APCs that have homed to the lymph nodes by similar mechanisms. The first step in T-cell activation is the recognition of specific peptides processed from viral proteins and presented in the grooves of MHC molecules on APCs. T cells often stay in contact with their APCs for about a 24 hour period (Huang et al., 2004), but studies have shown that only a brief 2 to 3 hour encounter with antigen is sufficient to stimulate a programmed T-cell differentiation series (Welsh, 2001). About 2 to 3 days after the original signaling event, T cells begin a proliferative expansion involving up to 12 to 15 divisions and at a rate as high as 3 to 4 divisions per day (Hand et al., 2007; Parish & Kaech, 2009). T cells will down regulate expression of CD62L, CCR-7, and IL-7 receptor (CD127), and up regulate expression of IL-2 receptor, CD44, and the antiapoptotic protein BclXL (Parish & Kaech, 2009). After 2 or 3 divisions, CD8 T cells will differentiate into cytokine-producing cytotoxic effector T cells. Their chromatin structure relaxes to make genes available for TCR-stimulated transcription factors, including Tbet and eomesodermin (Araki et al., 2008; Fitzpatrick et al., 1998). These cells acquire the ability to synthesize the antiviral and immunoregulatory protein IFNg, as well as other cytokines and chemokines, including MIP-1b and RANTES. As these CD8 T cells differentiate into CTL, they up regulate expression of the proapoptotic molecule Fas ligand (FasL) and develop cytoplasmic granules that contain the membrane-pore-forming molecule perforin and a variety of proapoptotic enzymes called granzymes (Strasser et al., 2009) (Fig. 2). On contact with virus-infected target cells, CD8 T cells will secrete cytokines, engage the target cell death receptor Fas, and discharge their granules, whose contents bind to target surfaces and deliver apoptotic signals.

Cd4 T-Cell-Mediated Help

CD4 Th cells usually orchestrate the production of cytokines that will shape the profile of the developing T cell response. The CD4 T-cell response can be typically categorized on the basis of their cytokine production as Th1 (favoring cellmediated immunity), Th2 (favoring antibody production), Treg (favoring immune suppression), and Th17 (favoring granulocyte-associated inflammation) (Reiner, 2009). During the generation of virus-specific T-cell responses, CD4 T cells produce cytokines such as IL-2 that will drive the optimal proliferation of both CD4 and CD8 T cells (Fig. 2). In the LCMV model, CD4 T cells are not required for the generation of virus-specific CTL during an infection, but CD4-deficient mice are unable to control infections with high doses of more virulent LCMV strains. The generation of CD8 CTL in the absence of CD4 T cell help may be attributed to the ability of CD8 T cells to secrete low levels of IL-2 during an LCMV infection. Although the IL-2 production by CD8 T cells may be sufficient for the short-term maintenance of CTL, it seems to be insufficient to resolve infections with highly virulent LCMV strains. This hypothesis is consistent with the finding that IL-2 deficient mice mount a weak LCMV-specific CTL response following infection. The need for T-cell help in the induction of IAV-specific CTL is dependent on the subtype of virus. An IAV subtype that induces costimulatory molecules (i.e., B7-2) on APC elicits CTL without a need for CD4 T cells. However, a subtype that requires CD4 help to evoke IAV-specific CD8 CTL failed to up regulate costimulatory molecules on infected APCs. During HSV-1 infection, mice lacking CD4 T cells generate frequencies of HSV-1-specific

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CTL precursors that are similar to that in wild-type mice, but only low cytolytic activity is detectable ex vivo. Thus, CD4 T-cell help may be necessary to sustain the expansion of CD8 CTL following the initial activation. In agreement, the addition of exogenous IL-2 restores the HSV-1-specific CD8 CTL response to wild-type levels in the absence of CD4 T cells (Szomolanyi-Tsuda et al., 2002). Whether or not CD4 T cells, their cytokines, and costimulatory molecules are required for the generation of CD8 T-cell responses during acute viral infections, programmed differentiation functions elicited by these processes seem to be important for the development of fully functional memory T cells. In the absence of CD4 T cells or CD40-CD40L interactions during the acute response, the memory cells that are generated have impaired proliferative responses to recall antigens. This is likely due to the ability of CD4 T-cell-produced IL-2 to affect the long term differentiation program of CD8 T cells early in their formative stages after initial encounter with antigen (Williams et al., 2006). Studies with LCMV-infected B cell knockout mice have additionally shown that CD4 T-cell responses are weak and CD8 T-cell memory is defective in the absence of B cells, which presumably provide some needed stimulation to T cells (Christensen et al., 2003).

T-Cell effector Systems: Antiviral Cytokines

Activated T lymphocytes have the ability to produce large quantities of antiviral cytokines, such as IFNg and TNF (Callan et al., 2000). Both CD4 and CD8 T cells synthesize these cytokines, suggesting that both T-cell populations can contribute to the resolution of infections with viruses that are sensitive to these effector cytokines. IFNg increases the expression of class I and class II MHC molecules and antiviral proteins, such as protein kinase R, 2–5 oligoadenylate synthetase and dsRNA-specific adenosine deaminase (Biron & Sen, 2001). A major effect of IFNg may also be to up regulate nitric oxide (NO) synthase, whose metabolic product, NO, can have antiviral properties against a wide variety of viruses, including HSV-1, murine cytomegalovirus, VSV, EBV, and pox viruses (Reiss & Komatsu, 1998). The expression of IFNg and TNF by T cells is tightly regulated and restricted to the period of contact with antigen. Removal of antigen induces the rapid shutdown of cytokine expression by the virus-specific T cells, which can be turned on again by the addition of antigen. This cyclic expression confines the secretion of cytokines to the infection site and reduces the impact on uninfected tissues (Slifka et al., 1999). The antiviral effects of cytokines like IFNg and TNF can occur without target cell lysis, this may be useful for the resolution of infections in vital organ systems, where a large amount of tissue destruction is not desirable. The noncytolytic control of HBV in the hepatocytes of HBVtransgenic mice is mediated by IFNg and TNF (Guidotti & Chisari, 1999). Cytokine-mediated antiviral protection has also been observed with many viruses. IFNg contributes to the control of HSV-1 infections at mucosal surfaces, but is minimally involved at cutaneous and neuronal sites. Thus, protective effects of cytokines produced by effector T cells appear to be dependent on many factors, including the site of the viral infection (Szomolanyi-Tsuda et al., 2002).

T-Cell effector Systems: Cell-Mediated Cytotoxicity

T cells have two separate systems that mediate cytolytic function. The first is the granule exocytosis pathway, and the second is the interaction of FasL (expressed on T cells) with Fas (expressed on targets). The granule exocytosis pathway is dependent on perforin and on granzymes A and B

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(Szomolanyi-Tsuda et al., 2002). These three components are expressed in T cells following activation and are then stored in membrane-bound, cytotoxic granules within the cytoplasm. Subsequent to the recognition of an infected cell, the granules migrate to the membrane region that interacts with the target cell and then fuse with the T-cell membrane, releasing perforin and granzymes. The free perforin will polymerize, forming a complex that creates pores in the target’s plasma membrane. The channels created by perforin allow the passage of granzymes into the cytosol of the target cell. The granzymes will induce apoptotic pathways that result in the eventual death of the infected cell. The granule exocytosis pathway is thought to be predominantly utilized by CD8 CTL, but perforin can also be detected in some CD4 T cells. Perforin-dependent cytotoxity associated with the granule exocytosis pathway is the primary mechanism for the control of LCMV, ectromelia virus, and IAV, as perforinknockout mice are severely impaired in their ability to clear these viruses (Szomolanyi-Tsuda et al., 2002). FasL is quickly and transiently up regulated on TCRstimulated T cells, and its ligation by Fas on target cells triggers a well-characterized apoptotic pathway in the target cell (Nagata, 1997; Strasser et al., 2009). This involves the aggregation of Fas-activation death domains (FADD), leading to the activation of caspases, and ultimately to apoptosis. An important consideration for FasL-mediated cytotoxicity is that the expression of Fas is not ubiquitous, and only Fasexpressing cells would be sensitive to the FasL-induced cell death. CD8 CTL clear IAV from the lungs of Fas-deficient lpr mice with delayed kinetics, but optimal clearance of IAV requires both Fas and perforin, implying that the two mechanisms can have an additive effect in the appropriate environment (Topham et al., 1997). It is thought that CD4 effector T cells preferentially utilize FasL-mediated cytotoxicity, but the ability of CD8 CTL to induce Fas-dependent apoptosis has also been clearly demonstrated. Studies employing in vivo cytotoxicity assays, whereby viral peptidepulsed target cells are delivered into virus-infected mice and assessed for rapid clearance in comparison to an unpulsed control, have shown redundant effects of both FasL and perforin mechanisms for both CD4 and CD8 T-cell dependent cytotoxicity (Byers et al., 2007; Jellison et al., 2005).

T-Cell invasion of Peripheral Tissue

The down regulation of homing receptors CD62L and CCR7 allows the activated T cells to leave the lymph nodes or spleen and migrate into the periphery where they can encounter virus-infected cells and control infections. Chemokines such as CXCL10 (IP-10) and CXCL5 are among those that help direct this process (Lane et al., 2006). When activated T cells migrate into an infection site, they hunt for infected cells by scanning the MHC molecules displayed on the surface. Once found, the clearance of infected cells can be a remarkably efficient process and accomplished by either direct cytolytic mechanisms or by the secretion of antiviral cytokines like IFNg. Ironically, the positive effects of clearing virus may be offset by the negative effects of virusinduced immunopathology. At the infection site, both CD4 and CD8 T cells can induce an inflammatory response that is similar to a delayed type hypersensitivity (DTH) reaction. This term is normally used to describe the delayed rather than the immediate inflammation that occurs on reexposure of a host to a T-cell immunogen, as can be seen in certain types of skin allergies, but a similar reaction can occur during a primary virus infection. This reaction involves the attraction of T cells and macrophages to the infection site and the release of inflammatory cytokines. These reactions can sometimes lead to severe damage of host tissue.

The classic example of this occurs with the relatively noncytopathic LCMV infection, which can induce a lethal T-cell-mediated leptomeningitis (Cole et al., 1972) and a severe T-cell-mediated hepatitis (Zinkernagel et al., 1986) in mice. There is a third possible consequence when an effector T-cell response collides with tissues expressing high viral loads. The T cells can become overstimulated and driven into apoptosis by Fas/FasL or TNF/TNFR mechanisms (clonal exhaustion) (Zhou et al., 2002). Alternatively, they can up regulate inhibitory molecules such as programmed death-1 (PD-1) and lag-3 and become functionally exhausted, where they cease cytolytic function and antiviral cytokine production (Blackburn et al., 2009). This serves little useful purpose for the host during infections with highly cytopathic and disseminating viruses, which will simply overwhelm and kill the host. However, infections with relatively noncytopathic viruses or viruses whose tropism is restricted by cell type may progress into persistent states. This phenomenon is found during high dose infections with LCMV in the mouse and with HCV and HIV infections in humans (Blackburn et al., 2009).

Silencing Phase of the T-Cell responses and Conversion into Memory

Following the clearance of viral antigen, the immune system begins turning off the antiviral response and establishing a virus-specific memory pool (Fig. 1). There are two waves of T-cell apoptosis during viral infections (Razvi et al., 1995). The first wave affects mainly previously established memory T cells and is driven by the high levels of IFNab occurring early in infection (Bahl et al., 2005). This may help to make room for the development of a vigorous new antiviral T-cell response. The second wave occurs after virus is cleared and after T cells have reached the end of their programmed proliferation pathway. At this “contraction,” most of the T cells in the spleen and lymph nodes die by apoptotic mechanisms or else disseminate into peripheral tissue, where they reside in a state more resistant to apoptosis (Marshall et al., 2001; Wang et al., 2003). This apoptotic decline in the T-cell response is at least partially mediated by a proapoptotic mitochondrial protein called Bcl-2 interacting molecule, or Bim, and not by Fas/FasL or TNF/TNFR interactions acting alone. However, it is severely curtailed in mice with combined Bim and Fas deficiency (Weant et al., 2008). During the progression of the T-cell response different T cells express different proportions of activation molecules that help dictate their fates. Cells destined to die express elevated levels of the adhesion molecule KLRG-1 and of the transcription factor Blimp-1 and reduced levels of Bcl-2 and CD127 (IL-7R) (Parish & Kaech, 2009). Activated CD8 T cells expressing high levels of CD127 and low levels of KLRG-1 can be transferred into naïve mice, and memory cells will be derived from those populations.

recall of Memory T Cells

After the contraction of the immune response, memory T cells, which are discussed extensively in other chapters in this volume, are found throughout peripheral tissues as well as in the spleen, lymph nodes, peripheral blood, and bone marrow. In general, their antigenic phenotypes and some functional properties differ with the anatomical site (Masopust et al., 2006; Ray et al., 2004; Wang et al., 2003). For example, “central” memory CD8 T cells, localized mostly in the lymphoid tissues, undergo low level IL-15- or IL-17-dependent homeostatic division, preserving their cell numbers for long periods of time in the absence of further antigenic challenge (Prlic et al., 2002). In the periphery,


“effector” memory cells tend to divide less, are more resistant to apoptosis, and express antigens that apparently give them compatibility with their environment. When stimulated with antigen, memory T cells release cytokines and acquire cytotoxic capacity more rapidly than naïve cells (Slifka & Whitton, 2001) and undergo a programmed proliferative expansion much like that of naïve T cells (Kim et al., 2002). The memory T cells respond to chemotactic factors and nonspecifically migrate into sites of infection, where they will remain in the presence of cognate viral antigen (Woodland & Kohlmeier, 2009). This rapid expansion of memory T cells, coupled with their memory B-cell counterparts, can lead to rapid clearance of virus, and, after resolution of the infection, to long-lasting high memory T-cell frequencies (Fig. 1). Vaccination schemes have been able to induce very high frequencies of memory T cells in peripheral tissues (Vezys et al., 2008), as the contraction phase of a memory response is less pronounced than that which occurs after an acute response (Harty & Badovinac, 2008).

B-CeLL reSPONSeS The importance of Antibody responses to Viruses

Sustained high levels of virus-specific antibodies after resolution of a viral infection provide the main mechanism to prevent reinfection of the host; but in many acute virus infections, such as with VSV, Sindbis virus, rotavirus, or noroviruses, antibodies are also important for the resolution of acute infection and recovery (Chachu et al., 2008; Szomolanyi-Tsuda et al., 2002). In humans, a comparison of fatal versus resolved cases of Ebola virus indicated a lack of virusspecific antibodies in the former group, and an early and robust IgG response in the latter, suggesting that quick and efficient IgG secretion may play an essential role in the survival from this viral disease. In acute virus infections that are controlled mainly by T cells, humoral immunity can play an important role in the long-term control of the infection, because antibodies can control reactivation or recrudescence of viruses that establish latency or persistence. Examples of this include persistent mouse hepatitis virus infection in the CNS, or mouse gamma herpesvirus 68 reactivation from latently infected macrophages and B cells (Szomolanyi-Tsuda et al., 2002). In addition, antibody-mediated clearance can serve as a back-up mechanism of defense in a partially immunocompromised host. For example, T cells can control murine PyV in normal and in B cell knockout mice, but T cell knockout mice will also recover from acute PyV infection due to the generation of T-cell-independent antiviral antibodies. The control of high doses of LCMV that overwhelm the virus-specific T cell responses also is dependent on intact humoral immunity, because mice deficient in B cells cannot clear this infection (Welsh et al., 2004). A similar situation is observed in mice infected with the mouse poxvirus ectromelia, where the CD8 T cells can not completely clear the virus in the absence of antiviral antibodies (Fang & Sigal, 2005). In addition, just as CD4 T-cell help is needed to generate strong B cell responses, B cells are necessary to induce strong memory CD4 and CD8 T-cell responses. LCMV-infected mice lacking B cells produce CD8 T cells low in IL-2 production and with poor memory recall responses (Christensen et al., 2003).

recognition of Viral Antigens by B Cells

In contrast to T cells, which recognize processed antigens presented as peptides on the MHC molecules of APC, B cells recognize epitopes on the unprocessed antigen. The specificity of the membrane-bound surface immunoglobulin

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B cell receptors (BCR) is directed against the three-dimensional structure of viral antigenic determinants on virus particles or on the surface of infected cells. Most of the viral epitopes recognized by B cells are nonlinear. These epitopes (also called conformational epitopes) consist of amino acids derived from different regions of the linear polypeptide chain, and they are formed as a result of the three-dimensional folding of the molecule. Denaturation of these proteins leads to loss of binding with the antibodies. In contrast, linear or nonconformational epitopes are not destroyed by denaturation. The contact surface between the binding site of the antigen and antibody is approximately 700A2 (Wilson & Cox, 1990). Three-dimensional structural studies of viral antigen-antibody complexes revealed that 15 to 20 residues on each molecule are in close contact. Change in only one residue can drastically reduce the affinity of antibody binding and result in the appearance of viral escape mutants that are resistant to neutralizing antibodies (Knossow et al., 1984). B cells function not only as antigen-specific effector cells activated by viruses but also as APCs. As naive B cells capture viral proteins through their cell-surface-bound BCR, they internalize and process the proteins into peptides, which get presented on MHC class II molecules to CD4 T cells (Kehry & Hodgkin, 1993; Parker, 1993). This allows for an exquisitely sensitive and effective mechanism for CD4 T cells to provide CD40L-dependent help for B cells expressing virus-specific BCR. However, under conditions of very high antigen load, B cells may present viral antigens on their class II MHC molecules irrespective of their BCR specificities. When this happens, activated virus-specific T cells can provide help to these B cells, leading to a polyclonal activation and proliferation of B cells not specific to the immunizing antigen. This can sometimes lead to the synthesis of antibodies directed against self-antigens. Moreover, engagement of CD4 T cells with B cells of nonviral specificities may distract the CD4 T cells from helping virus-specific B cells, thereby reducing or delaying virus-specific antibody responses (Hunziker et al., 2003; Jellison et al., 2007).

Kinetics of Antibody responses

The interaction of DCs, CD41 T cells, and B cells in the secondary lymphoid organs activates the humoral responses. These responses start with IgM secretion (Fig. 1 and 2). In mice infected with LCMV or PyV by day 3 to 4 postinfection, antibody-secreting cells (ASCs) appear in the spleen, as detectable by ELISPOT assays, and virus-specific IgM can be measured in the serum by ELISA (Welsh et al., 2004). The IgM molecules form pentamers via disulfide bonds. This structure greatly increases the avidity of the complexes to the antigen by linking the 10 binding sites of the bivalent pentamers of IgM molecules that may otherwise be of low affinity. Isotype switching occurs after the initial phase of antibody responses (Fig. 2). The activated B cells reacting to cytokine signals start to produce antibodies with the same antigen binding domains and specificity, but combined with another constant region backbone. This change has important consequences, because the biological function of antibodies largely depends on the constant region of the heavy (H) chain. In mice IgM, IgG2a (or IgG2c in C57BL/6 mice that harbor the Igh1-b allele, where the IgG2a gene is replaced by a very similar IgG2c) (Martin et al., 1998), and IgG2b are very efficient at complement fixation, followed by IgG3. The constant regions of IgG subclasses also differ in their binding affinities to Fcg receptors (FcgRI-III) expressed on various cell types. In mice FcgRI and FcgRII on monocytes and macrophages bind both IgG2a and IgG2b

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antibodies and mediate the phagocytosis of antibody-coated viral particles. FcgRIII, which are expressed on NK cells in addition to monocytes and macrophages, bind mainly IgG2a, IgG2b, and IgG1 antibodies, and have the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of virus-infected cells. IFNg, a cytokine known to promote isotype switching to IgG2a, is induced during many virus infections. Therefore, most antiviral IgG responses are predominantly of the IgG2a isotype in mice during acute infections (Hangartner et al., 2006). In contrast to the acute infection, however, mice persistently infected from birth with LCMV produce predominantly antiviral IgG1. In humans, IgG1 and IgG3 are the good complement fixing IgG isotypes, and they also bind efficiently to FcgRIII on NK cells and mediate ADCC. IgG1 is the predominant antiviral isotype in patients with acute HIV or HBV infections (Szomolanyi-Tsuda et al., 2002). B cells that complete isotype switching and secrete IgG start to accumulate in the spleen on day 4 or 5, and reach a peak on day 9 to 10 in LCMV-infected or PyV-infected mice (Fig. 1 and 2). Virus-specific serum IgG levels reach peak values on day 15 in LCMV-infected, and on day 21 in PyV-infected mice (Welsh et al., 2004). Although some B cells can switch isotypes outside the splenic follicles, the main sites for isotype switching and affinity maturation are specific structures in the follicles called germinal centers. Here, after initial proliferation, the activated B cells express activation-induced cytidine deaminase, an enzyme essential for both isotype switching and somatic hypermutation, and they up regulate surface antigens such as GL-7 and Fas. In the germinal centers, B cells are subjected to various rounds of somatic hypermutation and selection before they finally become antibody-secreting plasma cells (PC) or memory B cells (Klein & Dalla-Favera, 2008). The terminal differentiation into PC is initiated by a major switch in transcriptional programs directed by the transcriptional master regulator Blimp-1 (Shapiro-Shelef & Calame, 2005). The serum IgG levels usually do not decrease substantially after the acute responses in the case of systemic (intravenous or intraperitoneal) infections in mice and are maintained at a high level for the rest of the host’s life (Fig. 1) (Slifka & Ahmed, 1998). Similarly, live viral infections in humans can lead to high antibody levels that are sustained for several decades or even for a lifetime. For example, in humans, the half-lives of antiviral antibodies following measles, mumps, and rubella viral infections are estimated to be over 100 years and, after smallpox vaccination, about 92 years. In contrast, the same study revealed much shorter half-lives (11–19 years) for antibody responses to nonreplicating protein antigens, such as tetanus and diphtheria toxoids (Amanna et al., 2007). Many viruses persist at low levels in various organs, and the continuous antigenic stimulation of naïve and memory B cells and their differentiation into PCs may be responsible for maintaining the high antibody levels. In the absence of persisting viral antigen, when viruses such as IAV, VV, and LCMV are cleared within 2 weeks, antibody secretion by long-lived PCs residing in bone marrow may account for lifelong maintenance of serum antibody levels; elimination of memory B cells by irradiation did not result in the rapid decline of antiviral antibodies in mice previously infected with LCMV (Slifka & Ahmed, 1998). Studies using in vivo depletion of B cells in mice with monoclonal antibodies to CD20, a surface marker expressed on all mature B cells but not on PCs confirmed this model, as antigen-specific serum antibody and PC showed no reduction over a 4 month time period after the B-cell depletion (Ahuja et al., 2008;

DiLillo et al., 2008). Thus, continuous turnover and differentiation of memory B cells into PCs by nonspecific signals does not seem to be necessary for the maintenance of the PC population and for lifelong serological memory following virus infections. This remarkable longevity of the PCs in bone marrow is maintained by survival signals. Two cytokines of the TNF family, BAFF (B-cell activating factor) and APRIL (a proliferation inducing ligand) and IL-6 are thought to be important in establishing the “survival niche” for bone marrow PCs (Dorner & Radbruch, 2007). Macrophage migration-inhibitory factor (MIF), secreted by DCs in bone marrow, is also reported to provide essential survival signals for long-lived PCs (Sapoznikov et al., 2008). IgA is the main antibody isotype protecting mucosal surfaces. Switching to IgA requires the B-cell stimulating factors APRIL and BAFF (Castigli et al., 2005). Human antibody responses to localized mucosal infections are much more short-lived than responses generated by systemic infections, as they wane in a few months (Pulendran & Ahmed, 2006).

Affinities of Antiviral Antibodies

In most virus infections in mice, antibodies are detectable by ELISA at 3 to 5 days postinfection. Some of these early antibodies may already have neutralizing activity, such as in infections with VSV, rotavirus, yellow fever virus, or IAV (Szomolanyi-Tsuda et al., 2002). In contrast, LCMV, HBV, and HIV infections induce neutralizing antibodies only at a later phase, 50 to 150 days postinfection (Zinkernagel et al., 2001). Recombinant viruses made between VSV and LCMV by swapping their surface glycoprotein targets of neutralizing antibodies demonstrated that the timing of neutralizing antibody induction was determined by the surface glycoprotein itself, not by other properties of the virus (Hangartner et al., 2006). This finding suggests that the differences in the onset of neutralizing responses may be related, at least in part, to the differences in starting affinities of germline-encoded immunoglobulin sequences specific for the neutralizing epitopes of various viruses, as high affinity is thought to be necessary for an antibody’s ability to neutralize. Indeed, the average affinity of IgG antibodies to VSV G glycoprotein is quite high by day 6 postinfection and it does not further increase with time (Hangartner et al., 2006). The affinity of antiviral antibodies may be critical for their ability to provide protection. Formalin-inactivated measles vaccine induced low-affinity antibodies that, instead of protecting from disease, caused serious morbidity upon measles infection due to immune-complex-mediated illness (Polack et al., 2003). Similarly, immunization with formalin-inactivated RSV induced nonprotective antibody responses with severe pathological consequences in some of the immunized children reexposed to the virus. Recent studies have shown the lack of affinity maturation of antibody responses following the administration of formaldehydeinactivated RSV, in contrast to infection with the live, replicating virus. It was also shown that the activation of TLR and MyD88-mediated pathways in B cells by live RSV was essential for affinity maturation (Delgado et al., 2009).

T-Cell-independent and T-Cell-dependent Antiviral Antibody responses

The induction of antiviral antibody responses involves complex interactions of antigen-specific activated CD4 T cells and B cells. The delivery of the “cognate” T-cell help includes signaling via costimulatory molecules, such as CD28 and CD40L on activated T cells, and B7-1, B7-2, and CD40 on B cells (signal 2), and via cytokines


secreted by T cells (signal 3) (Kehry & Hodgkin, 1993). Because most viral antigens are proteins, and antibody responses to proteins usually depend on T-cell help, antiviral antibody responses were initially thought to be strictly T-cell-dependent (TD). Studies on PyV infection in mice lacking both ab and gd T cells clearly indicated, however, that a virus can behave as a T-cell-independent (TI) antigen (Welsh et al., 2004). These T cell knockout mice synthesized virus-specific IgM and IgG that controlled virus infection, and the mice did not develop an acute lethal myeloproliferative disease found in SCID mice lacking both T and B cells. The isotype-switched TI IgG responses were elicited only by live PyV infection, and not by immunization with polyvalent empty viral capsids or capsomeres. Thus, the repetitive nature of the viral capsid was not sufficient to induce isotype-switched TI antibody secretion. Other viruses, such as VSV, rotavirus, LCMV, PV, MCMV, and VV can also induce IgM and some isotype-switched (IgG or IgA) antibody responses in the absence of CD4 T-cell help, but the TI IgG responses are of much lower magnitude than the responses elicited in the presence of T-cell help (Szomolanyi-Tsuda et al., 2002). The studies with PyV demonstrated that TI antibody responses can be essential for surviving virus infection in a T-cell-deficient host, but they may also play a significant role in normal hosts that have intact T-cell functions. TI responses probably start earlier than TD antibody synthesis that requires T-cell activation (Garcia et al., 1999). Therefore, early control by TI antibodies may limit the damage of host tissues and reduce the virus load that the TD responses subsequently need to handle.

Participation of B-Cell Subsets in Antiviral Antibody responses

B cells in both mice and in humans can be classified into major subgroups with distinct roles in antiviral antibody responses. Innate B-1 B cells produce mostly “natural” antibodies, which are thought to represent only a narrow range of specificities. However, they have low neutralizing activity against some viruses, such as IAV and VSV (Hangartner et al., 2006) and these preexisting antibodies may provide the first line of humoral defense against viral invasion. B-1 B cells also participate in acquired immunity by mounting a virus-induced IgM responses to IAV (Choi & Baumgarth, 2008). Many viruses are not neutralized by sera from uninfected hosts and need to induce B-2 B cells to generate protective humoral immunity. Marginal zone B cells, which localize to the marginal sinuses of the spleen, can respond quickly and in a TI manner to pathogens, and they are thought to be mostly responsible for the early TI antibody responses (Song & Cerny, 2003). TD responses, which include the germinal center reaction and memory B-cell formation, are mostly generated by follicular B cells, which can recirculate throughout the body and represent the largest fraction of peripheral B cells. The separation of these roles by B-cell subsets, however, is not absolute, as marginal zone B cells can participate in TD responses (Song & Cerny, 2003) and follicular B cells can generate TI antibodies under some circumstances (Guay et al., 2009).

TLr and Antiviral B-Cell responses

TLR expression is very low or undetectable on human naïve B cells, but human memory B cells constitutively express TLRs and readily respond to TLR agonists (Chiron et al., 2008). In mice, resting B cells express most TLRs and, when treated with TLR ligands, they up regulate their expression of class II MHC and B7-2, making the B cells better APCs

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(Jiang et al., 2007). Moreover, both naïve and memory B cells from mice respond to stimulation with TLR ligands such as LPS and CpG oligonucleotides by polyclonal proliferation, irrespective of their BCR specificities. B1 and MZ B cells also readily differentiate into antibody-secreting PCs upon TLR stimulation, whereas B2 and follicular B cells do not. As B1 and MZ B cells are the major players in TI responses to pathogens, these findings suggest that TLR signals delivered by pathogens stimulate TI B cell responses (Genestier et al., 2007). TLR-mediated signals can affect the humoral TD immune responses at various steps and depending on the nature of the infecting virus. PyV infection initially induced quantitatively similar early IgM and IgG responses in mice deficient in MyD88, an adaptor molecule for TLR signaling, but antiviral IgG levels in the serum decreased after day 10 and the formation of long-lived bone marrow PCs was impaired. These studies suggested a B-cell-intrinsic defect in long-term humoral responses in the absence of MyD88mediated TLR pathways (Guay et al., 2007). This was similar to a defect found in a patient with a mutation in IRAK4, a kinase downstream of MyD88 (Day et al., 2004). IAV also induced diminished IgG responses in MyD88 knockout mice, tested after day 10 postinfection (Heer et al., 2007; Koyama et al., 2007). Treatment of B cells with TLR ligands such as CpG or LPS induces the expression of BAFF and APRIL, factors that strongly promote B-cell survival (Chu et al., 2007). A report describing TLR3-mediated BAFF induction by reovirus-infected salivary gland epithelial cells suggests that TLR-induced BAFF and APRIL production in other cell types also may contribute to enhanced B-cell survival (Ittah et al., 2009). TLR-mediated signals also affect isotype switching. IAVand PyV-infected mice defective in MyD88-mediated pathways made more antiviral IgG1 relative to IgG2a (Guay et al., 2007; Heer et al., 2007). In other studies, TLR9 ligation by CpG led to the induction of the Tbet transcription factor and subsequent isotype switching to IgG2a (Liu et al., 2003). The secretion of IFNab, which can be induced via TLR-dependent or TLR–independent mechanisms during virus infection, also promotes switching to IgG2a at the expense of IgG1 (Heer et al., 2007). It thus seems that TLRmediated signals play a major role in the maturation and survival of antiviral antibody responses.

Mechanisms of the Control of Virus infections by Antibodies

Antibodies exert their antiviral effects by a wide variety of mechanisms, acting at various stages of the viral life cycle (Fig. 5). Antibodies can neutralize viral particles by inhibiting their attachment to cells. This is most commonly achieved by direct binding of the antibody to the viral attachment protein and blocking its interaction with receptors on the cell surface. Antibody binding to a viral determinant distinct from the attachment site may also lead to conformational changes of the attachment site, preventing virus adsorption. Virus neutralization can also occur by aggregation or agglutination of the virus particles by antibodies, resulting in a reduction of infectious units. After the attachment of viral particles to the cell, neutralizing antibodies can interfere with virus penetration and uncoating by inhibiting the endocytotic internalization of virions and the fusion of the viral envelope with the cell membrane, respectively (Dimmock, 1993). Complement activation by virus-antibody complexes can further enhance the efficiency of antibody-mediated control of virus infections. Enveloped viruses can be lysed by antibody and complement. Moreover, both opsonization

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FIGURE 5 Antibody-mediated antiviral effector mechanisms. This figure depicts several antibody-dependent antiviral mechanisms, including (1) prevention of viral attachment by blocking the viral attachment site; (2) prevention of uncoating; (3) aggregation of viral particles; (4) blocking virus absorption by inducing conformational changes in the attachment site; (5) lysis of virionantibody-complement complexes; (6) opsonization; (7) lysis of virus-infected cells by antibody and complement; (8) antibody-dependent cell-mediated cytotoxicity; (9) inhibition of the release of virus particles; (10) intracellular inhibition of the viral life cycle. VR, virus receptor; V-ag, viral antigen; mw, macrophage; CR2, complement receptor.

of virus-antibody-complement complexes by macrophages via complement receptors and direct lysis of virus-infected cells displaying viral antigen determinants on the cell membrane by antibodies and complement are potential antiviral mechanisms (Dimmock, 1993). Antibodies binding to viral antigenic determinants on the surface of infected cells can be targeted by NK cells or macrophages via Fc receptors on these cells. This may lead to the lysis of the infected cells via antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC, mostly mediated by NK cells, has been clearly demonstrated in vitro in several human viral systems, such as with HIV (Ahmad & Menezes, 1996). Very little is known, however, of the in vivo contribution of ADCC to the control of virus infections in mice or in humans. Antiviral antibodies can facilitate the internalization of viral particles into various cell types, thus altering cell tropism and host responses. For example, antibody-adenovirus complexes are readily internalized via Fc receptors by macrophages which otherwise lack the CAR receptor for adenoviruses. Entry into macrophages and phagolysosomal targeting of the virus in these cells was reported to result in strong activation of TLR9-mediated pathways and, consequently, to enhanced innate immune responses such as IFNab secretion (Zaiss et al., 2009). Antibodies can also disrupt the viral life cycle inside the infected cells. Measles virus-specific antibodies were shown to suppress viral protein and RNA synthesis in infected cells in vitro. Antibodies specific for the surface E2 glycoprotein of the Sindbis virus protected mice from lethal CNS infection and cleared infectious virus from neurons in a noncytolytic manner. Cross-linking of E2 displayed

on the infected cell surface by the bivalent antibodies in vitro resulted in an inhibition of budding and release of the new virions. Subsequently, viral RNA and protein synthesis gradually decreased, and the infected cells recovered (Szomolanyi-Tsuda et al., 2002).

Some Antibodies are More useful than Others

In a given host, a wide variety of antibodies are produced with different specificities, affinities, and isotypes, and directed against different viral proteins, but only some of these antibodies have a potent antiviral effect. Studies with monoclonal antibodies specific to reovirus, VSV, IAV, and LCMV concluded that the in vitro characteristics of the antibodies, such as neutralizing ability or affinity, could not necessarily predict their utility in preventing or resolving virus infections in vivo (Szomolanyi-Tsuda et al., 2002). These findings suggest that some of the antiviral mechanisms mediated by antibodies in vivo may be different from the mechanisms studied in vitro. In fact, nonneutralizing antibody responses to the conserved internal nucleoprotein (NP) of IAV are protective against multiple IAV serotypes because of the conserved nature of the NP. Mechanisms involved in this NP-specific antibody-mediated protection include enhancement of DC maturation via immunecomplex formation, and, as a consequence, improvement of Th1 and CTL responses to the virus, and complementmediated lysis of virus-infected cells which display NP on their surface (Carragher et al., 2008; Yewdell et al., 1981). Similarly, nonneutralizing antibodies specific to the internal cell-associated nonstructural protein NS-1 of Japanese encephalitis virus can provide protection against infection (Krishna et al., 2009).


HeTerOLOGOuS iMMuNiTY iN VirAL iNFeCTiONS

The great majority of T cells proliferating during a viral infection is specific to viral antigens, but many virus-specific T cells also cross-react with unrelated antigens (Selin et al., 2006). The cross-reactive nature of T cells may be attributed to the flexibility of the TCR during antigen recognition, where the TCR shifts in conformation as it attempts to engage a peptide-MHC complex (Wu et al., 2002). Up to 10% of LCMV epitope-specific T cells may cross-react with uninfected target cells displaying a specific allogeneic MHC (Brehm et al., 2003). Similarly, part of the human CD8 T-cell response to Epstein-Barr virus (EBV) is directed against allogeneic human tissue (Burrows et al., 1997). The induction of allo-specific responses by viral infections may have detrimental effects for allogeneic transplants, as these cross-reactive T cells may enhance rejection. Of greater significance with regard to viral infections is the fact that similar or sometimes very different epitopes encoded by unrelated viruses can be recognized by crossreactive T cells, and such, T cells can contribute either to protective immunity or severe immunopathology (Selin et al., 2006). For example, IAV-specific human T cells have been shown to cross-react with antigens of HCV, EBV, and HIV. Heterologous immunity studies in a variety of mouse model systems have shown that cross-reactive T cells may alter immunopathology and either enhance or inhibit the clearance of virus. A cross-reactive but nonidentical viral antigen may stimulate the expansion of only a small subset of a memory T-cell pool produced in response to another virus, and that may result in an immunodominant yet narrow oligoclonal T-cell response that may be poor at clearing virus (Cornberg et al., 2006). Narrow oligoclonal responses in human infections have been shown to reflect poor prognosis and to correlate with the generation of epitope-escape viral variants. Notably, fulminant hepatitis in humans has been correlated with narrowly focused T-cell repertoires primarily directed against an HCV epitope cross-reactive with IAV (Urbani et al., 2005). Memory T-cell populations specific to a previously encountered virus may alter the immunodominance of the T cell response to an unrelated virus and ultimately also alter the subsequent immunological memory generated by that virus. However, the second virus may likewise alter the memory response to the first virus by skewing the first virus’ memory T-cell population due to cross-reactive expansion, and also by causing a general nonspecific reduction in preexisting memory. This generalized reduction in memory is caused by the IFNab-induced apoptosis of memory T cells that occurs early during viral infections (Bahl et al., 2005). Heterologous immunity may also apply to B-cell responses. It has long been known that immunity to one strain of IAV would influence antibody responses to other strains, whereby cross-reactive antigenic determinants would generate strong memory responses at the expense of primary responses to novel determinants on the challenge virus. This process has been referred to as the “original antigenic sin” of antibody responses, and much the same can occur for T-cell responses (Fazekas de St. Groth & Webster, 1966; Klenerman & Zinkernagel, 1998). Previously acquired humoral immunity to a heterologous dengue virus serotype may lead to a more severe form of dengue, known as dengue hemorrhagic fever. This antibody-dependent “immune enhancement” is mediated by complexes of virions and nonneutralizing crossreactive antibodies (Halstead, 1989). These complexes are adsorbed to and internalized by Fc receptor-bearing monocytes and macrophages with increased efficiency, resulting

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in a major increase in virus replication. This increased virus load can also be a potent stimulus for the reactivation of cross-reactive memory T cells that can mediate immunopathology, contributing to the severity of dengue hemorrhagic fever. Hence, B- and T-cell responses may collaborate under these conditions of heterologous immunity. We thank Keith Daniels for preparing the illustrations.

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20 Immune Responses to Persistent Viruses E. JOHN WHERRY AND PAUL KLENERMAN

OVERVIEW

particular, the impact of frequent antigen encounter—the consequences of this antigen encounter can be quite distinct in different infections. We will focus mainly on CD81 T cell responses. This is largely because these responses appear to play a crucial role in disease outcome across a range of persisting viral infections, and also because this area has developed rapidly in the past few years in both mouse models and in human studies. However, CD81 T-cell responses do not act alone, and the outcome of infection and the function of CD81 T cells is dependent on the concerted efforts of innate immunity and adaptive responses from CD41 T cells and B cells secreting antiviral antibodies. These responses are also discussed. Since our understanding of host–pathogen interactions during chronic infection was initiated through studies of LCMV infection in mice and this virus continues to provide the key model for high-level viral persistence, we will start with a discussion of immune responses during this infection. We will also examine two of the major human persistent infections, HIV and HCV. Because aspects of pathogenesis and the immune response for herpesviruses differ substantially from LCMV, HIV, and HCV, we will also discuss CMV, where long-term immunologic control is observed in both man and mouse, with a distinct pattern of memory CD81 T-cell differentiation.

As many as 40 million people worldwide are chronically infected with HIV (human immunodeficiency virus) and the prevalence of this infection in some parts of Africa can reach 40% of the population. More than 500 million people worldwide are infected with HBV (hepatitis B virus) and/or HCV (hepatitis C virus) and the rates of HIV/HCV coinfection are rising (Virgin et al., 2009). Herpesviruses are ubiquitous in humans and one could perhaps even consider viruses such as EBV (Epstein-Barr virus), CMV, (cytomegalovirus) and HSV-1 (herpes simplex virus type 1) commensals, at least in healthy individuals (Virgin et al., 2009). We are rapidly recognizing other persisting viruses and how they might contribute to disease. Identification of novel persisting viruses suggests they could have a role in the etiology of diverse diseases including cancer (Virgin et al., 2009). For the majority of these infections, immune responses play a critical role in control, or long-term containment of these infections. However, for some viruses like HIV, HBV, HCV in humans, or chronic LCMV (lymphocytic choriomeningitis virus) infection in mice, ineffective immune responses are associated with chronic infection and pathology (Virgin et al., 2009). In this chapter we will discuss some general principles regarding immune responses during chronic viral infections. The nature of these responses, however, is highly dependent on the type of virus and particular aspects of different infections. For persisting infections it is often difficult or impossible to define and describe aspects of in vivo viral dynamics and replication without also considering the impact of the immune response and vice versa. A broad consideration of viral pathogenesis and antiviral immune responses is therefore crucial to bear in mind when examining these persisting viral infections. For example, the impact of epitope mutation and escape from B- and T-cell responses is quite significant and important during infections with some RNA viruses, but plays less of a role during infection with DNA viruses. Also, tissue restricted replication can influence immune surveillance and the development of effective T- and B-cell responses. Thus, while there are common themes—in

PATHOLOGY DURING CHRONIC VIRAL INFECTION: WHOSE FAULT IS IT?

Virus replication can be cytopathic (i.e., directly causing cellular damage upon infection) or noncytopathic. In general, highly cytopathic viruses do not cause persisting infections since the ongoing cellular and tissue damage from the virus would not be tolerable by the host. Rather, highly cytopathic viruses are typically either cleared acutely (e.g., influenza virus) or kill the host (e.g., Ebola virus). Persisting viruses, therefore, are typically either conditionally cytopathic, or weakly/noncytopathic. Herpes viruses, through the use of different genetic programs (latency versus lytic replication), are conditionally cytopathic and typically only cause extensive cellular or tissue damage upon entry into the lytic cycle. Finally, viruses that are largely noncytopathic (e.g., LCMV, HBV, and possibly HCV) typically do not cause cellular or tissue damage directly themselves. In fact, infection of immunodeficient or neonatal mice with LCMV cause a

E. John Wherry, Department of Microbiology and Institute for Immunology, University of Pennsylvania, School of Medicine, 421 Corie Blvd., Room 312, Philadelphia, PA 19104. Paul Klenerman, Nuffield Dept of Medicine and NIHR Biomedical Research Centre Programme, Peter Medawar Building, University of Oxford, Oxford OX1 3SY, UK.

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life-long chronic infection with essentially no ill effects. The pathology upon infection with viruses like chronic LCMV or HCV is therefore largely immune-mediated, rather than virus-mediated. The ongoing, yet ultimately ineffective, immune response to viruses such as LCMV or HCV leads to prolonged tissue damage, and, in the case of HCV, the induction of a wound repair response that can ultimately lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Such immunopathology can also occur for other weakly or conditionally cytopathic chronic viral infections such as HIV and herpesviruses. Thus, both the viruses and the immune response they elicit can be responsible for the pathology associated with chronic viral infections. Mechanisms that attenuate the immune response in these settings such as immunological escape and T- and B-cell exhaustion certainly impair viral control, but might also play a role in reducing immunopathology.

STRATEGIES OF VIRAL PERSISTENCE

In discussing immune responses to persisting viruses, it is important to keep in mind the strategies exploited by different viruses to facilitate persistence. The immune response is capable of effectively controlling or eradicating a large number of viral infections and it does so by using a variety of effector mechanisms including cytotoxic T cells, antibodies, cytokines (from the innate and adaptive immune responses), among others. In general, for viruses that persist, three types of strategies exist to avoid immunological eradication (Table 1). (i) Immune ignorance: in this case, the immune system of the host is unaware of the persisting virus. Many examples of this type of immune evasion exist including infecting immunologically privileged sites, actively down regulating antigen presentation by disrupting the MHC class I or class II processing pathways, or true virologic latency (Virgin et al., 2009). (ii) Immune escape: here, high mutation rates and structural and functional flexibility in viral proteins allows especially RNA viruses to mutate epitopes recognized by B and T cells (Virgin et al., 2009). These mutations can decrease the effectiveness of ongoing immune responses during chronic infections and in the extreme case could allow complete escape of viral recognition by the immune system. Sometimes, but not always, these mutations can come at a fitness cost to the virus. (iii) Immune dysfunction: often generally termed “exhaustion,” immune dysfunction during chronic viral infections can take several forms (Shin & Wherry, 2007). T-cell exhaustion is classically defined by the loss of effector functions and proliferation that leads to ineffective control

TABLE 1

Strategies to facilitate persisting viral infection

Strategy

Mechanisms

Examples

Immunological ignorance

Infecting immunological privileged sites, down regulation of MHC, etc.

Herpes simplex virus, papillomavirus, CMV, EBV, etc.

Immunological escape

T- or B-cell epitope mutation, glycan shield, etc.

HIV, HCV, etc.

Immunological exhaustion

Loss of T- or B-cell function, active inhibition of immune responses, poor development of T- or B- cell memory.

LCMV, HIV, HCV, HBV, etc.

of persisting viral replication. However, there are other types of T-cell dysfunction that could occur during chronic infection including altered patterns of T-cell differentiation skewing responses away from functional T-cell memory, tolerance in situations where viral antigens are presented solely by nonprofessional antigen presenting cells, inhibition by regulatory T cells, and polyclonal B-cell activation. Finally, immune system dysfunction also encompasses the physical deletion or loss of virus-specific T cells that sometimes is observed during chronic infections with a high level of persistence (Moskophidis et al., 1993). Between ignorance, escape, and dysfunction, persisting viruses often outwit or outmatch the immune response. A given virus might, of course, utilize more than one of these strategies; a good example here would be HIV that can use latency, escape mutations, and T- and B-cell dysfunction. However, it is also important to point out that we often discuss mainly persisting viruses that cause overt disease (e.g., HIV, HCV, HBV) in the context of the immune response. For many chronic infections, persisting virus and immune control achieve a mutually acceptable balance between replication and antiviral immunity. In fact, while the focus of this chapter is on chronic viral infections that cause disease, it may be that the majority of persisting viruses coexist effectively with the immune system and cause little or no disease in healthy people (Virgin et al., 2009).

Lymphocytic Choriomeningitis Virus (LCMV)

LCMV provides a crucial paradigm for understanding antiviral immune responses as this viral infection has several distinct, clear-cut outcomes. LCMV is the prototypical Old World arenavirus and has been extensively used in studies to understand immune responses and immune system function (Buchmeier & Zajac, 1999). The virus has a small ambisense ssRNA genome encoding four proteins. Mice and other rodents are the natural host for LCMV, making it an excellent system to study host–pathogen interactions. One feature of LCMV that has made it such a widely used model is that different strains of the virus exist that lead to different outcomes upon inoculation into mice. For example, LCMV Armstrong infection or low dose infection with the WE strain results in an acute infection with virus control by 8 to 10 days postinfection and the generation of highly functional and protective memory B and T cells. Other strains, including clone 13, T1B, Traub, WE (high dose), and Docile, can cause chronic infections depending on the dose and route of infection. In order to understand the nature of responses in the setting of persistence, we will first discuss immune responses following acute LCMV Armstrong infection (i.e., infection that is rapidly controlled).

IMMUNE RESPONSES FOLLOWING ACUTE INFECTION

Optimal antigen stimulation of naïve CD81 (or CD41) T cells initiates extensive proliferation and differentiation of naïve T cells into effector T cells. The majority of these effector T cells dies over the subsequent 2 to 3 weeks of the response and a population of long-lived memory T cells is formed (Fig. 1) (Kaech & Wherry, 2007). Acute infection or vaccination normally results in the generation of potent, functional, and protective memory T cells. High quality memory T cells have several cardinal properties that distinguish them from naïve, effector, or even other subsets of memory-like T cells, and these properties allow memory T cells to confer protective immunity (Table 2). Chief among these memory T cell properties are: (i) the ability to rapidly reactivate effector functions including cytokine

20. Immune Responses to Persistent Viruses

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FIGURE 1 Host virus dynamics in acute resolving and persistent infection. The upper panel shows a setting where LCMV or HCV sets up acute infection that is then rapidly contained. In this case, robust and functional T-cell responses are induced and sustained over time. The lower panel shows a dynamic with long-term persistence of virus. In this case, T-cell responses are also induced but are not effectively sustained either physically or functionally. The immunologic correlates are shown on the right-hand side. These vary slightly between HCV and LCMV as indicated. The timescales are also different between the two viruses due to the very long period (weeks) after HCV infection prior to the initiation of the immune response.

and chemokine production and cytotoxicity, (ii) the reacquisition of the ability to home to secondary lymphoid tissues, (iii) a high proliferative potential, and (iv) the ability to persist long-term in the absence of antigen via IL-7 and IL-15-driven homeostatic proliferation (Kaech & Wherry, 2007). Acquisition of these memory T-cell properties occurs gradually over time as a population of memory T cells TABLE 2

develops following the effector phase of the primary immune response. While a model that has memory T cells gradually acquiring optimal memory T-cell properties in a linear fashion over time oversimplifies our current understanding of memory CD8 T-cell differentiation, such a model is useful to illustrate a few key points about the stages of memory CD8 T-cell differentiation. During the naïve to effector

Properties of functional memory T cells generated following acute infection or vaccination

Property

Example/Mechanism

Consequence

Rapid reactivation of effector function

Ability to produce antiviral cytokines and chemokines, and to kill within minutes to hours of stimulation

Begin eliminated virus prior to proliferation and very soon after secondary infection

Homing to 20 lymphoid organs

Reexpression of CD62L and CCR7

Allows memory T cells to scan professional APCs that home to lymphoid tissues. DC draining recently infected tissues can rapidly reactivate memory T cells.

High proliferative potential

Regain high capacity for division and reduced apoptosis upon stimulation perhaps by maintenance in a preactivated state of G0/G1

Shorter lag before division starts compared to naïve T cells, rapid burst of proliferation upon reinfection

Long-term, antigenindependent persistence

Use of IL-7 and IL-15 for homeostatic maintenance via slow, steady division

Maintains essentially constant numbers of memory T cells for years following primary infection. Essential for long-term protection by maintaining elevated numbers of antigen-specific T cells.

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(N to E) transition following initial T-cell activation, effector CD8 T cells acquire the ability to lyse infected cells and secrete antiviral cytokines upon antigenic stimulation (Kaech & Wherry, 2007). Following control of acute infection the majority (90–95%) of activated effector CD8 T cells dies by apoptosis. The remaining 5–10% form a pool of long-lived memory T cells (Kaech & Wherry, 2007). As these surviving effector CD8 T cells differentiate into memory T cells (E to M) they acquire a resting phenotype, but retain the ability to rapidly produce IFN-g and TNF (tumor necrosis factor) and to quickly reacquire cytotoxic activity when exposed to antigen (Kaech & Wherry, 2007). This E to M transition can even be further subdivided since the early memory CD8 T-cell pool resembles what have been termed effector memory T cells (TEM; CD62LLoCCR7Lo, low IL-2) while over time this TEM population gradually converts to a more central memory-like (TCM) CD8 T-cell population (CD62LHiCCR7Hi, high IL-2) (Kaech & Wherry, 2007) (Fig. 2). There are also T cells in the TEM population that are end stage or terminally differentiated and will not convert further to TCM (Kaech & Wherry, 2007). These terminal TEM have a longer half-life than true effector T cells, but ultimately are lost from the memory pool in the absence of persisting infection or antigen. For the long-term memory T cells, the TEM to TCM conversion that occurs in the absence of persisting antigen or infection reflects continued differentiation of memory CD8 T cells over time and the rate at which the conversion occurs can be determined by signals received during the first week of infection, including the strength of antigen stimulus (Wherry et al., 2003). An alternative model of memory T-cell differentiation suggests that different subsets of memory T cells (i.e., TEM and TCM)

represent separate lineages that are formed during primary infection and do not interconvert in the memory phase (Sallusto et al., 2004). While this remains a controversial area that is beyond the scope of this chapter, at least part of the discrepancy could relate to different definitions of TEM and TCM. In any case, once formed, it is the TCM population that is endowed with all the defining properties of memory CD8 T cells outlined above, and it is only this population that is capable of efficient antigen-independent homeostatic proliferation (Wherry et al., 2003).

MEMORY CD8 T-CELL DIFFERENTIATION DURING CHRONIC INFECTION

The scenario described above applies mainly to T-cell differentiation following acute infection with LCMV and other nonpersistent infections (e.g., vaccinia virus or influenza virus) or vaccinations. During chronic infection, memory T-cell differentiation can be dramatically altered. Chronic LCMV infection, for example, is accompanied by T-cell dysfunction or “exhaustion” that can vary from relatively mild to quite profound depending on the type and severity of the infection and the time of analysis. T-cell exhaustion was first defined as the persistence of virus-specific CD8 T cells during chronic infection that lacked effector function (Zajac et al., 1998). In these studies, virusspecific CD8 T cells could be identified by staining with MHC peptide tetramers, but these cells failed to produce antiviral cytokines (Zajac et al., 1998). Previous studies had demonstrated that high-dose infection with LCMV Docile strain could result in the physical elimination or deletion of antigenspecific CD8 T cells (Moskophidis et al., 1993). Subsequent

FIGURE 2 Distinct T-cell differentiation pathways associated with different virologic outcomes. After acute infection, if the virus is cleared effector T cells will eventually revert to a central memory phenotype (Top). If the virus is not cleared, two possible outcomes exist: either expansion of the effector memory pool, typically associated with subclinical virus persistence (Middle), or a program of T-cell exhaustion, typically associated with high level virus carriage (Bottom). The relationships between the phenotypes are not fully defined and the surface markers shown vary between infections and between mice and humans to some extent.


studies demonstrated that deletion often occurred for CD8 T-cell populations where the epitopes were presented at the highest levels (Shin & Wherry, 2007). These data are consistent with the idea that repetitive antigen encounter leads to modulation of CD81 T-cell function in this infection and that two distinct outcomes can occur, loss of effector functions, but T-cell persistence or physical deletion of virus-specific T cells. The persistence of dysfunctional CD8 T cells has subsequently been studied in considerable detail. CD8 T-cell exhaustion typically follows a hierarchy of loss of effector function with defects in the ability to produce IL-2, to proliferate, and to kill (at least ex vivo) occurring early, TNF production persisting longer and IFN-g and chemokine production lost only at very extreme stages of exhaustion (Shin & Wherry, 2007; Virgin et al., 2009) (Fig. 3). The physical deletion of virus-specific T cells noted in early studies appears to, in fact, be the final stage of exhaustion for cells receiving very strong stimulation. The severity of CD8 T-cell exhaustion depends on viral load, the duration of infection and the availability of CD4 T-cell help. CD8 T-cell exhaustion is also associated with dramatic changes in gene expression (Wherry et al., 2007). T-cell exhaustion is also accompanied by other phenotypic and functional changes (Table 3) (Wherry et al., 2007) and a key emerging characteristic of T-cell exhaustion is the elevated expression of inhibitory receptors (Crawford &

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Wherry, 2009). PD-1, an inhibitory receptor in the CD28 superfamily has received the most attention in this regard. This molecule is expressed by exhausted CD8 T cells and limits their function (Barber et al., 2006). Other inhibitory receptors (Crawford & Wherry, 2009) and nonreceptor pathways (Blackburn & Wherry, 2007) also appear to influence T-cell function during chronic viral infections and there is recent evidence for a similar paradigm for CD4 T cells (Brooks et al., 2005) and, in some settings such as HIV, virus-specific B cells (Moir et al., 2008). CD8 T-cell exhaustion can also result in a failure to develop the ability to undergo antigen-independent homeostatic maintenance, a cardinal feature of memory T cells generated after acute infections. The upregulation of receptors for IL-7 and IL-15 (CD127 and CD122) is a key event in normal memory T-cell differentiation that allows long-term antigen-independent persistence of memory T cells. During chronic infections, antigen-specific CD8 T cells express low levels of these receptors, fail to respond effectively to IL-7 and IL-15, and are largely incapable of undergoing antigen-independent homeostatic self-renewal (Shin & Wherry, 2007). As a result, if antigen is removed these virus-specific CD8 T cells fail to persist (Shin & Wherry, 2007). During chronic infections, instead of using IL-7 and IL-15, virus-specific CD8 T cells become “addicted” to antigen and require cognate TCR interactions

FIGURE 3 Stages of T-cell exhaustion. CD8 T-cell exhaustion is characterized by hierarchical stages of T-cell dysfunction in comparison with a resting memory cell. The top depicts the properties of a functional memory CD8 T cell generated following an acute/cleared infection or vaccination. The bottom depicts the stages or degrees of exhaustion that can occur when a virus persists. Decreased function and increased apoptosis are associated with higher viral load, lower CD4 T-cell help, and increased expression of inhibitory receptors such as PD-1.

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TABLE 3

Molecular pathways associated with T cell exhaustion during chronic viral infectiona

Type of defect

Examples/molecules

Potential consequence

Highly expressed inhibitory receptors

PD-1, LAG-3, 2B4, CD160, etc.

Inhibition of T-cell responses, reduced TCR signaling, limited effector functions and proliferation

Altered signaling

LCK, NFATc, IL-7R

Altered TCR and cytokine receptor signal transduction

Altered homing, migration, adhesion

CCR5, CCL5, CCL3, all increased, several integrins decreased

Enhanced inflammatory cell recruitment, altered adhesion

Transcription factor changes

Blimp-1, Eomes, etc

Altered differentiation state

Metabolic deficiencies

Ribosomal subunits, citric acid cycle

Reduced energy metabolism, defects in protein translation

Cell cycle, proliferation

Cell cycle checkpoint proteins

Poor proliferative responses

Increased apoptosis

Increased caspases, inhibitory receptors

Higher rates of cell death

a

Adapted from Wherry et al., 2007.

with peptide/MHC (major histocompatibility complex) for persistence in the original infected host (Shin et al., 2007). These antigen-addicted virus-specific CD8 T cells also remain CD62LLoCCR7Lo (i.e., TEM-like) and do not differentiate into the more stable CD62LHiCCR7Hi TCMlike cells observed to undergo normal antigen-independent homeostatic maintenance following acute infection (Shin & Wherry, 2007).

CD41 T CELLS

Following acute LCMV infection in CD41 T-cell deficient mice, the primary CD8 T-cell response is largely preserved and virus appears to be controlled (Matloubian et al., 1994). However, memory CD8 T-cell quality in the absence of CD4 T cells is compromised, demonstrating the critical role of CD41 T-cell help for optimal CD8 T-cell memory (Khanolkar et al., 2007). A critical role of CD41 T cells during chronic viral infection was first appreciated using chronic LCMV infection where a lack of CD41 T cells led to a dramatically reduced ability of antiviral CD81 T cells to contain the chronic infection (Matloubian et al., 1994). In addition, exhaustion of the CD81 T cells is more extreme in the absence of CD41 T-cell help (Zajac et al., 1998). In the setting of high viral load, CD41 T cells themselves may also undergo exhaustion (Brooks et al., 2005; Oxenius et al., 1998), though mechanistic details about CD41 T-cell exhaustion during chronic LCMV infection are not as well understood as for CD81 T cells.

B CELLS

Acute LCMV infection results in a robust LCMV-specific B-cell response (Hangartner et al., 2006) and the lack of LCMV-specific antibodies in CD41 T-cell deficient mice has been implicated in a failure to completely clear acute LCMV infection and the resulting memory CD81 T-cell defects (Planz et al., 1997). During chronic LCMV infection, virus-specific antibodies are also generated, but the persisting infection also appears to alter B-cell responses. First, virusspecific antibodies produced during chronic LCMV infection can lead to substantial immune complex deposition (Casali & Oldstone, 1983). Second, chronic LCMV infection can result in hypergammaglobulinemia and autoantibody production (Hunziker et al., 2002). Finally, the use of a B-cell receptor transgenic model indicated that while antiviral IgM responses were normal during chronic LCMV infection, the high antigen levels prevented optimal IgG responses by causing terminal differentiation of antigen-specific B cells into short-lived antibody secreting cells (Zellweger et al., 2006). Finally, antibody responses during persisting LCMV infection

can, in some cases, be improved by inhibiting or diminishing the virus-specific T-cell response (Recher et al., 2004). This effect is likely related to a reduction in immunopathology and preservation of lymphoid architecture that is normally damaged by the virus-specific T-cell response during chronic LCMV infection. Together with observations from other infections (see HCV and HIV below), these observations from the LCMV system suggest that the mechanisms of altered B-cell responses during persisting viral infections will be important to dissect further.

HIV CD81 T-Cell Responses

The CD81 T-cell response to HIV has been the focus of intense scrutiny since the earliest days of the epidemic. Studies of HIV seropositive donors using lymphocytes tested directly ex vivo from blood revealed substantial responses—detectable using even relatively insensitive chromium release assays (Walker et al., 1987). Such “fresh killing” is unusual in human viral infection, outside the setting of acute disease. Subsequent studies have refined both the targets of the CD81 T-cell response and the quality in some detail, although it remains the case that the nature of responses which determine the different clinical outcomes are generally poorly understood, with a few notable exceptions. The first epitope to be identified at the level of peptide was in a longterm nonprogressor (“007”) infected through contaminated Factor VIII. Nixon and colleagues (1988) identified a single peptide in gag, restricted by HLA-B27, which attracted a strong ex vivo response. HLA-B27 is associated with a reduced rate of progression to AIDS and it is likely that the response targeting this epitope is at least in part responsible for this slow disease course (Nixon et al., 1988). Later studies revealed that escape from this response was associated with loss of control over viremia, although multiple consecutive changes in the gag protein were required in order to facilitate the final escape mutation (Goulder et al., 1997). Studies starting in the early 1990s revealed a plethora of epitopes across the viral genome, present from acute infection onwards, and studies using MHC Class I tetramers have revealed the magnitude of these antigen-specific populations. Temporally, the emergence of CD81 T-cell responses specific for HIV was found to be associated with the control of viral load (Koup et al., 1994). However, why such broadly directed, high frequency, and apparently functional responses fail to control viremia in the long term remains a central question. Phillips and colleagues (1991) showed that many such CD81 T-cell responses were associated with mutations


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FIGURE 4 T-cell escape observed through an HLA “footprint.” Sequencing of dominant viral sequences in a cross-sectional manner can reveal prototypical escape mutations. Sequences are indicated according to a specific HLA (X in the diagram; e.g., B27). If an excess of mutations is seen at a specific site in the HLA-X group there is statistical evidence for T-cell-mediated immune pressure.

within the targeted epitopes, some of which affected peptide binding to MHC and others of which affected TCR interactions or were even TCR antagonists (Phillips et al., 1991). The significance of such mutation however was fiercely debated—critics pointed out that there is substantial variation throughout the genome so the effects could be stochastic, that multiple peptides are targeted, thus minimizing the impact of any single mutant and that the associations were unpredictable. However, a huge amount of subsequent work has shown that HLA-linked mutations driven by CD81 T cells are readily detected, including studies of large HIV1 populations, with in many cases a predictable pattern described as a “footprint” (Moore et al., 2002) (Fig. 4). The best examples of such responses derive from HLAB57-restricted CTL. Like B27, B57 is a protective allele. In this case, responses targeting multiple peptides, especially those in HIV gag, are associated with prototypic escape mutations. Such mutant viruses have been shown to possess fitness deficits; these mutations revert upon transmission to HLA-B57 negative hosts. Other mutations with minimal fitness costs revert very slowly, if at all, and consequently spread rapidly, in some cases leading to the virtual extinction of some epitopes over time (Leslie et al., 2004). This is one reason that such footprints may not be visible in population studies (Fig. 5). It is proposed that the combination of escape mutants driven by HLA-B57 imposes a fitness cost sufficient to protect against disease progression. The relative importance of this allele was highlighted in a whole genome association study (Fellay et al., 2007). Apart from issues relating to targeting and escape, the other main thrust of most investigation relates to the quality or phenotype of CD81 T cells. Like T cells specific for other persistent viruses, HIV-specific populations cannot revert to a central memory phenotype as they continuously reencounter antigen—thus, they tend to remain CD62L, CCR7, and IL7R low (Appay et al., 2008). Unlike LCMV, however, this process does not lead to a fully exhausted phenotype or deletion, but a partially exhausted T cell may exist. HIV-specific CD81 T cells do possess strong functional capacity for secretion of cytokine such as IFNg and TNF, but proliferative capacity is inversely correlated with viral load (Appay et al., 2008). Expression of PD-1 is high on HIV-specific CD81 T cells in persistent disease and correlates with viral load; blockade of PD-1 signaling in vitro reverses the proliferative defect (Kaufmann & Walker, 2009). Thus, PD-1 appears to modulate T-cell function in HIV. The process of exhaustion may be promoted by loss of antigen-specific CD41 T cell help, as seen in LCMV.

CD41 T CELLS

Depletion of the CD41 T-cell compartment is central to the pathogenesis of HIV. Loss of antigen-specific CD41 T cells affects many specificities, and HIV specific responses are included among these. Interestingly, CD41 T cells specific for HIV may be preferentially targeted in acute disease, since they are highly activated and thus provide an enhanced target for infection (Appay et al., 2008). Correlative studies have revealed an association between sustained CD41 T-cell responses to HIV and improved outcome of infection (Appay et al., 2008), although unlike for MHC Class I, there are no robust associations between specific MHC Class II molecules and protection. Also unlike for CD81 T cells, escape through mutation does not appear to be a major feature, although it has been reported (Harcourt et al., 1998). To what extent the loss of functional CD41 T-cell responses is entirely due to depletion has not been fully defined, but there is data to suggest that the process of functional exhaustion also occurs amongst HIV specific CD41 T cells. This may be reversed by blockade of inhibitory receptors (including PD-1) in vitro (Kaufmann & Walker, 2009). Studies using MHC Class II tetramers to define antigen-specific populations (which are much smaller than their MHC Class I restricted counterparts), revealed these may be maintained but with some reduced function (Scriba et al., 2005). Loss of polyfunctional CD41 T-cell populations—in particular, loss of IL-2 secretion—has been noted by many groups, and is reversed in vivo when virus load is lowered through effective antiretroviral therapy (Harari et al., 2004).

B-CELL RESPONSES

The impact of humoral responses against HIV has been the subject of much debate and dogma, but is not fully defined. The complexity of the antigen, the HIV GP120 glycoprotein, accounts for much of the confusion. There is no doubt that effective neutralizing antibody (Nab) responses are generated against this target, but immune escape occurs readily, through a combination of mutation of epitopes as for CD81 T cells and changes in glycosylation (“glycan shield”) (Wei et al., 2003). The consequence of this is somewhat different than for CD81 T cells, where mutations tend to become fixed once established. In envelope, a continuous generation of novel antibody responses has been reported, with associated recurrent escape; such experiments require careful evaluation of sequentially generated pseudoviruses to analyze “autologous” neutralization. The fitness consequences for the virus also

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FIGURE 5 HLA footprinting is limited by many variables. Although T-cell mediated selection may occur, the footprinting approach relies on the sequence of events indicated in the black diagrams. If a viral region is highly conserved, highly targeted by a single response, mutates under selection pressure and reverts upon transmission then a footprint is typically visible. However, intrinsic variation, geographical linkage of HLA and variant, failure of reversion, multiple selection events, or failure of selection can all lead to loss of power of the approach.

appear to be less severe than for some of the effective CD81 T-cell responses outlined above. However, there is evidence that broadly reactive and autologous Nabs persist in chronic infection, potentially contributing to the nonprogressor status of some “elite” controller groups (Scheid et al., 2009). A recent study indicates that functional defects might develop in HIV-specific B cells (Moir et al., 2008). In the study, B-cell “exhaustion” was characterized by poor proliferation and elevated expression of inhibitory receptors by HIVspecific, but not influenza-virus-specific B cells (Moir et al., 2008). In addition, in vivo blockade of the PD-1 pathway in SIV (simian immunodeficiency virus) infected macaques demonstrated a positive effect not only on T-cell responses, but also on SIV-specific antibody responses (Velu et al., 2008). It will be interesting to determine whether B-cell exhaustion, in addition to HIV escape from antibody, contributes to the poor humoral control of this virus.

HEPATITIS C VIRUS CD81 T-Cell Responses

Although, in principle, many of the same issues apply to HCV infection as they do to HIV infection—a high-level and persistent replication of a variable virus—in practice, the immune responses appear highly divergent. HCV, like

LCMV but unlike HIV, has two clear-cut virologic outcomes after infection (Fig. 1). On the one hand, a subset of subjects achieve long-term control of infection while, on the other, some subjects develop persistent viremia with associated liver inflammation and fibrosis. CD81 T-cell responses contribute to the control of infection, which occurs spontaneously in 20% to 50% of those infected, as well as contributing to the liver inflammation in the majority of cases where virus persists (Lauer & Walker, 2001). The first evidence that CD81 T-cell responses were critical to disease outcome came from experiments in the chimpanzee model, where acute infection could be readily studied and a strong temporal correlation between CD81 T-cell responses and initial viral control was observed (Cooper et al., 1999). Subsequent studies in humans revealed a similar association between the emergence of strong and sustained CD81 T-cell responses and control of infection (Bowen & Walker, 2005). Depletion studies in the chimpanzee revealed the importance of such CD81 T-cell populations (Bowen & Walker, 2005). In those where the virus persisted, immune escape was also observed (Bowen & Walker, 2005). Analogous to HIV, from initial studies in small cohorts, this phenomenon can now be viewed as HLA-associated “footprints” at a population level (Bowen & Walker, 2005). Also, MHC Class I associations with disease have been shown (interestingly, HIV, B27, and B57 are


associated with improved outcome after HCV infection), and the targeting of a dominant B27 restricted epitope with limited options for escape may account for some of the protective impact (Neumann-Haefelin et al., 2006). However, unlike HIV, if HCV persists CD81 T-cell responses wane rapidly in blood and become undetectable by conventional ex vivo assays in many cases. There appear to be many reasons for this. One is that responses may relocalize to the liver, where they are found to be enriched (Bowen & Walker, 2005). However, even in the liver they only appear to represent a minority of CD81 T cells and it is not clear to what extent this accounts for the loss in blood. Immune escape may potentially contribute to failure to maintain responses, although in fact, many responses that are detectable in chronic infection appear to be against mutant epitopes (from escape or from a previous infection with a distinct genotype). The process of exhaustion is likely to contribute to the down regulation of responses, and PD-1 expression is detectable on HCV-specific cells in chronic infection, especially within the liver (Radziewicz et al., 2006). Finally, the presence of regulatory T cell (Treg) populations, particularly enriched within liver tissue, might limit T-cell proliferation (Ward et al., 2007). The “black box” of the liver environment requires further unlocking before the critical mechanisms are revealed. One clue comes from the recent observation that HCV specific CD81 T cells express the C-type lectin CD161, a molecule associated with liver homing and also with Type 17 differentiation (Northfield et al., 2008).

CD41 T Cells

Unlike HIV, MHC Class II associations with disease outcome are readily observed for HCV and it is also clear that CD41 T-cell responses play a codominant role in disease outcome. In the chimpanzee model, depletion of CD41 T cells also leads to long-term persistence despite the maintenance of CD81 T-cell responses (Grakoui et al., 2003). In humans, very strong correlations exist between the presence of robust T-helper proliferative responses against viral nonstructural proteins and long-term containment (Semmo & Klenerman, 2007). However, since the virus does not infect CD41 T cells to any significant extent, the mechanisms involved in the failure of T cells in HCV are harder to explain. As for HIV and also for LCMV, in certain settings, exhaustion of CD41 T-cell populations can occur. Loss of IL-2 production in those cells found in chronic infection, as well as loss of proliferative capacity, have been noted (Semmo & Klenerman, 2007). Studies using MHC Class II tetramers have revealed long-term maintenance of antigenspecific populations in those with spontaneous clearance, compared with chronic infection where they are typically absent or just detectable ex vivo, but associated with weak functional capacity (Semmo & Klenerman, 2007). In those cases where they have been studied in acute disease, such populations disappear from blood relatively early, although quantitative and functional data on liver derived populations are sparse. Liver-infiltrating antigen-specific CD41 T cells have been found to secrete IL-10 (as have antigen specific CD81 T cells) (Semmo & Klenerman, 2007) and a substantial fraction of total CD41 T cell populations in the liver express Foxp3 and may represent regulatory cells (Semmo & Klenerman, 2007). The latter may contribute to down regulation of both CD41 and CD81 T-cell responses.

B Cells

Antibody responses to HCV have been investigated in some detail although as for HIV the huge variability of the envelope target (notably the hypervariable region of E2) has hampered interpretation of these results. Autologous neutralization of virus is observed in those who clear virus spontaneously,

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but also in those with chronic infection. However, in the latter, recurrent mutations may keep the virus “one step ahead” of the NAb response—the accumulation of escape mutations in the E2 gene during acute disease correlates with disease progression (Bowen & Walker, 2005). Overall, therefore, the ability of the virus to readily escape antibody may indirectly promote exhaustion of CD41 and CD81 T cells as in LCMV and HIV, and is a major challenge for antibody-based vaccine design. HCV also appears to drive some profound abnormalities in B-cell responses including mixed cryoglobulinemia, at least in some subjects (Charles et al., 2008). There could be similarities to the hypergammaglobulinemia observed during chronic LCMV infection or to B-cell exhaustion during HIV infection, but further studies are necessary. The impact of binding of the HCV envelope to its receptor CD81 (Bowen & Walker, 2005), a tetraspanin engaged in B-cell activation, is also still unclear.

CYTOMEGALOVIRUSES CD81 T-Cell Responses

Cytomegaloviruses infect a range of vertebrate hosts and establish persistent infection through evasion of host innate and adaptive immunity. Human and murine CMVs are normally well contained, however, and viral replication is found at extremely low levels within tissue. Upon immunosuppression, however, recrudescence and viremia is rapid, with significant clinical consequences (e.g., post bone marrow or solid organ transplantation). The favorable host virus balance in healthy subjects is achieved partly through the generation of substantial antiviral CD81 T-cell responses, which are among the most numerous of any measured. CD81 T-cell responses against murine CMV are elicited in acute disease and combine with NK-cell responses (especially in “resistant” Ly49H1 mouse strains such as C57BL/6) to contain infection. However, strikingly, during the subsequent weeks and months, specific responses appear to gradually accumulate in lymphoid and nonlymphoid organs over time (Karrer et al., 2003) (Fig. 6). This phenomenon of “memory inflation” is seen in resistant and nonresistant strains but restricted to certain epitopes. The dominance of the initial response is unrelated to the expansion of the inflating responses

FIGURE 6 Dynamics of T-cell responses during diverse types of persisting viral infections. T-cell frequencies and the dynamics of maintenance differ widely during chronic infections. Viruses that persist at a low level may be associated with T-cell memory inflation. This group includes viruses such as CMV and many others shown. Viruses where exhaustion may dominate include LCMV, HBV, and HCV, but this may also be true in many other settings and even for the same infection differing between specific epitopes.

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and thus the hierarchy of responses changes over time. Responses showing memory inflation take on additionally a striking phenotype, associated with reduction in costimulatory receptor expression (CD27/CD28lo), loss of lymph node homing (CD62L/CCR7lo), and retain expression of antiviral cytokines and Granzyme B (Sierro et al., 2005). Thus despite continuous antigen reencounter they do not appear exhausted in the manner of LCMV specific populations and retain strong proliferative capacity in vivo. In contrast to these inflationary responses, the majority of T-cell responses targeting other epitopes, despite the presence of continuous viral replication, return to a central memory phenotype and are found at stable levels with enrichment in lymph nodes (Sierro et al., 2005). Such noninflating populations are apparently not exposed to further peptide antigens, possibly through the protective activity of the inflating populations (some of which target genes under the immediate early promoter and which therefore may act as checkpoints), and possibly through the action of specific viral inhibitory pathways. However interestingly, deletion of three of the latter genes has little effect on the immune responses generated in vivo, possibly since much of the presentation occurs on uninfected APCs (i.e., through cross presentation [Gold et al., 2004]). We still have a lot to learn about the rules that govern this complex system, however, since inflating and noninflating populations may be found that target the same gene and are restricted by the same MHC allele. In humans, the same phenomenon of inflation has been observed (Komatsu et al., 2003), although given the extremely long periods of infection, the expansion of CMVspecific populations can be very large and can come to occupy a significant fraction of the total CD81 T-cell pool. Indeed, CMV seropositivity is associated with marked changes in the overall composition of CD81 T-cell subsets in the peripheral blood of healthy humans, which are similar to those seen associated with age (largely, accumulation of “effector memory” or terminally differentiated cells) (Northfield et al., 2005). CMV seropositivity is also associated with some excess mortality among the aged population and it has been proposed that immune senescence driven by CMV could contribute to this effect (Olsson et al., 2000).

CD41 T Cells

In comparison to the work on CD81 T cells, much less is known about the targets and phenotype of CD41 T-cell populations induced by CMV. Nevertheless, in humans these are very large populations, possessing strong functionality and an effector memory phenotype equivalent to their CD81 T-cell counterparts. In murine models, depletion of CD41 T cells has an effect similar to that of depletion of CD81 T cells on viral replication (although both require concomitant depletion of NK cells for viremia to occur) (Polic et al., 1998). Loss of CD41 T-cell responses to CMV is linked to failure to control the virus in those with progression to AIDS and also posttransplantation. Thus, like HCV, both CD81 and CD41 T cells have a balanced contribution to long-term virus control, although numerically, the CD81 T-cell responses vastly outnumber the helper populations. An interesting question that arises when thinking about the inflation of CMV-specific CD4 and CD8 T-cell populations is how much antigen-driven expansion of virus-specific T cells the immune system can accommodate. One concept is that there is a fixed amount of “space” in the T-cell compartment and that new antigen-specific T cells would displace preexisting specificities, leading to attrition of responses with antigen experience over time (Selin et al., 2006). This is often thought of as a bucket of fixed size that

must somehow accommodate all the necessary cells. Important details about this model relate to whether or not, for example, CD4 and CD8 T cells occupy the same niche in vivo and if naïve and memory T cells compete for the same space. This latter issue is quite relevant since with age and thymic involution the number of naïve T cells declines. However, recent studies suggest that the amount of space (both physical and conceptual) available to antigen-specific T cells may expand depending on the number of T cells it is necessary to accommodate (Vezys et al., 2009). In other words, with the addition of new memory T cells, the size of the whole bucket increases. While there is clearly more work to be done in this area, the implications of new research on this topic could have particular relevance to the inflationary CMV-specific T-cell responses and maintaining the flexibility to respond to other pathogens as we age.

B-CELL RESPONSES

Antibodies play an important role in containment of CMV viremia, although their role in the long-term containment of recrudescent virus within tissue is not so clearly defined. In humans, the presence of CMV-specific antibodies is an excellent predictive indicator of the likelihood of significant infection posttransplantation. In recipients of organs from seropositive donors, significant disease is much more likely among seronegatives, although superinfection is possible (Hughes et al., 2008). This is also clinically significant in the setting of maternal infection. Primary infection of the fetus is associated with a number of developmental defects, including deafness. Although maternal humoral immunity can protect against most in utero infections, CMV-affected infants can be born to seropositive mothers through infection with distinct viral genotypes (Boppana et al., 2001). This suggests some important limits to humoral immunity, although the molecular correlates of this have not been examined in the same detail as for HIV and HCV.

CONCLUSIONS

To some extent each immune response is unique to the pathogen or model used. However, some common themes do emerge, which will be summarized here. Exhaustion: While details differ from one infection to another, defects in CD8 T-cell effector function or memory formation have been observed in a number of mouse models of persisting infection (e.g., LCMV, murine gammaherpesvirus, mouse hepatitis virus, Friend leukemia virus, adeno-associated virus and polyoma virus, as well as primate models of SIV infection). In human disease, the best evidence for exhaustion may come from HCV, where responses may be very limited both in number and function. A similar outcome is observed in high-level carriage of HBV (Maini et al., 2000). Thus, in these settings, where the level of antigen reencounter is very substantial, the types of defects and exhaustion observed during chronic LCMV infection appear to dominate. In HIV infection, the impact of antigen reencounter is more subtle, although clear defects in specific functions such as proliferation are evident. Understanding the precise impact of T-cell functional defects, however, is made more complex by the impact of antigenic variation. Exhaustion may therefore play a critical role in a subset of chronic infectious diseases, perhaps in settings where immunopathology (such as readily occurs in the liver) is to be avoided. Although this argument is teleological, the striking similarities between some of the pathways involved in exhaustion (such as IL-10 and PD-1) and those involved in


induction of peripheral tolerance, suggest a shared evolutionary aim of protecting tissues. Inflation: While exhaustion has received a large amount of attention, it is only one of several outcomes of T-cell responses to persisting viruses. CMV demonstrates a setting where antigen reencounter leads to expansion of CD81 T-cell responses. Although these responses do not retain the features of resting “antigen-free” memory T cells and might have some similarities to the antigen-addicted T cells observed late following chronic LCMV infection (Shin et al., 2007), they do not resemble the functionally exhausted CD81 T cells found in mice with high level (i.e., viremic), chronic LCMV infection. Moreover, these inflationary CMV-specific T-cell responses show continuous expansion over time in vivo. This outcome may be a quirk of CMV biology, but a number of other responses show related profiles, including other herpesviruses, such as EBV, as well as parvovirus B19, and certain HIV responses (Isa et al., 2005). A number of features presumably need to come together for inflation to occur, including the quantitative degree of restimulation (at the level of the APC and features of the T-cell receptors and coreceptors), as well as the inflammatory context. The rules governing this behavior still need to be defined. Escape: Escape is a dominant feature of HCV and HIV, and can occur in most RNA viruses under appropriate settings, including LCMV. Escape was originally thought to represent a stochastic event, but turns out in most settings to be highly reproducible, with the virus exploring the available antigenic space to obtain a maximally fit mutant. This realization has allowed rapid progress to be made in the definition of escape in human “outbred” populations infected with highly variable pathogens. However, the overall impact of escape on the outcome of infection, outside a few really well-defined examples, is still not understood. Escape is also not seen in all epitopes and the differences between responses that drive viral evolution and disease outcome (“drivers”) and others that appear to passively follow antigenic load (“passengers”) remain to be fully defined. Regulation: Finally, regulation can either occur in a cellintrinsic manner via inhibitory receptors or through the actions of other cells such as Treg. Inhibitory receptors such as PD-1 clearly play an important role during a number of persisting infections and other inhibitory receptors pathways are likely to be important as well (Crawford & Wherry, 2009). Although the role of regulatory subsets has not been discussed here in much depth, there is still a potentially critical function for Treg during chronic viral infections. While the role of Treg in the LCMV model remains poorly understood, emerging studies appear to indicate a potential role for these cells (G. Punkosdy & E. Shevach, personal communication). In contrast, considerable work in the Friend leukemia virus infection model demonstrates a key role for Tregs (Iwashiro et al., 2001), and certainly in humans, there is a substantial accumulation of Tregs in the liver during chronic HCV infection (Ward et al., 2007). There is also a possible role for Treg during HIV infection (Kinter et al., 2007). Whether these differences are related to virus or tissue, or reflect the timing of infection (decades in the case of HCV) remain to be determined, but this represents an interesting area for the future, as the pathways that regulate these subsets and those of related cells such as Th17 cells are uncovered. Overall, persistent virus infections represent a major challenge for the future, both in terms of designing vaccines and therapies and in terms of defining the fundamental immunology. The key features outlined here provide a reasonable basis for exploring the responses to new viruses as they

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emerge, and for the design of new therapies for prevention and treatment of existing viruses. The latter provides huge challenges, but also potentially huge rewards.

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21 Acquired Immunity: Acute Bacterial Infections DENNIS W. METZGER

INTRODUCTION

pneumococcal infections were responsible for more than 90% of deaths during the 1918 influenza pandemic, which killed approximately 40 million individuals throughout the world (Morens et al., 2008). Because of this, pneumococcal infection became the focus of intense study in many laboratories following the 1918 influenza pandemic, including in the laboratory of Ostwald Avery, who used the bacterium to discover that DNA encodes genetic material, thus paving the way for Watson and Crick, and the ensuing genetic engineering revolution. Most importantly for this chapter, pneumococcus has been a focal point for efforts to develop effective antibacterial vaccines, initially against the major pneumococcal virulence factor, the polysaccharide capsule, and more recently, against bacterial outer membrane proteins. Thus, there has accumulated a wealth of information about the virulence of this pathogen as well as the means to induce acquired immunity that is effective in protection against bacterial disease.

Acute bacterial infections continue to represent a major challenge to human health. Such infections typically begin in mucosal tissues or the skin, become systemic, and induce cytokine production, which in extreme cases, can result in septic shock involving multiple organ failure. According to recent data from the World Health Organization for the year 2004, approximately 25% of worldwide deaths are still due to infections and acute respiratory infections, and kill over 4 million people every year. In addition, pneumonia and other respiratory infections continue to be the primary source of disability-adjusted life years, (i.e., healthy years lost to injury, illness, or premature death), which represent a significant economic burden. Antibiotics have historically been highly effective in treatment of bacterial infections but are losing their usefulness due to an increasing development of antibiotic resistance. In addition, antibiotics are not effective for treatment of septic shock once symptoms have developed. Another approach, which was routinely employed in the pre-antibiotic era and is a topic of resurging attention, is passive immunotherapy with immune serum (Casadevall et al., 2004). Nevertheless, the most effective approach for prevention of infection remains vaccination to induce protective acquired immunity. Acquired immunity to acute bacterial infections is the focus of this chapter. Bacterial toxins are well-known targets for vaccination to induce neutralizing antibody responses that prevent the symptoms of many acute bacterial infections. These include tetanus and diphtheria toxins. Remaining vaccines and those under development are focused primarily on polysaccharide capsules and outer membrane proteins, both targets for antibodies as well. Streptococcus pneumoniae will be extensively utilized throughout this chapter as a model for acquired immunity to extracellular bacteria. In addition, Francisella tularensis and Yersinia pestis are discussed as models for “intracellular” pathogens that cause acute bacterial infections. Pneumococcus is a common gram-positive pathogen that affects primarily children and the elderly. It can cause recurrent otitis medium, pneumonia, and meningitis, and is believed to result in over 1 million worldwide deaths annually, particularly in developing countries. Interestingly, secondary

ANTIBODY-MEDIATED PROTECTION

Acquired resistance to acute bacterial infections requires the interplay between humoral and cell-mediated immunity, including production of specific antibodies, activation of complement that can lead to bacterial killing and inflammatory responses, phagocytosis and killing by phagocytic cells, especially macrophages and neutrophils, and stimulation of T-cell help and cytokine production. These processes typically act in concert to ensure optimal protection so that they cannot be considered in isolation from each other. Nevertheless, all successful vaccines for human use rely upon induction of specific antibody production and the current focus of vaccine research remains on humoral immunity to induce protective effector mechanisms. Almost all acute bacterial infections are caused by extracellular bacteria, or bacteria that at least have an extracellular phase, and thus these pathogens are subject to antibody-mediated control. Indeed, congenital B-cell immunodeficiency in humans is primarily associated with susceptibility to repeated acute bacterial infections. In addition, before the advent of antibiotics and effective vaccines, passive immunotherapy with immune serum was the standard of care to prevent acute bacterial infection and remains a routine therapy for certain infections such as tetanus (Casadevall et al., 2004). The prevailing dogma in the field has been that

Dennis W. Metzger, Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York 12208.

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while humoral immunity plays a critical role in protection against extracellular pathogens, for “intracellular” pathogens that are believed to exploit the ability to replicate within cells to escape immune detection and elimination, cell-mediated immunity is more important. In turn, this notion has had a profound influence on the development of immune correlates of protection for pathogens such as Francisella tularensis and Yersinia pestis, even though in the pre-antibiotic age, patients suffering from tularemia and plague were successfully treated with xenogeneic immune serum. In spite of this conundrum, there is now convincing evidence that such organisms can replicate extracellularly and be targeted by humoral immunity. Indeed, while Y. pestis and F. tularensis can be found to replicate in vitro within macrophages, whether this in vitro phenomenon is reflected in vivo and the relevance of such intracellular replication to the high levels of virulence observed with these pathogens remain subjects of considerable debate (Metzger et al., 2007). For example, in vivo depletion of macrophages before infection has little overall effect on disease progression, although it does significantly influence antibody-mediated clearance by removing the effector cell responsible for opsonophagocytosis and bacterial killing (Kirimanjeswara et al., 2007).

THE ROLE OF ANTIBODY ISOTYPE IN PROTECTION: IgG VERSUS IgA

Clearly, the amount, avidity, and isotype of antibody can influence protective efficacy. It is generally believed that 0.15 mg/ml of serum antibody is indicative of a successful vaccination that will protect against acute bacterial infection. Most serum antibody is of the IgG isotype, which is capable of efficiently mediating opsonophagocytosis and complement fixation, as well as being transported across the placenta for protection of the developing fetus. Thus, IgG is believed to be the serum antibody isotype of choice due to its many effector functions. However, most bacterial infections target mucosal tissues during the initial stages of infection. Considering the fact that IgA antibody is by far the most abundant isotype in mucosal tissues and, indeed, humans produce greater amounts of IgA than all other immunoglobulins combined (over 3 grams daily), one might predict that IgA would be critical for protection against acute bacterial infections. Nevertheless, patients with congenital IgA immunodeficiency, which happens to be the most common congenital immunodeficiency disease in humans, are generally not immunocompromised and usually can clear infections as effectively as normal individuals (Burks & Steele, 1986; Carneiro-Sampaio & Coutinho, 2007; Conley et al., 2009). Protection in the absence of IgA is typically attributed to compensatory increases in the expression of secretory IgM (SIgM) and IgG at mucosal surfaces. Although most IgA-deficient patients are healthy, there is an increased incidence of several disorders in this population and these disorders tend to be localized to the respiratory tract. Of particular interest to this chapter, the most common infections associated with IgA immune deficiency include recurrent bacterial ear infections, sinusitis, bronchitis, and pneumonia. Interestingly, IgA immunodeficiency also has been found to be associated with an increased incidence of allergy. In a mouse model of Streptococcus pneumoniae infection, it has been demonstrated that protection can be transferred to mice with human IgA antibody (Steinitz et al., 1986). It has also been found that human polymeric IgA can mediate S. pneumoniae killing through complement receptors on phagocytes (Janoff et al., 1999). Immune deficient mice have been extensively studied in efforts to further understand the importance of IgA in

protective mucosal responses. These murine models include animals with genetic disruptions in the Igh-2 gene locus that encodes the a-H chain constant region as well as mice with disruptions in genes required for proper assembly of polymeric IgA and/or its transport across mucosal epithelia. Although human IgA deficiency appears to be caused by defects in regulatory mechanisms rather than in IgA constant region genes or genes involved in IgA transport, the murine models that have been developed do provide potentially important clues into the role of IgA in protection against microbial pathogens. A comparison of respiratory protection in wild-type and IgA2/2 mice against Shigella flexneri was performed by Way and colleagues (1999). An attenuated form of this pathogen that is defective in oxidase expression is 100-fold less active in mediating lethal disease but can induce mucosal immunity against the fully virulent bacterial strain. However, it was found that protection was not dependent upon IgA expression. A similar result was published by Murthy and colleagues (2004), who examined pulmonary protection against Chlamydia trachomatis. After intranasal infection of naïve animals, it was found that IgA1/1 and IgA2/2 mice contained equivalent amounts of pulmonary bacteria 10 days later. In both of the above studies, protection correlated with histological inflammation in the peribronchiolar areas of the lung and it was found that IgA2/2 mice demonstrated more extensive inflammatory changes in response to C. trachomatis infection than wild-type mice. Thus, IgA did appear to play a role in lung homeostasis and in reducing the extent of bacterial-induced inflammation after infection. IgA2/2 animals also expressed significantly higher levels of serum IgG antibodies, suggesting that these antibodies played an important role in protection. In contrast to the above studies, other groups have reported a pivotal role for mucosal IgA in protection from bacterial lung infection. Lynch and colleagues (2003) found that intranasal vaccination of IgA1/1 mice with pneumococcal polysaccharide conjugated to diphtheria toxoid induced protective immunity in adults against subsequent nasal carriage with S. pneumoniae type 14. The same vaccination regimen can protect neonatal mice against otitis media (Sabirov & Metzger, 2006). Such protection was not observed in IgA2/2 mice. Dependence upon mucosal IgA for protection against S. pneumoniae carriage was also seen using pIgR2/2 mice (Sun et al., 2004). Lack of protection in immunized IgA2/2 animals was directly correlated with the absence of antibody in nasal secretions. Furthermore, protection could be observed in IgA2/2 mice if the bacteria were first opsonized with serum antibody before intranasal challenge. It is important to note that the type 14 pneumococcal strain used in these studies did not induce inflammation in the respiratory tract, nor did it cause systemic infection when instilled intranasally (Sun et al., 2004). On the other hand, protection against intranasal challenge with S. pneumoniae type 3, a strain that does induce significant inflammation and systemic infection, did not require SIgA antibody (Sun et al., 2004). Similar findings have been recently obtained in Bordetella (Wolfe et al., 2007) and Francisella (Rawool et al., 2008) animal infection models. A potential caveat to the interpretation of these results is that the secretory component (the cleaved extracellular domain of pIgR) can inhibit adherence of some strains of S. pneumoniae to respiratory epithelial cells by direct binding to proteins on the bacterial cell surface, thus providing “innate” protection that is unrelated to IgA antibody specificity. Thus, from using IgA deficient animals it is clear that different laboratories have obtained disparate results and made opposite conclusions regarding the importance of IgA in protection against acute bacterial infections. However,

21. Acquired Immunity: Acute Bacterial Infections

in comparing these studies, it becomes apparent that it is the presence of inflammation that determines whether IgA is found to be necessary for any observed protection, (i.e., in the presence of inflammation, IgA deficient mice are protected from infection whereas in the absence of inflammation IgA deficient mice are not protected). This could best be explained by the fact that inflammation leads to increased dendritic cell/lymphocyte activation and subsequently, damage to the mucosal epithelial barrier, increased blood vessel permeability, and enhanced transudation of IgG antibodies from the bloodstream. Thus, inflammation could increase the efficacy of vaccination as well as protection upon pathogen challenge. Another variable involves the site of pathogen challenge. In the case of the lung, which has a high level of blood vessel penetration, and thus IgG transudation, inflammation would tend to obscure the requirement for IgA antibody. However, in the upper respiratory tract, in which few blood vessels are present and little IgG is transudated, a requirement for IgA would become more apparent. Thus, when immunization or pathogen challenge has occurred in the presence of significant inflammation in the upper respiratory tract, IgA has still been found to be critical for protection. Consistent with this model is the finding that IgA was not required for clearance of Bordetella from the lungs, but was essential in the upper respiratory tract where blood flow and inflammation is significantly lower (Wolfe et al., 2007). Importantly, with respiratory pathogens such as type 3 pneumococci, S. flexneri, and C. trachomatis, significant lung inflammation is typically observed very soon after challenge, but with others such as type 14 pneumococci, F. tularensis, and Y. pestis, inflammation is not observed or is significantly delayed. From the above discussion, it appears likely that the experimental model used and the amount of inflammation induced influences the apparent need for protective IgA. The ability of IgG to substitute for IgA likely relates, at least partially, to the site of infection, (i.e., IgA is required for protection of the upper respiratory tract while both IgA and IgG can be involved in protecting the lungs). Nevertheless, the human body expends a considerable amount of energy in producing 3 grams of IgA per day. It has been suggested that IgA is perfectly suited for protecting mucosal surfaces in a noninflammatory manner since it does not activate complement nor induce inflammatory reactions, and the studies in mice would tend to confirm this concept. Clearly, it would be preferable to contain infections in the respiratory tract (and other mucosal sites) before potentially serious inflammation develops. However, if endothelial and epithelial barriers are breached, IgG will transudate from the serum and serve as a back-up system to prevent potential blood-borne infection. What about IgA immunodeficient humans who generally do not show any significant effects of their immunodeficiency? First, it must be recognized that clinical IgA immunodeficiency is defined by the presence of ,50 mg/ml of serum IgA. Since we are only now beginning to understand the basis for this immunodeficiency in humans, but do know that a heavy chain genes are not disrupted, it is possible that IgA deficient individuals contain low levels of IgA that are sufficient to protect mucosal surfaces. In addition, considering the potential for compensation by SIgM and transudated serum IgG, these individuals may have subtle defects in protection that are not recognized. In fact, it is the subset of patients with defects in both IgA and IgG expression that fare the worst clinically, and IgA deficient mice tend to resemble this subset in showing an associated defect in isotype switching to immunoglobulin isotypes other than IgA. In summary, it is likely that IgA antibody provides an important first line of defense against infections of mucosal

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tissues. If these infections progress to a point such that significant inflammation occurs, epithelial and endothelial barriers will become compromised, IgG antibody will be transudated and this IgG will provide a second line of defense to prevent systemic, lethal spread of the infection. In the absence of IgG transudation, serum IgG will still provide an effective means for systemic protection and prevention of sepsis. Thus, the major role of respiratory IgA will be to provide protection against both morbidity and mortality, while IgG provides a back-up mechanism to ensure survival of the host. In the absence of inflammation, such as may occur in nasopharyngeal colonization with encapsulated bacteria, IgA will provide important protection by serving as “flypaper” to coat mucosal tissues and prevent overt infection, but IgG will be sufficient for survival under otherwise severe inflammatory (i.e., lethal) conditions.

REGULATION OF IgA PRODUCTION

Human IgA consist of two subtypes, IgA1 and IgA2. Understanding the mechanisms that control production of protective IgA at mucosal surfaces is an area of ongoing investigation. Cerutti (2008) has recently found that different mechanisms control expression of IgA1 and IgA2 in the intestinal tract. In essence, they have proposed that in gut Peyer’s patches, T cells releasing IgA-inducing cytokines such as TGF-b foster a germinal center reaction that includes somatic hypermutation and IgA class switch recombination. The resulting IgA1 B cells, primarily IgA11 B cells, are home to the gut lamina propria, where they differentiate into plasma cells that secrete high affinity IgA. These IgA11 effector B cells can also undergo sequential class switching to IgA2 expression, possibly in the lamina propria, by receiving T-cell-independent signals from bacterial-activated epithelial cells and dendritic cells. Such signals likely include BAFF and APRIL, and can induce class switching in both unmutated IgM1IgD1 B-1 cells derived from the peritoneum and in mutated IgM1IgD2 effector B cells from Peyer’s patches. Interestingly, mice have only one IgA subtype, which is most homologous to human IgA2. Whether the mechanisms that control mouse IgA production are similar to those that regulate human IgA2 expression is currently unknown. B cells must be able to home to the appropriate effector mucosal tissues such as the lung after activation; this is accomplished by the influence of selected chemokines and adhesion molecules. Interactions between a4b7 and MADCAM-1 appear to be critical for homing of B cells from the bloodstream to gut mucosal tissue, but only low levels of MADCAM-1 are present on endothelial cells in the respiratory tract. Homing to the respiratory tract appears to involve a4b1-VCAM1 interactions, the same interactions that are involved in recruiting systemic lymphocytes to sites of inflammation (Kunkel & Butcher, 2003). However, while CCR10 is up regulated on cells destined to home to the respiratory tract, it is absent on systemic lymphocytes (Kunkel et al., 2003; Morteau et al., 2008). Similarly, CCL28, the chemokine ligand for CCR10, is expressed preferentially by mucosal epithelial cells (Pan et al., 2000). Thus, CCR10–CCL28 interactions appear to cause trafficking of lymphocytes to mucosal tissues and a4b1VCAM1 interactions ensure homing to the respiratory (and incidentally, urogenital) tracts. This differential usage of homing receptors would explain early seminal findings showing that adoptively transferred, IgA-secreting B cells obtained from the respiratory tract preferentially traffic to recipient airways and show only low levels of trafficking to other organs, including the gut (Husband & Gowans, 1978; McDermott & Bienenstock, 1979; Rudzik et al., 1975; Weisz-Carrington

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et al., 1979). However, there are likely other regulatory factors involved since IgA-secreting B cells, but not IgM-secreting or IgG-secreting B cells, show preferential homing to mucosal tissues.

T-CELL-MEDIATED PROTECTION

In addition to B cells, T cells certainly play an important role in acquired immunity to acute bacterial infections. Their most obvious role is in helping B cells switch antibody isotype expression from IgM antibody to IgA and IgG antibodies, and in secretion of appropriate cytokines and chemokines to ensure proper activation and homing. However, it is possible that T cells also provide protective signals independently from humoral immunity. Indeed, again using S. pneumoniae as a model, there is evidence for protective mechanisms in humans other than antibodies. After the first 1 to 2 years of life, the incidence of bacterial disease and the duration of carriage occur simultaneously for multiple capsular serotypes, suggesting acquisition of a common immune response. Of significance, this can take place in young children. In mice, protection can occur independently from antibodies after vaccination with pneumococcal protein vaccine candidates; however, protection is dependent upon CD4 T cells (Basset et al., 2007). The effector phase of such CD4 T-cell-mediated protection seems to occur in an antigen-nonspecific manner (Trzcinski et al., 2008) and, in fact, it was recently shown by two groups (Lu et al., 2008; Zhang et al., 2009) that pneumococcus-specific CD4 T cells that express IL-17 (i.e., Th17 cells), are active in recruiting macrophages and neutrophils to the respiratory tract for enhanced elimination of bacteria. Thus, elicitation of the Th17 cell lineage can occur specifically through prior infection or vaccination, followed by recruitment of phagocytic cells that mediate their effector function in an antigenindependent manner. The role of phagocytic cells in enhancement of acquired immunity is described below.

INNATE IMMUNE MECHANISMS AS EFFECTORS OF ACQUIRED IMMUNITY

Innate effector mechanisms, including complement and phagocytic cells, act in concert with acquired immunity to mediate ultimate protection. In addition, TLR stimulation results in increased recruitment of T cell and phagocytes to the site of infection. The phagocytes that provide initial protection at mucosal sites are the tissue resident macrophages such as alveolar macrophages in the lung, which engulf and destroy invading pathogens. If this innate defense is overcome and inflammation ensues, monocytes and neutrophils are recruited from the bloodstream, become activated, and actively kill bacteria through expression of reactive oxygen and nitrogen species. While phagocytic cells can recognize nonopsonized bacteria through binding to scavenger receptors, opsonization by complement and/or antibody significantly enhances killing efficacy. Complement is an important component of innate defense against acute bacterial infection that can act in concert with acquired immunity through multiple mechanisms. Although complement-mediated lysis of antibody-coated red blood cells is a well-known phenomenon, humans deficient in the terminal components of the complement cascade are not more susceptible to bacterial infections other than Neisseria infections. Thus, this pathway may play a role in protection against some infections caused by gram-negative organisms, but it is likely that cell lysis is not a major means of bacterial elimination against the majority of acute bacterial pathogens, which, unlike Neisseria, have relatively thick cell walls that can resist insertion of the complement membrane attack

complex. Rather, it is probable that complement-mediated opsonization of bacteria by C3b, which becomes covalently attached to microbial surfaces, and recruitment of inflammatory cells by complement products such as C3a and C5a, are more critical for protection against the majority of acute bacterial infections. In this scenario, binding of specific antibody to microbes initiates the classical complement cascade, leading to generation of C3 complement fragments, among others, which then bind to cell receptors such as CD11b, CD21, and CD35 to promote phagocytosis as well as B-cell activation. The C5a fragment also stimulates adhesion of neutrophils to endothelial cells and at high doses, can stimulate the respiratory burst and production of reactive oxygen species to enhance microbe killing. Humans with congenital C3 immunodeficiency demonstrate increased frequency of serious infections with pyogenic bacteria, illustrating the importance of this complement component in acquired immunity to acute infection. Additional critical mediators for the effector phase of acquired humoral immunity are the Fc receptors on phagocytic cells. Phagocytic cells contain several receptors that recognize the Fc portion of IgG antibodies after forming complexes with specific antigen. These FcgRs are associated with a signaling molecule termed the common g chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM) on its cytoplasmic tail, and which links receptor clustering with activation of protein tyrosine kinases. This, in turn, stimulates cell activation and enhanced phagocytosis of the bound microbe. FcgR can further mediate antibodydependent cell killing (ADCC) by NK (natural killer) cells. Human phagocytic cells also express an FcR for IgA, termed CD89, which may cause cell activation and enhanced phagocytosis of IgA-opsonized bacteria in mucosal tissues. CD89 is likewise associated with the common g chain. However, mice do not express CD89, so that murine IgA cannot mediate protection through this pathway. This presents a significant mystery—IgA does not activate complement nor does it appear to enhance phagocytic uptake of bacteria, so how exactly does IgA mediate protection of the host in mucosal secretions? Invoking the term “microbial neutralization,” which is often ascribed to IgA’s effector function, is a fairly unsatisfactory term since it does not imply actual removal of the invader from the body. Clearly, more remains to be learned about the protective function of IgA. Of interest, important clues about the role of another mysterious antibody molecule, IgD, have recently emerged. IgD is expressed on the earliest mature B cell as an antigen co-receptor, together with IgM. However, there is little detectable IgD in the bloodstream and mice with a genetic defect in IgD production created through transgenic technology appear to be fully normal. Thus, IgD has been thought to be completely dispensable for host protection. However, Cerutti and colleagues (Chen et al., 2009) have recently discovered that human IgD is secreted into the upper respiratory mucosa and binds to bacteria that are present at this site. Circulating IgD was found by these investigators to bind to basophils through a calcium mobilizing receptor and cross-linking of IgD on basophils activated antimicrobial, proinflammatory, and B cell-stimulating programs. The authors concluded that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation at mucosal sites.

SYNERGY BETWEEN HUMORAL AND CELL-MEDIATED IMMUNITY

Results with various bacterial pathogens have shown protection by antibodies but also a significant contribution by T cells. Recent data (discussed above) have demonstrated


a role for protection against pneumococcal infection by Th17 cells, which mediate increased recruitment of neutrophils. In addition, it has also been shown that IFN-g (interferon-g) plays an important role in several bacterial infections. During lung infection with pneumococci, exogenous IL-12 treatment can stimulate IFN-g production, which induces TNF-a expression, and this, in turn, aides in recruitment of neutrophils that ultimately enhances survival (Sun et al., 2007). Similarly, protection against Y. pestis has been found to be dependent on IFN-g, TNF-a, and iNOS, and interactions between cell-mediated and humoral immunity (Parent et al., 2005, Smiley, 2008). In the case of Salmonella and Listeria, cell-mediated immunity plays a critical role in antibody-dependent bacterial clearance (Casadevall & Pirofski, 2006), and with F. tularensis, a similar interaction was found to be necessary for antibody-mediated protection (Kirimanjeswara et al., 2007). Thus, cell-mediated immunity can augment bacterial clearance but the specific mechanisms by which this is achieved are only beginning to be elucidated. With attenuated strains of F. tularensis, it has been shown that specific antibodies actually facilitate bacterial uptake by macrophages and enhance bacterial replication within the cells (Kirimanjeswara et al., 2008). However, when macrophages are first activated with IFN-g, such antibodyopsonized bacteria are rapidly killed. Without opsonization, IFN-g activated macrophages have only a bacteriostatic effect. Thus, antibody and IFN-g, a component of cell-

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mediated immunity, act synergistically in bacterial killing by macrophages. In vivo, antibodies are effective in CD42/2 or CD82/2 mice, suggesting that the presence of either the CD4 or CD8 T-cell population is sufficient for antibody-mediated protection against F. tularensis. Interestingly, antibodies are not effective against the highly virulent SchuS4 strain of F. tularensis. As few as 10 colony-forming units of F. tularensis SchuS4 can severely suppress host inflammatory responses in the lung for at least 72 to 96 hours after infection. During this time, bacteria escape from the lung environment into peripheral organs such as the spleen and liver. By hour 96, the bacterial burden in these organs reaches 108/organ, which appears to be a lethal level. Although IFN-g can be detected in the lungs 96 hours after infection, it is likely too late to allow antibodies to have any effect. Almost identical effects are seen after infection of mice with virulent strains of Y. pestis, which, like F. tularensis, is believed to express a relatively noninflammatory LPS on its cell membrane. Thus, one may predict that if inflammatory responses could be up regulated early during the infection, this would allow adoptively transferred antibodies to mediate bacterial clearance and the host to survive infection. In fact, our own, unpublished data have demonstrated that IFN-g-activated macrophages are fully capable of in vitro killing of antibody-opsonized F. tularensis SchuS4. In conclusion, it appears that for many, if not all acute bacterial infections, synergy between humoral and cellmediated responses is critically required for inducing effective acquired protection (Fig. 1).

FIGURE 1 Synergy between humoral and cell-mediated immunity. Antibodies in complex with target bacteria bind to Fc receptors on macrophages. However, for effective bacterial killing, the macrophages must be first activated by IFN-g that is produced by bacteria-specific Th1 cells. In the absence of macrophage activation, antibody may actually enhance infection with bacteria that can replicate within macrophages, such as F. tularensis and Y. pestis. Th17 cells also increase protection by recruitment of neutrophils and other innate effector cells to the infection site.

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BACTERIAL TARGETS FOR INDUCTION OF ACQUIRED IMMUNITY

Bacterial capsules are very effective virulence factors that allow encapsulated bacteria such as S. pneumoniae to inhibit phagocytosis. Pneumococci further evade protective humoral immunity by changing their capsule subtypes such that over 90 capsular polysaccharide serotypes are now described. Other bacterial species such as Neisseria meninigitis and Haemophilus influenzae similarly contain multiple capsule subtypes, although variability occurs to a lesser extent with these pathogens. Nevertheless, the ability to change capsule serotype represents a major obstacle to development of effective vaccines for induction of universal, protective acquired immunity. The approach currently taken is to target those capsule serotypes that are most prevalent, at least in developed countries. However, the medical community is increasingly observing “serotype replacement” (i.e., an increasing incidence of infections by bacteria with less common capsular serotypes not included in the vaccine). An additional problem with polysaccharide vaccines is that they consist of T-independent antigens that do not stimulate sufficient T-cell help and, thus, only weakly induce B-cell isotype switching, affinity maturation, and, most importantly, memory development (Mond et al., 1995). Furthermore, humans do not develop antibody responses to polysaccharides until around 2 years of age (Siegrist & Aspinall, 2009). The reason for this delay in responsiveness

remains unclear despite significant amounts of research in animal models over several years; it results in leaving infants particularly susceptible to infections by encapsulated bacteria, which cause recurrent episodes of otitis media that can progress to pneumonia, meningitis, and sepsis. In an attempt to overcome this problem, Robbins and colleagues (Schneerson et al., 1980) took advantage of principles developed in the 1960s and 1970s by investigators such Mitchison (1971), who performed experiments using mouse models of immunization with hapten-carrier antigens. In these early studies, it was found that antibody responses to small molecules (haptens) could only be effectively induced when they were covalently coupled to larger protein antigens (carriers). The antibodies produced could then bind to the free hapten molecules, demonstrating that the haptens were antigenic, but by themselves not immunogenic. This concept was exploited to initially develop protective vaccines against the capsular polysaccharide of H. influenzae, type b (Hib), a common cause of childhood infections. The isolated polysaccharide antigen was covalently attached to a protein carrier such as tetanus toxoid, diphtheria toxoid, or Haemophilus outer membrane protein. The same principle was subsequently applied to the now standard pneumococcal and meningococcal conjugate vaccines, and is being developed for other encapsulated bacteria. The basis for the ability of polysaccharide conjugate vaccines to induce effective antibody responses is illustrated in Fig. 2.

FIGURE 2 Induction of IgG and IgA polysaccharide-specific antibodies by conjugate vaccines. A vaccine containing bacterial capsular polysaccharide (PS) covalently attached to a carrier protein (CRM) binds to PS-specific B cells through surface immunoglobulin receptors. The complexes are then internalized and the CRM carrier molecule is degraded into peptide fragments within B-cell lysosomes. These processed peptides are then bound to MHC class II molecules and presented to T cells. The T cells become activated and generate signals to the B cells to initiate immunoglobulin class switching. The result is production of PS-specific IgG and IgA antibodies as well as enhanced B-cell memory.


The advent of polysaccharide protein conjugate vaccines has led to significantly improved protection against encapsulated bacteria such as Hib and S. pneumoniae in both children and adults. Notably, these vaccines have substantially reduced the incidence of invasive disease and, thereby, rates of death from these bacteria. However, as stated above, the polysaccharide conjugate vaccine approaches are still subject to serotype replacement and this is becoming increasingly observed, particularly with the pneumococcal vaccines. In addition, since most pathogens enter the body at mucosal sites, it would be ideal to target vaccines to these “first responders,” but most vaccines including the polysaccharide conjugate vaccines are given intramuscularly, a route that induces systemic immunity (and hence, protects against systemic disease) but is relatively less effective at inducing protection at mucosal sites such as the respiratory tract. Investigations into developing mucosal vaccination procedures are discussed below. Another approach that is of great interest is to employ outer membrane proteins as vaccine targets. For pneumococci, there are several potential targets, including pneumococcal surface protein A (PspA), PspC, autolysin, and pneumolysin. Similar vaccine candidates have been identified for various other bacterial pathogens. The advantages of such proteins as vaccine targets include the fact that they are shared among various serotypes, thus overcoming the problem of limited serotype coverage and potential serotype replacement. In addition, such vaccines should be less expensive to produce since they

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are protein antigens that do not require chemical conjugation and are likely to be highly immunogenic, including in young children, thus capable of inducing effective immune memory. However, bacterial outer membrane proteins can also change their epitope structures through antigenic shift, such as is currently being observed with Bordetella. Further advances in the area are expected as clinical trials are ongoing.

MUCOSAL ADJUVANTS FOR INDUCTION OF ANTIBACTERIAL-ACQUIRED IMMUNITY

As stated above, nearly all pathogens enter the body through mucosal tissues, yet induction of acquired immunity against bacteria in humans is routinely achieved by parenteral vaccination, which is typically not an ideal route for induction of mucosal immune responses. Thus, there is a continuing interest in developing procedures for direct mucosal vaccination. However, mucosal tissues are designed not to mount immune responses to inhaled or ingested particles, therefore, there is a requirement for adjuvants to overcome this “tolerance” barrier. Several adjuvants have been tested in animal models, including bacterial toxins, TLR agonists, and immune modulators (Wilson-Welder et al., 2009). Bacterial enterotoxins such as cholera toxin have been found to be particularly potent mucosal vaccine adjuvants in animals (Liang et al., 1989; Wu & Russell, 1993). Unfortunately, these molecules can be highly toxic to humans

FIGURE 3 A model for polymicrobial synergy between influenza virus and S. pneumoniae in the lung. During recovery from influenza, approximately 1 week after initial infection, virus-specific T cells are recruited into the pulmonary tract. These T cells secrete IFN-g, which causes alveolar macrophages to express greater levels of MHC molecules, and also down regulates expression of certain scavenger receptors such as MARCO. The result is that adaptive immunity to the influenza virus is increased through more efficient antigen presentation. However, decreased expression of MARCO causes reduced recognition of nonopsonized pneumococci, leaving the host severely susceptible to secondary bacterial infection.

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and even variant molecules that have been engineered to induce less toxicity can still cause significant neurological sequelae (Couch, 2004). Thus, several laboratories are investigating other approaches to achieve protective acquired immunity in mucosal tissues, including incorporation of vaccine candidates with nanospheres, CpG, or cytokines. With the regard to the latter, IL-12 is a particularly attractive molecule that is a strong inducer of IFN-g expression (Metzger, 2009) and, thus, can mediate enhanced synergy between humoral and cell-mediated immunity, which, as illustrated above, is critical for optimal protection against bacterial pathogens (Fig. 1).

DETRIMENTAL EFFECTS OF ACQUIRED IMMUNITY ON ACUTE BACTERIAL INFECTIONS

The focus of this chapter has been on the protective effects of acquired immunity. However, in some instances, acquired immunity can actually be detrimental. This is clearly seen with immediate hypersensitivity reactions but can also occur during microbial infections. Specifically, increased occurrence of acute bacterial infections following viral infections is a well-known clinical problem. Such co-infections are often seen with influenza virus and S. pneumoniae, the two pathogens that cause the majority of the respiratory infections in humans. Although influenza infection alone may cause pneumonia, secondary bacterial pneumonia, which leads to excess morbidity and mortality during typical influenza pandemics including the major pandemic of 1918– 1919, is a common cause of severe disease (McCullers, 2006; Stiver, 2004). Indeed, recent retrospective studies by Fauci and colleagues (Kuiken & Taubenberger, 2008; Morens et al., 2008) of published autopsy case reports, found that over 90% of deaths during the 1918 influenza pandemic likely resulted from secondary bacterial pneumonia. Similarly, most deaths in the 1957 influenza pandemic were due to secondary bacterial pneumonia, although the number of negative autopsy lung cultures was somewhat higher compared to 1918 cultures, probably due to the availability of antibiotics (Louria et al., 1959). In one study, 75% of confirmed fatal cases of influenza had bacteriological and histological evidence of bacterial pneumonia, mainly due to S. aureus or S. pneumoniae (Louria et al., 1959). The remaining 25% of fatal cases appeared to be caused directly by influenza viral pneumonia. The reason for such increased susceptibility to secondary bacterial infection following influenza has remained unknown for several years, although recent findings indicate that it is due to the host antiviral adaptive immune response (Sun & Metzger, 2008). Specifically, IFN-g produced by influenza-specific T cells that are recruited into the lung following viral infection has been found to down regulate expression of the MARCO scavenger receptor on alveolar macrophages that is critical for recognition and clearance of nonopsonized pneumococci. In essence, by focusing on generating protective adaptive immunity against an ongoing viral infection, the host becomes temporarily susceptible to bacterial infections (Fig. 3). The fact that secondary bacterial infections are responsible for a significant proportion of deaths during influenza pandemics has led to a recent call for stockpiling pneumococcal conjugate vaccines as a part of a plan for influenza preparedness (Klugman & Madhi, 2007). However, if the increased susceptibility to secondary infection is indeed due to defective innate lung antibacterial immunity, it is not clear whether preexisting acquired immunity will overcome this defect, a question that is currently being investigated.

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22 Acquired Immunity: Chronic Bacterial Infections ANDREA M. COOPER AND RICHARD ROBINSON

INTRODUCTION

of the immune response and also, of course, be better able to manage disease caused by chronic bacterial infection and chronic inflammatory diseases in general. In this chapter we will consider the acquired cellular response to bacterial challenge with a focus on members of the genus Mycobacterium. In some cases, chronic bacterial infection results from immunodeficiency, whereas in other cases the host generates a normal immune response but the pathogen has the means to either circumvent or survive the most potent antibacterial mechanisms mounted. In either case, determination of the mechanisms mediating survival of both host and pathogen will highlight the pathways by which the immune response in induced, expressed, and regulated under a variety of conditions.

Discussion of immunity to infectious disease most often invokes a model wherein the rapid success of the immune response destroys the pathogen; in the converse of this model, if the pathogen is not controlled then death ensues. In the successful immune response, the pathogen grows, is recognized by the innate response, acquired specific immunity is induced, and the pathogen is eliminated, which is followed by the contraction of the immune response and the establishment of acquired specific memory populations capable of responding rapidly to a subsequent infection (Fig. 1A). In this chapter, we will discuss what happens when the immune response is capable of controlling a bacterial pathogen but not eliminating it. In this case, acquired immunity is defined not as the response capable of eliminating the pathogen but the response that allows survival of the host. In the case of chronic infection, the presence of the pathogen can continue to stimulate the innate and acquired response and thereby drive activity in the effector arm of the immune response (Fig. 1B). This constant stimulation may result in one of several levels of acquired response: continued high-level activity (curve A), fluctuating activity depending upon bacterial burden (curve B), or low-level activity (curve C). Regardless of activity level, the constant stimulation of the response provides a unique challenge to the host, as a balance between control of the pathogen and control of the pathogenic effects of the immune response must be maintained. In addition, the persistence of the pathogen continuing to stimulate the effector response could lead to exhaustion (curve D). The presence of ongoing infection and inflammation is also likely to severely interfere with the generation of acquired specific memory (Fig. 1B). The continued interaction between the pathogen and the host can result in several important consequences: sustained inflammatory responses; disrupted tissue architecture; altered antigen-presentation; and exhaustion of the acquired cellular population and the need for extensive regulatory pathways that may limit protection. It is by determining the mechanisms by which both the host and the pathogen manage this chronic interaction that we will learn more about the consequences of chronic stimulation

WHY DOES CHRONIC INFECTION OCCUR?

Bacteria evolve under the pressure of natural selection and it is likely that inadvertent invasion in a vertebrate host followed by fortuitous expression of genes that promote growth within the host allowed the earliest pathogens to become established. What then selects for chronic infection? One issue that limits the success of pathogens is their ability to spread to other hosts. In pathogens that cause rapid death of the host, spread of the disease is limited due to the reduced movement of the infected host through the population. In contrast, pathogens that do not cause rapid death can more easily spread throughout populations as they are exposed to more potential hosts. While both lifestyles are utilized by pathogens, one genus of bacteria has occupied the “chronic” niche very successfully; these are members of the Mycobacterium genus with the master being Mycobacterium tuberculosis, which is thought to have infected approximately one-third of the world’s population (Dye, 2006). How then does an acute or chronic lifestyle result in success of the pathogen? We can compare the acutely infectious Y. pestis and the chronic M. tuberculosis. Y. pestis passages between fleas and humans as well as between humans, and thus spreads either by infecting other humans immediately by the pneumonic route or by infecting fleas by inoculation through blood feeds. The second route of transmission means that spread can occur regardless of the speed of death in the human host. In this way, the acutely infectious Y. pestis can reach N generations rapidly within the host and still spread efficiently (Fig. 2). In contrast, the pathogen M. tuberculosis

Andrea M. Cooper and Richard Robinson, Trudeau Institute, Inc, 154 Algonquin Ave., Saranac Lake, NY.

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acquired iMMuNiTY TO MicrOBiaL iNFecTiONS

FIGURE 2 Chronic infection allows for increased transmission. A bacterial pathogen with multiple transmission strategies such as Y. pestis can afford to grow rapidly and kill one host (the human) as the alternative hosts provide a reservoir and the possibility of further spread. In contrast, in the absence of an alternative vector for transmission, it is advantageous for bacteria to persist within a host and thereby maximize the exposure of the infected host to target hosts (e.g., M. tuberculosis).

FIGURE 1 Chronic bacterial infection influences the host immune response. When the acquired cellular response of the host rapidly eliminates an invading bacterial population, it rapidly contracts and a memory population capable of rapidly responding to reinfection is generated (a). In a chronic infection (b), there are several potential outcomes for the host response that depend upon bacterial burden, the level of immunosuppression from the bacteria, the extent of immunoregulation, and the inflammatory environment. The response could maintain a high level (long dashed line, A), fluctuate in response to bacterial number (dot/dash line, B), contract to a level capable of controlling bacterial growth (short dash, C), or become depleted as the bacterial infection persists (short to long dash, D). In tuberculosis, it appears that curve C is the pattern of host response; however, curve D may also apply as the infected host ages.

is usually transmitted only by the aerosol route from human to human and must therefore depend upon extensive exposure to other humans in order to maintain consistent dispersal and maintenance within any population. To achieve this exposure level, the long-term survival of the host combined with sufficient tissue damage in the lung to promote transmission is important. One way for M. tuberculosis to achieve this would be by replicating slowly to avoid killing the host and by modulating the host immune response to generate the kind of damage to the lung that will result in an infectious cough. In this way, a chronic bacterial infection results in the same number of generations as in the acute model while still allowing for extensive transmission to occur. The relative incidence of M. tuberculosis and Y. pestis associated disease in the world suggests that the chronic model is a very successful option for a pathogen.

Whether there has been common evolution between the human host and M. tuberculosis is beyond the scope of this chapter but it appears that the human populations with the longest exposure to M. tuberculosis exhibit the classic chronic infection profile whereas naïve isolated populations exhibit a disease profile more akin to an acute infection with rapid growth of the bacteria and limited immune control (Hurtado et al., 2003). These data suggest that chronicity of infection is selected for in the human population as a preferable outcome compared to rapid early death. The nature of the inflammatory response to M. tuberculosis in the absence of acquired immune response suggests that the pathogen makes use of the acquired response to generate the damage required for transmission. Specifically, in the absence of CD4 T-cell immunity, humans develop granulocytic lesions that do not develop into the classical lesions thought to promote transmission (de Noronha et al., 2008). It is interesting to postulate that coevolution of the bacteria and host in this disease has resulted in a chronic disease with improved transmission as a result of immune responses. Indeed, if tuberculosis were not such a devastating disease, one could almost say that the interaction between humans and M. tuberculosis was developing into a commensal one.

IMMUNE RESPONSES TO CHRONIC BACTERIAL PATHOGENS

Extensive discussion of the innate response to bacteria is undertaken elsewhere in this book and our focus here is on the elements of the acquired immune response that mediate both immunity (i.e., control of the bacterial growth) as well as the immunopathologic consequences that are often concomitant with expression of immunity to chronic bacterial pathogens. While innate responses can control many infections and any compromise in the innate response is highly detrimental to the survival of the host, the acquired response is essential to

22. acquired immunity: chronic Bacterial infections

expand and regulate the effector response when immediate control of an invading pathogen is not achieved. The other crucial element of the acquired response is that it has the capacity to generate specific memory and thereby provide rapid and effective protection against rechallenge. All three of these functions (expansion, regulation, and memory) are affected by persistence of the chronic bacterial pathogen. The acquired response generates the specificity required to target the effector arm of the immune response through recombination of a limited number of genes to generate Tand B-cell receptors as well as somatic hypermutation to generate highly specific antibody. These specific receptors are capable of recognizing both self- and nonself antigen, however, the majority of these antigens are not dangerous. It is therefore necessary for specific induction and regulation pathways of the acquired response to exist. The induction of the specific response is initiated by pattern recognition receptors on surveillance cells that recognize classes of molecules associated with pathogens or abnormal tissue. These receptors recognize not specific molecules, but patterns associated with nonhost macromolecules or host molecules only freely available when an abnormal situation has occurred within the host. A crucial element of this response is that it is flexible such that when a mannose-expressing molecule from M. tuberculosis binds the C-type lectin DC-SIGN on dendritic cells, it is immunostimulatory, but when a fucose-expressing ligand from Helicobacter pylori binds DC-SIGN, the effect is immunosuppressive (Gringhuis et al., 2009) (Fig. 3). When the response to ligation of the pattern recognition receptors is stimulatory, the surveillance cells can migrate, produce cytokine, express novel surface molecules, and present antigen to T cells within the confines of the draining lymph

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node. Upon stimulation, these T cells can acquire many functions including direct antipathogen activity, activation of infected cells, mediating the death of infected cells, and promoting the expansion and increased specificity of the B-cell-derived antibody response (Fig. 3). Thus, while the innate response recognizes the dangerous nature of the invasion event via the pattern recognition receptors, it is the acquired response that expands, regulates, and remembers the most effective effector mechanisms capable of mediating immunity against the invading organism. In addition to the nature of the pathogen, it is the location of the pathogen within the host that defines the type of immune response induced. Indeed, bacterial pathogens that cause chronic infection can occupy many niches within the host and these various niches require distinct effector functions in order to clear or control the bacteria. The niche is defined by the specific organ and the location of the bacteria within that organ; thus, while H. pylori inhabits the mucoid lining of the stomach in close proximity to the epithelial cells (Blaser & Atherton, 2004), M. tuberculosis initiates an inflammatory response and lives within the recruited phagocytes within the lung (Cooper, 2009). The niche necessarily defines the acquired response required to control the pathogen such that intracellular bacteria require T-cell immunity whereas extracellular bacteria require the exquisite selectivity of antibody responses in order to be targeted and attacked by innate responses. Bacteria can change the niche that they occupy. For example, M. ulcerans begins as an intracellular pathogen—as do most other pathogenic members of the Mycobacterium genus—but becomes extracellular as disease progresses (Demangel et al., 2009; Portaels et al., 2009; Silva et al., 2009).

FIGURE 3 The nature of acquired immune response to chronic bacterial infection. The nature and extent of the acquired cellular response to chronic bacterial infection depend upon a variety of factors. The nature of the bacterial stimulus will determine the initial and extended activation of the acquired response by determining the levels of APC activation and inflammatory cytokines. The ability of the bacteria to affect the architecture and function of the lymph node as well as the site of infection greatly influences the continued activation of the acquired specific responses as well as defines the ability of the protective cellular response to be expressed.

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How then does the chronic presence of a bacterial pathogen influence these functions of the acquired immune response? The answer to this question may lie in the level of stimulatory activity driving the innate response and thus the level of support for the continued expression of acquired specific effector function. A bacterium that is continuously growing and releasing molecules that stimulate the pattern recognition receptors of the innate response will be highly inflammatory and will require strong regulatory activity by the host in order to limit collateral damage to tissue; it is also likely that the memory response will be poorly induced. In contrast, a bacterium that is not growing and/or does not release foreign stimulatory molecules will require limited regulation of the effector immune response and may allow for a memory response to become established (Fig. 3).

T-CELL IMMUNITY TO CHRONIC PATHOGENS

As discussed above, acquired specific cellular immune responses act to augment and expand the killing mechanisms expressed by the components of the innate immune system. These mechanisms are numerous and diverse and are tailored to the nature of the challenge. It is the specificity of the antigen-specific effector cells (both B and T cells) that provides the targeting of immunity, however it is the variety of T-cell phenotypes that determines the flexibility of the effector arm of immunity and which underpins its ability to deal with chronically persisting bacteria. In the past, basic studies of immunological responses to inert antigens served to define the basic nature of acquired cellular immune responses and indeed the simplicity of these responses was crucial in allowing mechanisms to be dissected. In contrast, more recent studies, made possible by the development of tools capable of dissecting the nature of the T-cell response during an infection, have highlighted the truly complex nature of acquired immunity. In this respect, study of the immune response to chronic infections has played, and will continue to play, a major role. Indeed, the classical description of functional subsets of T helper 1 (Th1) and T helper 2 (Th2) cells capable of playing a decisive role in the outcome of infection came from the study of chronic infection of BALB/c mice with Leishmania major (Mossman & Coffman, 1989). Over the last 20 years, while the Th1/Th2 T-cell populations have become paradigmatic, more sophisticated tools and a greater number of immunologists choosing infection models for their studies have resulted in the demonstration that there are a range of phenotypic outcomes for T-helper cells and that these outcomes do not always represent an end stage differentiation (Zhou et al., 2009). Importantly, the epigenetic modifications that allow for the flexibility of antigen-specific T cell subsets has been more completely defined (Wei et al., 2009). In addition, while T cells can follow a defined pathway that promotes a specific phenotype and function, during chronic infection, the establishment of a predominant subset is not the usual outcome. The flexibility of the acquired cellular response is not surprising as the interaction between host and pathogen alters over time and the host must be able to respond to these changes. Thus, as a pathogen enters the host, it may exist as an intracellular entity and thereby require effector T cells that recognize and lyse infected cells or perhaps effectors that are capable of initiating or expanding the killing mechanisms of the infected cell. A chronic pathogen will have some mechanisms whereby it overcomes these initial killing activities or may become extracellular. Once

the bacteria become extracellular, other control pathways must be initiated. For instance, a pathogen within a host cell that is unable to kill the bacterium can be exposed to attack by causing the host cell to lyse. This lysis of host cells can result in tissue damage such as is seen in tuberculosis, wherein cavitary lesions occur. In this case, one may imagine that sealing off the damaged tissue would be an important activity of the acquired response and indeed effector cells that mediate accumulation of mononuclear cells which surround the lysed cells (i.e., granuloma formation) are crucial to limiting the pathogenesis of some chronic infections. Extracellular bacteria may be targeted by effector cells that can directly lyse the bacteria or which bring in innate cells such as granulocytes capable of lysing, damaging, or expelling pathogens that have been targeted by specific antibody. The two niches described above (intracellular and extracellular) are associated with the Th1 and Th2 subsets respectively, with Th1 mediating host cell activation and granuloma formation, whereas the Th2 subset mediates damage and expulsion of extracellular parasites.

T-CELL PHENOTYPES

Antigen-specific T cells exhibit a range of functions which affect the ability of bacteria to persist in vivo. One of the most commonly measured activities of these T cells is the release of cytokines, which allow for short-range communication between the T cell and other host cells. The production of IFN-g (interferon-g) is a crucial effector function of T cells specific for pathogens that undertake an intracellular lifestyle. This IFN-g, when directed to an infected cell, allows the host cell to alter its gene-expression and its phenotype. Specifically, macrophages that have taken up bacteria but have not become activated fail to control the growth of the bacteria and therefore provide a privileged site for replication. Such uptake can occur by the use of specific receptors that prompt phagocytosis without prompting initiation of activation at the same time. In contrast, macrophages that receive a signal from IFN-g recognize the phagocytic event as potentially dangerous and initiate bactericidal or bacteristatic activities. This activated phenotype is typified by maturation of the phagosome to phagolysosome, generation of toxic oxygen and nitrogen radicals, and the removal of the phagosome from the nutrient pathways of the host cell. In addition to activating phagocytes, IFN-g can also induce expression of chemokines, which are small molecules that serve to recruit and organize cells of the immune system to work most efficiently. IFN-g promotes a mononuclear influx of cells and can override the induction of neutrophilic inflammation mediated by T-helper cells producing the inflammatory cytokine IL-17 (Cruz et al., 2006). IFN-g also acts to drive B cell class-switching and, thereby, the production of IgG2a and IgG3. Other cytokines that are induced during chronic infection are IL-4 and IL-10, which can down regulate the IFN-g response by limiting the expression of the master regulator of Th1 induction (Tbet) or by limiting the production of the Th1-supporting cytokine IL-12 by the antigen-presenting cells (APC). The expression of IL-4 by T cells is dependent upon the master regulator GATA-3 and this, in turn, is counter-regulated by IFN-g. This type of counter-regulation between effector T-cell subsets by cytokine action on transcription factor expression appears to be a general mechanism whereby the flexibility of the immune response is maintained over time (Zhou et al., 2009). This type of crossregulation is particularly important in chronic infections where establishment of a predominant T-cell phenotype


may serve to limit the flexibility of the response. It is likely that this type of flexibility is crucial for the survival of the host when a pathogen cannot be eradicated.

PROBLEMS WITH MEMORY IN THE PRESENCE OF INFECTION

The impact of ongoing infection on the establishment of memory populations is not yet defined. As discussed above, the level of immune activation may well determine the extent to which antigen-specific cells are able to establish a long-lived memory population (Fig. 3), however, this has yet to be shown. There are two simple models that may describe what happens to memory cell populations during chronic infection. The first is that there is a range of cell fates initiated at activation such that the extent of activation signal defines whether a cell becomes a full-fledged effector or goes directly to a resting effector cell capable of becoming a memory cell. Alternatively, cells may be activated similarly, but as the cell divides, the fate of the cell is decided such that one becomes a short-lived effector while the other becomes a memory cell (Fig. 3). In the first case, chronic infection may interfere with memory cell population development, as there would be continued high levels of activation that would preclude establishment of resting cells capable of becoming memory cells. In the second case, a memory cell population would continue to be established as long as cell division occurred and thus chronic infection would serve to drive memory as well as effector populations. In both cases, the environment where the cell is induced and recruited will also determine the size of the memory population as a function of the cell’s ability to respond and survive within an inflammatory site (Fig. 3).

THE IMPACT OF CHRONIC INFECTION ON ACQUIRED CELLULAR RESPONSES

There are two main areas where chronic infection with bacteria can affect acquired cellular immunity. The first is in induction of new antigen-specific cells over time and the second is the expression of function of these cells. In both cases, the presence of bacteria alters the environment within which induction and expression of function occur. Induction of antigen-specific cells depends upon the ability of specific cell types to interact and this depends upon the correct expression of specific molecules both in time and space. Thus, while there is antigen, antigen presenting cells, and likely some naïve antigen-specific cells within any peripheral tissue during infection, the ability of these cells to interact is greatly reduced by the fact that they may not be colocalized in time and space. This is where the lymphoid system provides a location, wherein the interaction between specific cell types and antigen is increased, allowing for selective induction of antigen-specific immunity. Often during chronic bacterial infection and particularly with members of the genus Mycobacterium, the lymph node becomes infected and inflamed, which then has the potential to alter the interaction between cells; this has been shown for viral infections (Mueller et al., 2007a, 2007b). During chronic tuberculosis, antigen-specific cells are newly induced but at a reduced rate compared to the initial expansion of cells (Winslow et al., 2003). The cause of this reduction may be ascribed to active suppression of immunity by M. tuberculosis but could, at its simplest, be a result of the disruption of the node structure and interference with the normally nurturing environment of the node. As bacteria are limited by antibiotic treatment, the level of antigen-specific cellular responses increases in tuberculosis patients and whether this is due to improved node structure and function is not yet known.

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In addition to an effect on the stroma of the node, the presence of M. tuberculosis within the lymph node will result in an increased accumulation of inflammatory macrophages, thereby altering the levels of cytokines such as IL-12, IL-23, and IL-10, as well as the expression of the homeostatic chemokines that regulate the structure and function of the lymph node. The expression of specific cytokines is required for the generation of specific effector function among newly activated cells and it is these cytokines that can alter the phenotype of the response. For example, in tuberculosis, members of the IL-12 family of cytokines have been implicated in protective immune responses and, indeed, expression of IL-12p70 throughout chronic infection is required to maintain control of bacterial growth. Specifically, even if a protective type 1 response is induced and expressed, this response fails to maintain protection if IL-12 is lost (Feng et al., 2005). Further, the production of IL-23, while required for expression of an optimal IL-17 response to M. tuberculosis, does not appear to be required for protection but in its absence, the nature of the chronic inflammatory response is altered (Khader & Cooper, 2008a). Thus, the relative levels of IL-12p70 and IL-23 within the infected lymph node is likely to have an impact on the induced effector function of T cells activate within that node. Despite infection of the draining lymph nodes, newly activated T cells are induced during chronic infection, albeit at a low number. These cells and indeed fully differentiated effector T cells must then accumulate within the lung or other infected site to mediate protection. During initial expression of immunity, the inflammatory site is simple in structure with a small number of newly recruited monocytederived phagocytes being associated with limited numbers of granulocytes and lymphocytes. As the inflammatory site develops, however, the size of the phagocyte areas increases and novel structures develop, which have the potential to interfere with granuloma integrity. In addition, as the phenotype of the antigen-specific effector cells may change as a function of infection of the lymph nodes, the nature of the inflammatory response may also alter. In the case of tuberculosis, there is a progressive development of nascent bronchus-associated lymphoid tissue (BALT) with large areas of B cells (Gonzalez-Juarrero et al., 2001). The function of these areas in the protective response is not known; however, B cells can affect protective immunity during high bacterial burden infections (Maglione & Chan, 2009). As the numbers of cells and the complexity of the granuloma increases, it becomes a more complex issue for the effector T cell to contact the infected host phagocyte. The ability of the effector cell to travel through the many uninfected inflammatory phagocytes is likely to be dependent upon the correct temporal and spatial expression of chemokines within the granuloma. While this pattern has not yet been fully defined, it appears that homeostatic chemokines important in B cell migration, such as CXCL13, may influence the ability of T cells to leave the immediate vicinity of the vessels from which they emerged and migrate within the granuloma (Khader et al., 2009). In addition to an increased complexity within the infection site resulting from the chronic nature of infection, the presence of other cells within the site can reduce the efficacy of protective cells. Thus, when large numbers of phagocytes are present within a lesion, they can greatly change the cytokine environment, as well as generate large amounts of toxic, tissue-damaging molecules. Thus, a preponderance of IFN-g-activated macrophages within a lesion will produce toxic radicals that will limit the viability of effector T cells within the lesion (Cooper et al., 2002). If the macrophages produce a lot of IL-10, this will down regulate the

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local cellular response and limit immune-mediated damage. If neutrophils are the dominant cell type, they have the capacity to create necrotic areas within tissue that are not accessible to effector T cells.

ACQUIRED CELLULAR IMMUNITY TO CHRONIC BACTERIAL INFECTION AS ILLUSTRATED BY THE GENUS MYCOBACTERIUM Mycobacterium tuberculosis

This very successful pathogen occupies a predominantly intracellular location within inflammatory phagocytes in the lung. It has also been shown to be within granulocytes and to exist in an extracellular location within the liquefied centers of lung lesions. During initial infection it is thought to migrate to the draining lymph node of the infection site (usually the aerosol exposed lung), whereupon it induces a significant level of effector T-cell activation. This induction has been shown clearly in a mouse model and is extrapolated to be the case in humans due to the extent of antigenspecific T-cell responses able to be measured in exposed and diseased individuals (Cooper, 2009). Using gene-deficient mice, the essential elements of bacterial control and survival of a M. tuberculosis induced death have been shown to be the ability to mount an antigen-specific, MHC class II restricted, T-cell response capable of macrophage activation via cytokine mediated instruction (North & Jung, 2004). In humans, the failure of HIV-infected individuals to successfully limit the progression of tuberculosis indicates that a similar requirement for antigen-specific immunity exists. Many individuals are exposed to M. tuberculosis and likely harbor bacteria throughout their lives but fail to develop any sign of disease (Dye, 2006). Their exposure is deduced from the fact that they express an antigen-specific response to M. tuberculosis antigens and the fact that these responses are extremely long lived. These exposed individuals do not have disease but could be considered to be chronic hosts of this pathogen. Why some people develop disease and others do not is beyond the scope of this chapter but it is at least in part due to dose and living conditions, as the incidence of disease is higher when people are closely confined and malnourished (Dye, 2006). In almost all vertebrate hosts, initial infection is followed by a period of bacterial growth, which occurs in the absence of acquired cellular immunity (Dannenberg & Collins, 2001). Once acquired immunity is expressed, bacterial growth ceases and the subsequent development of disease occurs without significant growth of bacteria. In the mouse model and in humans, this disease is predominantly seen in the lung and it is the expression of acquired specific cellular responses within this organ that is likely to define the nature of disease (North & Jung, 2004). While the lung is important in pathogenesis of disease, the expression of acquired immunity is, in fact, essential in other organs, as mice deficient in acquired immunity as well as humans with HIV exhibit a profound disseminated disease with multiple organ involvement (Cooper, 2009; North & Jung, 2004). One may conclude that the expression of acquired immunity in the nonlung tissue is required for survival while the expression of this activity within the lung is required for the immunopathological nature of the disease. The quality of the T-cell response in mice and humans can be determined by examination of ex vivo responses to antigen. The response most often measured is the production of the cytokine IFN-g; this is due to the requirement for this cytokine in control of bacterial growth. If IFN-g is

totally absent, then bacteria grow continually and a granulocytic lesion develops with rapid death of the host being the consequence. If IFN-g is present but there is no antigenspecific immunity, then bacterial growth is modestly limited compared to the absence of IFN-g, however this is short lived and bacteria overcome the host almost as rapidly as in the total absence of the cytokine. Together, these data suggest that targeted delivery of high levels of macrophageactivating cytokine is required to successfully control intracellular bacterial growth and to allow development of a chronic infection. Another macrophage-activating cytokine, TNF-a (tumor necrosis factor-a) is also essential for control of bacterial growth because, in its absence, bacterial growth is as rapid as in the absence of IFN-g. Again, there is a requirement for antigen-specific immunity as TNF-a is produced in the absence of MHC class II, but it is not sufficient to establish chronic infection. That macrophage activation is required for control of bacterial growth is demonstrated by the progressive growth of bacteria in mice lacking elements of macrophage function, such as the inducible nitric oxide synthase enzyme (North & Jung, 2004). Thus, in the absence of these elements of immunity, chronic infection does not occur. It is also clear, however, that these elements of immunity are insufficient to clear the bacteria. The chronic nature of M. tuberculosis infections suggests that there must be regulation of the acquired response in the lung such that while bacterial control is established, the protective response is limited in order to control tissue damage to this essential organ. Indeed, regulatory T cells are induced during the mouse model of tuberculosis and they appear to limit the acquired response to a modest degree (Scott-Browne et al., 2007). They are induced in an IFNg-dependent manner, suggesting that, during chronic infections it is the nature of the effector response that defines the nature of the regulatory response (Koch et al., 2009). In contrast to the paradigmatic Leishmania major model there is no strong Th2 response that counter-regulates the Th1 response during M. tuberculosis infection, as in the absence of the defining Th2 cytokine, IL-4, there is little alteration to the protective response in the mouse model (North & Jung, 2004). IL-10, a cytokine capable of limiting macrophage activation, can limit the protective response to M. tuberculosis to a modest degree if it is induced, however, it is not always present (Cooper, 2009). T cells capable of producing cytokines, such as IL-17, which recruits neutrophils and regulates their function, are also induced following M. tuberculosis infection both in mice and humans (Khader & Cooper, 2008b). These cells do not appear to be of significance during a primary infection but as disease becomes chronic or as antigen load increases, these cells are associated with increased granulocytic accumulation and increased pathology (Khader & Cooper, 2008a). There is a contraction of the effector T-cell population following the establishment of static bacterial burden such that the numbers of antigen-specific T cells capable of producing cytokine is reduced from the peak but is maintained above background levels (Winslow et al., 2003). Whether this reduction is due to altered node structure, a reduction in antigen availability, or a result of inhibition of antigen presentation is not yet clear, however, the fact that this reduction is not associated with increased bacterial burden suggests that the reduced number of antigen-specific cells is sufficient to maintain the control of bacterial growth. The delivery of exogenous antigen by means of repeated challenge with related bacteria at a distinct site does not result in improved control of bacterial growth nor does it result in increased numbers of protective IFN-g producing T cells, but rather results in an increase in the pathologic


response within the lung lesion (Cooper, 2009). It is plausible therefore that the contraction of the effector response may be required to limit the development of this pathologic response. This is an area that needs to be addressed in order to better identify and separate the protective response from the pathologic response. Once we have separated these responses, we will be better able to independently modulate them and thereby improve vaccination.

Mycobacterium ulcerans

Mycobacterium ulcerans lies third in the table of disease-causing agents and exhibits both similarities to and differences from the other members. While its mode of transmission is not yet fully defined, it is prevalent in children, is associated with water, and may be transmitted by insect bite (Silva et al., 2009); the pathogen itself is considered a descendent of M. marinum (Demangel et al., 2009). Unlike M. tuberculosis, M. ulcerans is a disease of the skin and bone and can be either self-limiting or can progress to a significant and devastating disease. The initial lesions caused by M. ulcerans are contained and not ulcerated, the bacterium is intracellular, and there is a significant T-cell response that looks like the response to other intracellular Mycobacteria (Silva et al., 2009). As disease progresses, bacteria are predominantly extracellular and cause a devastating necrotic disruption of the skin, with lesions that can become so large that they threaten the life of the host (Demangel et al., 2009). As the disease progresses, the acquired response in the form of cellular immunity is unlikely to have a significant impact within the necrotic tissue. It is important to note, however, that acquired cellular immunity as induced by bacille Calmette Guerin (BCG) vaccination appears to have a protective effect both in humans and in mouse models (Demangel et al., 2009; Silva et al., 2009). This suggests that either the intracellular stage is important in disease progression or that BCG induces some humoral response that is protective during the extracellular phase. Treatment involves dual therapy with rifampicin and streptomycin and surgical removal of the lesion. It has been proposed that M. ulcerans is immunosuppressive first as a result of its ability to limit the function of dendritic cells, and second due to the reported cytotoxic effect it has on host cells of many types (Demangel et al., 2009). The cytotoxicity effect is associated with the ability of the bacteria to generate a macrolide, called mycolactone, that is coded for by a virulence plasmid not seen in M. marinum (Demangel et al., 2009). The lysis of host cells by mycolactone is also thought to contribute to the development of the necrotic lesion, as M. ulcerans strains lacking this plasmid are not ulcerative. How then does the acquired response interact with this pathogen? Despite the inhibitory activity of M. ulcerans on dendritic cells, antigen-specific cellular and humoral responses can be detected in humans and mice (Demangel et al., 2009; Silva et al., 2009). That some element of the acquired response can be protective against this unusual pathogen is demonstrated by the ability of BCG vaccination to protect against disease (Silva et al., 2009). The ability of the pathogen to limit the acquired response is shown by several lines of evidence. Upon treatment with antibacterial agents the acquired immune response becomes more prevalent within the lesions, with lesions moving from the necrotic state to a strong mononuclear granulomatous response after 8 weeks of antibiotic treatment (Schütte et al., 2009). In this scenario, it would appear that as bacteria are killed or energetically limited by the antibiotic, the ability of the pathogen to interfere with the ability of the acquired response to contain the bacteria and the development of the necrotic lesion is reduced. In addition, the systemic inhibition of IFN-g production seen in this disease is resolved

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following removal of the lesional site by surgery (YeboahManu et al., 2006). Immunosuppression caused by M. ulcerans has also been suggested to depend upon the type of helper T-cell response induced by this pathogen; thus, while healthy exposed individuals exhibit a strong Th1 response to M. ulcerans in vitro, patient Th1 responses are reduced compared to the healthy controls (Gooding et al., 2001, 2002). One might think that the classical Th1/Th2 balance may be in effect here, and indeed in Australian populations an increase in Th2 cytokines is seen in affected individuals compared to controls (Gooding et al., 2002); however, this has not been seen in other populations (Westenbrink et al., 2005). Direct studies on the lesion have also demonstrated that higher Th1 responses are seen in the nodular lesion, whereas IL-10 is increased in the necrotic lesion (Prévot et al., 2004) and systemically in patients with necrotic lesions (Phillips et al., 2006). These data suggest that, as for M. tuberculosis, the induced cellular response is largely Th1 in nature and that any immunosuppression is not mediated by cross-regulation of this type 1 response by type 2 responses. The role of regulatory cells has not been extensively explored in this disease but it is possible that, as for M. tuberculosis infections, a population of IFN-g-dependent regulatory T cells may be induced. The relative importance of cytokine cross-regulation, regulatory T-cell control, and the active cytotoxic activity of the bacteria in regulating the acquired response to M. ulcerans has yet to be fully defined. The role of antibody in limiting M. ulcerans infection is not clear. Antigen-specific humoral responses are seen in the majority of patients and exposed individuals (Dobos et al., 2000; Gooding et al., 2001). Interestingly, although IgG antigen-specific responses do not allow discrimination between patients and exposed controls, the IgM response is highly specific and sensitive for detection of active disease (Okenu et al., 2004). Intriguingly, there is an association between the presence of IgG specific for salivary gland extracts of the proposed aquatic vector for M. ulcerans and the ability of exposed individuals to resist development of necrotic lesions (Marsollier et al., 2007). In other mycobacterial diseases, antibody and B cells are largely ignored as mediators of protective immunity, however, there is interesting evidence supporting a role for B cells and/or immunoglobulin in tuberculosis (Cooper 2009; Maglione & Chan, 2009). The potential role of B cells and antibody in pathogenesis of Buruli ulcer should receive more attention. In sum, M. ulcerans can induce humoral and cellular immunity but appears to limit the expression of this immunity by lysis of host cells. Whilst the bacterium is intracellular, it is likely that cellular immunity is able to limit the progression of disease, however, once extracellular it is not clear whether antibacterial activity by the host is protective. The role of antibody in this extracellular phase is not known, although the extracellular phase does allow for increased levels of IgM. As for the other mycobacterial diseases, IFN-g is associated with protective responses and vaccination provides modest protection against disease progression.

Mycobacterium leprae

Mycobacterium leprae occupies a unique intracellular niche during its chronic phase as it has a predilection for Schwann cells, the cells that surround and protect nerve axons. It gains entry to these cells as a result of laminin a2 providing a linker between specific bacterial molecules and the abdystroglycan on the basement membrane of Schwann cells. M. leprae causes a spectrum of disease, the nature of which is associated with a range of acquired cellular responses. Specifically, there is a multibacillary (lepromatous) disease, which consists of high numbers of disseminated bacteria and low

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acquired immunity, as well as paucibacillary (tuberculoid) disease wherein lesions have low numbers of bacteria but there is a strong acquired cellular response. A curious aspect of the multibacillary disease is the reversal reaction wherein acquired cellular responses improve and immune-mediated pathologic damage of the nerves occurs. Clearly there is a great deal of complexity in the Leprosy model and our understanding of the acquired cellular response is limited by the fact that there is no tractable animal model of the disease. In humans, a type 1 delayed type hypersensitivity response is associated with protection from bacterial growth but when a reversal reaction occurs, it is this type 1 hypersensitivity that appears to mediate the nerve damage as a bystander effect (Walker & Lockwood, 2008). So, as in tuberculosis, the generation of antibacterial activity is associated with potentially damaging immunopathology. Therefore, there is an advantage to both the host and the pathogen to limit the acquired immune response. The immunoregulatory pathway in leprosy is not yet defined; however, the genetic predisposition of people with specific alleles of the PARC2 and PACRG genes suggests that these E3 ligases play a role. Specifically, there is a variant in the promoter region shared by these genes that is associated with an increased risk of leprosy (Mira et al., 2004). These E3 ligases are part of the ubiquitination system and are associated with immunosuppression both in terms of reduced macrophage antibacterial activity and also as a mediator of T-cell anergy (Schurr et al., 2006). The specific mechanism, whereby these ligases augment the development of leprosy, is not yet clear. The role of feedback pathways has been analyzed in animal models, and it appears that when IFN-g is absent, the consequences to bacterial growth are minimal; however, the inflammatory response is greatly increased (Cooper et al., 2002). It appears that IFN-g acts to limit T-cell accumulation in mycobacterial disease by inducing apoptosis in the T cells (Li et al., 2007) and this activity is limited by the immunity-related GTPase lrgm1 (Feng et al., 2008). In addition to the E3 ligases, human genetic studies have also implicated lymphotoxin-a in susceptibility to this disease. Specifically, those harboring a low-producing mutant allele for lymphotoxin-alpha are more susceptible to early onset leprosy (Alcaïs et al., 2007). Mechanistic analysis of the function of the lymphotoxin-a in a mouse model suggests that the absence of this cytokine results in very reduced granulomatous response with few lymphocytes and a more rapid expansion of the bacterial burden over time (Hagge et al., 2009). It is possible that the absence of this cytokine leads to reduced T-cell activation, as it is involved in the coordinated interaction of cells of the immune system. Alternatively, its absence may reduce the ability of already activated T cells to accumulate within the granulomatous site, due to the failure to induce specific chemokines. The consequence of HIV infection on the development of leprosy is that, unlike in tuberculosis, there is little impact of HIV-induced immunodeficiency on the incidence of leprosy and indeed the granulomatous response and the clinical spectrum of the disease are unaltered in coinfected individuals (Ustianowski et al., 2006). It has been proposed that the difference between the impact of HIV on tuberculosis and leprosy lies in the nature of the T-cell response with the T cell in tuberculosis being more highly activated and therefore more susceptible to the detrimental effects of HIV infection (Ustianowski et al., 2006). Despite the limited impact of HIV on disease susceptibility, the increased availability of highly active antiretroviral therapy has resulted in immune reconstitution disease in leprosy in much the same way as it has in tuberculosis (Batista et al., 2008; Ustianowski et al., 2006).

All of the mycobacteria appear to induce an antigenspecific response in vivo. They all also appear to induce a level of immunosuppression and/or immunoregulation, which, while not significantly limiting the ability of the host to control the bacteria, does influence the type of inflammation at the lesional site.

CONCLUSIONS

The response of the host to chronic infection needs to be modulated to avoid long-term damage to the host and to be flexible in order to accommodate changing bacterial activity. Rapid induction of responses is essential to limiting the expansion of chronic bacteria. This rapid induction needs to be followed by the establishment of regulatory activity that limits the extent of the cellular response while also limiting damage to the host. Once chronic infection is established, the nature of the cellular response may have to change, as the bacterial niche may change. Determining the constituents of the acquired cellular response to chronic bacterial infection will allow for improved diagnostics, immunotherapeutics, and vaccines.

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23 Acquired Immunity: Fungal Infections LUIGINA ROMANI

INTRODUCTION

commensalism on human skin and body surfaces without necessarily causing disease (Romani, 2001). Despite the fact that human beings are constantly exposed to fungi, fungal diseases, though disparate in nature, are relatively rare. This indicates that fungi, capable of colonizing almost every niche within the human body, must possess particular adaptation mechanisms (Cooney & Klein, 2008; Hube, 2009) of coexistence, which deviates into overt disease under conditions of either too low or too high immune responses. The most common of the human diseases caused by fungi are the opportunistic fungal infections that occur in patients with defective immunity. Candida species remain the fourth most important cause of hospital-acquired bloodstream infections. Invasive aspergillosis, mostly caused by Aspergillus fumigatus and A. terreus, and other mold infections, are a leading cause of infection-related death in hematopoietic stem cell transplant recipients. Fungal diseases include type I hypersensitivity, the most prevalent disease caused by airborne fungi, and a large number of other illnesses, including allergic bronchopulmonary mycoses, allergic chronic sinusitis, hypersensitivity pneumonitis, and atopic eczema/ dermatitis syndrome (AEDS; formerly atopic dermatitis) (Levin, 2009). Sensitization to molds has been reported in patients with asthma and allergic bronchopulmonary aspergillosis is frequent in patients with asthma and cystic fibrosis (Denning, 2006). There is evidence that fungal sensitization also contributes to autoreactivity against self-antigens due to shared epitopes with homologous fungal allergens (Romani & Puccetti, 2008). Although Malassezia yeasts are a part of the normal skin microbiota, they have been associated with a number of diseases affecting the human skin, such as pityriasis versicolor, folliculitis, seborrheic dermatitis and dandruff, atopic AEDS, psoriasis, and—less commonly— with other dermatologic disorders (Levin, 2009). The complex relationships of fungi with the vertebrate immune system is partly due to some prominent features. Among these, beside genomic microvariation (Odds & Jacobsen, 2008) to adapt to environmental abiotic stress conditions (Cooney & Klein, 2008; Hube, 2009), the ability to reversibly switch from one form to the other in infection may have resulted in an expanded repertoire of cross-regulatory and overlapping antifungal host responses at different body sites. Examples are the thermally dimorphic fungi (H. capsulatum, P. brasiliensis, C. immitis, and

Although Th1 responses driven by the IL-12 (interleukin 12)/ IFN-g (interferon-g) axis are central to protection against fungi, the paradigm has been revisited with two new T cell populations entering the scene: the Th17 cells involved in inflammatory responses, and the T regulatory cells (Tregs), which down tune immune responses to avoid damage to the host. Some degree of inflammation is required for protection, particularly in mucosal tissues, during the transitional response occurring between the rapid innate and slower adaptive response. However, progressive inflammation worsens disease, limits protective antifungal immune responses, and ultimately prevents pathogen eradication. In this scenario, Th17 cells may play an inflammatory role previously attributed to uncontrolled Th1 responses, while the capacity of Tregs to inhibit aspects of innate and adaptive antifungal immunity is required to regulate the balance between tolerogenic and inflammatory responses. The enzyme indoleamine 2,3-dioxygenase (IDO) and tryptophan metabolites contribute to immune homeostasis by inducing Tregs and taming heightened inflammatory responses. The new findings support a view in which the immune system tailors protective responses to suit infecting fungi while limiting host damage through resistance and tolerance, two types of host defense mechanisms that contribute to the best fitness in response to fungi. Hopefully, targeting potentially harmful inflammation could be translated into new medical practices.

THE MULTIFACETED INTERACTION OF FUNGI WITH MAMMALIAN HOSTS

The kingdom of fungi comprises over 1. 5 million fungal species, 150 to 400 of which are associated with a wide spectrum of diseases in humans and animals. Most fungi (such as Histoplasma capsulatum, Paracoccidioides brasiliensis, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Pneumocystis jirovecii, and Sporothrix schenckii) are ubiquitous in the environment and probably have gained their pathogenic potential in environmental niches. Some, including Malassezia spp. and Candida albicans, establish lifelong Luigina Romani, Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy.

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B. dermatitidis), which transform from saprobic filamentous molds to unicellular yeasts in the host; the filamentous fungi (such as Aspergillus spp.) that, inhaled as unicellular conidia, may transform into a multicellular mycelium; and some species of Candida, capable of growing in different forms such as yeasts, blastospores, pseudohyphae, and hyphae (Fig. 1). Thus, in the context of the antagonistic relationships that characterize the host–pathogen interactions, the strategies used by the host to limit fungal infectivity are necessarily disparate; in retaliation, fungi have developed their own elaborate tactics to evade or modulate host defenses and to survive (Romani, 2004; Shoham & Levitz, 2005). However, because fungal diseases are rare, a stable host– parasite interaction is a likely condition for most, potentially pathogenic, fungi. This condition requires that the elicited immune response be strong enough to allow host survival

FIGURE 1

with or without pathogen elimination and to establish commensalism/persistency without excessive proinflammatory pathology. Therefore, the balance of proinflammatory and anti-inflammatory signaling is a prerequisite for successful host/fungal interactions and requires the coordinate actions of both innate and adaptive immune systems (Romani, 2004; Romani, 2008a; Romani & Puccetti, 2006a). This chapter attempts to position the new findings on acquired immunity and fungi within the conceptual framework of a two-component antifungal response that includes resistance (i.e., the ability to limit fungal burden) and tolerance (i.e., the ability to limit the host damage caused by either the immune response or other mechanisms). Evolutionarily conserved from plants to vertebrates (Schneider & Ayres, 2008), this new concept may help to define the best fitness in response to fungi and its integration into new medical practices.

The pathogenesis of typical fungal infections.

23. Acquired Immunity: Fungal Infections

RESISTANCE AND TOLERANCE TO FUNGI

Resistance and tolerance are two types of host defense mechanisms that increase fitness in response to fungi (Zelante et al., 2009a). In experimental candidiasis and aspergillosis, both defense mechanisms are activated through the delicate equilibrium between Th1 cells, which provide antifungal resistance mechanisms, and regulatory T cells (Tregs) limiting the consequences of the associated inflammatory pathology. Indeed, while some degree of inflammation is required for protection, particularly at mucosal tissues during the transitional response occurring between the rapid innate and slower adaptive response, progressive inflammation worsens disease and ultimately prevents pathogen eradication. The inflammatory response is initially mediated by cells of the innate immune system followed by a later adaptive immune response, which responds to the signals originated by the innate immune system. The decision of how to respond will still be primarily determined by interactions between pathogens and cells of the innate immune system, but the actions of T cells will feed back into this dynamic equilibrium to suppress overzealous innate responses. Although Th1 responses driven by the IL-12 /IFN-g axis are central to protection against fungi, it is also an undisputed fact that patients with inborn deficits in the IL-12/IL-23/ IFN-g loop do not demonstrate increased susceptibility to most infectious agents, including fungi, with few exceptions (Romani et al., 2008a). This finding implies that other cytokine pathways may also play a role. The new entry, the Th17 pathway, playing an inflammatory role previously attributed to uncontrolled Th1 cell reactivity and Tregs and capable of fine-tuning protective antimicrobial immunity in order to minimize harmful immune pathology, have become an integral component of the immune response to fungi. The enzyme IDO and kynurenines pivotally contribute to this delicate balance by providing the host with immune mechanisms adequate for protection without necessarily eliminating fungal pathogens or causing an unacceptable level of tissue damage. In their capacity to induce Tregs and inhibit Th17, IDO and kynurenines pivotally contribute to cell lineage decision in experimental fungal infections and reveal an unexpected potential in the control of inflammation, allergy, and Th17-driven inflammation in these infections. In this context, the Th17 pathway, which down regulates tryptophan catabolism, may instead favor pathology and serves to accommodate the seemingly paradoxical association of chronic inflammation with fungal disease (Romani et al., 2008b).

Bidirectional influences between infection and immunerelated pathology have been known to also exist in chronic mucocutaneous candidiasis (CMC), a primary immune deficiency presenting as an inability to clear C. albicans yeasts, which persist in recurring lesions of the skin, nails, and mucous membranes (Lilic, 2002). Although occasionally associated with autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (a condition of dysfunctional T-cell activity), CMC encompasses a variety of clinical entities, the pathogenesis of which is largely unclear. CMC patients often develop endocrine and inflammatory disorders, which suggests deregulation of the inflammatory and immune responses. These observations highlight a truly bipolar nature of the inflammatory process in infection, at least by specific fungi. Early inflammation prevents or limits infection, but an uncontrolled response may eventually oppose disease eradication. This condition is crucially exemplified by recent findings in chronic granulomatous disease (CGD) mice, in which an intrinsic, genetically determined failure to control inflammation to sterile fungal components determines the animals’ inability to resolve an actual infection with A. fumigatus (Romani et al., 2008a). A main implication of these findings is that, at least in specific clinical settings, it is an exaggerated inflammatory response that likely compromises a patient’s ability to eradicate infection and not an “intrinsic” susceptibility to infection that determines a state of chronic or intractable disease. Clinically, severe fungal infections occur in patients with immune reconstitution syndrome (IRS), an entity characterized by local and systemic reactions that have both beneficial and deleterious effects on infection (Singh & Perfect, 2007). Intriguingly, IRS responses are also found in immunocompetent individuals and after rapid resolution of immunosuppression, indicating that inflammatory responses can result in quiescent or latent infections manifesting as opportunistic mycoses. This likely reflects the association of the severity of disease with high levels of proinflammatory cytokines in patients with paracoccidioidomycosis (Corvino et al., 2007). Thus, although host immunity is crucial in eradicating infection, immunological recovery can also be detrimental and may contribute towards worsening disease in opportunistic and nonopportunistic infections.

FUNGAL RECOGNITION AND IMMUNE ACTIVATION Innate Immune Receptors

FROM IMMUNE PROTECTION TO IMMUNE RECONSTITUTION SYNDROME-LIKE DISEASES

Unresolved infection and inflammation are major epigenetic and environmental factors that contribute to chronic diseases, autoimmunity, and, in specific settings, to an increased risk of cancer. Fungi can exploit or subvert a host’s inflammatory response and thus affect carriage and pathogenicity (Romani & Puccetti, 2008). A hyperinflammatory response does, in fact, enhance virulence in Saccharomyces cerevisiae and Aspergillus nidulans infections. In the normal skin, the fungus Malassezia down regulates inflammation via TGF-1 (transforming growth factor 1) and IL-10, and establishes itself as a commensal (Ashbee, 2006). In contrast, in atopic dermatitis and psoriasis, the skin barrier acts to enhance release of allergens and molecules involved in hyperproliferation, cell migration, and disease exacerbation. Therefore, an inflammatory circle seems to be at work, the manipulation of which may offer strategies to control or prevent exacerbations of these diseases.

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Most of the innate mechanisms are inducible upon infection, and their activation requires specific recognition of invariant evolutionarily conserved molecular structures shared by large groups of pathogens by a set of pattern recognition receptors (PRRs) that directly recognize fungal molecules including Toll-like receptors (TLRs), C-type lectin receptors (CRLs), and the galectin family (Netea et al., 2008; Romani, 2004; Ruas et al., 2009). For instance, the CLR Mincle, an FcRg-associated activating receptor that senses damaged cells, recognizes Malassezia, and plays a crucial role in the inflammatory response to the fungus (Yamasaki et al., 2009). Implicated in fungal sensing are also NODlike receptors (NLRs) that sense nonmicrobial danger signals—that is, xenocompounds or molecules that, when recognized, alert the immune system of hazardous environments, perhaps independently of a microbial trigger—and form large cytoplasmic complexes called inflammasomes that link the sensing of fungal products and metabolic stress to the proteolytic activation of the proinflammatory cytokines IL-1b and IL-18. The NALP3 inflammasome has been

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associated with several autoinflammatory conditions and can direct adaptive immune responses (Chen et al., 2009). Receptors on phagocytes not only mediates downstream intracellular events related to clearance, but also participate in complex and disparate functions related to immunomodulation and activation of immunity, depending on cell type. Therefore, in order to achieve optimal activation of antigen-specific adaptive immunity, it is first necessary to activate the pathogen-detection mechanisms of the innate immune response. A number of cell wall components of fungi may act through several distinct PRRs, each activating specific antifungal programmes on phagocytes and dendritic cells (DCs) but cooperating for full immune cell activation (Dan et al., 2008; Netea, 2008). The environmental set up probably affects PRR functioning, given that the optimal ability of cells to phagocytose fungi is observed in the environment where a pathogen is naturally encountered (Behnsen et al., 2007). However, another function of the innate immunity that is emerging is that it also has a role in sterile inflammation— that is, inflammation caused by endogenous ligands. In this regard, TLR activation itself is a double-edged sword, as members of the TLR family are involved in the pathogenesis of autoimmune, chronic inflammatory disorders such as asthma, rheumatoid arthritis, and infectious diseases. Thus, by hyperinduction of proinflammatory cytokines, by facilitating tissue damage or by impairing protective immunity, TLRs might paradoxically promote the pathogenesis of infections. Not surprisingly, therefore, the exploitation of PRRs may also provide a mechanism to divert and subvert host immune responses by fungi (Netea et al., 2004).

Dendritic Cells

As sentinels of the immune system, DCs scan their environment for the presence of pathogens. DCs sense pathogens either directly or indirectly via endogenous factors such as cytokines and chemokines, which are produced by other cell types in response to infection. Although indirect signals in the form of endogenous factors alert DCs, direct activation of DCs by microbial components is crucial for the induction of primary T-cell responses. For years, DC biologists have oscillated between two seemingly antagonistic ideas: functional specialization (division of labor) of DC subsets and plasticity (multitasking). More recently, a third hypothesis is gathering support: cross talk between functionally distinct DC subsets. This reveals a previously unappreciated hierarchy of organization within the DC system, and provides a conceptual framework to understand how cooperation between functionally distinct, yet plastic, DC subsets can shape adaptive immunity and immunological memory (Pulendran et al., 2008). DCs are uniquely adept at decoding the fungus-associated information and translating it into qualitatively different adaptive T-cell immune responses (Romani et al., 2002; Romani & Puccetti, 2006b). The finding that methamphetamine, whose chronic abuse has reached epidemic proportions, potently inhibits fungal processing and killing by DCs (Talloczy et al., 2008), points to the unique and indispensable role of DCs in antifungal immunity. DCs are capable of internalizing different fungal morphotypes through different receptors and forms of phagocytosis. The ability of a given DC subset to respond with flexible activating programs to the different stimuli, as well as the ability of different subsets to convert into each other confer unexpected plasticity to the DC system (Romani et al., 2002; Romani & Puccetti, 2006b). As a matter of fact, the capacity of DCs to initiate different adaptive antifungal immune responses depended upon specialization and cooperation between DC subsets

(Montagnoli et al., 2008; Romani et al., 2002; Romani & Puccetti, 2006b) as well as the activation of distinct intracellular signaling pathways (Bonifazi et al., 2009). A number of PRRs determine the functional plasticity of DCs in response to fungi and contribute to the discriminative recognition of the different fungal morphotypes. This process exemplifies the importance of PRRs not only in direct early immune responses, but also in orchestrating the adaptive immunity (Romani & Puccetti, 2006b). The results are consistent with the view that the exploitation of distinct recognition receptors in DCs may determine the full range of the host’s immune relationships with fungi, as shown with C. albicans (Romani et al., 2002; Romani & Puccetti, 2006a; Romani & Puccetti, 2006b). Indeed, fungus-pulsed DCs translated fungus-associated information to Th1, Th2, Th17, and Treg cells in vitro and in vivo upon infusion of different DC subsets (Montagnoli, 2008). For instance, the infusion of plasmacytoid DCs in bone marrow-transplanted mice resulted in the concomitant Th1/Treg cell priming eventually leading to fungal growth restriction, limited inflammatory pathology and, interestingly, transplantation tolerance (Montagnoli et al., 2008). These results, along with the finding that fungus-pulsed DCs could reverse T cells anergy of patients with fungal diseases, may suggest the utility of DCs for fungal vaccines and vaccination (Awasthi, 2007; Montagnoli et al., 2008). A wealth of evidence indicates that DC immunogenicity/ tolerogenicity is not a characteristic of a specific subset or lineage of DCs, but an environmentally acquired feature. In this regard, the tryptophan metabolic pathway pivotally contributed to DC regulation, such that tolerance and Treg induction could be mediated by DCs expressing IDO (Orabona et al., 2004). In response to fungi, IDO expression conferred tolerogenic properties to DCs (Zelante et al., 2009b) such that Candida-pulsed, IDO-expressing gut DCs ameliorated experimental colitis (Bonifazi, 2009). Thus, multiple and functionally distinct receptor/signaling pathways in DCs ultimately affecting the local Th:Treg balance could be successfully exploited for either commensalism or pathogenicity, a finding suggesting a high degree of coevolution of mammalian hosts and symbiotic fungi. By subverting the morphotype-specific program of activation of DCs, innate environmental factors and fungi themselves qualitatively affect DC functioning and Th/Treg selection in vivo, ultimately impacting fungal virulence. Thus, the model accommodates the concept of virulence as an important component of fungus fitness in vivo within the plasticity of immune responses orchestrated by DCs. Indeed, impaired DC maturation and function have been associated with disease in patients with CMC (Ryan et al., 2008).

ACQUIRED ANTIBODY- AND CELL-MEDIATED IMMUNITY

The success of passive antibody in preventing and treating fungal infections in experimental models and certain vaccines that elicit protective antibody, strongly indicate that some antibody responses can make a decisive contribution to host defense to medically important fungi (Pirofski & Casadevall, 2006). Protective antibodies have now been described for C. albicans, C. neoformans, A. fumigatus, Pneumocystis spp., and H. capsulatum. In recent years, two antibodies have entered clinical evaluation for fungal diseases. In addition to classical mechanisms of antibody action, additional mechanisms have been revealed, including inhibition of growth and germination, biofilm formation, direct antifungal effects, and alteration of intracellular trafficking of fungi (Pirofski & Casadevall, 2006; Shi et al., 2008).


A consensus has now emerged that the inability of immune sera to mediate protection against fungi reflects inadequate amounts of protective antibody and/or the simultaneous presence of protective and nonprotective antibodies. Nonetheless, much remains to be learned about the nature of protective antibodies and the relationship between the natural antibody response and resistance and susceptibility to fungal pathogens, since antibody responses can be a marker of disease rather than immunity. Additionally, there is currently insufficient evidence to indicate how antibodies mediate their protective effects at the different body sites in infections. Serological and skin reactivity surveys indicate the development of acquired cell-mediated immunity (CMI) to fungi. Lymphocytes from healthy subjects show strong proliferative responses after stimulation with fungal antigens and produce a number of different cytokines (Romani et al., 2008a). For dimorphic fungi, the initial exposure either is asymptomatic or results in mild infection that confers protective immunity. In contrast, the severity of the disease correlates with the degree of the impairment of CMI and elevated levels of antibodies. For C. neoformans, the high prevalence of antibodies to cryptococcal antigens in normal individuals suggests that primary infection is followed by fungal growth restriction and concomitant immunity, as recently demonstrated in experimental cryptococcosis. As a matter of fact, direct inhibition of T-cell proliferation by fungal polysaccharides may underlie the defective CMI of patients with persistent cryptococcal infections (Yauch et al., 2006). Underlying acquired immunity to C. albicans, such as the expression of a positive delayed type hypersensitivity (DTH), is demonstrable in adult immunocompetent individuals. There is extensive plasticity in the T-cell response to fungi. The heterogeneity of the CD41 and CD81 T-cell repertoire may account for the multiplicity and redundancy of effector mechanisms through which T lymphocytes participate in the control of fungal infections. These may include direct antifungal activity, apoptosis, and complex effector functions resulting from the dynamic interactions between T cells bearing selected members of the Vb families of the T-cell receptor (Romani, 2004 ). The functional plasticity is such to uncover vaccine potential in conditions of immunodeficiency. The flexible program of the T lymphocytes also implicates the production of a number of mediators, including cytokines that are instrumental in mobilizing and activating antifungal effectors, thus providing prompt and effective control of infectivity once the fungus has established itself in tissues or spread to internal organs. Therefore, host resistance to fungi appears to be dependent on the induction of cellular immunity, mediated by T lymphocytes, cytokines, and a number of effector phagocytes.

Th1/Th2/Th17 Cells

Generation of a dominant Th1 response driven by IL-12 is essentially required for the expression of protective immunity to fungi. Through the production of the signature cytokine IFN-g and help for opsonizing antibodies, the activation of Th1 cells is instrumental in the optimal activation of phagocytes at sites of infection (Fig. 2). Therefore, the failure to deliver activating signals to effector phagocytes may predispose patients to overwhelming infections, limit the therapeutic efficacy of antifungals and antibodies, and favor persistency and/or commensalism (Romani, 2004). Immunological studies in patients with polar forms of paracoccidioidomycosis demonstrate an association between Th1-biased reactivity and the asymptomatic and mild forms

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of the infection, as opposed to the positive correlation of Th2 responses with the severity of the disease and poor prognosis. Patients with inborn errors of the IL-12/IL-23/ IFN-g-mediated immunity are susceptible to disseminated paracoccidioidomycosis (Romani et al., 2008a). IL-4 acts as the most potent proximal signal for commitment to Th2 reactivity that dampens protective Th1 responses and favors fungal allergy. IL-4 may both deactivate and activate phagocytes and DCs for certain specialized functions; for instance, it may inhibit the antifungal effector activities of phagocytes, yet may promote IL-12 production by DCs (Romani, 2004). Thus, the most important mechanism underlying the inhibitory activity of IL-4 in infections relies on its ability to act as the most potent proximal signal for commitment to Th2 reactivity that dampens protective Th1 responses and favors fungal allergy. In atopic subjects and neonates, the suppressed DTH response to fungi is associated with elevated levels of antifungal IgE, IgA, and IgG. However, susceptibility to fungal infections may not always be associated with an overt production of IL-4 (Lilic, 2002) (Fig. 2). Over the past several years, the demise of a Th1/Th2 dichotomy paradigm has been accompanied by a renaissance in probing the basic tenets of CD41 T-cell biology. As a result, instead of only two distinct “fates” for developing T cells, research has identified alternative fates and more flexibility in T-cell cytokine production than previously envisioned (Zhou et al., 2009). Th17 cells are now thought to be a separate lineage of effector Th cells contributing to immune pathogenesis previously attributed to the Th1 lineage (Kaufmann & Kuchroo, 2009). They produce unique cytokines (IL-17, IL-17F, IL-21, and IL-22) and express transcription factors distinct from Th1 and Th2 cells. Naïve mouse and human CD41 T cells activated in the presence of TGF-b and IL-6 express the transcription factor RORgt (retinoid-related orphan receptor g t) and become Th17 cells that are stabilized by DC-derived IL-23 and amplified by IL-1 and IL-21. IL-6, IL-21, and IL-23 all induce phosphorylation of the signal transducer and activator of transcription 3 that has multiple binding sites on the IL-17A promoter. In contrast, TGF-b, with IL-2, up regulate expression of the transcription factor FoxP3 (forkhead box P3) and activate “inducible” Tregs, which suppress immune responses. Several lines of evidence further support the notion of a reciprocal relationship between FoxP31 Tregs and Th17 cells. In addition, since TGF-b can induce both RORgt and FoxP3, it is interesting that the level of TGF-b and the presence of cytokines such as IL-6 and IL-21 dictate whether expression of RORgt/ RoRa or FoxP3 predominate and, depending on the level of free transcription factors, determine whether T cells will differentiate into the Treg or Th17 phenotype (Kaufmann & Kuchroo, 2009). Emerging data on the mechanism by which Th17 cells induce tissue inflammation suggest that IL-Th17 cells first infiltrate the site of tissue inflammation and then recruit other proinflammatory effector T cells (including Th1 cells) and innate cells (including neutrophils) to sites of tissue inflammation. As IL-17 receptors are widely expressed on parenchymal/tissue cells and IL-17 induces production of IL-1, IL-6, TNF (tumor necrosis factor), matrix metalloproteinases, IL-8, and chemokines, these mediators coordinate infiltration of other cell types to the site of inflammation and mediate massive tissue inflammation at the site where IL-17 is abundantly produced. Th17 cells are induced in fungal infections through TLR- and non-TLR-dependent signaling (De Luca et al., 2007; Heninger et al., 2006; Kleinschek et al., 2006;

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FIGURE 2 Th cell subsets in response to fungi. The figure shows the orchestration of Th/Treg cell subsets differentiation, their transcription factors, cytokine production, and possible effector/ regulatory functions in fungal infections. DCs, dendritic cells. See text for details.

Leibundgut-Landmann et al., 2007; van de Veerdonk et al., 2009; Zelante et al., 2007). Th17 are present in the human T-cell memory repertoire to C. albicans (Acosta-Rodriguez et al., 2007; Fenoglio et al., 2009) and A. fumigatus (Bozza et al., 2009), and defective Th17 cell differentiation has been linked to CMC in patients with primary immunodeficiencies (Milner et al., 2008). However, both positive and negative effects on immune resistance have been attributed to Th17 and IL-17 receptor (IL-17R) signaling in experimental fungal infections (Conti et al., 2009; Huang et al., 2004; Zelante, 2007). Thus, the role of IL-17 and Th17 cells in immunity versus pathology in fungal infections and diseases remains controversial (Zelante

et al., 2009a). It is likely that the protective versus diseasepromoting effect of the IL-17/Th17 pathway may depend on the stage and site of infection. Early in infection, IL-17A may exert some forms of antifungal resistance via defensins and neutrophils (Conti, 2009). Later, the failure to down regulate microbe-induced expression of IL-17A could eventually be one major link connecting infection with chronic inflammation (Zelante, 2007) (Fig. 2). The mechanisms that linked inflammation to chronic infection have been credited to the offending potential of IL-17A that, although promoting neutrophil recruitment, impeded the timely restriction of neutrophil inflammatory potential, thus preventing optimal protection to


occur (Romani et al., 2008b). IL-17A also activated the inflammatory program of neutrophils by counteracting the IFN-g-dependent activation of IDO, known to limit the inflammatory status of neutrophils, as well as by inducing the release of metalloproteinases and oxidants, which likely accounts for the high inflammatory pathology and tissue destruction associated with Th17 cell activation. These new findings provide a molecular connection between the failure to resolve inflammation and lack of antifungal immune resistance and point to strategies for immune therapy of fungal infections that attempt to limit inflammation to stimulate an effective immune response. More generally, the Th17 pathway could be involved in the immunopathogenesis of chronic fungal diseases where persistent fungal antigens may maintain immunological dysreactivity. As a matter of fact, IL-17A neutralization increased fungal clearance, ameliorated inflammatory pathology, and restored protective Th1 antifungal resistance, a finding that points to the therapeutic utility of immunomodulatory strategies aimed at reducing Th17-driven hyperinflammation in fungal infections (see Table 2). However, despite the excitement raised by the new findings, much remains to be learned, including the dependency of Th17 cells on the plasticity of human CD41 T-cell differentiation and the relative contribution of the various populations of IL-17-producing cells to the pathogenesis of infections and diseases caused by the different fungi. In this regard, Th17 cells also produce IL-22, a member of the IL-10 family of cytokines, which has been shown to play a more important role than IL-17 in host defense in the lung and gut (Zenewicz & Flavell, 2008). Our recent findings suggest that the IL-23/IL-22/defensins pathway is crucially involved in the control of fungal growth at mucosal and nonmucosal sites in both candidiasis and aspergillosis, particularly in conditions of Th1 deficiency. Interestingly, memory IL-221CD41 cells specific for C. albicans are present in humans (Liu et al., 2009) and are defective in patients with CMC (Eyerich et al., 2008). Thus, further tweaking the Th17 model, Th17 may exert its protective role in fungal infections through IL-22. However, IL-22-producing Th17 cells failed to confer the same level of resistance to reinfection and to inflammatory pathology conferred by the Th1/Treg axis (De Luca et al., 2010). Thus, in the relative absence of protective Th1/Treg, IL-221Th17 cells may fulfill the role of a protective response that exploits primitive effector defense mechanisms of antifungal resistance. This finding suggests that functionally distinct “modules” of immunity evolved to provide resistance (i.e., ability to limit fungal burden), or tolerance (i.e., the ability to limit the host damage in response to fungi) (Zelante et al., 2009a).

TABLE 1

295

INDUCING TOLERANCE Via Tregs

Immune responses to fungi are modulated by one or more types of cells that perform a regulatory function. Tregs with anti-inflammatory activity have been described in fungal infections of both mice (Hori et al., 2002; Lazar-Molnar et al., 2008; McKinley et al., 2006; Montagnoli et al., 2002; Montagnoli et al., 2006; Deepe & Gibbons, 2008) and humans (Cavassani et al., 2006) (Table 1). Some cells with this function, such as CD41 Foxp31 natural Tregs (nTregs), originate in the thymus and preexist prior to infections, whereas others may be induced as a consequence of infection (iTregs) or in conditions of impaired costimulatory signaling and in the presence of deactivating cytokines and drugs. As already discussed, a reciprocal relationship has been described between the development of Foxp31 Tregs and effector Th17 cells, so that naïve T-cell activation in the presence of innate stimuli divert iTreg generation to Th17 generation. A number of clinical observations suggest an inverse relationship between IFN-g and IL-10 production in patients with fungal infections. High levels of IL-10, negatively affecting IFN-g production, are detected in chronic candidal diseases (Eyerich et al., 2007), in the severe form of endemic mycoses and in neutropenic patients with aspergillosis (Romani & Puccetti, 2006a). However, solid evidence demonstrating a causal role for IL-10 in susceptibility to fungal infections are lacking. Recently, it has been suggested that rather than causing the infection, IL-10 production may be a consequence of the infection (Romani & Puccetti, 2006a). This would predict that, in the case of chronic fungal infections, characterized by a state of chronic inflammation, IL-10 could be the homeostatic host-driven response aimed at keeping inflammation under control (however possible this is). With pathogens like fungi that have a complex pathogenesis, multiple types of regulatory cells could influence the outcome. In experimental fungal infections, fungal growth, inflammatory immunity, and tolerance in the respiratory or the gastrointestinal mucosa were all controlled by the coordinate activation of nTregs—limiting early inflammation at the sites of infection—and pathogen-induced iTregs, which regulated the expression of adaptive Th immunity in secondary lymphoid organs. Thus, a fine balance is established between different Treg subsets, effector components of immunity, and fungi. However, as the Treg responses may handicap the efficacy of protective immunity, the consequence of Treg activity is not only less damage to the host but also to fungal persistence. Because both the recovery of C. albicans from the gastrointestinal tract and the

Regulatory T cells in fungal infectionsa

Fungi

Species

Effects of Tregs on immunity and pathology

Reference

Candida albicans

Mouse

Antifungal Th1 cell responses limited; immunopathology controlled; required for memory responses

Montagnoli et al., 2002

Paracoccidioides brasiliensis

Human

Increased frequency of Foxp31 Treg cells in the blood and fungi-induced granuloma; increased suppressive activity in vitro

Cavassani et al.,2006

Aspergillus fumigatus

Mouse

Local recruitment of Treg cells to the lung; neutrophils controlled through IDO


Histoplasma acapsulatum

Mouse

Protective immunity inhibited

Lazar-Molnar et al., 2008 Deepe and Gibbons, 2008

Pneumocystis carinii

Mouse

Dampened inflammation and lung injury

McKinley et al., 2006

a

IDO, indoleamine 2, 3-dioxygenase.

296


detection of underlying Th1 reactivity, such as DTH and lymphoproliferation, can fluctuate in healthy subjects, it is likely that Tregs mediate tolerance to the fungus at the site of colonization. This may have allowed fungal persistence and the occurrence of memory immunity to a commensal. CMC, although encompassing a variety of clinical entities (Lilic, 2002), has been associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, a condition in which Treg induction is defective (Ryan et al., 2005). The different Treg cell populations may have the capacity to influence the emergence or function of one another. This was best illustrated in murine aspergillosis where, early in infection, inflammation was controlled by the expansion, activation, and local recruitment of nTregs suppressing neutrophils, whereas late in infection, and similarly in allergy, tolerogenic iTregs inhibited Th2 cells and prevent allergy to the fungus (Montagnoli et al., 2006). The level of inflammation and IFN-g in the early stage set the subsequent adaptive stage by conditioning the IDO-dependent tolerogenic program of DCs and the subsequent activation and expansion of tolerogenic Tregs preventing allergy to the fungus. Therefore, regulatory mechanisms operating in the control of inflammation and allergy to the fungus are different but interdependent as the level of the inflammatory response early in infection may impact susceptibility to allergy in conditions of continuous exposure to the fungus. nTregs, by affecting IFN-g production, indirectly exert a fine control over the induction of late tolerogenic iTregs. Thus, a unifying mechanism linking natural Treg cells to tolerogenic respiratory Treg cells in response to the fungus is consistent with the revisited “hygiene hypothesis” of allergy in infections—that is, an early reduction in microbial burden may predispose to allergy and may provide, at the same time, mechanistic explanations for the significance of the variable level of IFN-g seen in allergic diseases and asthma and for the paradoxical worsening effect on allergy of Th1 cells. Collectively, these observations suggest that the capacity of Tregs to inhibit aspects of innate and adaptive immunity is pivotal in their regulatory function and further support the concept of “protective tolerance” to fungi, implying that a host’s immune defense may be adequate for protection without necessarily eliminating fungal pathogens—which would impair immune memory—or causing an unacceptable level of tissue damage (Romani & Puccetti, 2006a).

Via IDO

IDO has a complex role in immunoregulation in infection, pregnancy, autoimmunity, transplantation, and neoplasia (Grohmann et al., 2003). As already mentioned, IDOexpressing DCs are regarded as regulatory DCs specialized to cause antigen-specific deletional tolerance or induction of CD41CD251 Tregs. These findings establish a mutual interaction between DCs and Tregs for the upkeep of

immunological tolerance. In experimental fungal infections, IDO blockade greatly exacerbated infections, the associated inflammatory pathology, and swept away resistance to reinfection as a result of deregulated innate and adaptive immune responses caused by the impaired activation and functioning of suppressor CD41CD251 Tregs producing IL-10 (Romani & Puccetti, 2006a). More recently, while capable of inducing the Foxp3-encoding gene transcriptionally, tryptophan catabolites were also found to suppress the gene encoding RORgt, the Th17 lineage specification factor (De Luca et al., 2007). Thus, regulation of iTregs/ Th17 balance at the host/pathogen further emphasizes the pivotal role of tryptophan catabolism and IDO in tolerogenesis and prevention of inflammation and allergy. The IDO mechanism has revealed an unexpected potential in the control of inflammation not only in infection but also in airway allergy, a condition in which tolerogenic DCs could have a protective function. IDO expression is paradoxically up regulated in patients with allergy or autoimmune inflammation, a finding suggesting the occurrence of a homeostatic mechanism to halt ongoing inflammation. As already discussed, a unifying mechanism linking nTregs to tolerogenic iTregs via IDO appears to be at work in murine allergic aspergillosis. Recent data have confirmed the protective role of the IDO/Tregs axis in fungal allergy (Grohmann et al., 2007). In this model, modulation of tryptophan catabolism via the glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand, GITRL, inhibited Th2-cell responses and allergy and induced the expression of Foxp31 Tregs through mechanisms dependent on IDO induction by components of the noncanonical NF-kB signaling pathway (Grohmann et al., 2007). Thus, induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids (Grohmann et al., 2007). The implication for IDO in immunoregulation in fungal infections has several important implications. As C. albicans is a commensal of the human gastrointestinal and genitourinary tracts and IFN-g is an important mediator of protective immunity to the fungus, the IFN-g/IDO axis may accommodate fungal persistence in a host environment rich in IFN-g. In its ability to induce Th1 immunity within a regulatory environment and to prevent Th17 development, IDO expression may correlate with the occurrence of local tolerogenic responses. Alternatively, the high levels of IL-10 production may be a consequence of IDO activation by the fungus, impairing antifungal Th1 immunity and thus favoring persistent infection. The fact that fungal hyphae, more so than yeasts, activate the expression of IDO further suggests that differential sensing of fungal morphotypes through distinct recognition receptors may promote distinct immune responses. In addition, fungal hyphae, more so than yeasts, may promote tolerance and thus contribute to commensalism and eventually to

TABLE 2 Prospectives on anti-inflammatory strategies in fungal diseases a Strategies

Mechanisms

References

IL-17/IL-23 antagonists

Prevention of pathogenic inflammation

Zelante et al., 2007

Exogenous kynurenines

Promotion of Tregs and inhibition of Th17

Romani et al., 2008b

IDO targeting in allergy

Promotion of Tregs and inhibition of Th2

Grohmann et al., 2007

Induction of protective tolerance via Th1/ Tregs


1

IDO DCs as fungal vaccines in transplantation a

DCs, dendritic cells; IDO, indoleamine 2,3-dioxygenase.


immunoevasion. Because germinating Aspergillus conidia promote inflammatory responses by subverting tolerance (Montagnoli et al., 2006), the central role of tolerance at the fungus/host interface and the pivotal role of tryptophan catabolism in the tolerant state are both emphasized. Ultimately, the manipulation of Tregs by fungi gives further support to the notion that gut microbiota may function as a major regulator of immunological tolerance both locally and at distant sites.

CONCLUSIONS: TRANSLATING IMMUNITY INTO CLINICAL PRACTICES Vaccine Development

With the exception of a killed spherule vaccine against coccidioidomycosis, no fungal vaccine trials have ever been conducted (Cutler et al., 2007; Dan et al., 2008). However, the level of our understanding of fungal–host interactions has progressed to the point where vaccines against both primary fungal pathogens and the prevalent opportunistic fungi are becoming a reality (Cutler et al., 2007). The experiments involving protective antibody responses against b-1,3-glucan suggest that a universal fungal vaccine could be developed (Cassone, 2008). The mechanisms of immunity vary from antibody-mediated immune responses to cell-mediated responses, and even a combination of both of these main arms of the acquired immune system. Controlled stimulation of Th17 cells could be of value for design of novel vaccination strategies aimed at inducing antifungal defense mechanisms.

Immunomodulation

New discoveries in the field of fungal immunology have offered new grounds for a better comprehension of cells and immune pathways that are amenable to manipulation in patients with or at risk of fungal infections (Segal et al., 2006). Our increasing understanding of the basic mechanisms that dictate development and function of Th17 cells, as well as our better knowledge of how Th17/Tregs regulate each other as well as other immune and nonimmune cells, provides guidelines for rational design of novel immunomodulatory therapies that limit inflammation in order to stimulate an effective immune response. Tryptophan metabolites and Th17 inhibitors are likely candidates as potent regulators capable of taming overzealous or heightened inflammatory host responses to the benefit of pathogen control and host survival (Table 2). Notwithstanding the redundancy and overlapping repertoire of antifungal effector mechanisms, the pivotal role of different types of Tregs in the control of Th1/Th2 inflammatory responses, as well as in Th17 antagonism, suggests that manipulation of Tregs could be a promising therapeutic approach devoid of risks associated with interference with homeostatic mechanisms of the immune system. In this regard, the potential of the anti-TNF antibody infliximab to induce anti-inflammatory cytokines IL-10 or TGF-b via retrograde signaling or through induction of a certain subset of Tregs is of interest (Deepe & Gibbons, 2008). However, how Tregs contribute to the deadly disseminate histoplasmosis that occurred in a gene-therapy trial designed to deliver a TNF-a antagonist for inflammatory arthritis is not known (Frank et al., 2009).

A Change of Strategy

Infectious agents can induce autoimmune diseases but paradoxically can also suppress allergic and autoimmune disorders. A central question is to determine whether fungal

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exposure/colonization contributes to the burst of pathogenic autoimmunity or alternatively (but not mutually exclusive), whether dysregulation precedes, if not promotes, diseases caused by fungi. In this context, in their ability to subvert the inflammatory program through the activation of the IL-23/Th17 axis, fungi may eventually lead to immune dysregulation, including allergy and autoimmunity (Romani & Puccetti, 2008). However, their ability to activate Tregs may represent a mechanism whereby dysregulated immunity is prevented in a manner similar to “protective” microbiota of the gastrointestinal tract. The future will tell us whether, as with cancer (Gatenby, 2009), trying to control the disease may prove a better plan than striving to get rid of the microbes we live with. This study was supported by the Specific Targeted Research Project “SYBARIS” (FP7-HEALTH-2009), and the Italian project PRIN prot.2007KLCKP8_004.

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24 Acquired Immunity to Intracellular Protozoa PHILLIP SCOTT AND ELEANOR M. RILEY

INTRODUCTION

protozoans: Plasmodium, Leishmania, Toxoplasma gondii, and Trypanosoma cruzi. These represent the most important intracellular protozoans causing disease in humans, and, as such, they are the most studied of intracellular parasites. Although each of these parasites live within cells, they differ substantially in their life cycles. Leishmania exist in two major forms, the promastigote—a flagellated organism found in the sand fly—and the amastigote, which develops following entry of promastigotes into phagocytic cells (macrophages and dendritic cells) of the host. T. cruzi is transmitted by reduviid bugs and, similar to Leishmania, they can invade cells and transform to amastigotes. However, T. cruzi can invade almost any cell, and after several rounds of division the organisms transform to trypomastigotes that are released into the circulation. Toxoplasma has a more complicated life cycle than either Leishmania or T. cruzi. Within the gut epithelial cells of the cat, the organisms undergo sexual multiplication, resulting in the production of oocytes that become infective after being shed in the feces. While Toxoplasma can multiply sexually only in the cat, many vertebrate species can be infected, and in these animals the parasites invade the epithelium and transform to tachyzoites, a rapidly multiplying form of the parasite. These organisms then disseminate and can invade any cell in the body. Once an effective immune response is generated, a slowly multiplying form, termed the bradyzoite, is able to survive within cysts. Thus, Leishmania, T. cruzi, and Toxoplasma all infect macrophages and dendritic cells, although T. cruzi and Toxoplasma are not restricted to these cells. Although still an intracellular protozoan parasite, Plasmodium exhibits a quite distinct life cycle from Leishmania, T. cruzi, and Toxoplasma. The sexual phase of the life cycle occurs in the mosquito vector with infected mosquitoes injecting sporozoites into the skin, which then migrate to the liver and undergo several rounds of asexual (mitotic) replication inside hepatocytes before emerging as merozoites, which invade red blood cells (RBCs). Further, mitotic replication inside RBCs leads to the clinical symptoms of malaria and eventually generates gametocytes that are infectious to mosquitoes. As a consequence of the similarities in life cycle, and specifically the importance of macrophages and dendritic cells as host cells, the immune responses to Leishmania, T. cruzi, and Toxoplasma will be discussed together, and malaria will be considered separately.

The control of pathogenic intracellular protozoa is dependent upon both an innate and acquired (or adaptive) immune response. Innate immune responses play an important role by controlling the early replication of parasites, giving time for acquired immunity to develop, although innate immune responses are rarely able to provide sufficient protection to prevent disease. Rather, it is the antigen-specific expansion of clonal T- and B-cell populations, which then marshal a variety of effector functions, that leads to the control of parasites. However, the distinction between innate and acquired immunity is not clear cut, as not only does the innate immune response influence the type of effector responses that develop, but the effector mechanisms invoked by clonally expanded lymphocytes are often mediated by cells considered part of the innate immune response. There are several reasons why pathogens fail to be eliminated following infection. Some organisms may invade the host silently and fail to provoke a strong immune response. More often than not, however, infections elicit a response. But to be effective it must be the appropriate response, and while there are many effector mechanisms available to combat infection, not all of them are effective against any one particular parasite. Moreover, the development of certain immune responses will impede the development of others. For example, the cross-regulation between Th1 and Th2 type immune responses is well established. Thus, initiating the appropriate immune response is a critical step in controlling any pathogen, and in some cases pathogens fail to do so. However, intracellular protozoa (similar to other intracellular pathogens) often provoke the type of immunity that would be best suited to control these organisms—such as the induction of a Th1-type immune response—and yet these parasites still cause chronic infections. Understanding how protective immunity can be induced, as well as knowing the mechanisms used by these intracellular parasites to avoid destruction, is critical for the development of new therapies as well as vaccines. In this chapter we will investigate the acquired immune responses important in the control of four intracellular Phillip Scott, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104. Eleanor M. Riley, Department of Infectious and Tropical Diseases, London School of Tropical Medicine and Hygiene, London, UK.

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INTRACELLULAR PARASITES OF PHAGOCYTIC CELLS: LEISHMANIA, TOXOPLASMA GONDII, AND TRYPANOSOMA CRUZI Initiation of Immunity

The initiation of an acquired immune response is dependent upon the recognition of the pathogen as foreign, which leads to the production of inflammatory cytokines that promote the increased function of antigen presenting cells, as well as production of cytokines (such as IL-12 [interleukin-12]) that will promote the development of T cells, important for an effective immune response. Studies with mice lacking MyD88, an adaptor protein associated with Toll-like receptor (TLR) function, demonstrate that recognition of TLR ligands is critical in promoting resistance to these intracellular parasites. Several parasite TLR ligands have been described, such as glycophosphatidylinositol (GPI) anchors in the case of T. cruzi and Toxoplasma (as well as Plasmodium) and a lipophosphoglycan (LPG) in the case of Leishmania, which activate macrophages and dendritic cells via TLR2 (Gazzinelli et al., 2004; Liese et al., 2008; Yarovinsky, 2008) (see chapter 18). However, other TLRs most likely also contribute to initiating the immune responses, since the phenotype of TLR2 knockout mice, when infected with these parasites, is not as severe as that observed with MyD88-deficient mice. For example, Toxoplasma produces profilin, which activates cells via TLR11 (Yarovinsky, 2008), and DNA from T. cruzi and Leishmania can also activate cells via TLR9 (Liese et al., 2008; Shoda et al., 2001). In the main, similar pathways have also been demonstrated in human cells, although, as human cells do not express TLR11, recognition of T. gondii likely differs between mice and humans. There remains, moreover, a great deal to be learned about the parasite molecules triggering these responses and the signaling pathways leading to activation of innate cells. For example, while the major transcription factors mediating activation of innate cells belong to the NF-kB family, studies in NF-kB knockout mice infected with Toxoplasma or Leishmania have shown that whereas these mutant mice often demonstrate increased susceptibility to infection, this is more often due to a failure in the adaptive immune response (e.g., T-cell proliferation), than to defects in the initial innate immune responses (Mason et al., 2004). These unexpected results highlight the complexity involved in TLR signaling. Ongoing studies are focused on defining how TLRs mediate their effects during parasitic infections, as well as how synergy between multiple TLRs and other pattern recognition receptors may influence the degree of activation and the subsequent adaptive immune response. IL-12 is the critical cytokine for promoting the development of Th1 cells following infection with intracellular protozoa. Consequently, IL-12 deficient mice are unable to develop a Th1 response and exhibit extreme susceptibility to Leishmania, Toxoplasma, and T. cruzi. Several phagocytic cells can make IL-12, including dendritic cells (DCs), macrophages, and neutrophils. DCs, due to their production of IL-12 as well as their superior ability to present antigen to naive T cells, play a pivotal role in initiating the immune response to intracellular parasites. By acting on proliferating T cells within the lymph nodes or spleen, IL-12 facilitates epigenetic changes that lead to a stable Th1 phenotype. Where the parasites come into contact with DCs will depend on their site of entry. In general, DCs most likely contact antigen in the tissues and then migrate to secondary lymphoid organs to meet naïve T cells. For example, in the case of Leishmania, using 2-photon intravital imaging dermal DCs can be visualized taking up parasites after

infection (Ng et al., 2008). These DCs may then migrate to the draining lymph nodes and initiate the immune responses, although DCs resident within the lymph node or migrating into the lymph node from the blood may also take up parasites (or parasite antigens) that have traveled to the draining lymph node and contribute to the response. While DCs are critical for initiating the immune response, the first cells to enter a site of inflammation are granulocytes. Neutrophils have often been associated with protection, but it is becoming clear that parasites can use them for their own benefit. For example, following infection with Leishmania major, neutrophils have been shown by 2-photon intravital imaging to rapidly enter the site of infection and take up many of the parasites (Peters et al., 2008). Rather than promoting the immune response, however, it appears that neutrophils transfer the parasites to DCs and macrophages without activating those cells. Consistent with this idea, neutrophil depletion prior to infection of mice with L. major leads to increased control of the parasites, although this may depend upon the host and parasite strain or species (Peters & Sacks, 2009; Ritter et al., 2009). These results and others have prompted a reevaluation of the role that granulocytes play in the immune response to parasites. Complicating the analysis is that the involvement of neutrophils during infection has usually been demonstrated by depletion studies using antibodies against Gr1, a molecule expressed on neutrophils but now known to be expressed on other cell types, including DCs and macrophages. Thus, many of these studies will need to be reinterpreted, and the role of other Gr11 cells will need to be defined (Egan et al., 2008).

T-Cell Dependent Control of Intracellular Protozoa

Immunity to intracellular parasites is largely dependent upon T cells, which can be demonstrated experimentally by adoptive transfer. Thus, both CD4 and CD8 T cells play a role in the immunity exhibited against Leishmania, Toxoplasma, and T. cruzi, but to varying degrees. The differences in the strategies that that these parasites use to survive within phagocytic cells may help determine the dominance of the CD4 or CD8 T-cell response during these infections. For example, T. cruzi parasites secrete a lytic enzyme that releases parasites from an endosomal compartment into the cytoplasm, thus escaping fusion with the lysosome. Toxoplasma creates its own parasitophorous vacuole, and thus never fuses with the endosomal compartments containing class II, but the parasitophorous vacuole interacts with the endoplasmic reticulum allowing parasite molecules to gain entry into the class I pathway (Roy et al., 2006; Goldszmid et al., 2009). In contrast, Leishmania survive and multiply within class II1 phagolysosomes, and never escape into the cytoplasm. The immunologic consequences of these differences are that proteins from both T. cruzi and Toxoplasma appear to more readily enter the class I pathway, leading to the generation of a CD81 T-cell response that contributes to protection. Due to cross presentation however, CD8 T cells are also activated following Leishmania infection and therefore play a role in promoting resolution of disease (Scott et al., 2004). Furthermore, all of these infections are associated with activation of CD41 T cells, which contribute to resistance. Parasites have played a key role in studies investigating how Th1 and Th2 cell subsets develop and are maintained. Th1 and Th2 cells, defined by their ability to produce IFN-g and IL-4 respectively, mediate dramatically different immune responses. For example, Th1 cells produce IFN-g that activates macrophages to produce nitric oxide that increases killing of Leishmania, Toxoplasma, and T. cruzi, while macrophages exposed to the IL-4 produced by Th2 cells become

24. Acquired Immunity to Intracellular Protozoa

alternatively activated, and appear better suited to control extracellular helminth parasites. Experimental studies with L. major has played a particularly prominent role in defining the factors participating in the development of Th1 and Th2 cells, since, unlike most other intracellular pathogens, these parasites can induce either a Th1 or Th2 response, depending upon the strain of mouse infected. Thus, L. major infection in many inbred mouse strains is associated with a Th1 response, while infection of BALB/c mice leads to a dominant Th2 response, and consequently a fatal outcome (Scott et al., 2004). The divergent responses seen in L. major infected mice continues to provide a convenient model to test the role of cytokines, transcription factors, and various cell types in the development of Th1 and Th2 cell subsets. While the Th1/2 paradigm has been the mainstay of cellular immunology for more than 20 years, recent studies indicate that CD4 T cells can be divided into additional subsets based on differential cytokine production and/or transcription factor expression. There remains a great deal to know about the development and stability of these new subsets, but they clearly can influence the outcome of infection. For example, T cells making the cytokine IL-17 (Th17 cells) and expressing the transcription factor RORgT are important in certain types of autoimmunity, and have recently been implicated in susceptibility of BALB/c mice to L. major, since BALB/c IL-17 deficient mice are more resistant to L. major (Lopez Kostka et al., 2009). This increased resistance is associated with decreased neutrophil accumulation, further implicating neutrophils as cells that promote susceptibility rather than resistance. In contrast to L. major infections in IL-17 deficient mice, it has been reported that when IL-17R deficient mice are infected with Toxoplasma, they are more susceptible, which was attributed to a defect in neutrophil accumulation at sites of infection (Kelly et al., 2005). However, IL-17 can play a pathogenic role in Toxoplasma infection. Thus, mice that are unable to down regulate IL-17 production due to the absence of IL-27 signaling develop severe neuroinflammation (Stumhofer et al., 2006). A variety of cells with regulatory function also have an impact on the outcome of the infection with intracellular protozoa. These include CD41 T regulatory cells expressing Foxp3, Th1 cells making IL-10, as well as macrophages making IL-10 or TGF-b. In the case of Leishmania, T regulatory and IL-10 producing T cells, as well as macrophages producing IL-10 promote increased susceptibility to the parasites (Anderson et al., 2008; Belkaid et al., 2002; Maroof et al., 2008; Miles et al., 2005) (see chapter 35). Thus, IL-10 deficient BALB/c mice become resistant to L. major (Kane & Mosser, 2001), and following infection of IL-10 deficient C57BL/6 mice with low doses of parasites the immune response is able to eliminate all of the parasites (Belkaid et al., 2002); IL-10 is also important in human leishmaniasis, where nonhealing infections have also been associated with IL-10 (Nylen & Sacks, 2007). On the other hand, while Toxoplasma and T. cruzi infected IL-10 deficient mice control parasite replication efficiently, these mice succumb due to a pathologic immune response associated with toxic levels of TNF-a, IFN-g, and IL-12 (Gazzinelli et al., 1996; Hunter et al., 1997). Thus, depending on the parasite, the balance between a protective immune response and immunopathology can be easily disrupted. The fact that L. major parasites do not induce such severe immunopathology in IL-10 deficient mice may be an indication that they are less able to induce proinflammatory cytokines than either Toxoplasma or T. cruzi. The differential development of Th1 and Th2 cells following infection of different strains of mice with L. major

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has been very useful in exploring how T cell subsets develop and are regulated, although as discussed above factors other than a dominant Th2 response may be important in susceptibility to L. major. In addition, immune responses to other species of Leishmania clearly differ from those seen in mice infected with L. major. For example, mice that normally resolve L. major infections develop chronic infections with New World species (such as L. mexicana or L. amazonensis) (McMahon-Pratt & Alexander, 2004). This susceptibility is not due to an exaggerated Th2 response, but due to the inability to induce a strong Th1 response. Similarly, susceptibility to L. donovani, the agent causing fatal infection in humans, is not associated with a Th2 response (Kaye et al., 2004). Taken together, these observations demonstrate that multiple mechanisms may promote increased susceptibility to Leishmania. What is consistent, however, is that protection is uniformly associated with IFN-g mediated immunity. Infection with Leishmania, Toxoplasma, and T. cruzi is associated with substantial resistance to reinfection. This immunity involves the generation of memory T cells, as well as the maintenance of effector cells due to the persistence of low numbers of parasites once the disease has resolved. For example, following L. major infection, a pool of antigen-reactive central memory CD41 cells are generated that can be maintained in the absence of parasites (Zaph et al., 2004). These cells provide some level of protection, although the presence of persistent parasites appears to be important for maintaining optimal immunity (Belkaid et al., 2002). When mice are infected with T. cruzi, they also generate a population of stable central memory CD81 T cells, which can be maintained without parasites (Bustamante et al., 2008). These results suggest that at least some memory T cells can be maintained without persistent parasites, an important point in considering the development of a vaccine. However, under normal conditions, individuals who have resolved an infection with Leishmania, Toxoplasma, or T. cruzi have low levels of parasites that persist long-term. Thus, the immunity observed in these individuals is more akin to the concomitant immunity observed in tumor systems, and is most likely mediated by effector T cells that are continually generated from either naïve T cells or central memory T cells by the presence of persistent parasites (Fig. 1).

Activation of Macrophages and Dendritic Cells

The key cytokine important for resolution of leishmaniasis, American trypanosomiasis, or toxoplasmosis is IFN-g, and although IFN-g plays many roles in the immune response, its major function in this context is the activation of macrophages (and DCs) to exhibit increased killing capacity. This activation is enhanced by other cytokines (such as TNF-a) and TLR ligands. IFN-g regulates a large number of genes, which control several different pathways associated with microbicidal activity. The induction of nitric oxide synthase (iNOS, NOS2), leading to nitric oxide production, is a major pathway leading to parasite elimination. However, other molecules also contribute to killing intracellular pathogens. For example, one of the most studied has been NADPH phagocyte oxidase, mediating the respiratory burst associated with the generation of several toxic oxygen molecules, including superoxide anion, hydrogen peroxide, and peroxynitrite. These molecules can synergize with NO to eliminate intracellular parasites. Another important family of molecules associated with macrophage killing are the immunity-related GTPases (IRGs; also known as p47 GTPases) (Taylor et al., 2004). Studies in knock out mice lacking IRG proteins indicate that several of them are

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FIGURE 1 Maintenance of immunity in the presence of persistent parasites. Several protozoal infections resolve their disease without eliminating all of the parasites, but nevertheless contain the parasites at low numbers and are resistant to reinfection. This resistance can be maintained by the continual generation of effector T cells from naïve T cells, a central memory pool of T cells, as well as from resting effector T cells.

required to control Toxoplasma, Leishmania major, and Trypanosoma cruzi (Taylor et al., 2004). How they mediate their protective role is still an area of active investigation, but the localization of the IRG proteins to parasite-containing vacuoles suggests that they may be acting to disrupt the parasite environment within the host cell (Zhao et al., 2009). In addition to these induced immune responses, innate differences in macrophage function can modulate the outcome of infection. This is best illustrated by the mutation in the proton efflux pump, termed Nramp1 (Slc11A1), which leads to increased susceptibility to several intracellular parasites including Leishmania (Blackwell et al., 2003). While most of our understanding of the mechanisms associated with killing intracellular parasites comes from studies with macrophages, other cells play a role. Indeed, recent studies suggest that many of the cells infected with Leishmania are TNF-iNOS producing DCs, rather than macrophages (De Trez et al., 2009). Moreover, since Toxoplasma and T. cruzi can invade many different cell types, other mechanisms can also contribute to control of these organisms. There are several ways that intracellular parasites avoid being eliminated by activated phagocytes. For example, in the case of L. major infections in BALB/c mice, a dominant Th2 response develops, with IL-4 and IL-10 inhibiting classical macrophage activation. Alternatively, other Leishmania parasites may fail to induce sufficient Th1 cells, and thus fail to achieve sufficient levels of IFN-g to eliminate the parasites. However, even when a strong Th1 response is induced by intracellular parasites, there are mechanisms that dampen the microbicidal activity of infected cells. For example, infection of macrophages with Leishmania leads to inhibition of many cellular functions, including IFN-g inducible signaling pathways (see chapter 36). As discussed above, the induction of regulatory cells producing immunosuppressive cytokines (such as IL-10 and TGF-b) contribute to the chronicity of these infections. Moreover, while TLR activation induces the development of classically activated macrophages, expression of the arginine hydrolytic enzyme arginase 1 (Arg1) is simultaneously induced; the significance of the presence of Arg1 in modulating the immune response to Toxoplasma was shown in mice where it was deleted, which led to more efficient control of the parasites (El Kasmi et al., 2008). Finally, the ability of infected cells to be activated depends upon interactions between Th1 cells making IFN-g and the infected

cell, and, using intravital imaging, it was recently shown that in leishmanial lesions not all infected cells interact with infiltrating T cells (Filipe-Santos et al., 2009). Why this is the case is unclear, but it provides one explanation for why parasites may fail to be completely eliminated in the face of a robust Th1 response.

Other Effector Mechanisms Controlling Intracellular Protozoa

An understanding of how Leishmania, Toxoplasma, and T. cruzi are controlled by the adaptive immune response leads to the conclusion that IFN-g mediated activation of infected phagocytic cells is critical. While CD8 T cells with the potential to be cytolytic are generated in all of these infections, the evidence suggests that their primary role is associated with the production of IFN-g. In leishmaniasis, while CD8 T cells are important in controlling low dose infections, IFN-g deficient CD8 T cells are not protective (Scott et al., 2004). Similarly, in Toxoplasma infections the importance of IFN-g, rather than perforinmediated cytolysis, was demonstrated in adoptive transfer experiments (Wang et al., 2004), and even when Toxoplasma infected cells were lysed by cytotoxic T lymphocytes, the parasites were not killed (Yamashita et al., 1998), further indicating that this pathway may not be important in CD8 T-cell function with these infections. Each of these infections induces a substantial antibody response. In Toxoplasma infections, antibody contributes to the long-term control of the parasites (Frenkel & Taylor, 1982), and participates in vaccine-induced immunity, although the importance of antibody is secondary to cellular responses (Sayles et al., 2000). Similarly, in the chronic phase of T. cruzi infection, antibodies are believed to help control the trypomastigotes circulating in the blood (Brener & Gazzinelli, 1997). However, in the case of Leishmania, antibodies not only do not appear to play any protective role during any stage of the infection, but, in fact, may play a detrimental role. Thus, as discussed above, when Leishmania parasites are opsonized by antibody and taken up by macrophages, IL-10 production is promoted (Miles et al., 2005). The importance of this in vitro observation is demonstrated in studies with FcR deficient mice, which exhibited increased resistance to Leishmania (McMahon-Pratt & Alexander, 2004; Scott et al., 2004).


MALARIA

Naturally acquired pre-erythrocytic immunity (i.e., immunity to sporozoites and liver stages) in humans is slow to develop and incomplete as evidenced by the high rates of blood stage infection in people in living in endemic areas, the very slow age-related decline in parasitemia, and the fact that even adults living in highly endemic areas will periodically develop low levels of blood stage infection. By comparison, immunity to blood stages appears to be more efficient and develops more quickly, such that immunity to the very severe consequences of infection (severe anaemia, metabolic acidosis, and cerebral malaria) is typically achieved within the first 5 to 8 years of life in highly endemic settings and all episodes of clinical malaria become less frequent and less debilitating with increasing age and are accompanied by markedly lower parasite densities. Typically, adults in endemic areas are able to limit parasite densities to levels below the threshold for triggering a clinical episode. Acquired immunity to malaria is frequently described as being either antiparasite immunity, encompassing effector mechanisms that kill and clear parasites or parasite-infected cells, or clinical (or antitoxic) immunity (i.e., mechanisms that minimize the inflammatory immune response that contributes to malaria pathogenesis without necessarily removing infected cells) (Fig. 2). Infected hosts also generate “transmission blocking” immune responses to antigens expressed on gametocytes—the mosquito infective stage of the life cycle—that act within the mosquito gut to prevent differentiation and development of sexual stage parasites. It is widely believed that acquired immunity to malaria rapidly wanes in the absence of frequent reinfection. In fact, a recent extensive review of the evidence suggests that antiparasite immunity is long lived but that clinical immunity may be quite short lived, such that previously immune individuals who become infected after

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spending long periods in nonendemic areas may develop mild-to-moderate symptoms of malaria despite having very low parasitemia; importantly, their risk of dying or developing severe complications of infection are very low (Struik & Riley, 2004).

Initiation of Immunity

As for other protozoa, malaria parasites encode ligands for pathogen-recognition receptors, which trigger activation of dendritic cells and macrophages and initiation of antigen presentation to T cells. For intraerythrocytic parasites, these ligands include glycosylphosphatidylinositol (GPI), which forms the membrane anchor of many parasite surface proteins and which is a ligand for TLR2/MyD88 (Gowda, 2007) and either hemozoin, which is an insoluble ironcontaining crystalline by-product of hemoglobin digestion, or microbial DNA trapped within the hemozoin, which triggers TLR9 (Parroche et al., 2007) and the Nalp3 inflammasome (Dostert et al., 2009) (see chapter 18). PRR (pattern recognition receptor) ligands for other life cycle stages of the parasite have not yet been identified, and it is possible that the lack of potent PRR ligands may explain the lack of inflammation induced by sporozoites, liver stages, and gametocytes and their comparatively poor immunogenicity. Alternatively, the relatively poor immunogenicity of sporozoites, liver stages, and gametocytes may result from the very small amounts of protein that are presented to the immune system. For example, each mosquito bite delivers only tens or hundreds of nonreplicating sporozoites, which, in turn, generate tens or hundreds of infected liver cells, but it is comparatively easy to induce high levels of protective immunity to malaria by injecting (by needle or mosquito bite) tens of thousands of radiation-attenuated sporozoites (Epstein et al., 2007).

FIGURE 2 Age/transmission-dependent acquisition of antimalarial immunity. Immunity to malaria is acquired gradually over time; in endemic areas, this means that immunity is acquired as a function of age (number of years exposed). Resistance to severe disease and death is acquired most rapidly. Immunity to mild clinical disease is acquired next and correlates with the induction of immune responses that limit parasite density. Immunity to infection, per se, as evidenced by less frequent episodes of asymptomatic, low density infection, is eventually acquired but in most individuals is only partial, meaning that episodes of infection can persist throughout life but rarely cause clinical signs or symptoms. Different immune mechanisms are believed to provide clinical and antiparasitic immunity (see text). Diagram adapted from original by B. M. Greenwood, London School of Hygiene and Tropical Medicine.

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Two TCR-transgenic mouse lines, one for an MHC class I/CD81 T-cell-restricted epitope from the major surface protein, the circumsporozoite (CS) protein, of P. yoelii sporozoites and one for an MHC class II/CD41 T-cell-restricted epitope from P. chabaudi merozoite surface protein (MSP)-1 are allowing very detailed dissection of the processes leading to CD81 and CD41 T-cell priming in liver and blood stage infections. Key observations for CD81 T cells that confer immunity by killing parasites in the liver include: (i) naïve CS-specific T cells can acquire effector function within 48 hours of infection (Sano et al., 2001); (ii) these activated CD81 T cells inhibit further antigen presentation and T-cell expansion by killing DCs (Hafalla et al., 2003); (iii) IL-4 signaling is essential to maintain liver stage specific memory CD81 T cells (Morrot et al., 2005); and (iv) CD81 T-cell effector function does not require IFN-g (Chakravarty et al., 2008) (see chapter 46). Priming of CS-specific T cells occurs in the lymph nodes draining the site of the mosquito bite (Chakravarty et al., 2007). Similarly, for blood stage immunity, we now know that MSP-1specific CD41 T cells provide help for antibody production, confer protection in an antibody-dependent manner, and that CD11c1 CD81 and CD82 DC populations provide distinct signals for T-cell differentiation. The same model has revealed that only CD82 DCs are able to induce the IL-4- and IL-10-secreting T cells, which are associated with the switch from the Th-1-dominated to the Th-2/ Tr-1-dominated response that is linked to successful resolution of blood stage infection (Sponaas et al., 2006; Stephens et al., 2005). Priming of adaptive responses to blood stage malaria occurs in the spleen, but gradual accumulation of hemozoin-containing macrophages leads to destruction of the normal splenic architecture (Achtman et al., 2003) and may exacerbate the problems of DC–T cell and T cell–B cell communication described below. Although blood stage malaria infections do induce adaptive T- and B-cell responses, there is a significant body of evidence—most convincingly from murine malaria infections—that antigen presentation becomes increasingly impaired as the infection progresses (see chapter 35). DCs actively phagocytose P. chabaudi infected red blood cells (iRBCs) leading to DC maturation, IL-12 secretion, and CD41 T-cell activation (Ing et al., 2006; Sponaas et al., 2006) but accumulation of hemozoin subsequently, and most evidently at the time of peak parasitemia, down modulates DCs leading to reduced T-cell proliferation, migration, and cytokine secretion. Impaired DC–T cell interactions have been demonstrated by live cell imaging and suggest that lack of costimulation may be the primary defect (Millington et al., 2007), but there is also evidence for inhibition of cross-presentation of heterologous antigens by malariaexposed DCs (Wilson et al., 2006). As a consequence, initiation of primary immune responses to irrelevant antigens is markedly impaired during the course of an acute malaria infection. However, it is unlikely that a temporary inability to initiate primary immune responses explains the more generalized immune suppression associated with clinical malaria, and research is ongoing to identify the causes of, for example, the increased susceptibility of malaria patients to secondary bacterial infections. In human systems, incubation of DCs with RBCs infected with P. falciparum clones that induce adherence of iRBC to the DC (via binding to CD36 or thrombospondin) reduces their subsequent response to LPS (lipopolysaccharide) (Urban et al., 1999) and this has been interpreted as evidence of malaria-induced immunosuppression, however, the long incubation times (.48h) in these experiments make it impossible to know whether the inhibited state is

preceded by a state of activation (as might be expected for DCs). Analysis of plasma cytokines in individuals undergoing experimental malaria infections reveals up regulation of cytokines such as IL-6, IL-12, and TNF within hours of first detection of parasite RNA in the circulation, indicating effective activation of macrophages or DCs, although it was noted that after ex vivo stimulation of blood mononuclear cells with P. falciparum iRBC, the vast majority of IL-12 secreting cells were macrophages rather than DCs (Walther et al., 2006). Hemozoin is known to activate some human monocyte functions and to suppress others (Schwarzer et al., 2008); again, the timing of these events is likely to be crucial with the gradual accumulation of hemozoin leading to a progressive loss of monocytic inflammatory potential.

Cell-Mediated Effector Mechanisms

Both CD41 T cells and CD81 T cells are known to play important roles in antimalarial immunity. CD81 T cells are believed to be mainly involved in immunity to liver stage parasites, whereas CD41 T cells likely play major roles in both liver and blood-stage immunity (Fig. 3). As described above, the CS protein is a known target of CD81 T cells; a second sporozoite surface protein (SSP2, more commonly known as thrombospondin-related adhesive protein or TRAP) and liver stage specific antigen-1 (LSA-1) are also well-documented targets for cell-mediated immunity. It was initially assumed that CD81 T cells functioned as cytotoxic T lymphocytes (CTLs), directly binding to malarial peptide/MHC class I complexes on the surface of the infected hepatocyte and either lysing the cell (in a perforin- and granzyme-dependent manner) or inducing the hepatocytes to undergo apoptosis (Hoffman et al., 1989), but a study showing that elimination of P. berghei liver stages was independent of both of these pathways (Renggli et al., 1997) caused the focus to switch to IFN-g, IL-12, and NO-mediated mechanisms (Doolan & Hoffman, 2000). Recent studies however have provided evidence that both IFN-g-producing CD81 T cells (Jobe et al., 2007) and classical CTLs (Trimnell et al., 2009) are involved in cellmediated immunity to liver stage parasites. In some, but not all, mouse-parasite strain combinations, CD41 T cells can contribute to, or are essential for, liver stage immunity and, in some strain combinations, CD41 T cells are required during the induction phase of the response (Doolan & Hoffman, 2000); these marked differences between mouse strains in the mechanisms of immunity suggest that similarly diverse mechanisms of liver stage immunity are likely to confer protection after infection/vaccination of outbred human populations, making it difficult to identify robust biomarkers of protective immunity. Overcoming the poor immunogenicity of subunit (recombinant protein or synthetic peptide) sporozoite and liver stage antigens has been a major focus of pre-erythrocytic stage vaccine research for more than 20 years, with limited success leading researchers to revert to attenuated live sporozoites in the hope of making a vaccine that is both protective and logistically feasible (Speake & Duffy, 2009). As with pre-erythrocytic stages of malaria infection, the exact mechanisms of acquired protective immunity to blood stages are unclear, although the general consensus is that IFN-g from CD4 T cells induces macrophage activation and that the phagocytic capacity of these macrophages is enhanced by opsonization of parasites or iRBC by specific antibody. In truth, however, clear evidence of a protective role for IFN-g is limited to studies in the P. chabaudi mouse model, where parasite clearance is markedly delayed in the absence of IFN-g (Stevenson et al., 1990; van der Heyde et al., 1997). In P. yoelii infections, parasite clearance is


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FIGURE 3 Antimalarial immune mechanisms. The immune effector mechanisms that are believed to confer immunity to different parasite life cycle stages are shown. This summary is based on the weight of evidence from very large numbers of experimental studies in animal models, in vitro studies with human cells, and immunoepidemiological studies. In no case is the actual mode of protection definitively known and there are currently no absolute immune correlates of protection against malaria infection or disease.

unaffected in some IFN-g-deficient animals (Couper et al., 2007) but seems to be required in others (van der Heyde et al., 1997), and ablation of IFN-g pathways has a similarly variable effect in P. berghei infections (Waki et al., 1995; Greig, Riley, & Couper, in preparation). On the other hand, there is considerable evidence from mouse models that overproduction of IFN-g is harmful, leading to weight loss, anemia, liver damage, and a neurological syndrome that shares many of the key features of human cerebral malaria (Artavanis-Tsakonas et al., 2003; de Souza & Riley, 2002). Blood stage malaria vaccines designed to induce strong, CD41 T-cell-mediated IFN-g secretion have been associated with severe pathology (e.g., Makobongo et al., 2003). It is now abundantly clear that acquiring the ability to regulate CD41 and CD81 effector T-cell responses to blood stage malaria antigens, by production of “antitoxic” antibodies (see below) or by induction of regulatory populations of T cells, is a key aspect of a protective immune response (Riley et al., 2006). In both mice and humans, IL-10 seems to be the most important regulatory cytokine and, during acute infection at least, is derived from Tr-1 or Th-1 cells rather than from classical endogenous Foxp31 T-regulatory cells (Couper et al., 2008; Walther et al., 2009) (see chapter 35).

Antibody-Mediated Immune Effector Mechanisms

Despite being intracellular for much of their life cycle, malaria parasites are not totally invulnerable to the effects of antibodies (Fig. 3). Sporozoites are extracellular, and thus exposed to antibodies during their migration from the connective tissue of the dermis and subdermis via the lymph and/or blood to the liver. Although extracellular for very short periods of time (a few seconds at most), the numerous evasion strategies that have evolved suggest that merozoites

are also susceptible to antibodies. Just as importantly, however, the parasite needs to modify its host cell by exporting to the erythrocyte surface membrane an array of parasiteencoded proteins that modify the permeability, deformability, and adhesive characteristics of the RBC to facilitate parasite growth, differentiation, and sequestration in deep tissues (Maier et al., 2009). These parasite-encoded proteins represent potential targets for antibody-mediated destruction or clearance of infected cells. In humans, antibodies to the CS protein are naturally acquired in response to infection, but although the prevalence of antibodies and their average titer increase with age and increasing exposure to malaria (Hoffman et al., 1986), both prevalence and mean titers of anti-CS protein antibodies are much lower than for antibodies to blood stage parasite antigens, indicating that the CS protein is naturally rather poorly immunogenic. In rodent models of malaria infection and sporozoite vaccination, antibodies to the CS protein and to the other major sporozoite surface protein, TRAP, can immobilize sporozoites in the skin (Vanderberg & Frevert, 2004), preventing them from reaching blood vessels and leading instead to their clearance through the lymphatic system. It was demonstrated many years ago, however, that antibody cross-linking of the surface antigens of sporozoites can lead to sloughing of the surface coat proteins (the so-called circumsporozoite precipitation reaction) (Potocnjak et al., 1980), which may offer a means for the parasite to evade the effects of antibodies. While there is in vitro evidence that human antibodies can inhibit sporozoite invasion of liver cells (Hollingdale et al., 1984) and such antibodies can be induced by vaccination (Okitsu et al., 2007), only those human sera with extremely high titers of naturally acquired antisporozoite antibodies were

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able to mediate this effect (Hoffman et al., 1986), suggesting that it is not a particularly effective mechanism of naturally acquired immunity. Anti-CS antibodies can opsonize sporozoites, mediating their phagocytosis by human monocytes (Schwenk et al., 2003). Recent immunoepidemiological data indicate that children with high titers of IgG antibodies to the CS protein, TRAP, and a liver stage antigen, LSA-1, are less susceptible to P. falciparum infection than children who possess lower levels of these antibodies; importantly, significant resistance was only seen in children with high titers of antibodies to all three antigens, suggesting that these antibodies act via different and synergistic mechanisms (John et al., 2008). Merozoites, the invasive form of blood stage parasites, are less vulnerable to antibody-mediated immune effector mechanisms than are sporozoites, as they are extracellular for a much shorter period of time (seconds rather than minutes). Nevertheless, the erythrocyte invasion process is complex, with numerous different molecular interactions involved in attachment to the red cell membrane, orientation of the apical complex towards the point of attachment, and invagination of the erythrocyte membrane (Cowman & Crabb, 2006). Each of the many parasite molecules involved in this process is a potential target for antibodies that interrupt essential receptor-ligand interactions, preventing merozoite entry into the erythrocyte and allowing them to be taken up by macrophages. Antibodies to these various invasion-associated antigens are commonly found in serum from malaria exposed individuals—often at quite high titers—but there is little direct evidence that they are able to block invasion. Affinity purified human antibodies (Egan et al., 1999) or antibodies induced by vaccination (Miura et al., 2009) have been shown to inhibit growth of erythrocytic parasites in vitro and this correlates with protective efficacy of the vaccine in some animal studies (Singh et al., 2006). Assessing the invasion inhibitory capacity of naturally acquired antibodies in human serum is more difficult (as the serum contains antibodies to many different malaria antigens), but assays using transgenic P. falciparum parasites in which the protein of interest has been replaced by a functionally homologous but antigenically distinct protein from another malaria species are beginning to facilitate these investigations (John et al., 2004). Monoclonal antibodies to various merozoite surface or apical complex proteins are able to block erythrocyte invasion in vitro and are useful tools for dissecting the epitope specificity of, and the effector functions mediated by, protective antibodies. A particularly exciting example of this approach is the recently reported study in which human chimeric IgG1 and IgG3 antibodies have been engineered to contain the variable regions from mouse monoclonal P. falciparum MSP1-specific antibodies with known invasion inhibitory activity (Lazarou et al., 2009). The IgG1 antibodies, but not the IgG3 antibodies, induced NADPH-mediated oxidative bursts and degranulation of human neutrophils. In addition to inhibition of erythrocyte invasion, antibodies to merozoites or erythrocyte-associated proteins opsonize parasites for phagocytosis by monocyte/macrophages; human IgG3 antibodies have consistently been found to be more effective than other IgG subclasses, and the extent of opsonization has been linked to protective immunity (Groux & Gysin, 1990; Tebo et al., 2002). Opsonization and phagocytosis are good examples of synergy between cellular and humoral arms of the immune response as activation of monocyte/macrophages by cytokines such as IFN-g enhances their ability to engulf antibody-coated iRBC; for example, FcRg-mediated, antibody-dependent phagocytosis of P. berghei iRBC is seen in wild-type but not in IFN-g-deficient animals (Yoneto et al., 2001).

Parasite-encoded erythrocyte surface proteins appear to be highly immunogenic with antibodies specific for the infecting clone of parasites being induced after a single infection. In vitro, antibodies to iRBC surface-expressed proteins block adherence of iRBCs to vascular endothelial cells (recently reviewed by Hviid, 2010) and it is widely assumed that this prevents mature, schizont-infected RBCs sequestering in postcapillary venules of vital organs where they contribute to the pathology of malaria infection. Phagocytosis of opsonized iRBCs is also observed. Infected cells that are unable to sequester are more vulnerable to clearance (e.g., in the spleen); avoidance of this antibody response has driven the evolution of antigenic polymorphism and clonal antigenic variation such that incoming infections are not recognized by preexisting antibodies (Hviid, 2010). The best evidence that antibodies to clonally variant surface antigens are associated with protective immunity comes from studies in African children where preexisting antibodies to the variant expressed on a current malaria infection were linked to a reduced risk of developing severe malaria (Bull et al., 1998), and from pregnant women in whom the presence of antibodies to particular antigenic variants that confer the ability of iRBCs to sequester in the placenta are associated with resistance to the syndrome known as pregnancy-associated malaria (Hviid, 2004). Antibodies to circulating, intraerythrocytic gametocytes (the sexual stages of malaria parasites) are induced in mammalian hosts and are taken up by mosquitoes as they feed. In the mosquito midgut, the gametocytes emerge from their protective host cell membrane and are exposed to these serum antibodies. Serum IgG antibodies to gametocyte surface proteins induce complement-mediated lysis of gametes (Healer et al., 1997) and complement fixing isotypes of antigamete antibodies in immune human serum are linked to blocking of parasite differentiation within the mosquito (Healer et al., 1999; Roeffen et al., 1996).

CONCLUSIONS

Intracellular protozoan parasites continue to cause devastating diseases in a large number of people. And in spite of a substantial amount of work, a successful vaccine for any of them remains an elusive goal (see chapter 46). Why is this the case? There are probably several answers, and certainly the complexity of these organisms contributes to the problem. However, for those intracellular parasites of phagocytes, failure may largely be due to the fact that success may require inducing and maintaining a strong cell-mediated immune response, rather than relying solely on antibodies. Unfortunately, at a fundamental level, how memory for cellmediated responses is induced or maintained is not clear. Even in the case of malaria, where both humoral and cellmediated immune responses are important for resistance, the problem may lie not only with understanding the biology of the parasites, but with limited understanding of how long-term immunity is generated. Thus, it is likely that it will be from studies that focus on the biology of the parasites and the host immune responses, both in experimental animals and in humans, that success in developing vaccines for these parasites is likely to be found.

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25 Acquired Immunity to Helminths DAVID ARTIS AND RICK M. MAIZELS

INTRODUCTION

immune components will help us achieve the optimal balance of immune responsiveness to maximize immunity while avoiding deleterious immunopathological consequences.

Helminth parasites are multicellular worms belonging to the Nematode, Trematode, and Cestode taxa which have coexisted with the vertebrate immune system for a long evolutionary time. Consequently, an impressive array of host innate and adaptive immune mechanisms have developed to control helminth infections and the pathologies they engender. Despite these defenses, a number of important parasitic helminths have become adept at evading immunity and continue to pose major health problems in humans and animals. In humans, the pattern of helminth infections amply illustrates the key characteristics of these parasites and the courses of disease they cause. Most species do not multiply in the mammalian host, and helminth infections accumulate through repeated transmission, reflecting both chronic susceptibility and remarkable parasite longevity (often .10 years). Infections can reach very high prevalence, particularly among children, and even today over 25% of the human population carries one or more helminth parasite species (Hotez et al., 2008). Infections are often overdispersed, with a small number of highly infected carriers, and polymorphisms controlling susceptibility or resistance to helminth infections are now being identified (Quinnell, 2003). In this chapter, we focus on adaptive immunity to helminth parasites, first in the context of naturally acquired immunity in human populations, and subsequently in terms of experimental studies. Laboratory models for the study of protective immunity, in both intestinal tract or tissue systems, present common and specific features; in general, the Th2 arm of cellular immunity is seen to orchestrate the antihelminth immune response, acting through pivotal inducer and activating cytokines including IL (interleukin)-4, IL-5, IL-9, and IL-13. These mediators promote effector mechanisms ranging from antibodies, macrophages, and granulocytes, to epithelial cell responses that have less prominence in microbial and protozoal infections (Anthony et al., 2007). In the final part of the chapter, we will consider how understanding the different roles of key

IMMUNITY TO HELMINTH PARASITES IN HUMANS

The strongest indication that acquired immunity to helminth infections may develop in humans comes from the changing patterns of infection with age. Peak intensities of schistosome infection, for example, are observed in juveniles, whereas older individuals are more likely to have low-level or undetectable infections (Bundy & Medley, 1992). Moreover, the higher the level of transmission in a community, the younger the age of peak infection intensity, implying that immunity is acquired following a certain quantum of exposure to the parasite (Woolhouse, 1992). While intensity declines with age in all common helminth infections, prevalence is often asymptotic, indicating a degree of acquired immunity that limits the survival of new waves of parasites and/or reduces the fecundity of those that are able to establish themselves. With each helminth species following different developmental and migratory pathways within the host and using different transmission life history strategies, unanswered questions remain about the targets and locales for protective immunity in humans and how partial immunity may act to limit, but not eliminate, the presence of parasitic organisms. The most common group of human helminth parasites are the soil-transmitted geohelminths, principally the gastrointestinal nematodes Ascaris, hookworm (Ancylostoma and Necator species), and Trichuris (Hotez et al., 2008). These infections show the classic age-intensity profile, which declines after the childhood years (Bundy & Medley, 1992). To explore whether intensity is related to immune responsiveness in humans, Turner and colleagues (2003) measured cytokine responses of peripheral blood T cells challenged with Ascaris antigen among individuals above the age of peak intensity. Within this subgroup, intensity (measured by fecal egg counts) was inversely related to the strength of the Th2 cytokine (IL-4, IL-9, IL-10, and IL-13) response. The same investigators also conducted treatment/ reinfection studies, in which therapeutic drug clearance of gastrointestinal helminths was performed and worm burdens were assessed 8 to 9 months later, after natural reacquisition

David Artis, Department of Microbiology, University of Pennsylvania School of Medicine, Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, 314 Hill Pavillion, 380 South University Ave., Philadelphia, PA 19104-4539. Rick M. Maizels, Centre for Immunity, Infection and Evolution, and Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, EH9 3JT, United Kingdom.

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of infection from the contaminated environment (Jackson et al., 2004). In this setting, the degree of resistance to reinfection with either Ascaris or Trichuris again correlated with Th2 cytokine responses (in this case, IL-5 and IL-13), as well as with the IL-4-dependent IgE isotype (Bradley & Jackson, 2004). Schistosomes are the most prevalent tissue-dwelling helminths of humans, with adult worms residing in the mesenteric vasculature (e.g., S. japonicum, S. mansoni) or the bladder wall (e.g., S. haematobium). Again, Th2 immunity appears to be critical in minimizing worm burdens, as first indicated by IgE and eosinophil studies, and subsequently by cytokine assays (summarized by Walter et al., 2006). In classic treatment/reinfection studies, those most resistant to reinfection following chemotherapy had significantly higher IgE levels in both S. haematobium (Hagan et al., 1991) and S. mansoni (Dessein et al., 2004) endemic areas. Eosinophils, a characteristic feature of the Th2 response to helminths, have long been associated with enhanced killing of larval schistosomes (Capron et al., 1982), with recent studies demonstrating greater immunity in eosinophilic humans (Ganley-Leal et al., 2006). At the cytokine level, Th2 polarization is evident in resistant individuals (assessed through T-cell clones specific for larval schistosome antigens), while susceptible patients’ cells are more IFN-g/Th1 biased (Dessein et al., 2004). The importance of IL-13, in particular, also emerges from genetic studies, in which polymorphisms raising the level of IL-13 responsiveness are associated with greater resistance to schistosomal infection (Dessein et al., 2004). In lymphatic filariasis and onchocerciasis, adult parasites reside in the lymphatics and skin respectively, while releasing microfilariae for onward transmission through bloodfeeding vectors. Immunity to filarial nematodes is not readily apparent in human populations, for two interrelated reasons: responsiveness is greatly dampened by these parasites, and most of the exposed population carry infection for a long period of their life. In general, infection intensities increase through adolescence, but reach a stable plateau level of infection once the peak is attained (Day et al., 1991). This illustrates an intriguing example of “concomitant immunity” in which the existing adult worm load is tolerated but new waves of invading larval parasites are thought to be eliminated. In individuals with detectable circulating microfilariae, suppression of T-cell reactivity can be profound (Maizels & Yazdanbakhsh, 2003), so that both Th1 (IFN [interferon]-g) and Th2 (IL-5) signature cytokine responses to parasite antigens are lost (Sartono et al., 1997). Reactive individuals, however, may progress to dermal or lymphatic pathology, which has very recently been linked to heightened antiparasite Th1 and Th17 responses (Babu et al., 2009), suggesting by default that Th2 immunity may be the most beneficial to protect humans from filarial nematode infection. Overall, these studies show a convincing quantitative protective effect of Th2 immunity in suppressing hyperinfection in endemic populations, accompanied by some uncertainty of how, if ever, sterile immunity to helminths may be achieved. But the question is raised of why Th2 immunity fails to be sufficiently mobilized in so many individuals to attain an immune status. Part of the answer lies in the ability of parasites to profoundly modulate the host immune system, so that immunity is only expressed after a long-haul process of overcoming parasite immunoregulation. The restoration of peripheral T-cell responsiveness to parasite antigens after curative drug therapy indicates that live parasites actively down modulate host immune capacity. Interestingly, IL-10 responses are not reduced and indeed antibodies to IL-10 or TGF-b (transforming growth factor b) are able to rescue the in vitro proliferative response of T cells from infected

patients (King et al., 1993). Moreover, one of the few immune responses to be greatly enhanced in filariasis and schistosomiasis is production of IgG4 isotype antibodies, which are known to be promoted by IL-10 (Satoguina et al., 2005). In the treatment/reinfection studies mentioned above, individuals with high IgG4:IgE ratios did not display immunity to reinfection, arguing that helminth-induced IgG4 will act as a blocking antibody to protect parasites from immune attack. Both IL-10 and TGF-b are also closely linked to regulatory T cell (Treg) activity, which is now becoming more evident in a number of human helminth infections (e.g., in filariasis [Babu et al., 2006]). It is possible that Tregs act directly to impede immune clearance of parasites (as indicated in animal models discussed below), and that an unfavorable excess of Tregs in human infection may take a long time to correct. Alternatively, the action of Tregs may primarily be to limit the pathological consequences of infection, as suggested by the finding that lymphatic filariasis patients who progress to inflammatory sequelae such as lymphedema have reduced Treg expression (Babu et al., 2009). Resolution of these questions will be critically important in designing future intervention studies to promote protective immunity in humans.

ANIMAL MODELS OF HELMINTH INFECTION

Our understanding of acquired immunity to helminths is based on a rich substrate of animal models, most importantly, the mouse (Table 1). Some human pathogens, including S. mansoni, are fully infective in murine models, which reproduce many of the clinical features of infection such as hepatic fibrosis. While many other human parasites (such as the common intestinal worms) are not infective to rodents, closely related species are available that parasitize mice and therefore provide excellent model systems. As will be detailed below, studies across a range of helminth infections have uncovered specific mechanisms that are important in different settings; hence, while some general features of protective immunity to helminths can be identified, the exact combination of factors that mediate elimination differ between the various species in question. Arguably, the most important factor in dictating which immune mechanisms are in play is the host tissue niche occupied by the parasite. At this stage, our understanding of the mechanisms of immunity is still relatively fragmented. Studies using genetic deletion or blockade of cytokines and their receptors have helped delineate which components are required for immunity and which are dispensable. Advancing towards a more holistic picture of the most effective combinations requires more painstaking work and more consideration of the physiological settings in which protective immunity will be required to operate in vivo.

ACQUIRED IMMUNITY IN THE GASTROINTESTINAL TRACT CD41 Th2 Cell-Dependent Immunity to Intestinal Nematode Parasites

As discussed in chapters 19 to 24, immune-mediated control of viral, bacterial, fungal, and protozoan pathogens is associated with the development of pathogen-specific CD41 or CD81 T-cell responses that are characterized by either the production of proinflammatory cytokines (including IFNg, IL-17A, and TNF [tumor necrosis factor]a) and/ or direct cytotoxic activity. In contrast, studies in murine model systems have identified a critical role for CD41 Th2 cells that produce IL-4, IL-5, IL-9, and IL-13 in resistance to intestinal nematode infection. The results of these studies

25. Acquired Immunity to Helminths TABLE 1

Major human helminth parasites and corresponding model systems

Species

Common name; disease

Taxon

Human infections

Ancylostoma caninum/ Necator americanus

Hookworms; anemia

Nematoda

580 million

Ascaris lumbricoides

Common roundworm; ascariasis

Nematoda

800 million

Brugia malayi/ Wuchereria bancrofti

Filariasis; Nematoda elephantiasis

120 million

Echinococcus granulosus/E. multilocularis

Hydatid cyst; echinococcosis

Cestoda

.3 million

Onchocerca volvulus

River blindness; onchocerciasis

Nematoda

20 million

Schistosoma Schistosomiasis; Trematoda mansoni/ Bilharzia S. japonicum/ S. haematobium

200 million

Trichinella spiralis Pork worm, Trichinosis

Nematoda

1000s p.a.

Trichuris trichiura Whipworm; trichuriasis

Nematoda

600 million

a b

315

Mouse model species

Life cycle

Heligmosomoides Ac/Na/Nb: skinpolygyrus; penetrating larvae, Nippostrongylus migrate through brasiliensis lungs to gut; Hp: oral ingestion of larvae, remains intestinala – Fecal-oral transmission of eggs; larvae hatch in stomach but migrate through lungs and return to gut Litomosoides Mosquito transmission of sigmodontis larvae through skin; migration to lymphatics; release of newborn microfilariae into blood Both directly Ingestion of eggs by infective humans (or mice) as intermediate host, harbor protoscoleces in cysts which infect definitive canid hosts – Blackfly transmission of larvae through skin; parasites remain subcutaneous; and release newborn microfilariae into skin Cercariae invade from S. mansoni aquatic snail, migrate directly through lung to infective vascular sites (Sh: bladder wall; Sm, Sj: hepatic portal vein)b Directly Larvae in undercooked infective meat; adults in intestine release newborn larvae which encyst in muscle T. muris Fecal-oral transmission of eggs

Notes Closely related to ruminant parasites (e.g., Haemonchus contortus)

Closely related to pig roundworm Ascaris suum

Less prevalent human species include Loa loa and B. timori

Among most lifethreatening of helminth infections

Blindness results from microfilariae in eye; generalized dermatitis

Pathology largely due to eggs either in egress (e.g., through bladder wall) or lodged in liver Infects very wide range of host species

Ac, Ancylostoma caninum; Na, Necator americanus; Nb, Nippostrongylus brasiliensis; Hp, Heligmosomoides polygyrus. Sh, Schistosoma haematobium; Sm, S. mansoni; Sj, S. japonicum.

support the reported correlations between expression of Th2 cytokines, type 2 cytokine-mediated effector mechanisms and immunity to helminths in humans (discussed above), and identify functional roles for Th2 cell-derived cytokines in expulsion of intestinal nematode parasites. The mechanisms of recognition of helminth parasites by the innate immune system remain poorly defined. However, current models suggest that intestinal epithelial cells (IECs) and professional antigen presenting cells such as dendritic cells (DCs) and macrophages are critical in initiating and regulating antiparasite immune responses (Perrigoue et al., 2008). These cell types have been proposed to directly recognize nematode parasites and parasite-derived ES (excretory-secretory) products via a number of mechanisms including recognition of nematode glycans, proteases, and chitin.

In addition to direct innate recognition, nonspecific tissue damage created by intestinal nematode parasites may provoke danger signals that influence innate responses to infection. Th2 cells express IL-4, IL-5, IL-9, and IL-13 and their differentiation is influenced by a number of factors including IL-4 itself, the Notch pathway, and IEC-derived cytokines which, as shown in Fig. 1, include IL-17E (IL-25), IL-33, and thymic stromal lymphopoietin (TSLP) (Saenz et al., 2008). Recent studies suggest all these cytokines play an important role in initiating a program of gene expression required for the development of Th2 cytokine responses. For example, IL-17E can be produced by Th2 cells, IECs, monocytes, and granulocytes and appears to be particularly important in the development of antiparasite Th2 responses following exposure to Trichuris muris or Nippostrongylus

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FIGURE 1 Initiation and regulation of parasite-specific Th2 cell responses following exposure to intestinal nematode infection. Following infection with intestinal nematode parasites, intestinal epithelial cells express a wide range of chemokines and immunoregulatory cytokines, including IL17E, thymic stromal lymphopoietin (TSLP), and IL-33. These cytokines can promote expression of IL-4 and/or IL-13 in granulocyte populations including mast cells (MC), eosinophils (EOS), and basophils (BASO). IL-17E and TSLP can also create a permissive environment for Th2 cell differentiation by limiting expression of proinflammatory cytokines in dendritic cells (DCs). Naïve CD41 T cells are activated in the draining mesenteric lymph nodes and, in the presence of the appropriate signals, differentiate into host protective Th2 cells that express IL-4, IL-5, IL-9, IL-13, and IL-17E.

brasiliensis infections. Eliciting the expansion of non-B or non-T cells that produce abundant amounts of IL-4 and IL-13 is one mechanism through which IL-17E has been proposed to promote immunity to infection. IEC-derived IL-33 and TSLP are also important in not only influencing the development of Th2 cell responses following T. muris infection, but also appear to be dispensable in the context of other intestinal nematode infections. TSLP is expressed by epithelial cells, keratinocytes, and granulocytes and promotes Th2 cell differentiation by simultaneously limiting expression of proinflammatory cytokine production by DCs and inducing IL-4 expression in mast cells and basophils. In addition to expressing high levels of IL-4, emerging studies suggest basophils also coexpress MHC class II and costimulatory molecules and can act as antigen presenting cells that promote Th2 cell differentiation (Sullivan & Locksley, 2009). In contrast, IL-33 expression appears to be restricted to epithelial cells, macrophages, and DCs, however, it acts on multiple granulocyte populations to elicit IL-4 production. Associated with production of cytokines and chemokines by IECs, activation and recruitment of basophils, eosinophils, mast cells, NK (natural killer) cells, and NK T cells are hallmarks of the early immune response to intestinal nematode infections. All of these cell lineages have been proposed as early sources of IL-4, IL-13, and/or TSLP that promote and sustain optimal Th2 cell responses following exposure to intestinal nematodes (Stetson et al., 2004) (see Fig. 1). The importance of Th2 cells and IL-4 in expulsion of intestinal nematodes was first demonstrated by Urban and Finkelman following challenge with Heligmosomoides polygyrus infection of mice (Finkelman et al., 1997). Subsequent studies employing IL-4 deficient mice or anti-IL-4 monoclonal antibody treatment of normally resistant wild-type mice

confirmed an important role for IL-4 in immunity to T. muris (Finkelman et al., 1997; Grencis, 1997a). Although initial expression of Th2 cytokines can occur in the absence of IL-4, it appears that maintenance of optimal Th2 cell responses require IL-4-IL-4Ra signaling that results in the phosphorylation of STAT-6. The transcription factor GATA-3 is also critical in the differentiation of Th2 cells. However, expulsion of N. brasiliensis and Trichinella spiralis was unaffected by depletion or genetic deletion of IL-4, suggesting a redundancy in its protective role against infection. Consistent with this, subsequent studies identified that the requirement for IL-4 in immunity to T. muris was dependent on mouse genetic background. For example, C57BL/6 IL-42/2 mice were susceptible to infection while BALB/c IL-42/2 exhibited normal expulsion of parasites (Cliffe & Grencis, 2004). Although independent of IL-4 itself, a number of studies highlighted the importance of IL-4Ra and STAT-6 expression in immunity to multiple intestinal nematode parasites. The IL-4-related cytokine, IL-13, shares the IL-4Ra chain and, similar to IL-4, promotes STAT-6 phosphorylation. Indeed, the importance of IL-13 in immunity to intestinal nematode infection, independent of IL-4, was subsequently demonstrated in a number of nematode infections. Mice deficient in IL-13 exhibit impaired expulsion of N. brasiliensis, T. spiralis, and T. muris infections. In most cases, mice lacking both IL-4 and IL-13 exhibit even slower expulsion of parasites than IL-13 deficiency alone, highlighting the cooperative nature of the IL-4 and IL-13 pathways (Grencis & Bancroft, 2004). These findings are supported by the demonstration that mice deficient in STAT-6 signaling exhibit delayed expulsion following exposure to N. brasiliensis, T. spiralis, and H. polygyrus infection. Notably, although STAT-6 expression was important for immunity to most intestinal nematodes, deletion of STAT-6 had variable effects

25. Acquired Immunity to Helminths

on the magnitude of the antiparasite Th2 cell response, suggesting STAT-6 influences both Th2 cells and the effector response elicited against specific parasites. In addition to IL-4 and IL-13, other Th2-associated cytokines such as IL-3, IL-5, and IL-9 can influence immunity to some, but not all, intestinal nematode infections. For example, although the mechanisms of action remain unclear, administration of exogenous IL-3 could promote immunity to T. spiralis and infections with Strongyloides species. IL-5 appears to play a minor contributory role in the response following primary H. polygyrus infection, although expulsion of N. brasiliensis, T. spiralis, T. muris, Strongyloides spp., and secondary H. polygyrus appears to be independent of IL-5 (Grencis, 1997b). A more substantial role for IL-9 in immunity to intestinal nematode infection was demonstrated in the context of T. muris and T. spiralis infections. Transgenic overexpression of IL-9 resulted in enhanced expulsion of both parasites, whereas blockade of IL-9 responses resulted in protracted T. muris infection. In the case of T. spiralis infection, protective effects of IL-9 appear to be partially dependent on mast cells (Pennock & Grencis, 2006). Collectively, the emerging paradigm in the development of protective immunity following intestinal nematode infection is that IEC-derived cytokines such as IL-17E, IL-33, and TSLP play a critical role in the early innate events that promote the development of Th2 cells (Artis, 2008; Artis & Grencis, 2008). IECs can influence DC activation and provoke production of IL-4 and IL-13 from nonlymphocyte sources including basophils, eosinophils, and mast cells (Fig. 1). Depending on the infection, NK and NK T cells may also produce IL-4 and/ or IL-13. These cell populations subsequently influence the differentiation of Th2 cells that express IL-3, IL-4, IL-5, IL-9, and IL-13 (Stetson et al., 2004). All these Th2 cell-derived cytokines can influence immunity to infection, with a central role of IL-4/IL-13 signaling through IL-4Ra and STAT-6 (Finkelman et al., 2004; Urban et al., 1992).

Th2 Cytokine-Dependent Immune Effector Mechanisms in Expulsion of Intestinal Nematode Parasites

While the development of adaptive immunity to intestinal nematodes is dependent on parasite-specific CD41 Th2 cells, the immune effector mechanisms that mediate expulsion of nematodes appear to be nonparasite-specific in nature. Rather, Th2 cytokine-dependent intestinal inflammation and associated alterations in tissue physiology are general features associated with expulsion of most intestinal nematodes. Following their development in draining lymph nodes, some parasite-specific Th2 cells recirculate to intestinal tissue where they elicit local production of cytokines, chemokines, and other inflammatory mediators that promote recruitment of myeloid cell lineages including mast cells, basophils, eosinophils, and alternatively activated macrophages (Else, 2002). Associated with this inflammatory cell infiltrate, Th2 cytokines derived from either Th2 cells or infiltrating myeloid cells provoke significant changes in intestinal physiology that are a common response associated with expulsion of most nematodes. For example, Th2 cytokines elicit alterations in smooth muscle contractility and dysregulated IEC function including proliferation, differentiation, permeability, and ion exchange (see Fig. 2). These inflammatory responses and associated changes in tissue physiology, rather than having a direct cytotoxic on the parasites or parasite-infected cells, are thought to create a local habitat unsuitable for optimum survival of intestinal nematodes. Consistent with this hypothesis, most intestinal nematodes are expelled from their immune host live and can survive in nonimmune hosts upon surgical transfer.

317

Although stereotypic Th2 cytokine-dependent inflammatory and physiologic changes in the intestine are associated with immunity to most intestinal nematodes, the relative importance of each effector mechanism appears to depend on the biology of specific nematode parasites. In the following sections, the influence of Th2 cytokine-dependent regulation of mast cells, alternatively activated macrophages (AAMac), and IEC function on expulsion of specific nematode parasites will be discussed.

MAST CELLS

The influence of mast cells on immunity to intestinal nematode infection is species specific. For example, Th2cell-dependent optimal expulsion of primary T. muris, N. brasiliensis, and H. polygyrus infections appears to be independent of mast cells. In contrast, mast cells are critical for rapid expulsion of T. spiralis that inhabits an intracellular niche in the small intestine (see Table 1). Similar to other nematode parasites, rapid expulsion on challenge infection is associated with a robust CD41 Th2 cell response, and stereotypic Th2 cytokine-associated inflammation, including elevated serum IgE responses, peripheral blood eosinophilia, goblet cell hyperplasia, altered IEC proliferation, changes in intestinal muscle contractility, and mucosal mast cell hyperplasia. Grencis et al. (1993) systematically dissected these responses and revealed that intestinal mast cells are essential for efficient expulsion of T. spiralis. In infected mice and rats, there is a strong temporal association between intestinal mast cell hyperplasia and worm expulsion (Miller, 1996). Mast-cell-deficient mice (W/Wv or WSh/Sh) show a delayed worm expulsion (Ohnmacht & Voehringer, 2010) and depletion of mast cells in infected mice using anti-ckit or anti-stem cell factor (SCF) antibody significantly delays worm expulsion. Secretion of mast cell proteases, in particular mouse mast cell protease 1, MMCP1, appears to be critical for the host protective effects of mast cells as mice deficient in MMCP1 exhibit delayed worm expulsion (Pennock & Grencis, 2006). Intestinal mastocytosis in T. spiralis-infected mice is regulated by hematopoietic growth factors such as SCF and by CD41 TH2 cell-derived cytokines including IL-3, IL-4, and IL-9. SCF appears to regulate proliferation, differentiation, and migration of mast cell precursors in bone marrow. Upon exit from bone marrow, IEC-derived chemokines, including CCL2, promote recruitment of mast cells into the intraepithelial compartment of the intestine where local production of TGF b regulates expression of MMCP1. Secretion of mast cell-derived proteases, including MMCP1, is thought to promote worm expulsion through regulation of IEC tight junction proteins that results in elevated epithelial cell permeability and increased secretion of fluid into the lumen (McDermott et al., 2003). Coupled with Th2 cytokine-dependent changes in smooth muscle contractility, mast cell responses are thought to promote a “weep and sweep” response, rendering the IEC interface between the host and parasite unsuitable for colonization (see Fig. 1).

ALTERNATIVELY ACTIVATED MACROPHAGES

Macrophage activation and effector functions, such as expression of iNOS, have been traditionally associated with expression of proinflammatory cytokines and immunity to bacterial and protozoan pathogens. However, recent findings highlight the heterogeneity and potential plasticity of macrophage responses. Activation and recruitment of AAMac are a hallmark of Th2 cytokine responses associated with intestinal nematode infection and allergy (Maizels

318


FIGURE 2 Th2 cell-mediated immune effector mechanisms in expulsion of intestinal nematode infection. Th2 cell-derived cytokines can promote granulocyte activation including expression of IL-4, MHC class II, costimulatory molecules, and mouse mast cell protease (MMCP). In addition, Th2 cytokines can promote alternative activation of macrophages (AAMac) that express chitinases, chitinase-like molecules (YM-1), arginase-1, and RELMa that can promote parasite expulsion, wound healing, and can limit the magnitude of Th2 cytokine responses. Th2 cells can also influence nonhematopoietic cell lineages, including promoting smooth muscle contractility and alterations in intestinal epithelial cell (IEC) proliferation, differentiation, and migration. Therefore, parasitespecific CD41 T cells elicit nonparasite-specific inflammation and changes in intestinal physiology that create an environment that is unfavorable for parasite persistence. The relative importance of each pathway depends on the particular species of nematode parasite that infects the host.

et al., 2004). Alternatively activated macrophages can be induced by IL-4, IL-13, IL-10, and IL-21 and are characterized by expression of multiple genes, including arginase-1, RELMa, chitinases, and chitinase-like molecules such as Ym-1 (Nair et al., 2006). The expression of chitin in the cuticle of nematodes suggests that chitinases or chitinase-like molecules may be important immune effector molecules in the expulsion of intestinal nematodes, while other AAMacassociated genes may have potent immunoregulatory and tissue-protective functions. A host-protective role for AAMac in immunity to intestinal nematodes was first illustrated by Gause and colleagues following infection with the tissue-dwelling nematode H. polygyrus (Anthony et al., 2007). This is a natural intestinal nematode infection of mice that is commonly used as a model for human hookworm infection. Primary infections with H. polygyrus occur following ingestion of the larval parasites that transiently invade the mucosa before establishing a luminal niche (see Table 1). Primary infections are typically chronic, however most experimental studies employ drug clearance of primary infection and analysis of subsequent secondary responses. The importance of AAMac in immunity to secondary or challenge H. polygyrus infection was revealed by chemical depletion of macrophages. Inhibition of arginase-1 had a similar effect on impairing resistance to infection,

suggesting a critical role for AAMac-derived arginase activity in worm expulsion (Anthony et al., 2007; Patel et al., 2009). Whether other AAMac-associated genes, including chitinases or RELMa, contribute to immunity to the tissue-dwelling phase of H. polygyrus and related parasites has not been examined to date. In addition to a direct role in immunity to intestinal nematode infection, AAMac exhibit potent immunoregulatory and wound healing functions in the context of Th2 cytokine-mediated inflammation. For example, AAMac are critical in limiting potentially lethal intestinal inflammation associated with experimental S. mansoni infection and RELMa derived from eosinophils and AAMacs can limit the magnitude of Th2 cytokine responses following infection with multiple intestinal helminth infections. RELMa also induces collagen deposition from tissue-resident myofibroblasts, suggesting that this molecule could play a role in wound healing associated with intestinal nematode infection (Artis, 2006). Given that helminth parasites are large and undergo developmental stages that require either a transient intracellular stage or a tissue-invasive stage, it is not surprising that these infections are associated with a marked inflammatory response and significant tissue damage. Therefore, the immunoregulatory and wound healing properties of AAMac can also be considered hostprotective (see Fig. 2).


TH2 CYTOKINE-MEDIATED REGULATION, IEC PROLIFERATION, AND DIFFERENTIATION

In addition to recruitment of inflammatory cells, expulsion of most intestinal nematodes is commonly associated with dramatic alterations in IEC proliferation, turnover, and/or differentiation. Until recently, these changes in IEC function were thought to be a pathologic “off-target” effect associated with worm expulsion, however, it is now clear that Th2 cytokine-dependent regulation of IECs plays a critical contributory role in expulsion of some nematode parasites. Each crypt within the intestine is an individual proliferative unit, with pluripotent stem cells located near the base of the crypt dividing by asymmetric division to undergo self-renewal and give rise to a number of cell lineages, including Paneth cells, enteroendocrine cells, and goblet cells. Migrating IECs are shed at the luminal surface of the crypts and the rates of IEC proliferation, migration, and shedding are a tightly regulated process that is critical for tissue homeostasis. Trichuris spp. inhabit a partially intracellular niche within IECs of the cecum and proximal colon and appear to continually invade new cells in order to counter the constant proliferation, migration, and shedding of IECs. Recent studies indicate that Th2 cytokine-dependent changes in IEC proliferation, migration, and differentiation contribute to expulsion of T. muris and perhaps other intestinal nematode parasites. Most inbred strains of mice expel larval stages of T. muris between days 17 to 21 post infection. As discussed above, immunity to infection is dependent on non-T-cell-derived cytokines, including TSLP, IL-17E, and IL-33 that promote CD41 Th2 cell production of IL-4, IL-9, and IL-13, all of which are important in optimal expulsion of the parasite. Immunity to infection is associated with elevated serum IgE levels and the presence of AAMacs, mast cells, and eosinophils in the lamina propria of the intestine. However, it is clear that worm expulsion can occur independently of mast cells, eosinophils, and AAMacs as expulsion is normal in animals depleted of these cell lineages. Further, adoptive transfer of purified CD41 T cells into lymphocyte-deficient mice is sufficient to induce worm expulsion indicating that B cells and antibody production are not necessary for expulsion of T. muris (Else & Finkelman, 1998). Characteristic changes in the intestinal epithelium are associated with expulsion of T. muris, including changes in IEC proliferation, turnover, and differentiation into goblet cells, and recent studies implicate these physiologic changes in IEC biology in the expulsion of the parasite. For example, in mice expelling their parasites, almost a doubling of the rate of IEC turnover occurs whereas in animals that do not expel the worms, only a slight elevation in turnover is apparent. These changes in IEC turnover appear to be regulated, at least in part, by IL-13 (Cliffe et al., 2005). In contrast, in genetically susceptible strains that harbor persistent infection, IFNg counter-regulates the potential protective TH2 cytokine response and slows down the IEC turnover rate through induction of the chemokine CXCL10 (Artis & Grencis, 2008). Accordingly, a slow moving epithelium is established, presumably promoting maintenance of parasites. Critically, blockade of CXCL10 in susceptible mice raised the turnover rate of IECs that was associated with worm expulsion. Based on these findings, a model of “epithelial escalator” was proposed in which Th2 cytokines promote expulsion of the parasite via rapid IEC turnover that propels the parasite from its favored intraepithelial niche (Artis & Grencis, 2008) (Fig. 2). In addition to alterations in IEC turnover, increased differentiation of goblet cells is a hallmark of expulsion of

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many intestinal nematodes. Goblet cells secrete mucus, trefoil peptides, and other bioactive molecules including intelectins and resistin-like molecule-beta (RELMb/FIZZ2) (Artis, 2006). In the steady state, goblet cell-derived mucus is known to create a physical barrier at the mucosal surfaces of the intestine and a number of studies have demonstrated that intestinal-dwelling nematodes do not establish, are impeded from doing so, or can be physically trapped in secreted mucus. There are a number of reports that expression of mucin genes, including Muc-2 and Muc-3, is up regulated following intestinal nematode infection. Changes in glycosylation status of mucins also occur following intestinal nematode infection, although at present, there is limited evidence to support a direct role for mucins in expulsion of intestinal nematode parasites. Similarly, goblet cells express intestinal trefoil factor 3 (TFF3), chloride channel, calcium activated 3 (mCLCA3, Gob5), and intelectins. Although expression of these molecules is up regulated following infection with a number of intestinal nematode parasites and Th2 cytokines appear to regulate their expression, a direct role for these molecules in worm expulsion remains undefined. In contrast, goblet cell-derived RELMb appears to play both an immunoregulatory and host protective role following intestinal nematode infection. RELMb expression is restricted to IECs in the intestine and production can be regulated by a number of factors including Th2 cytokines. Maximal secretion of RELMb into the intestinal lumen is associated with worm expulsion following exposure to multiple intestinal nematode infections including N. brasiliensis, T. spiralis, and T. muris. Rapid expression of RELMb is also a hallmark of Th2 memory responses following secondary challenge with T. muris. Secreted RELMb was found to associate with the chemosensory apparatus of some nematodes and to interfere with host-finding behavior. Therefore, it has been hypothesized that RELMb may contribute to worm expulsion through disorientation of the parasites (Artis, 2006). Recent studies demonstrated that RELMb2/2 mice exhibit impaired immunity to N. brasiliensis, confirming an important role in immunity to infection (Herbert et al., 2009). Critically, although the magnitude of the antiparasite immune response appears to be regulated by RELMb, expulsion of T. muris and T. spiralis appear to be independent of RELMb, highlighting the potential redundancy that operates in the context of effector mechanisms involved in expulsion of nematode parasites. Taken together, studies in diverse parasitic nematode infections have demonstrated multiple Th2 cytokine-dependent effector mechanisms including mastocytosis, recruitment of AAMacs, and regulation of IEC permeability, proliferation, turnover, and differentiation. Therefore, as discussed above, parasite-specific CD41 Th2 cell responses elicit nonspecific “modular” responses characterized by type 2 inflammation and changes in intestinal physiology, all of which can create an unfavorable environment for this diverse group of parasites (Fig. 1).

ACQUIRED IMMUNITY TO TISSUE-DWELLING HELMINTHS

Immunity to parasites within the internal tissues of the body operates very differently than in the gastrointestinal locale. However, the generation of a dominant Th2 response forms a strong common theme between intestinal helminths and those parasitizing the tissues and, as described above, cells of the innate immune system are instrumental in shaping the mode of T-cell reactivity. The distinction between intestinal helminths and those living in the various somatic organs

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of the host is further blurred by the fact that the larval stages of many gut-dwelling helminths traverse the tissues (e.g., in hookworms, larvae penetrate the skin before migrating to the lung and reaching the stomach via the esophagus). Moreover, intestinal parasites can encompass significant tissue-dwelling phases such as the larval forms of H. polygyrus, which temporarily encyst in the submucosal muscle wall (see Table 1). Even entirely tissue-dwelling helminths may occupy different niches as they progress through the developmental cycle, such as seen with the lung-stage schistosomulae and vascular plexus adult schistosomes. Hence, a key issue is when and where tissue stages of helminth parasites may be intercepted and killed by the host immune system. Where parasites are directly skin-penetrating, there is evidence of localized trapping. For example, serum antibodies from animals vaccinated with irradiated A. caninum L3 were able to inhibit larval migration through the skin in vitro (Fujiwara et al., 2006), while mice that overexpress IL-5 mounted a subcutaneous eosinophil-rich response that trapped the majority of N. brasiliensis larvae at the site of entry (Daly et al., 1999). Such reactions are unlikely in the naïve host however and, in primary schistosome infections, the skin phase is thought primarily to serve as a stimulus to systemic Th2 immunity, which acts to eliminate schistosomals at the subsequent lung stage (Mountford & Trottein, 2004). In many tissues—particularly the lungs, liver, and gut— the immune system forms granulomas around parasitic organisms. In the case of secondary infection with H. polygyrus, as discussed above, these focus AAMac to eliminate parasites while still in the submucosal wall (Anthony et al., 2006). In the liver, however, where granulomas form around trapped Schistosome eggs, they protect the host from the toxic effects of egg secretions but do not mediate immunity to the adult parasites remaining in the portal vasculature (Anthony et al., 2007). Quite possibly, effective killing of helminths in tissue sites is achieved by direct actions of hematopoietic cells and granuloma formation occurs when killing cannot be accomplished. Arguably, the closest representation of immune-mediated lethality can be visualized in chamber experiments, in which helminths are implanted in cell-permeable constructs and killing can be correlated with ingress of eosinophils and other host cells (Rotman et al., 1996). Certain helminth parasites inhabit the vascular and lymphatic systems. Although it is difficult to observe immune killing in these dispersed organs, substantial data has been built up on immune-dependent clearance of Brugia microfilariae (Mf) from the bloodstream. While innate immune attack can be mediated by eosinophils, the adaptive immune response is dependent both on antibody production and on FcR expression, indicating that antibody-dependent cellular cytotoxicity (ADCC) like mechanisms are in play in removing these parasites from the bloodstream (Gray & Lawrence, 2002). Because xid mice deficient in T-independent IgM responsiveness cannot control blood microfilaremia and other mutant mice with IgM priming defects are similarly susceptible, evidence argues for an exclusive role for IgM in immunity to bloodstream Mf. These three examples of skin, muscle wall, and vasculature serve to illustrate the diversity of biological environments and immunological mechanisms that are likely to be mobilized against helminth parasites. However, as in the intestinal locale, a unifying component remains the CD41 T cell; hence mice deficient in any gene linked to CD41 T-cell development (including RAG, MHC class II, or CD4 itself) are highly susceptible to infection and are generally unable to express any form of protective immunity. In contrast, the CD81 population (as well as other, less conventional T-cell

types) are rarely implicated in acquired immunity. Within the CD41 population, it is often assumed that Th2 cells are the mediators of immunity to helminths; however, this is not always the case. For example, vaccine-induced immunity to the cercarial larvae of S. mansoni in mice requires a Th1 response in the dermis (James & Glaven, 1989), while immunity to larvae of the cestode Taenia crassiceps is Th1-mediated and IL-12p35-deficient animals are highly susceptible to infection (Rodriguez-Sosa et al., 2003). Interestingly, in both schistosomiasis and cestode infections, an early Th1 response, which can play a protective role, appears to be supplanted by a later dominant Th2 response, which is not so effective at eliminating parasites (Pearce & MacDonald, 2002; Zhang et al., 2008). This scenario stands in contrast to the more general observation in nematode infections that disruption of Th2-associated genes such as IL-4, IL-13, or STAT-6 will drastically impair antihelminth immunity, as described above. As mentioned above, a further common feature with intestinal immunity is the partnership between adaptive and innate components which first dictates the mode of T-cell subset differentiation, and comes again into play when nonparasite-specific cells act to eliminate helminths in the tissues (Cadman & Lawrence, 2010). This relationship has been most clearly expounded in murine schistosomiasis, in which dendritic cells selectively induce a Th2 response to schistosome egg antigen (Pearce & MacDonald, 2002). In addition, in murine filariasis models, AAMacs are also able to induce Th2 responses, a contribution likely to play increasing significance in the upkeep of Th2 reactivity during the chronic phase of infections (Maizels et al., 2004). Most recently, a role for basophil-derived IL-4 in the initiation of Th2 responsiveness to tissue helminths has become clearer, with the discovery that both S. mansoni and the tissue cyst-forming Echinococcus multilocularis release ligands that stimulate basophils to release this cytokine through nonantigen-specific interactions with surface-bound IgE (Schramm et al., 2007). The generation of Th2 immunity in this way can, in a manner that differs exquisitely according to the precise parasite/tissue combination in question, mediate immune killing of helminths. For example, in mice carrying a genetic deletion of eosinophils, N. brasiliensis had a much greater propensity to reach the lungs and progress to the gut (Knott et al., 2007), consistent with earlier work from the same group showing that constitutively eosinophil-rich IL-5 overexpressing mice permitted fewer larvae to reach the intestinal site. However, the same eosinophilic mice could not attack Toxocara canis larvae, and earlier studies had established that eosinophil-deficient mice mounted equivalent vaccineinduced immunity to S. mansoni (Sher et al., 1990). Interestingly, in an elegant illustration of the importance of parasite niche, eotaxin2/2 mice in which eosinophils cannot enter the peritoneal cavity show increased killing of blood microfilariae of Brugia malayi, alongside failure to kill peritoneal parasites (Simons et al., 2005). Hence, eosinophils can mediate immunity to some extent, and against some parasites, but not in every case. Indeed, as evidence is now emerging that eosinophils may secrete proteins that dampen Th2 responses (Pesce et al., 2009a, 2009b), and survival of T. spiralis is actually reduced in the absence of eosinophils (Fabre et al., 2009), it can be appreciated that eosinophils may strongly influence the outcome of infection in alternative ways, depending crucially on the precise setting in question. Neutrophils are often assumed to be exclusively involved in antibacterial immunity, but in some instances have proven essential in antihelminth immunity. One example is in the mouse filarial parasite, Litomosoides sigmodontis.


Both IFN-g-deficient and IL-5-deficient mice suffer increased worm and microfilarial loads, with doubly deficient mice being highly susceptible (Saeftel et al., 2003). In this instance, TNF-a appears to be an essential mediator that activates neutrophils for antiparasite immunity and there is a requirement for Th1/Th2 synergy rather than antagonism for the successful resolution of this infection. Several other examples have been reported, in which neutrophils are important in acting through either an antibody-dependent or antibody-independent mechanism (reviewed by Cadman & Lawrence, 2010) suggesting that, as with eosinophils, there are defined settings in which the action of neutrophils will prove to be crucial for immunity. Macrophages have long been known to attack schistosome larvae through classical activation and the production of nitric oxide (James & Glaven, 1989), but the newcomer to our understanding of innate cell response to helminth infection is certainly the AAMac, as discussed above. These cells mediate immunity to the tissue-invasive stage of H. polygyrus (Anthony et al., 2006), but in other contexts, such as the thoracic cavity niche parasitized by adult L. sigmodontis, the role of AAMac is more in dampening host T-cell reactivity (Taylor et al., 2006). Indeed the AAMac phenotype has been strongly associated with immune suppression as with the dampening of liver pathology in schistosomeinfected mice (Pesce et al., 2009a, 2009b) and with the potent antiproliferative suppressive effects in filarial infections (MacDonald et al., 1998). Hence, as with other innate cell types, the contribution of macrophages depends entirely on the context of host signaling and helminth biology. Where innate cells can effectively deal with tissue helminth infections, they will have been stimulated and armed by the adaptive immune system; cytokines from both T cells and B cells play crucial roles in achieving the necessary level of response (Wojciechowski et al., 2009), even if antibodies are not crucial in many cases. To date, most studies have used mMT B-cell-deficient mice, which while being generally more susceptible to helminth infection, do not help identify the alternate roles of B cells as antibody or cytokine producers (or indeed, as APCs [antigen-presenting cells]). More exacting studies have employed secIgM2/2 mice deficient only in IgM secretion; such mice are more susceptible to infective larvae of B. malayi, and data indicate a role for IgM in mediating macrophage adherence to the surface of the infective larval stage (Rajan et al., 2005). Perhaps surprisingly, in view of the evidence from human studies discussed above, there is conspicuously little evidence that IgE fulfills a protective role in the mouse, possibly because of the more restricted expression of IgE Fc receptors on murine leukocytes, including eosinophils.

CONCLUSIONS

Successful resolution of helminth infections requires the appropriate mode and degree of immune responsiveness if full immunity is to be achieved without severe pathological consequences. This intricate balance between protection against infection and forestalling immunopathology can too often go awry, as in the 5% to 20% of the helminthinfected population progressing to severe disease such as hepatosplenic fibrosis in schistosomiasis, blindness in onchocerciasis, and elephantiasis in lymphatic filariasis. The vital importance of striking this balance may be reflected in the number of regulatory controls and checkpoints in the immune system that are invoked (including regulatory T and B cells, alternatively activated macrophages at the cellular level, and IL-10 and TGF-b at the cytokine level). It is entirely plausible that the halting nature of human

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immunity to helminths can be ascribed to the strength of regulatory restraints that have evolved to minimize pathological outcomes. This promises to be a fascinating area for future research, and one that may allow us to understand human responsiveness to helminths and to intervene in an appropriate and beneficial manner. The authors’ laboratories are supported by the National Institutes of Health, the Burroughs Wellcome Fund, the National Institute of Diabetes and Digestive Kidney Diseases, the Crohn’s and Colitis Foundation of America, the University of Pennsylvania (D.A.), and by the Wellcome Trust and Asthma UK (R.M.). We thank David Hill (University of Pennsylvania) for excellent illustrative work on our figures.

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25. Acquired Immunity to Helminths Rotman, H. L., W. Yutanawiboonchai, R. A. Brigandi, O. Leon, G. J. Gleich, T. J. Nolan, G. A. Schad, and D. Abraham. 1996. Strongyloides stercoralis: eosinophildependent immune-mediated killing of third stage larvae in BALB/cByJ mice. Exp. Parasitol. 82:267–278. Saeftel, M., M. Arndt, S. Specht, L. Volkmann, and A. Hoerauf. 2003. Synergism of gamma interferon and interleukin-5 in the control of murine filariasis. Infect. Immun. 71:6978–6985. Saenz, S. A., B. C. Taylor, and D. Artis. 2008. Welcome to the neighborhood: epithelial cell-derived cytokines license innate and adaptive immune responses at mucosal sites. Immunol. Rev. 226:172–190. Sartono, E., Y. C. M. Kruize, A. Kurniawan-Atmadja, R. M. Maizels, and M. Yazdanbakhsh. 1997. Depression of antigen-specific interleukin-5 and interferon-g responses in human lymphatic filariasis as a function of clinical status and age. J. Infect. Dis. 175:1276–1280. Satoguina, J. S., E. Weyand, J. Larbi, and A. Hoerauf. 2005. T regulatory-1 cells induce IgG4 production by B cells: role of IL-10. J. Immunol. 174:4718–4726. Schramm, G., K. Mohrs, M. Wodrich, M. J. Doenhoff, E. J. Pearce, H. Haas, and M. Mohrs. 2007. IPSE/alpha-1, a glycoprotein from Schistosoma mansoni eggs, induces IgEdependent, antigen-independent IL-4 production by murine basophils in vivo. J. Immunol. 178:6023–6027. Sher, A., R. L. Coffman, S. Hieny, and A. W. Cheever. 1990. Ablation of eosinophil and IgE responses with anti-IL-5 or anti-IL-4 antibodies fails to affect immunity against Schistosoma mansoni in the mouse. J. Immunol. 145:3911–3916. Simons, J. E., M. E. Rothenberg, and R. A. Lawrence. 2005. Eotaxin-1-regulated eosinophils have a critical role in innate immunity against experimental Brugia malayi infection. Eur. J. Immunol. 35:189–197. Stetson, D. B., D. Voehringer, J. L. Grogan, M. Xu, R. L. Reinhardt, S. Scheu, B. L. Kelly, and R. M. Locksley.

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Pathology and Pathogenesis

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26 Pathology and Pathogenesis of Bacterial infections WARWICK J. BRITTON AND BERNADETTE M. SAUNDERS

intRodUCtion

with rich nutrients and rapidly replicate. The immediate or innate host immune responses rapidly eradicate the infection in the case of most bacteria, but bacterial pathogens have either specific virulence mechanisms or immune evasion strategies, which allow them to persist for hours to days, and to disseminate to other organs. The continuing innate immune responses and subsequent adaptive immune response causes the recruitment and activation of leukocytes, which may control the infection but at the expense of necrotic damage to the infected host tissues. In addition, some bacteria secrete exotoxins, which lyse host cells or bind to specific receptors resulting in characteristic damage to the host (discussed in chapter 12, this volume). Examples of exotoxins, which directly damage host cells, are the poreforming toxin of Staphylococcus aureus and streptolysin O from Streptococcus pyogenes (Group A) or pneumolysin from Streptococcus pneumoniae, both of which damage cell membranes leading to lysis.

Bacterial pathogens induce potent innate and adaptive immune responses, which, in the majority of situations, are able to eradicate an infection. This often is associated with inflammatory damage to the infected tissues, and the balance of the virulence of the pathogen and the timing and intensity of the host immune responses to the pathogen determines the extent of the pathological damage to the host. Extracellular and intracellular bacterial pathogens induce different types of immune responses and have varying strategies for evading the host immune responses. As a consequence, this can cause different patterns of immunopathology. Some organisms can survive within a specialized cellular niche despite a strong immune response and such bacteria establish chronic infection, often in the absence of overt clinical disease. Impairment of the host immune response may result in unrestrained replication of bacterial pathogens and this may lead to exaggerated damage of the infected tissue or to a different pattern of immunopathology to that developing in an immunocompetent host. This chapter will summarize the different types and mechanisms of pathology caused by bacteria once they cross mucosal or cutaneous barriers, and either multiply in extracellular spaces or take up residence within host cells, and illustrate how different types of immune responses can cause a variety of pathological damage to the host. Detailed analysis of mucosal immunity and the effects of the gastrointestinal bacterial flora on the host will not be discussed. Further details on the pathogenesis of individual bacterial pathogens and the types of host immune responses are included in preceding chapters.

immediate inflammatory Response to infection

The characteristic pathological feature of infection with extracellular bacteria is the early recruitment of neutrophils and their activation via pattern recognition receptors, which recognize conserved bacterial ligands (see chapter 16, this volume). This promotes the phagocytosis and killing of the invading microbes, but also causes the death of host cells resulting in pus formation. The recruitment of granulocytes and mononuclear phagocytes is triggered by the activation of soluble components of the innate immune system and the release of chemokines and proinflammatory cytokines by epithelial cells and resident macrophages.

Complement and Inflammation

Pathology oF eXtRaCellUlaR BaCteRial inFeCtions

The complement system is composed of over 20 different proteins and can be activated directly by extracellular bacteria in multiple ways. First, the soluble mannose-binding lectin (MBL) can bind to terminal mannose units on bacterial surfaces and activate the MBL-associated serine protease-1 and protease-2 and cleave C4 and C2 in the classical pathway of complement activation (Ip et al., 2009). This results in the activation of C3 and C5 convertases, the production of the C3a and C5a proinflammatory fragments, and the binding of C3b and downstream complement fragments to the bacteria. Bacteria also provide surfaces on which C3b is stabilized by factors B and D, leading to the activation of C3 via

Bacteria rapidly colonize the skin and mucosa of the upper airways, gut, and genital tract of mammals following birth, and the host is soon exposed to many bacterial pathogens. Minor abrasions of the skin or mucosal surfaces permit bacteria to enter the extracellular spaces where they are provided Warwick J. Britton and Bernadette M. Saunders, Centenary Institute, Locked Bag No 6, Newtown, 2042; and Discipline of Medicine, Sydney Medical School, University of Sydney (D06), Sydney, 2006, NSW, Australia.

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the alternate complement pathway. Further, some bacteria can activate the classical pathway in an immunoglobulinindependent manner via the effects of SIGN-R1, a cell surface C-type lectin receptor with homology to human DC-SIGN (Kang et al., 2006). SIGN-R1 is expressed on marginal zone macrophages and binds C1q and the polysaccharide of S. pneumoniae leading to the activation of classical complement pathway and the C4-dependent deposition of C3b on S. pneumoniae. The products of complement activation, C3a and C5a, bind receptors on macrophages, neutrophils, and mast cells stimulating the release of chemokines, cytokines, histamine, and leukotrienes, which increase the expression of adhesion molecules on endothelial cells and promote leukocyte recruitment. The phagocytosis of complement-labeled bacteria is enhanced by the binding of C3b and inactivated fragments of C3b to complement receptors on neutrophils and macrophages. In addition to the four well-defined complement receptors for C3 fragments, a fifth complement receptor, CRIg, a member of the Ig superfamily, mediates the complement-dependent clearance of extracellular bacteria by phagocytic Kupffer cells in the liver (Helmy et al., 2006). Different bacterial pathogens can activate individual components of the innate defense system, and the complement system may channel the responses from different pattern recognition receptors to a limited number of common effector mechanisms that eliminate the pathogens (Roozendaal & Carroll, 2006).


Antimicrobial peptides (AMPs) are rapidly induced by bacterial infections at epithelial surfaces including the skin, lung, and gut. These are small (12–50 amino acids) peptides with a positive charge and amphipathic structure, and were first characterized because of their direct antibacterial effects (Lai & Gallo, 2009). In humans, this includes cathelicidin or LL-37, and multiple a-defensins and b-defensins, which are stored in the granules of neutrophils and Paneth cells, or produced by epithelial cells, keratinocytes, or macrophages. These AMPs kill both gram-positive and gram-negative bacteria and show some selectivity in that some mammalian AMPs have maximum effectiveness against specific groups of bacterial pathogens relevant to the tissue where those AMPs are expressed. It is increasingly recognized that mammalian AMPs have broader effects on the innate immune response, and they can stimulate chemotaxis, cytokine release, angiogenesis, and wound healing (Lai & Gallo, 2009). For example, LL-37 is chemotactic for neutrophils, monocytes, and T cells, while individual human a-defensins (NHP-1 and NHP-2) and b-defensins (hBD-3 and hBD-4) recruit monocytes and macrophages and hBD2 is chemotactic for mast cells. AMPs also exert indirect chemotactic activity by stimulating chemokine and cytokine release from monocytes and keratinocytes. As a result, AMPs are major contributors to the inflammatory responses and host defense against bacterial infections at epithelial surfaces (Hiemstra, 2007). Components of the bacterial cell wall activate both cell surface and intracellular pattern recognition receptors, in particular, Toll-like receptors (TLRs) on neutrophils, monocytes, and epithelial cells to release proinflammatory cytokines and chemokines, which further amplify the inflammatory response to infection. For example, lipopolysaccharide in the cell walls of gram-negative bacteria is the major ligand for TLR4, and flagellin is the ligand for TLR5. Activation of these TLRs stimulates a signaling cascade through the MAPK (mitogen-activated protein kinases) and NF-kB pathways leading to cytokine and chemokine release. The family of nucleotide-binding oligomerization domain (NOD)-like receptors (NLR) provide intracellular sensors to detect microbial products. Cell-wall-derived peptidoglycan

from gram-positive bacteria activates the intracellular NLR pathway leading to the formation of the inflammasome, a collection of proteins that assemble and activate caspase 1, which in turn cleaves pro-IL(interleukin)-1b to release of the proinflammatory cytokine IL-1b.

Chemokines and Inflammation

The interactions of chemokines and their receptors are critical for the recruitment of neutrophils and monocytes and the inflammatory response to infection (Charo & Ransohoff, 2006). Chemokines are produced by resident macrophages, epithelial cells, and keratinocytes in response to the direct effects of bacterial products, as well as AMPs and proinflammatory cytokines such as TNF (tumor necrosis factor) and IL-1b. There is extensive redundancy in the binding and function of the .50 human chemokines and their receptors, but examples of chemokines important for antibacterial responses are CXCL8 (IL-8) and CCL2 (MCP-1). CXCL8 is the prototype CXC chemokine, which binds to the CXCR1 and CXCR2 receptors on neutrophils, and attracts these cells to the site of bacterial infection and stimulates the respiratory burst to form reactive oxygen intermediates (ROI) and release of granules (Charo & Ransohoff, 2006). CCL2 binds to CCR2, and with other members of MCP family (CCL7, CCL8, and CCL13) is important for the recruitment of monocytes, as well as immature dendritic cells and T cells. Mice lacking CCR2 have reduced influx of inflammatory monocytes during Listeria monocytogenes and Mycobacterium tuberculosis infection with delay in DC (dendric cell) migration from the site of infection and T-cell activation, and this results in reduced control of these infections (Serbina et al., 2008). Proinflammatory cytokines amplify the chemokine response. For example, deficiency in the TNF leads to reduced chemokine production and delayed monocyte recruitment during mycobacterial infections, resulting in failure to form granulomas and inability to control mycobacterial infections (Roach et al., 2002).

Neutrophils and Inflammation

Activation of neutrophils at the sites of extracellular bacterial infections by cytokines, such as TNF and IL-1b, the complement fragments C3a and C5a, and directly by bacterial ligands promotes phagocytosis of the bacteria, fusion of phagosomes with lysosome and bacterial killing by lysosomal proteases. The activation of phagosome oxidase or NADPH oxidase results in assembly of functional enzymes in the phagosome membrane, which pumps ROI into the phagosome-lysosome where they are catalyzed by lysosomal myeloperoxidase to produce highly reactive sodium hypochlorite. The combination of ROI and proteases kill the engulfed bacteria, but the extracellular release of ROI and proteases produces local necrosis of the infected tissues and the signs of inflammation. The accumulated dead neutrophils and tissue destruction produces pus, the pathological hallmark of infection with virulent extracellular bacteria. The importance of neutrophil recruitment and activation to the control of extracellular bacterial infections is highlighted by two primary immunodeficiencies. In leucocyte adhesion deficiency the absence or functional deficiency of the CD11b chain of the integrin, LFA-I, results in the failure of neutrophils to adhere to and cross the endothelium of infected tissues, so that affected children experience severe progressive, bacterial infections strikingly in the absence of pus formation. In chronic granulomatous disease, mutations in the p90 or p40 chains of phagosome oxidase prevent the formation of functional enzyme and the production of ROI, which are essential for the efficient killing of phagocytosed bacteria. This results in repeated episodes of severe bacterial, as well as fungal, and other infections, and instead of

26. Pathology and Pathogenesis of Bacterial Infections

the resolution of the acute inflammatory response usually observed with extracellular bacterial infections, granulomatous infiltrates develop in infected organs leading to further tissue destruction.

Contribution of th17 t Cells to the inflammatory Responses

Th17 cells, a recently defined subset of ab CD41 T cells, are activated early during the course of bacterial infections at mucosal surfaces and are important for recruitment of neutrophils to the site of infection (Dubin & Kolls, 2008). Naïve CD41 T cells develop into Th17 cells when stimulated by bacterial peptide/MHC class II complexes on DCs in the presence of IL-6 and TGF-b, and the subsequent expansion of Th17 cells in vivo during infection or autoimmune diseases is dependent on the heterodimeric cytokine IL-23. Th17 cells express the transcription factor RORgt and produce IL-17A, IL-17F, IL-22, TNF, and IL-6 (Weaver et al., 2007). IL-17A and IL-17F stimulate chemokine production from macrophages and epithelial cells leading to the influx of neutrophils, while TNF promotes chemotaxis and also activates neutrophils and macrophages. Although Th17 cells were first characterized as proinflammatory T cells in experimental autoimmune diseases, IL-17 is required for host defenses against extracellular bacterial infections. For example, IL-17 is essential to control pulmonary infection with Klebsiella pneumoniae, Pseudomonas aeruginosa, and S. pneumoniae in mice, and limit damage to the lungs (Dubin & Kolls, 2008; Lu et al., 2008). In addition, gd T cells also produce IL-17, and these may also contribute to the early recruitment of leucocytes to mucosal surfaces during bacterial infections. Recently IL-17 and IL-22 have been found to regulate AMP production by epithelial cells in the gut and lung, highlighting the interactions of innate and adaptive immune responses to control bacterial infections (Kolls et al., 2008).

amplification of the inflammatory Responses by specific antibodies

The adaptive antibody response to bacterial pathogens (described in chapter 19) greatly amplifies the initial inflammatory response to infection, but also enhances bacterial killing mechanisms leading to clearance of infection and resolution of the inflammation. Specific IgM antibodies are produced within 5 days of infection, and on binding bacteria activate C1q and the complement cascade leading to increased opsonization of bacteria by phagocytes and leukocyte recruitment. Under the influence of bacteriaspecific T cells, particularly follicular helper T cells within the germinal centers of draining lymph nodes, the activated B cells switch to produce high affinity IgG, which is even more effective at amplifying the host response. In addition to complement activation and efficient neutralization of bacterial toxins, IgG attached to bacteria binds through its constant region to a family of four Fcg receptors on phagocytes and other leucocytes (Nimmerjahn & Ravetch, 2008). This IgG-FcgR binding further increases the phagocytosis of bacteria, but importantly provides an additional mechanism of activation of neutrophils and macrophages. FcgR-I and FcgR-III have immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domain of the common signaling g chain, and phosphorylation of tyrosine in these domains activates a signaling cascade through phospholipase C and the RAS-RAF mitogen-activated kinase pathways, resulting in increased production of proinflammatory molecules. Mice deficient in the common g chain of FcgRI and FcgRIII show reduced control of some bacterial infections, but also decreased inflammation in antibody-mediated of autoimmune disease. The FcgRs also function as regulators

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of immune responses (Nimmerjahn & Ravetch, 2008). For example, FcgRIIB contains a different immunoreceptor tyrosine-based inhibitory motif (ITIMs) in its cytoplasmic domain, and IgG binding to FcgR-IIb on B cells results in down regulation of antibody production.

Pathological outcomes of extracellular Bacterial infection

The pathology caused by extracellular bacterial infection is due to a combination of neutrophil-driven inflammation and the cytotoxic effects of specific bacterial toxins. The inflammatory responses usually resolve following the clearance of the bacteria, either through the combined effects of antibody and innate immune responses or by intervention with antimicrobial therapy. Removal of antigen is the major factor in down regulating antibody and T-cell responses, but a variety of factors interact to prevent excessive tissue damage, including intrinsic controls on the expansion of T and B cells, induction of regulatory T cells, and complement regulatory proteins, which limit the effects or increase the removal of activated complement components. Specific antibodies, in particular high affinity IgG, which readily penetrates the extravascular space, are also important for neutralizing bacterial toxins at the site of infection and preventing the life-threatening effects of exotoxins at distal sites, such as the neuromuscular toxin from Clostridia tetani and cardiac effects of diphtheria toxin. The primacy of antibodies in the control of extracellular bacterial infections is demonstrated by the profound susceptibility of children and adults with primary antibody deficiency to sinopulmonary, meningeal, and other bacterial infections (Tarzi et al., 2009). The recurrent bacterial infection in these individuals is often accompanied by relentless tissue destruction leading to bronchiectasis, sinusitis, and chronic purulent ear disease reminiscent of the preantibiotic era.

Colonization and Biofilm development

Some bacterial pathogens adapt to the host environment and form biofilms, which permit the chronic colonization of the host and ongoing pathological damage to infected organs. A range of bacteria can form biofilms directly on epithelial surfaces or on implanted foreign material, such as intravenous cannulas and vascular grafts, however P. aeruginosa has a particular propensity for biofilm formation (Wagner & Iglewski, 2008). P. aeruginosa is an opportunistic pathogen of immunocompromised hosts, particularly cystic fibrosis subjects, but also patients with burns and neutropenia. P. aeruginosa has multiple characteristics that contribute to its ability to colonize and cause chronic infections, including its capacity to form biofilms, inherent antibiotic resistance, multiple virulence factors and metabolic versatility. Biofilms are populations of bacterial cells enclosed in an extracellular matrix composed of polysaccharides, secreted proteins, and bacterial DNA and cellular debris attached to an epithelial or foreign surface. In cystic fibrosis, P. aeruginosa responds to environmental signals through quorum sensing and switches to a mucoidforming variant and forms biofilms, which provide a survival advantage for the bacteria. The bacteria are shielded from phagocytes, and the secreted alginate scavenges free radicals and protects from defensins. As a result, the emergence of mucoid-secreted P. aeruginosa in cystic fibrosis patients is associated with lung damage and increased mortality.

Bacteria-induced shock syndromes

Bacterial infections can have profound pathological effects systemically at sites distant to the primary infection. The molecular pathogenesis of the classic sepsis syndromes induced by bacterial infections has been unravelled with

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the identification of TLRs as critical pathogen recognition receptors for bacterial products; TLR4 for endotoxin or LPS from gram-negative bacteria and dimers of TLR1/2 and TLR2/6 as the receptors for LTA, bacterial peptidoglycan and lipoproteins from gram-positive bacteria (van der Poll & Opal, 2008). For example, LPS binds to TLR4 and the coreceptor MD2 on mononuclear phagocytes and DCs and signals through MyD88-dependent and MyD88independent pathways to activate NF-kB and stimulate the release of chemokines and production of ROI and NO resulting in local inflammation and the release of the proinflammatory cytokines, TNF, IL-1b, and IL-6, which have both local and extensive distal effects. These effects include vasodilation of blood vessels through the activation of inducible NO synthase and endothelial injury, reduced cardiac output, hypotension or “septic shock”, hypoglycemia, and intravascular coagulation leading to multiorgan damage (van der Poll & Opal, 2008). Gram-positive bacteria can cause a different form of toxic shock syndrome (TSS) through the production of exotoxins. TSS is an acute, multisystem illness characterized by hypotension and multiorgan failure, which develops early in the course of a infection with toxin-producing strains of S. aureus or Streptococcus pyogenes (group A Streptococcus, GAS). These produce protein toxins, which act as superantigens and stimulate polyclonal T-cell activation (Lappin & Ferguson, 2009). Staphylococcal TSS can develop following vaginal infection with S. aureus, which was associated in the 1980s with the use of tampons or as a complication of primary staphylococcal infection or in patients with burns or surgical procedures. Streptococcal TSS is commonly associated with invasive soft tissue GAS infections, but can occur with up to 13% of GAS infection from any source. The responsible toxins belong to a family of 40 bacterial superantigens, produced mainly by S. aureus and S. pyogenes strains, but also Mycoplasma arthritidis and Yersinia pseudotuberculosis (Fraser & Proft, 2008). These protein toxins stimulate the rapid and extensive activation of polyclonal populations of T cells, resulting in the massive release of cytokines that cause systemic inflammation. The staphylococcal and streptococcal superantigens bind as unprocessed intact proteins to the MHC class II molecule on antigen presenting cells (APCs) and to a region of the T-cell receptor separate to the conventional peptide binding, usually on the Vb chain. This binding stimulates the clonal expansion of T cells bearing the characteristic Vb T-cell receptors, up to 20% to 30% of T cells, as compared to the 0.01% T cells activated through conventional TcR signaling (Fraser & Proft, 2008). The expanded T cells rapidly produce large quantities of IL-2, IFN-g, and lymphotoxin-a, contributing to more T-cell recruitment and to the stimulation of proinflammatory cytokines, such as TNF and IL-1b from APCs. The resulting cytokine storm produces similar effects to LPS-induced sepsis and the degree of activation of proinflammatory pathways, as reflected by NF-kB activation, correlates with the clinical severity of TSS (Lappin & Ferguson, 2009).

immune Complex-Mediated Pathology

The development of clinically distinctive pathological syndromes at different sites to the original infection have long been recognized as a consequence of bacterial infections. The two best defined syndromes, rheumatic fever and glomerulonephritis, follow group A streptococcal infections, but each has a different pathogenesis. Rheumatic fever and subsequent rheumatic heart disease are caused by autoimmune mechanisms, while post-streptococcal glomerulonephritis (PSGN) is due to immune complex-mediated pathology. PSGN is a syndrome of edema, hypertension,

hematuria, and proteinuria developing 1 to 4 weeks after GAS infections of the skin or pharynx, associated with the pathological features of acute proliferative glomerulonephritis (Naicker et al., 2007). PSGN develops after infection with select Lancefield M type GAS strains with nephritogenic potential. Originally attributed to molecular mimicry between streptococcal and renal components, recent studies have identified the formation in situ and the deposition of immune complexes in the glomeruli as the cause (Rodriguez-Iturbe & Batsford, 2007). Streptococcal antigens bind with IgG to form soluble immune complexes of 300 to 500 kDa, which deposit in the kidney and activate the classical complement pathway through C1q leading to the release of the proinflammatory fragments C3a and C5a, which bind to and activate neutrophils and macrophages and initiate glomerulonephritis. Interestingly, however, only two-thirds of PSGN patients have circulating immune complexes and there is no clear correlation between the presence or the level of circulating immune complexes and the clinical or pathological severity of PSGN (RodriguezIturbe & Batsford, 2007). Therefore, attention has focused on the formation of antigen body complexes in situ with subsequent activation of complement. Cationic protein antigens can cross the negatively charged glomerular basement membrane, and then react with anti-GAS IgG to form the subepithelial deposits typical of PSGN. Complement activation then leads to the patchy deposition of C3b on the antigen-antibody deposits, fall in serum C3 and C4 components, and recruitment of neutrophils and monocytes into the affected glomeruli. Recently, the streptococcal pyrogenic exotoxin B, SpeB, which is a cationic cysteine protease, and its precursor, zSpeB, have been identified as the potential nephritogenic antigen. SpeB is the most abundant secreted protein of all strains of GAS, is highly cationic, and has been colocalized with IgG and C3b in the glomerular deposits of PSGN patients. High levels of anti-SpeB antibodies are associated with PSGN, and are not present in patients with uncomplicated GAS infections or acute rheumatic fever, suggesting that the anti-SpeB antibodies contribute to subepithelial deposits (Rodriguez-Iturbe & Batsford, 2007). The nephritis-associated streptococcal plasmin receptor (NAPlr) also contributes to the inflammation by binding to glomerular basement membrane and activating proteases to digest extracellular matrix. Other chronic bacterial infections may also release soluble antigens, which bind with specific IgG to form circulating immune complexes, which are deposited in the glomeruli. This may occur during infective endocarditis, which is caused by a variety of bacteria including Streptococci, and during chronic infection of implanted foreign grafts.

Pathology oF intRaCellUlaR BaCteRial inFeCtions

Intracellular bacterial pathogens are a diverse group of organisms, which have evolved unique defense strategies that facilitate their survival within host cells. They cause significant human disease, including gastrointestinal diseases following Listeria monocytogenes and Salmonella spp. infections and chronic diseases such as tuberculosis (TB) and leprosy. Control of these pathogens requires both the activation of an antigen-specific T-cell response and the concurrent development of an appropriate inflammatory response, essential to recruit antigen-specific T cells and macrophages to surround and contain bacilli infected cells to limit dissemination of the infection. However, this inflammatory response, while essential for containment and elimination of infection, is often characterized by the development of


damaging tissue pathology. The pathological response that develops following intracellular infection is influenced by numerous host and pathogen factors.

Rapidly growing intracellular Bacteria Listeria monocytogenes

L. monocytogenes is a rapidly growing gram-positive bacterium. Control of listerial infection relies on a robust innate immune response in which neutrophils are essential, however, DCs, NK (natural killer) cells, and macrophages, particularly Kupffer cells, all contribute to optimal immunity. Depletion of neutrophils in mice leads to uncontrolled bacterial replication, marked tissue damage, and is rapidly fatal. Indeed, neutrophils are essential to control the initial phase of many acute intracellular bacterial pathogens, including Salmonella, Burkholderia, and Yersinia species (Conlan, 1997; Wiersinga & van der Poll, 2009). A strong CD81 T-cell response is required to clear Listeria infection and provide immunity to secondary challenge. Multiple cytokines are also critical for development of the inflammatory response to Listeria infection. TNF, particularly soluble TNF, plays an important role in establishing a coordinated immune response, through regulated recruitment of inflammatory cells, stimulation of adhesion molecule expression on endothelial cells, and induction of chemotaxis (Musicki et al., 2006). Humans on anti-TNF therapy show a marked increase in susceptibility to Listeria infection, as do other immunocompromised individuals. Human listeriosis presents as three typical clinical manifestations, gastroenteritis, material-fetal neonatal listeriosis, or cerebral infections. The success of Listeria as a human pathogen is in part due to its capacity to cross multiple host barriers and to invade nonphagocytic cells. Multiple virulence factors including internalin A and B, and invasins have all been shown to interact with specific cellular membrane receptors to induce bacterial internalization (Seveau et al., 2007). Cerebral infections include meningitis, meningoencephalitis, brain stem encephalitis, and brain abscess. Studies in mice have indicated that an CD11b1Ly-6cHi specific subset of monocytes, become parasitized in the bone marrow and play a key role in transporting bacteria into the CNS (Serbina et al., 2008). How these cells are recruited to the CNS is yet to be elucidated, but once there, Listeria is able to infect surrounding cells including endothelial cells and neutrons. Brain invasion via infection of endothelial cells and a neuronal route have also been demonstrated, often leading to rapid inflammation in the CNS with devastating outcomes (Drevets & Bronze, 2008).

Chronic intracellular Bacteria: Mycobacteria Mycobacterium tuberculosis

M. tuberculosis is the most important chronic bacterial infection in humans, and is responsible for 9 million cases of TB and 1.9 million deaths annually. Intriguingly, the majority (90%) of the 2 billion infected humans have latent TB infection (LTBI) and do not develop clinical disease, a testimony to the effectiveness of the complex antimycobacterial cellular immune response, which restrains the pathogen within granulomas, the pathological hallmark of mycobacterial infections. Innate immune responses are inadequate to control pathogenic mycobacteria, and the details of acquired immunity to mycobacteria are discussed in chapter 21. However, before considering the pathology of tuberculosis it is necessary to review the major features of host immunity to this pathogen. M. tuberculosis is an obligate intracellular slow-growing pathogen of macrophages, and key to its success is that natural infection usually occurs by aerosol infection with low numbers of bacteria infecting

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alveolar macrophages. This allows a nidus of infection to be established in a permissive environment over 2 to 3 weeks before there is sufficient bacterial load to allow infected DCs to migrate to the draining lymph nodes where they activate T lymphocytes. The major protective T-cell subset against mycobacteria are CD41 T cells, as evidenced by the profound susceptibility of HIV coinfected subjects to TB, either as progressive primary TB or markedly increased reactivation of LTBI, and as confirmed in experimental TB (Cooper, 2009). A broad range of other T subsets are also activated during M. tuberculosis infection, including MHC class Irestricted CD81 T cells in both humans and mice, nonclassically restricted CD81 T cells in humans, CD1-restricted CD42 CD82 (double negative, DN) ab T cells and gd T cells (Behar & Boom, 2008; Ottenhoff et al., 2008). Activation of Th17 T cells also occurs early during murine TB, and Th17 cells are present in subjects with LTBI (Cooper & Khader, 2008). The proinflammatory cytokines, IL-17A, IL17F, IL-22, and TNF produced by these cells stimulate early chemokine synthesis and the recruitment of CD41 T cells to the site of M. tuberculosis infection. Th17 cells are down regulated by IFN-g produced from Th1-like T cells, however in the absence of IFN-g, BCG-specific Th17 cells contribute a small protective effect against M. tuberculosis (Wozniak et al., 2006). Mycobacteria-specific CD41 and CD81 T cells and other lymphocytes migrate to the site of primary infection in the lung where they activate M. tuberculosis infected macrophages and freshly recruited monocytes to kill the mycobacteria. Studies in gene deficient mice and human and mouse macrophages have identified cytokines essential for control of M. tuberculosis, including IFN-g, IL-12, TNF, LTa, and IL-1b (reviewed in O’Garra & Britton, 2007; Cooper & Khader, 2008). The major cytokine for activating macrophages to kill mycobacteria is IFN-g, which acts through the STAT-1 pathway to stimulate phagolysosomal fusion and the production of ROI, RNI, and LRG-47. The critical role for IFN-g is confirmed by the profound susceptibility of infants with defects in IFN-g signaling to infection with attenuated Mycobacterium bovis (BCG) and avirulent nontuberculosis mycobacteria (NTM) (Abel & Casanova, 2006). Nevertheless, although IFN-g is essential, it is not sufficient to kill M. tuberculosis, and synergistic stimulation with TNF and IFN-g is required for maximum mycobacterial killing in murine macrophages, and 1,25(OH) vitamin D3 and GMCSF also contribute to the activation of human macrophages (O’Garra & Britton, 2007; Maglione & Chan, 2008). Despite the breadth of T-cell responses and killing mechanisms, M. tuberculosis has a diversity of mechanisms that manipulates the macrophage responses and ensures the survival of some mycobacteria (reviewed in Britton & Triccas, 2008; Pieters, 2008). The persistence of mycobacteria leads to continuing antigenic stimulation, the further influx of CD41 and CD81 T cells into the site of infection and chronic stimulation of M. tuberculosis infected macrophages leading to the formation of granulomas. Before considering the pathology of these, it is important to note that although the mouse model of M. tuberculosis infection has been very instructive, there are differences in the acquired immune response to the M. tuberculosis between humans and mice. These include the prominence in the human cellular responses of M. tuberculosis specific CD81 T cells restricted by nonclassical MHC Class I molecules (Ottenhoff et al., 2008), the fact that IL-12 is not essential for granuloma formation in humans as IL-12-deficient subjects develop granulomas to NTM (Abel & Casanova, 2006), and differences in macrophage killing mechanisms. For example, activation of inducible nitric oxide synthase (iNOS) is critical for mycobacterial killing in murine macrophages, but iNOS is poorly

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induced by IFN-g in human macrophages in vitro, and the role of NO in mycobacterial killing in humans is debated. In fact, cytokine-activated human macrophages are unable to kill M. tuberculosis in vitro, and the most effective means for killing the organism is the induction of apoptosis of the infected human macrophages. An example of apoptosis-mediated killing of M. tuberculosis by human macrophages is the activation of the purinergic receptor P2X7, a ligand-gated calcium channel by ATP. ATP-binding rapidly induces a calcium flux in infected cells, activation of phospholipase-D2 and phagolysosomal fusion leading to mycobacterial killing and the induction of apoptosis (Lammas et al., 1997; Fernando et al., 2007). Nonfunctional polymorphisms in P2X7 are associated with increased susceptibility to TB disease, confirming this pathway contributes to the control of M. tuberculosis infection in humans. Vitamin D-dependent antimicrobial killing also plays a more significant role in human macrophages. Activation of TLR2 on human monocytes induces the enzyme, which converts 25(OH) vitamin D3 into active 1,25(OH) vitamin D3, and also up regulates the vitamin D receptor (VDR) (Liu et al., 2007). The effects of increased VDR signaling include synergistic activation of macrophages with IFN-g and induction of the human antimicrobial peptide, cathelicidin (Liu et al., 2007). Polymorphisms in VDR are associated with TB and leprosy, and interact with decreased levels of vitamin D to further increase susceptibility to TB (Wilkinson et al., 2000).

Pathology of Tuberculosis Infection

The pathological characteristic of human tuberculosis is the formation of granulomas around M. tuberculosis infected macrophages in the lungs and other organs. The availability of small animal models of tuberculosis has allowed the dissection of the cellular and molecular events responsible for granuloma formation. The immunopathological response of the guinea pig to M. tuberculosis most closely resembles the human response, as granulomas in the guinea pig develop central necrosis and cavitation, however, the availability of gene deficient mice and immunological reagents has permitted the most detailed analysis of the development of granulomas and protective cellular immunity against M. tuberculosis in mice (Orme, 2003; Saunders et al., 2008). Following low-dose aerosol infection of the relatively resistant C57BL/6 mice with M. tuberculosis, the initial inflammatory infiltrate around infected alveolar macrophages includes recruited monocytes and a small number of neutrophils, which along with NK cells may provide a degree of transient restraint on bacterial replication (Pedrosa et al., 2000; Feng et al., 2006). But the infection is not controlled, and by day 11 to 14 postinfection in the mouse infected DCs migrate to the draining lymph nodes and activate both CD41 and CD81 T cells (Wolf et al., 2008). These antigen-specific T cells are recruited back to the lung from day 17 to form cellular aggregates of macrophages and lymphocytes, and M. tuberculosis specific IFN-g production becomes detectable in the lungs. The influx of CD41 and CD81 T cells continues up to 8 weeks, and by 4 weeks postinfection, the progressive increase in mycobacterial numbers in the lungs is halted by IFN-g-dependent activation of infected macrophages, which in the mouse is associated with the induction of iNOS (Cooper, 2009). The bacterial load drops 50% to 90% to a stable plateau level, but despite this strong T-cell response, activated macrophages are unable to eradicate all the mycobacteria, and the bacilli persists at the same level until late in the course of the infection when M. tuberculosis overwhelms the host response. From 6 weeks, the granulomas develop a more organized structure with close apposition of infected macrophages, larger epithelioid

cells and CD41 T cells, which are present throughout the lesions. There are fewer CD81 T cells and these are scattered mostly at the periphery of the granulomas. B cells are also recruited into the infected lung and form aggregates at the edge of the granulomas. With time, the lymphocytes form dense blocks within the granulomas, which accumulate foamy macrophages containing cholesterol, and progressively more of the lung becomes infiltrated. Bands of fibrosis develop around and within the granulomas, contributing to the containment of the infection for up to 9 months, but eventually the granulomas break down and fatal infection supervenes. Granuloma formation is in part regulated by genetic factors. For example, some mouse strains (DBA/2, CBA/J), which are more susceptible to M. tuberculosis, develop looser granulomas composed mainly of macrophages with fewer T cells and display increased tissue necrosis and impaired control of bacterial growth (Saunders et al., 2008). Although cavities do not normally develop in murine TB, one highly susceptible mouse strain develops with necrotizing lung lesions and rapid progression following aerosol M. tuberculosis infection. The super susceptibility locus (sst1) responsible for this effect on chromosome one contains the intracellular resistance gene 1 (lpr1), which exerts its effect through macrophages (Pichugin et al., 2009). The cytokine and chemokine requirements for granuloma formation have been dissected in mice. CD41 T cells, the production of IFN-g and IFN-g-controlled effector molecules, such as iNOS and the small GTPase, LRG-47, are required for granulomas to develop (Saunders & Britton, 2007; Cooper & Khader, 2008). In addition, TNF, which is derived from infected macrophages and infiltrating T cells, is essential for both the generation and maintenance of granulomas and the control of bacterial replication in M. tuberculosis and M. leprae infection (Flynn et al., 1995; Bean et al., 1999; Hagge et al., 2009). TNF regulates chemokine production and chemokine receptor expression by infected macrophages and is necessary for the migration of leukocytes through tissues and the colocalization of infiltrating lymphocytes and macrophages to form granulomas (Roach et al., 2002). Mycobacteria-specific T cells develop normally in TNF deficiency, but in the absence of the microenvironment of granulomas, T cells are unable to activate mycobacterial killing in infected macrophages and the animal’s rapid demise. The TNF-signaling required to form granulomas is very local, as membrane-bound TNF is sufficient for the formation of granulomas and the control of infection for some months (Saunders et al., 2005). The related TNFSF (TNF super family) member, soluble LT-a, is required for granuloma formation independently of TNF in both experimental M. tuberculosis and M. leprae infections, although the pattern of dysregulated inflammation varies between TNF and LT-a deficient mice in chronic leprosy infection (Hagge et al., 2009). There is an absolute requirement for TNF for the suppression of M. tuberculosis infection in humans, as dramatically illustrated by the rapid reactivation of active disease in subjects with LTBI receiving anti-TNF therapy for rheumatoid arthritis or other autoimmune diseases (Wallis et al., 2004). Reactivation occurred with 3 months in more than 50% of those receiving anti-TNF monoclonal antibodies, indicating that sustained TNF signaling is essential to maintain the necessary chemokine gradients and integrity of the granuloma in LTBI as well as acute TB. Although the TB model has been very informative, there are significant differences in the patterns of granulomas in human and murine TB (Saunders et al., 2008). First, the granulomas in human TB lesions are more structured with a central region of epithelioid cells and macrophages surrounded by a layer of CD41 T cells with a cuff of CD81 T cells.


Multinucleate giant cells are a feature of human TB granulomas, but rarely are present in murine TB. Fibrosis is more prominent forming a more defined outer layer around human granulomas, which often calcify as chronic lesions. Aggregates of B cells also occur throughout the infected lungs in human TB. The second difference is that human TB granulomas typically develop a central core of caseous necrosis. This may be related to increased cellular necrosis or apoptosis of macrophages and epithelioid cells following cytokine activation in human granulomas and/or reduced central blood supply to the fibrotic granulomas. Thirdly, the central region of the human granuloma becomes hypoxic, in contrast to the murine granuloma, which maintains normal oxygen saturation (Tsai et al., 2006). This hypoxia induces a change in gene expression in the persisting M. tuberculosis, which switches to anaerobic metabolism and can survive as dormant bacilli within the granuloma for decades. The hypoxia can also increase cellular necrosis leading to a slow increase of the caseous center within the expanding granuloma. If this necrotic lesion erodes into a bronchial passage, then a cavity will form, providing the access for spread of M. tuberculosis by the respiratory route. Analysis of surgically removed cavities from TB patients demonstrated the absence of T cells and T-cell macrophage interactions at the luminal surface of the necrotic cavity, contributing to the unrestrained replication of bacilli within macrophages (Kaplan et al., 2003).

Pathology of leprosy infection

Mycobacterium leprae is an obligate intracellular pathogen of macrophages and Schwann cells and is the cause of leprosy, a slowly progressive infection of skin and peripheral nerves resulting in significant disability worldwide (Britton & Lockwood, 2004). The bacterium cannot grow in in vitro cultures, but M. leprae replicates slowly in mouse footpads and in armadillos, and this discovery provided sufficient organisms to permit analysis of its structural and antigenic components and sequencing of its genome (Scollard et al., 2006). M. leprae is remarkably inert in host tissues, and leprosy is an important example of how host immune responses alone can be responsible for development of disease manifestations. The diverse clinical and pathological features of leprosy form a spectrum of disease, which is determined by the pattern of cellular immune responses to M. leprae (Britton & Lockwood, 2004; Scollard et al., 2006). The majority of M. leprae-infected individuals develop strong T-cell responses, which activate macrophages to kill the intracellular pathogen, and no evidence of disease. In less than 5% of subjects the organisms persist and multiply slowly within dermal macrophages and Schwann cells within dermal nerves and superficial nerve trunks, eventually leading to clinical skin lesions or peripheral neuropathy. In tuberculoid leprosy, the vigorous cellular immune response is associated with a small number of welldemarcated skin lesions with central anesthesia, pallor, and dryness and few demonstrable acid-fast bacilli, and involvement of 1 to 2 nerve trunks. The dermis contains highly structured granulomas with central epithelioid cells and multinucleate giant cells, usually without caseous necrosis, surrounded by IFN-g-producing CD41 T cells and an outer well-defined cuff of CD81 T cells. Circulating M. leprae specific CD4 T cells produce IL-2, IFN-g, and TNF, while specific antibody responses are weak or absent. The Th1promoting cytokines, IL-12 and IL-18, are highly expressed in tuberculoid lesions along with the activation markers, CD25 and CD45RO. Tuberculoid lesions also contain gd T cells and DN ab T cells, which recognize mycobacterial LAM and mycolic acid. These nonpeptide antigens are presented by CD1, and CD11CD831 DCs are present in

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dermal granulomas in tuberculoid, but not lepromatous leprosy, suggesting CD1-restricted T cells may limit the growth of M. leprae. CD41 T cell lines isolated from tuberculoid lesions display cytolytic activity and reduce mycobacteria viability in infected macrophages. They release the AMP, granulysin, which has direct antimycobacterial activity in vitro and colocalizes with CD41 T cells in tuberculoid lesions, suggesting it contributes to control of M. leprae infection (Ochoa et al., 2001). Patients at the lepromatous pole of the leprosy spectrum have multiple skin lesions of variable morphology, generalized infiltration of the skin, and involvement of multiple nerves. They have no demonstrable T-cell responses to M. leprae, although cellular responses to other pathogens including M. tuberculosis are retained, but develop high levels of specific antibodies to M. leprae phenolic glycolipid-I and other antigens. The dermis is replaced by sheets of M. leprae-laden, foamy macrophages, with no well-organized granulomas and few CD41 and CD81 T cells. Lepromatous lesions express transcripts for IL-4 and IL-10. Most leprosy patients have the intermediate types of borderline-tuberculoid (BT), mid-borderline, and borderline-lepromatous (BL) leprosy. Progression from BT to BL leprosy is associated with a reduction in Th1-like T-cell responses, increasing bacillary load, more frequent skin and nerve lesions, and higher antibody levels. A central conundrum in leprosy is what determines whether a patient develops tuberculoid or lepromatous leprosy. There is evidence for a variety of possible mechanisms, including immune deviation from a Th1 to Th2 pattern of T-cell response, deletion of M. leprae-reactive T cells, and the suppressive effect of regulatory T cells in lepromatous disease, but there is no unifying explanation for why such major differences in host response develop in some infected individuals (Britton & Lockwood, 2004; Scollard et al., 2006). Patients with borderline leprosy may experience spontaneous increases in T-cell reactivity to M. leprae with increased IFN-g and TNF production, resulting in increased cell-mediated inflammation in skin lesions and nerves causing loss of nerve function. These type I, or reversal reactions, occur in at least one-third of borderline leprosy patients and are responsible for significant nerve damage in leprosy. Erythema nodosum leprosum (ENL) is a severe generalized inflammatory reaction affecting 50% of lepromatous leprosy patients and is characterized by painful skin nodules, with the pathology of panniculitis, fever, neuritis, iritis, and glomerulonephritis. Circulating immune complexes were originally implicated in the pathogenesis of ENL, but these are not reproducibly associated with ENL. High levels of serum TNF are a feature of ENL and one of the effects of thalidomide, which is dramatically effective in this condition, is to reduce TNF levels as well as the inflammatory lesions (Britton & Lockwood, 2004; Scollard et al., 2006).

Pathology of Mycobacterium ulcerans infection

A different pattern of pathology develops in the skin infection caused by Mycobacterium ulcerans, termed Buruli ulcer (Wansbrough-Jones & Phillips, 2006). In this case, the tissue destruction is caused not by host immune response but by a potent toxin, mycolactone, secreted by the Mycobacterium. Mycolactone is large polyketide synthesized by a gene complex encoded by a giant plasmid (Stinear et al., 2004), and it is proposed that this gives the Mycobacterium a competitive advantage to survive in the salivary glands of water insects. Following M. ulcerans infection of skin through abrasions or possibly insect bites, the mycolactone causes necrosis of cells in the subcutaneous tissue and minimal inflammatory response around the extracellular mycobacteria,

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leading to large ulcers with undermined edges. These ulcers extend and then heal with scarring to cause significant disability. Studies in humans and mice infected with M. ulcerans suggest that in patients with active ulcerating disease, the mycolactone suppresses phagocytosis of M. ulcerans by macrophages and development of a protective Th1 T cell response, but that in the nodular or healing forms of disease, IFN-g-secreting CD41 T-cell responses develop, activate macrophages to kill the bacilli, and promote granuloma formation (Wansbrough-Jones & Phillips, 2006).

aUtoiMMUne inFlaMMatoRy daMage indUCed By BaCteRial inFeCtions

Bacterial infections may trigger autoimmune responses, which cause serious autoimmune disease some weeks or months after the infection. The best-defined mechanism for these responses is molecular mimicry, when epitopes on molecules on the bacterial pathogen are shared with host molecules on particular organs, so that antibacterial antibody or T-cell responses cross-react with the host tissues causing inflammatory damage. Two clearly defined syndromes, which are caused by cross-reactive T cells and antibodies respectively, are rheumatic fever with resulting chronic rheumatic heart disease (RHD) and Guillain-Barré syndrome (GBS). Rheumatic fever develops following infection with rheumatogenic strains of GAS, and is characterized by a migratory inflammatory polyarthritis, fever, clinical carditis, and occasionally, rash or nodules (Carapetis et al., 2005). Recurrent episodes of rheumatic fever are associated with repeated carditis and inflammation of the cardiac valves, leading to slowly progressive RHD with narrowed or leaking valves and cardiac failure. Extensive studies have identified the surface M protein as the major GAS protein, which stimulates cross-reactive T-cell responses (Cunningham, 2004). The streptococcal M protein and cardiac myosin have structural similarities, and CD41 T cells from the heart valves and blood of RHD patients recognize M protein and myosin. Further, immunization of Lewis rats with M protein results in cross-reactive T-cell responses to the M protein and myosin, and the development of carditis and valvulitis. As myosin is not present in cardiac valves, the initial damage to the valves may be due to M proteinreactive T cells recognizing laminin in the valvular basement membrane. Antibodies which cross-react between GAS cell wall components and valvular tissue may also help initiate the inflammatory reaction in heart valves. Discovery that the cardiac-reactive epitopes are located in the N-terminal region of the M protein and the GAS-specific protective antibody determinants are in the C-terminal region led to the development of epitope-based anti-GAS vaccines based on the C-terminal region, which are free of cardiac complications (Batzloff et al., 2006).

gBs

GBS is a demyelinating inflammatory neuropathy presenting as neuromuscular paralysis with rapidly progressive weakness in the limbs and possibly respiratory and facial muscles, with pain and paraesthesia. Preceding infection with Campylobacter jejuni, a cause of gastroenteritis, is recognized as the most common trigger for the onset of GBS and can occur in up to 30% patients, while other pathogens, such as Haemophilus influenzae Epstein-Barr virus and cytomegalovirus, are also known precipants (van Doorn et al., 2008). Half of GBS patients have serum antibodies to a variety of gangliosides present in human peripheral nerves, such as GMI and GD1a, and these antibodies cross-react

with lipo-oligosaccharide antigens present on the surface of C. jejuni. Gangliosides are sialic acid containing glycosphingolipids, and the gangliosides recognized by antibodies from GBS patients have a specific distribution in lipid rafts within peripheral nerve membranes and contribute to the structure and maintenance of the cell membrane. Binding of the antigangliosides antibodies to sites on the peripheral nerves activates the complement cascade, and the subsequent deposition of complement is an essential component of the inflammatory damage to the Schwann cells and nerve axons. Different types of antiganglioside antibodies are associated with different clinical variants of GBS; for example, anti-GM1 or anti-GD1a antibodies occur more commonly in patients with acute motor axonal neuropathy and antiGC1b antibodies in GBS patients with involvement of the extraocular muscles (Kaida et al., 2009). The type of lipooligosaccharide expressed by isolates of C. jejuni influences the specificity of antiganglioside antibodies, which develop in infected subjects, and the subsequent clinical variant of GBS. For example, C. jejuni strains with GM1-like and GD1a-like lipo-oligosaccharide have been isolated from patients with pure motor GBS (Kaida et al., 2009). This is a striking example of the specificity of molecular mimicry triggered by bacterial infections. The molecular mechanisms of other clinically recognized syndromes following bacterial infections, such as reactive arthritis following infection with Chlamydia trachomatis, Shigella spp., or Salmonella spp., are yet to be defined.

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27 Helicobacter pylori: the Role of the Immune Response in Pathogenesis KAREN ROBINSON AND JOHN C. ATHERTON

INTRODUCTION

always results in chronic inflammation of the gastric mucosa. Mononuclear cell and neutrophil infiltration, accompanied by high levels of chemokines and proinflammatory cytokines occurs throughout the lifetime of the infected host (Yamaoka et al., 1997). Gastritis is usually asymptomatic but for some people, after prolonged periods, it can lead to gastric atrophy (loss of specialized gastric glands and reduced acid secretion), which is a precursor to the development of further premalignant pathological changes and gastric adenocarcinoma (Fox & Wang, 2007). Whether ulceration occurs in the duodenal or gastric mucosa is largely dependent upon the site of H. pylori colonization and inflammation (Atherton & Blaser, 2009) (Fig. 1). In antral-predominant gastritis, inflammatory cytokines suppress somatostatin production from G cells, leading to increased gastrin production because somatostatin is a negative regulator of gastrin. These cytokines may also directly stimulate G cells to release gastrin. Hypergastrinemia increases gastric acid output, leading to duodenal epithelial damage and the formation of gastric metaplasia in the duodenum. Once the duodenal epithelial cells have been replaced by those of the gastric type, H. pylori is able to colonize the duodenum and trigger inflammation. Antralpredominant gastritis is therefore associated with increased risk of duodenal ulceration. In contrast, in pan-gastritis or corpus-predominant gastritis, acid output is reduced because proinflammatory cytokines, especially interleukin-1beta (IL-1b) and tumour necrosis factor alpha (TNFa), inhibit the secretion of acid from parietal cells in the corpus. Feedback causes hypergastrinemia, which is associated with increased epithelial cell proliferation, gastric gland atrophy, and increased expression of cancer-associated factors and antiapoptotic mediators (Konturek et al., 2003). This combination in the inflamed hypochlorhydric stomach of increased epithelial cell proliferation, DNA-damaging compounds such as reactive oxygen and nitrogen species, and an induced resistance to apoptosis is thought to facilitate the accumulation of mutations and thus an increased risk for carcinogenesis (Fox & Wang, 2007). The association between H. pylori infection and the development of noncardia gastric cancer is very strong, and H. pylori was classified as a biological carcinogen in 1994.

Helicobacter pylori was discovered less than 30 years ago by Barry Marshall and Robin Warren, who won a Nobel Prize for their work in 2005 (Marshall & Warren, 1984). This pathogen colonizes and persists in the mucosa of the human stomach and in some individuals causes peptic ulcers or gastric cancer. The gram-negative spiral-shaped bacilli are microaerophilic, fastidious, and slow-growing in vitro. H. pylori is able to survive in the low pH environment of the stomach by using its unipolar flagella to migrate rapidly into the gastric mucus layer, and through the expression and release of urease. This enzyme hydrolyzes urea in interstitial fluid to generate ammonium ions and to buffer the bacterial periplasm against gastric acid. H. pylori infections are extremely common, and approximately half of the world’s population have the bacteria in their stomachs (Taylor & Blaser, 1991). The prevalence of infection varies and is associated with poor socioeconomic status and crowded living conditions, particularly during childhood. Most people become colonized as young children, and this persists for life unless treated with antibiotics (Malaty, 2007). The prevalence of infection has declined over the last 30 years, particularly in developed countries where less than 40% of the population are infected, compared to 80% to 90% in developing countries (Taylor & Blaser, 1991). This is thought to be because fewer children are becoming infected, most likely because of improved sanitation and living conditions. Transmission of H. pylori is not yet completely understood, although it is known to require close interpersonal contact. The bacteria are not normally culturable from human feces, but can easily be isolated from the mouth after gastroesophageal reflux. The infection is acquired in childhood from family members or from other children, most probably via the oral–oral route (Malaty, 2007).

H. PYLORI MEDIATED DISEASES

Only 10% to 20% of infected individuals have symptomatic disease (Malaty, 2007), but colonization with H. pylori Karen Robinson, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, United Kingdom. John C. Atherton, Nottingham Digestive Diseases Centre Biomedical Research Unit, University Hospital, Nottingham, NG7 2UH, United Kingdom.

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FIGURE 1

Patterns of gastritis and their links with disease

Gastric cancer is the fourth most common type of cancer and the second leading cause of cancer-associated death (Parkin et al., 2005). The asymptomatic nature of most H. pylori infections means that clinical manifestations appear at a late stage and the prognosis is then poor. Five-year survival rates are very low (less than 20%) despite good standards of health care. There is evidence to suggest that clearance of the infection, before premalignant lesions have developed, can protect against gastric cancer (Fox & Wang, 2007). Therefore, an important challenge is to design strategies for much earlier diagnosis. Apart from gastric adenocarcinoma, H. pylori is also the major causative agent of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori infection induces lymphoid follicle formation and, in some cases, neoplastic cells form and spread from the follicular marginal zone.

In cases where MALT lymphoma is limited to the gastric mucosa, this condition frequently resolves following antibiotic eradication of the infection. In more severe cases, the cells migrate to regional lymph nodes and more distant mucosal sites (Du & Atherton, 2006). The development of disease as a consequence of H. pylori infection is influenced by human genetic differences (Table 1), virulence factors expressed by the colonizing strain (Table 2), and environmental factors such as smoking and concurrent infections. The immune response to infection may be affected by each of these. Infected SCID mice, which essentially lack B and T cells, are heavily colonized by H. pylori but do not develop gastric inflammation (Smythies et al., 2000). The immune response, therefore, plays a major role in determining the level of inflammation and occurrence of disease.

TABLE 1 Host immune response gene polymorphisms and their associations with disease Gene (position of SNP or polymorphism) IL-1B (231, 2511) IL-1RN (tandem repeats) TNFA (2308) IL-8 (2251) IFNGR2 (Exon 7-128) IL-7R (11560) IL-5 (2745) IL-17F (17488) IL-10 (21082, 2819, 2592) TGFB (2509, 1869) TLR4 (1299, 1399)

Association with disease Increased risk of gastric ulceration and cancer Increased gastric cancer risk Increased gastric cancer risk Increased gastric cancer risk Increased gastric cancer risk Increased gastric cancer risk Reduced gastric cancer risk Increased gastritis Unclear. Some evidence of increased gastric cancer risk Increased gastric cancer risk More severe gastritis, increased risk of pre-cancer

27. Helicobacter pylori: the Role of the Immune Response in Pathogenesis

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TABLE 2 H. pylori virulence factors with known effects on the immune response Virulence factor cag PaI and CagA

VacA

Known effects on innate or adaptive immunity Association with increased Th1, IL-12, and IL-18 responses (Yamauchi et al., 2008; Takeshima et al., 2009). Induction of IL-8 and defensins by epithelial cells (Boughan et al., 2006). Suppresses T-cell activation (Gebert et al., 2003; Boncristiano et al., 2003). Induces T cells to express IL-12p40 (Takeshima et al., 2009).

OipA

Stimulates increased epithelial cell inflammatory responses (Yamaoka et al., 2004).

DupA

Increased epithelial cell IL-8 responses (Lu et al., 2005). Promotes Th1 responses, activates neutrophils, and stimulates monocytes and macrophages (Montecucco & de Bernard, 2003).

HP-NAP

HOST DEFENSES AND THE DEVELOPMENT OF DISEASE Innate Immunity

A wide variety of innate host defense mechanisms are chronically induced by H. pylori on the gastric mucosa, including the expression of proinflammatory and antibacterial factors by gastric epithelial cells (Boughan et al., 2006; Yamaoka et al., 1997). Immune and inflammatory cells are continuously recruited to the gastric mucosa, and H. pylori stimulates innate responses from these infiltrating cells also (Gobert et al., 2004). The innate response has an important influence upon disease severity, as it affects the level of inflammation and tissue damage, bacterial colonization density, and the type and magnitude of adaptive immune responses (Boughan et al., 2006; Yamaoka et al., 1997).

Epithelial Cells

Most H. pylori cells are found in the deep gastric mucus, close or adherent to the epithelial cell layer. The main cellular interaction of H. pylori is thus with gastric epithelial cells and this is likely to be a major initiator and ongoing stimulus of the immune response to the infection. All H. pylori infections interact with epithelial cells to some extent, for example with epithelial Toll-like receptors (TLRs), although the bacteria may have evolved to minimize this, as discussed later. Although all H. pylori strains cause ongoing gastric inflammation, the severity of this gastritis differs profoundly between individual infections, and a major driver of this is whether specific virulence factors are expressed by the infecting strain (Atherton & Blaser, 2009). As expected, more virulent strains stimulate more profound proinflammatory epithelial cell signaling and are associated with higher levels of gastritis and a higher risk of H. pylori-associated disease. Risks of both duodenal ulcers and gastric cancer are usually higher with more virulent strains. This is because it is the level of inflammation more than its distribution that is affected by virulence factors; individuals with high-level

Association with disease cag PaI1 strains are associated with increased risk of peptic ulcer disease and gastric adenocarcinoma (Ogura et al., 2000; Atherton & Blaser, 2009). s1/i1 forms are associated with increased risk of peptic ulcer disease and gastric adenocarcinoma (Atherton et al., 1995; Rhead et al., 2007). OipA “on” associated with increased risk of peptic ulcer disease (Yamaoka et al., 2004; Yamauchi et al., 2008). Increased risk of duodenal ulcer (Lu et al., 2005). Contributes to gastric inflammation (Montecucco & de Bernard, 2003).

antral inflammation have increased risk of duodenal ulceration and those with high level pan- or corpus-predominant inflammation have increased risk of gastric ulceration or gastric adenocarcinoma (Blaser & Atherton, 2004). H. pylori virulence factors have potential importance for individual and population management of the infection: screening for and treating pathogenic strains before they cause disease may be a sensible approach. Individual virulence factors and their epithelial interactions are discussed below.

The cag Pathogenicity Island (PaI) and CagA

The most important and best characterized H. pylori virulence determinant is a group of about 30 genes present in some but not all strains, called the cag PaI. Encoded within this is a type IV secretion system (T4SS), a conduit that connects bacterial with epithelial cell cytoplasm and allows “injection” of a bacterial protein, CagA, into the epithelial cell (Odenbreit et al., 2000). cag1 strains stimulate epithelial cells to secrete a range of proinflammatory cytokines and chemokines, most notably the neutrophil chemoattractant IL-8. They also induce profound gastritis in the H. pylori-infected Mongolian gerbil model (compared with cag-disrupted isogenic mutants which induce very little) and are associated with high level gastritis and increased ulcer and cancer risk in humans (Atherton & Blaser, 2009; Ogura et al., 2000). Bacterial-epithelial cell coculture experiments, where different genes in the cag PaI are inactivated, show that induction of proinflammatory cytokines is through two main mechanisms, one independent of and one dependent on the translocated effector protein CagA. The CagA-independent mechanism occurs by way of small amounts of soluble peptidoglycan products leaking into the epithelial cell and being recognized by the intracellular pattern recognition receptor (PRR), Nod1 (Viala et al, 2004). This activates the transcriptional activator NF-kB, leading to transcription and release of proinflammatory cytokines and chemokines and of antimicrobial peptides (Boughan et al., 2006). CagA itself is activated in the epithelial cell by src and abl

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kinases through tyrosine phosphorylation of CagA at specific motifs. Activated CagA then also activates NF-kB, as well as stimulating other signaling systems affecting tight junctions, cell polarity, and the cytoskeleton (Atherton & Blaser, 2009). This is important because different H. pylori strains have different forms of CagA with different types and numbers of tyrosine phosphorylation motifs: European and American strains have from 0 to 4 motifs and those with more motifs cause more profound epithelial cell changes and are associated with more inflammation and a higher risk of disease in humans (Argent et al., 2004; Higashi et al., 2002). East Asian strains have a “super-activating” motif, which may contribute to the very high rate of H. pylori-associated gastric cancer in Japan and parts of China (Higashi et al., 2002). Whether the CagA-dependent or CagA-independent signaling mechanisms are more important in human pathogenesis is unclear, but CagA transgenic mice develop gastric cancer with little inflammation, implying that in this model at least, CagA is a necessary factor (Ohnishi et al., 2008).

The Vacuolating Cytotoxin, VacA

Nearly all H. pylori strains produce VacA, but it is polymorphic and only some forms are toxigenic (Atherton et al., 1995). VacA is a pore-forming toxin that has multiple effects on epithelial cells, including vacuole formation (from uptake of pores into endosomes), apoptosis, disruption of cellular junctions, and effects on the cytoskeleton (Montecucco & de Bernard, 2003). It is unclear which of these effects are important in vivo, but VacA-rich H. pylori extracts and purified VacA cause ulcers in a mouse model, and mice lacking the VacA receptor, receptor protein tyrosine phosphatase b (RPTPb), do not develop ulcers (Fujikawa et al., 2003). In models, VacA appears predominantly to cause epithelial damage rather than inflammation, although, in humans, strains with toxigenic forms of VacA are associated with increased inflammation as well as increased risk of peptic ulceration and gastric adenocarcinoma (Rhead et al., 2007).

Blood Group Antigen-Binding Gene A2 (babA2) and Adhesins

Adhesion of H. pylori to epithelial cells is a prerequisite for disease, but some strains adhere better than others. The best-characterized adhesin affecting strain virulence is BabA (encoded by babA2). This binds specifically to Lewisb on gastric epithelial cells (Boren et al., 1993). Presumably, strains that are closely adherent to epithelial cells can deliver other virulence factors more efficiently; certainly babA21 strains are associated with higher levels of inflammation and higher risk of disease than other strains (Gerhard et al., 1999).

Outer Inflammatory Protein Gene A (oipA)

oipA is either transcribed or not in H. pylori strains depending on the number of CT repeats at its 59 end, a gene regulatory mechanism called slipped strand mispairing (Yamaoka et al., 2000). In vitro assays show that oipA enhances proinflammatory cytokine transcription in epithelial cells, but through different mechanisms to cag PaI genes, including through phosphorylation of the transcriptional activator STAT1 (Yamaoka et al., 2004).

Duodenal Ulcer Promoting Gene A (dupA)

This recently discovered gene is a homolog of a T4SS component, but is not part of the cag PaI (Lu et al., 2005). In some published H. pylori genome sequences, it is surrounded by genes that potentially encode other proteins comprising a complete T4SS machinery. Further characterization

is required, but it is associated with increased levels of IL-8 and inflammation in human gastric mucosa. Interestingly, in most (but not all) reports, it is associated with an increased risk of duodenal ulceration but a reduced risk of gastric cancer (Lu et al., 2005), raising the possibility that it may predispose specifically to antral-predominant inflammation. Like other virulence factors, it is described as inducing IL-8 release from epithelial cells, although the mechanism is unknown.

Interactions Between Virulence Factors

Although virulence factors are not genetically linked in H. pylori strains, they tend to be present or absent en masse, implying that strains benefit from either possessing all virulence factors or no virulence factors (Atherton & Blaser, 2009). Strains possessing virulence factors are closely interactive with the host epithelium and presumably benefit from this (perhaps by gaining nutrients), but cause disease, in part by stimulating a more profound immune response. Strains lacking virulence factors are less interactive, less immunostimulatory, but are much less pathogenic. Interestingly, although indirect effects of virulence factors are proinflammatory, direct effects on immune cells are often inhibitory, at least in vitro (Gebert et al., 2003). This is discussed further below, and raises the possibility that virulent strains may persist despite their interactive lifestyle, at least in part by down regulating immunity.

Infiltrating Leukocytes

Gastric epithelial cell cytokines and chemokines released in response to H. pylori induce granulocyte, monocyte, and lymphocyte migration into the inflamed mucosa. High densities of these cells are associated with more severe inflammation and pathology. Where the innate response to H. pylori results in tissue damage, immune and inflammatory cells in the lamina propria are exposed to bacteria and bacterial components, which heightens the response further (Wilson & Crabtree, 2007).

Neutrophils

The human H. pylori-infected gastric mucosa is chronically infiltrated by a variable number of neutrophils (Allen, 2001) recruited by epithelial and inflammatory cell chemokine responses. These cells contribute to the inflammatory response by secreting cytokines and releasing tissue-damaging factors from neutrophilic granules. They also perform a bactericidal role, where phagosomes containing engulfed bacteria are fused with lysosomes and intracellular granules. Within phagolysosomes, bacteria are exposed to a range of bactericidal effector mechanisms including enzymes to degrade cell walls and proteins, antimicrobial peptides to lyze cells, and reactive oxygen and nitrogen species (ROS and RNS) to induce DNA damage. It has been shown that Helicobacter infected neutrophil-depleted mice exhibit similar colonization densities and severities of gastritis to normal infected animals, implying that neutrophils play only a minor role in protective immunity (Ismail et al., 2003). A recent study using vaccinated mice however, showed that depletion of the neutrophil response prevented immune-mediated reductions in bacterial colonization (DeLyria et al., 2009). This indicates that vaccine protection may be mediated by T-cell induction of gastric neutrophil infiltration. The H. pylori factor HP-NAP is reported to interact directly with neutrophils to induce ROS production via NADPH oxidase, as well as inducing degranulation and release of other tissue damaging factors (e.g., myeloperoxidase and matrix metalloproteinases) (Montecucco & de Bernard, 2003).

27. Helicobacter pylori: the Role of the Immune Response in Pathogenesis

Macrophages

Macrophages play several roles in the innate response to H. pylori (as reviewed by Wilson & Crabtree, 2007). Their importance in disease pathogenesis was demonstrated by the fact that macrophage-depleted mice had a significantly reduced H. pylori gastritis severity (Kaparakis et al., 2008). Phagocytosis is an important antibacterial mechanism, however, H. pylori is able to evade this. The engulfed bacteria survive inside phagosomes that fuse together into megasomes rather than fusing with lysosomes to induce bactericidal activity. The megasomes provide a protected intracellular niche and possibly even contribute to the persistence of infection (Allen, 2001). The bacteria also disrupt NADPH oxidase activity and cause extracellular release of toxic ROS rather than concentrating it within phagosomes. H. pylori is able to neutralize the released ROS via catalase activity (Wilson & Crabtree, 2007), however, chronic exposure to ROS is likely to result in host cell DNA damage and favor cancer development. When macrophages are activated in the presence of type 1 cytokines (e.g., TNFa, IL-12, and IL-18), they secrete more proinflammatory factors and have enhanced bactericidal activity compared to those activated in the presence of T-helper 2 associated cytokines. This includes increased production of nitric oxide, which has been shown to kill H. pylori in cocultures with macrophages. In order to counteract these bactericidal effects, the bacteria express arginase (Gobert et al., 2001). Other H. pylori factors known to have stimulatory effects on monocytes and macrophages include HP-NAP (Montecucco & de Bernard, 2003) and heat shock protein 60 (Gobert et al., 2004).

Dendritic Cells

Increased numbers of gastric mucosal dendritic cells (DCs) are present in H. pylori infected humans and mice (Ninomiya et al., 2000). It is not known whether projections from DCs can extend between gastric epithelial cells to take up bacteria from the lumen, as is known to occur in the intestine. Possibly, gastric DCs take up bacteria and bacterial components that pass through the epithelium following tissue damage from inflammation, or virulence factor mediated loosening of epithelial cell tight junctions. Studies have shown that in mice, Peyer’s patches in the intestine are an important inductive site for H. pylori immunity, where the bacteria are taken up through M cells into the underlying DC-rich area of specialized lymphoid tissue (Nagai et al., 2007). In vitro, human DCs are activated in response to incubation with H. pylori, leading to up-regulated expression of MHC class II and costimulatory molecules, and the secretion of inflammatory cytokines including IL-12, IL-6, IL-8, IL-10, IL-1b, and TNFa (Guiney et al., 2003; Kranzer et al., 2005). It is unclear whether the presence of a functional cag PaI influences the ability of H. pylori strains to stimulate innate responses from DCs, as published reports are conflicting (Guiney et al., 2003; Kranzer et al., 2005).

Natural Killer and Natural Killer T Cells

CD561CD32 natural killer (NK) cells are present in the gastric mucosa and respond to incubation with H. pylori or its secreted products (e.g., urease) by secreting cytokines such as interferon-gamma (IFNg) and TNFa (Yun et al., 2005). Besides this, NK cells produce perforin and granzymes to induce bacterial cell lysis but, as with reactive oxygen and nitrogen, this may also cause damage to host cells. NK T cells, expressing CD3 along with NK cell markers CD56, CD94, or CD161, are present in the human gastric mucosa. O’Keeffe and colleagues recently reported increased frequencies of CD31CD1611 NK T cells in the

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gastric epithelium of H. pylori-infected patients (O’Keeffe et al., 2008). The function of these cells during H. pylori infection is still unclear.

Inflammatory Factors and Disease Risk

Carcinogenesis occurs in rodent models when Helicobacter infections lead to higher levels of inflammation (Fox & Wang, 2007). The bacteria have direct proinflammatory effects on epithelial cells, inducing particularly high levels of IL-8 expression, which chemoattracts neutrophils and lymphocytes. Proinflammatory cytokines, especially IL-1b and TNFa, have major effects on gastrin and gastric acid production (as mentioned previously), and are therefore important determinants of H. pylori-induced disease (Fox & Wang, 2007). In addition, H. pylori-induced cell signaling leads to expression of proinflammatory and angiogenic factors such as IL-8, IL-1b, IL-6, TNFa, and cyclooxygenase-2 (COX-2) (Kitadai et al., 2003). Many studies have shown that polymorphisms in cytokine genes have significant effects on the risk of peptic ulceration and gastric adenocarcinoma development (Lochhead & El-Omar, 2007) (Table 1). Gene polymorphisms, which predispose an individual to increased production of proinflammatory cytokines and/ or lower levels of anti-inflammatory factors, are associated with a significantly greater risk of disease. Reactive oxygen and nitrogen intermediates produced during inflammation may directly induce mutations by causing DNA damage and inhibiting DNA repair mechanisms (Jaiswal et al., 2001). They also activate host signaling pathways and have angiogenic activity.

Pattern Recognition Receptors

Many innate immune mechanisms are triggered by the engagement of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) present on infectious organisms. NOD1, the intracellular receptor stimulated by cag PaI1 strains, is a PRR that recognizes a component of gram-negative bacterial peptidoglycan (Viala et al., 2004). TLRs are a family of cell-surface PRRs, each binding different PAMPs. Activation of PRRs results in the expression of proinflammatory cytokines and other cancerassociated genes, and is thought to play an important role in gastrointestinal carcinogenesis (El-Omar et al., 2008). Studies using TLR-transfected cell lines have shown that H. pylori can stimulate proinflammatory gene expression via TLR2, TLR4, TLR5, and TLR9. Compared to other bacteria such as E. coli, however, H. pylori PAMPs, including LPS and flagellin, have very poor TLR-stimulating activity (Lee et al., 2003). In addition, the PRRs on gastric epithelial cells are not easily accessible since they are expressed on the basolateral rather than the apical surface during infection. This avoidance of a more profound inflammatory response in the gastric mucosa probably aids H. pylori’s lifelong persistence. Once bacteria have penetrated the mucosa, however, they can more easily interact with PRRs on immune and inflammatory cells. Using mice deficient in TLRs 2, 4, 7, and 9, it was recently shown that the inflammatory response of DCs to a lysate of H. pylori was mediated via TLR2, and, to a lesser extent, TLR4. H. pylori DNA also activated DCs via TLR9 (Rad et al., 2009). H. pylori heat shock protein 60 has been shown to activate TLR2 on human monocytes (Gobert et al., 2004). PRR activation and downstream signaling has an important impact on the response to infection. As with inflammatory cytokine genes, polymorphisms in PRR genes also influence disease risk. For example, polymorphisms in TLR4 have been associated with more severe gastritis and increased risk of developing precancerous pathology (El-Omar et al.,

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2008). In mouse models, TLR9 expression by DCs has been shown to control the extent of the lymphocyte response and gastric neutrophil infiltration in response to H. felis (Anderson et al., 2007). NOD1-deficient mice were found to be colonized to significantly higher densities than their wild-type counterparts, and this was thought to be due to differences in expression of antimicrobial peptides such as beta-defensin 4 (Boughan et al., 2006; Viala et al., 2004).


Small cationic antimicrobial peptides of two main groups, defensins and cathelicidins, are secreted as part of the innate immune response to H. pylori. In addition to being bactericidal effectors, these molecules can also act as signaling molecules, for example, by inducing epithelial cell chemokine secretion and functioning as a chemoattractant for immune and inflammatory cells (Wehkamp et al., 2007). Elevated levels of human beta defensins (hBD) 2 and 4, and alpha defensins 1, 2, and 3 are present in the gastric tissues of H. pylori-infected patients. A microarray study on infected rhesus macaques showed that defensin responses are largely dependent on presence of the cag PaI in the colonizing strain (Hornsby et al., 2008). In humans, H. pylorimediated hBD-2 (but not hBD-3) expression by gastric epithelial cells is dependent upon NOD1 and the cag PaI (Boughan et al., 2006).

Acquired Immunity Antibody Responses

Robust mucosal and systemic IgA and IgG antibody responses are detected in H. pylori-infected individuals (Wilson & Crabtree, 2007), but the role of antibodies in mediating protection is questionable since B-cell deficient mice have successfully been immunized (Garhart et al., 2003). Some studies suggest that IgA can block infection, since anti-H. pylori IgA in human breast milk is associated with delayed colonization of infants (Thomas et al., 2004). Human studies have also shown that the presence of a strong IgA response to infection is associated with a significantly increased risk of gastric ulcer and gastric cancer development (Knekt et al., 2006). Why such associations exist is unknown but a possible explanation is that serum IgA bound to penetrating bacteria blocks the activation of complement and phagocytosis, leading to higher colonization densities, increased exposure to toxins, and tissue damaging and angiogenic inflammatory responses. The antibody response to H. pylori is known to contribute to pathogenesis by triggering autoimmunity. Molecular mimicry of host antigens by H. pylori drives the production of antibodies, which react with homologous human antigens such as Lewis blood group antigens on gastric epithelial cells and the parietal cell H1, K1-ATPase (D’Elios et al., 2004). Such parietal cell-reactive antibodies are commonly found in sera from infected individuals, and these could potentially trigger local inflammation and tissue damage.

T-Cell Responses

T-cell responses are thought to be key regulators of the immune response and to be involved in the development of pathology and disease, because the balance of T-cell subsets present influences the level of the inflammatory response and H. pylori colonization density. H. pylori virulence factors are associated with both positive and negative effects on T-cell activity (Table 2). An important T-cell suppressor (at least in vitro) is VacA, which inhibits T cell activation. VacA-induced anion channel formation inhibits the passage of calcium into the cell. In the absence of this signal, cellular pathways are disrupted, the transcription factor NF-AT does

not translocate to the nucleus, and IL-2 secretion is inhibited (Gebert et al., 2003). A second mechanism for VacAmediated inhibition of T-cell activity is driven by activation of a MAP kinase signaling pathway, which leads to disrupted actin polymerization and, hence, effects on the cytoskeleton (Boncristiano et al., 2003). In addition, expression of the costimulatory molecule, B7-H1, by gastric epithelial cells is elevated during H. pylori infection in vivo. Engagement of this molecule also suppresses T-cell activity (Das et al., 2006). A strong concurrent immunological stimulus may also skew the immune system in favor of or against proinflammatory and cancer-associated T-cell responses. Coinfections of H. felis and an intestinal parasite in mice increased Th2 responsiveness and reduced gastric pathology, whereas coinfections with Toxoplasma gondii increased Th1 responses and induced more severe gastric inflammation and premalignant pathology (Fox & Wang, 2007). The role of the different subsets of T-cells in H. pylori-mediated disease is discussed in detail below.

CD41 T-Helper Cell Responses

Although the T-cell response to H. pylori infection includes both CD41 and CD81 cells, the main focus has been on the CD41 T-helper response. T-helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs) have all been found in H. pyloriinfected gastric mucosa (Caruso et al., 2008; Robinson et al., 2008) (Table 3). Th1 responses are thought to play the major role in disease development and vaccine-induced protective immunity, and therefore these cells are the best studied. Th1. The importance of Th1 cells in H. pylori-mediated inflammation was demonstrated using IFNg- and IL-12deficient mice, where significantly reduced levels of gastritis were observed in infected animals compared to wild-type controls (Akhiani et al., 2002; Smythies et al., 2000). The Th1 response is associated with a reduced density of H. pylori gastric colonization, and it is also required for a successful immune-mediated reduction in bacterial numbers following vaccination (Akhiani et al., 2002). The presence of peptic ulcer disease and severity of gastritis are associated with higher numbers of IFNg-secreting Th1 cells in the infected human gastric mucosa (Robinson et al., 2008; Wilson & Crabtree, 2007), indicating a possible role in inducing human disease. Work published by Guiney and colleagues (2003) showed that incubation of human DCs with H. pylori in vitro induced a potent IL-12 response but little or no IL-6 or IL-10. This cytokine environment would preferentially stimulate the differentiation of naïve T cells into Th1 cells during induction of an anti-H. pylori immune response. The presence of a predominant Th1 response in the infected human gastric mucosa indicates that immune-mediated clearance of an infection may be possible, but strategies for vaccination using mouse models have not been very successful so far (Wilson & Crabtree, 2007). H. pylori expresses several virulence factors that are known to stimulate or enhance Th1 responses (Table 2). There have been inconsistencies in the reported effects of the cag PaI upon cultured human dendritic cells (Guiney et al., 2003; Kranzer et al., 2005), however, it is clear that infections with cag PaI1 strains are associated with increased mucosal Th1-associated cytokine responses (e.g., IFNg, TNFa, IL-12, and IL-18) (Takeshima et al., 2009; Yamaoka et al., 1997; Yamauchi et al., 2008). Th1 responses are also influenced by HP-NAP, which may also inhibit Th2 responses in allergy models or parasite coinfections in mice (D’Elios et al., 2009). Similarly, OipA and VacA are reported to stimulate Th1-associated cytokine responses (Takeshima et al., 2009; Yamauchi et al., 2008).

27. Helicobacter pylori: the Role of the Immune Response in Pathogenesis TABLE 3

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Association of T-helper subsets with H. pylori induced inflammation, pathology, and colonization

Feature

T-helper subset Th1

Th17

Th2

Inflammation

More severe inflammation and pathology

Less severe inflammation; may counterbalance damaging Th1 responses

Inhibition of inflammation and pathology

Pathology

High levels associated with peptic ulceration and gastric cancer

Increased inflammation, neutrophil infiltration, and pathology, although may have some suppressive effects by inhibiting Th1 High levels associated with peptic ulceration and gastric cancer

Low levels associated with peptic ulceration; high levels associated with rapid cancer progression

Vaccination efficacy Colonization

Required for successful vaccination in mice Reduced colonization densities in mice and humans

High levels associated with lack of peptic ulceration; responses may switch from Th1 to Th2 during gastric cancer development Not required for successful vaccination in mice Increased colonization densities in mice

Required for successful vaccination in mice Reduced colonization densities in mice

Th2. Th2 cells have been detected in the infected human gastric mucosa (Robinson et al., 2008) but their role in protective immunity and disease pathogenesis is uncertain. It has been shown that Th1 responses predominate in individuals with active gastritis, but there is a strong Th2 response in those with gastric cancer or precancerous pathology (Ren et al., 2001). The reasons behind these associations remain unknown, and it is possible that the patients with cancer or precancer had Th2 responses at earlier stages in their infections. Vaccine studies have shown that Th2 responses are not necessary for protective immunity in mice (Garhart et al., 2003). Interestingly, gastritis in mice is more severe in IL-4 deficient mice, implying that the Th2 response plays a role in suppressing inflammation (Smythies et al., 2000). Th17. Information on the role of Th17 cells in Helicobacter infections has only just begun to emerge. IL-17A and IL-17F responses lead to neutrophil accumulation and, since neutrophilic gastritis is a prominent feature in H. pylori infection, Th17 cells have aroused a great deal of recent interest (Caruso et al., 2008). H. pylori-infected IL-17-deficient mice had significantly reduced levels of gastric mucosal neutrophils (Shiomi et al., 2008). Higher than normal numbers of Th17 cells are present in infected human gastric mucosa. The extent of the neutrophil infiltrate correlates with IL-17 expression, where the highest concentrations are found at ulcer sites (Mizuno et al., 2005). IL-23, an important factor for the differentiation and expansion of Th17 cells, plays a role in several intestinal inflammatory diseases and in cancer development. This cytokine is also highly expressed in H. pylori-infected human gastric mucosa (Caruso et al., 2008). Surprisingly, anti-inflammatory effects of IL-17A on H. pylori-induced gastritis have also been reported. Administration of IL-17A neutralizing antibody to infected mice actually increased Th1 cytokine expression and the severity of gastritis (Otani et al., 2009). The Th17 response may therefore limit gastric inflammation and pathology by inhibiting the differentiation and/or activity of Th1 cells. Two recent studies have demonstrated a requirement for IL-17 in the induction of vaccine-induced protective immunity. An influx of CD41IL-171 T cells into the gastric mucosa was observed in mice immunized prior to H. felis

Treg

Inhibits vaccine efficacy in mice Increased colonization densities in mice and humans

infection, and the administration of IL-17 neutralizing antibodies inhibited clearance of the infection and reduced gastric inflammation (Velin et al., 2009). Elevated IL-17 responses in immunized mice correlated with increased infiltration of neutrophils in the gastric mucosa. Neutralizing antibody treatment to deplete the neutrophil response in immunized mice prevented the reduction in bacterial colonization (DeLyria et al., 2009), indicating that the protective effect of Th17 cells is mediated via induction of gastric neutrophilia. Regulatory T cells. H. pylori-infected human gastric mucosal tissues have been shown to contain large numbers of regulatory T cells (Tregs). These cells suppress inflammation, DC maturation and activation, and also inhibit the activity of effector T cells by a variety of mechanisms, including engagement of receptors (e.g., CTLA-4), secretion of suppressive cytokines (e.g., IL-10 and TGFb), and metabolic disruption (e.g., by adenosine release). Therefore, it is unsurprising that the Treg response is associated with reduced inflammation and pathology, suppressed antibacterial immune responses, and thus increased bacterial colonization loads (Harris et al., 2008; Lundgren et al., 2005; Rad et al., 2006; Robinson et al., 2008). Most CD41CD25hi Tregs in human gastric tissues are IL-101, and the infected human gastric mucosa also contains elevated expression of Treg-associated genes such as IL10, TGFb1, and FOXP3. There are also high levels of CTLA-41 Tregs (Lundgren et al., 2005; Robinson et al., 2008). A recent paper reported that CD39 and CD73, which suppress the function of effector T cells via the generation of adenosine, are expressed by gastric Tregs (Alam et al., 2009). H. felis-infected CD73 deficient mice had significantly more severe gastritis and reduced colonization loads than wildtype controls, demonstrating a role for this Treg mechanism in Helicobacter infection. We previously reported that low numbers of human gastric Tregs are associated with occurrence of peptic ulceration, hence these cells may be protective against the development of H. pylori-mediated disease (Robinson et al., 2008). A study on the Treg response in infected children (who generally do not develop ulcers) also showed that gastric Treg cell responses were increased and gastric pathology was reduced compared to infected adults (Harris et al., 2008).

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Increased levels of Tregs are commonly found in patients with cancer, as tumors themselves may produce factors to increase the numbers of these cells. Tregs inhibit antitumor immunity and so are associated with increased cancer progression and poor prognosis. Consistent with this, increased levels of Tregs have been found in gastric adenocarcinoma tissue compared with surrounding normal tissue, and Tregs from patients with gastric adenocarcinoma suppressed the gastric antitumor cytotoxic T-cell response (Lundin et al., 2007). Mouse infection studies, using antibody-mediated Treg depletion or adoptively transferred Tregs, have shown that the response modulates gastric inflammation and inhibits protective immunity so that a high-level chronic infection is maintained (Rad et al., 2006; Raghavan et al., 2003). Secretion of IL-10 is an important mode of action for H. pylori-induced Tregs, as IL-10 deficient mice can only transiently be colonized and they respond with far more severe inflammation than wild-type animals (Chen et al., 2001). Adoptively transferred Tregs from IL-10 deficient mice were not able to modulate H. pylori-induced gastritis.

CD81 Cytotoxic T Cells

Increased numbers of CD81 cytotoxic T cells are found in the gastric mucosa of infected humans. The presence of duodenal ulcers is associated with elevated numbers of activated CD81 cells in the peripheral blood (Figueiredo Soares et al., 2007). Elevated levels of CD81 cells are also present in the stomachs of H. pylori-infected mice. These cells play a role in H. pylori inflammation and disease, but this has been less well studied. Human gastric mucosal CD81 memory T cells can be reactivated in culture with H. pyloriexposed antigen presenting cells (Azem et al., 2006), and they may express inflammatory cytokines such as IL-17 (Caruso et al., 2008). In addition, more severe gastric inflammation (mediated by CD81 T cells) was observed in H. pylori-infected CD41 cell-deficient mice, indicating that their activity is usually regulated by the T-helper or Treg response (Tan et al., 2008).

CONCLUSIONS

A detailed knowledge of the immune mechanisms required for clearance of H. pylori infection is vital for vaccine development. The increasing prevalence of antibiotic resistant strains, especially in countries where gastric cancer is common, is now driving the need for a vaccine. Unfortunately, the vaccine strategies tested so far have been largely unsuccessful (Wilson & Crabtree, 2007). A detailed knowledge of the immune response is also important if we are to understand why certain people develop symptomatic disease whereas the majority do not. Humans have coevolved with H. pylori and thus the immune system is programmed to take account of stimuli from the bacteria present in the stomach. There are reports that infection is associated with a lack of atopic disease and asthma (Atherton & Blaser, 2009; D’Elios et al., 2009), therefore, it is possible that a Helicobacter-free person is missing some of the immunological cues needed for good health. A targeted approach to H. pylori management may be sensible, focusing on identifying those people at greatest risk of developing H. pylori-associated disease, and ensuring that antibiotic treatment is successful for those colonized by the more pathogenic strains.

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28 Pathogenesis of Helminth Infections THOMAS A. WYNN AND JUDITH E. ALLEN

INTRODUCTION

Eggs that lodge in the tissues, particularly the liver, gut, and bladder wall, are the main cause of pathology. The gastrointestinal nematodes represent the most abundant parasites, infecting over one-third of the human population and a much higher percentage of animals, both wild and domestic. Some of these species develop entirely within the gut, while many, such as Ascaris and the hookworms have lung-migrating stages. Finally, the tapeworms, although the most poorly studied, can lead to some of the most severe diseases caused by helminths, with species that form cysts in the tissues, including the brain and eyes. With a few exceptions, these infections are chronic, with individual adult parasites living as long as 30 years, with many of these infections causing anemia, intestinal inflammation, wasting, poor physical health, and cognitive impairment. Although effective treatments exist for most of these diseases, deficiencies in production and distribution of therapeutic agents have contributed to the relentless scourge of helminthmediated disease in the tropics.

Parasitic worms that infect people and animals cover an enormous phylogenetic spectrum within the animal kingdom. Nematodes (round worms) and platyhelminths (flukes and tapeworms) come from two highly distinct phyla. However, because of their common ability to infect mammals, all of these parasites are classified as helminths. It is remarkable that parasitism as a strategy for survival has evolved independently numerous times within the different branches of the animal kingdom, most particularly among the nematodes (Blaxter, 2003). This diversity among parasitic species manifests itself not only in fundamental differences in biochemistry and structure, but also in life histories, including places of residence or migration both outside and within the mammalian host. There is not an organ in the body that cannot be infected by one of these metazoan invaders, and each parasite uses a diverse range of mechanisms to subvert or utilize the host response to successfully complete their objective—reproductive success. With genome sizes little more than half the size of our own, the complexity of the potential interactions between host and parasite are enormous. Helminths of key public health importance illustrate this complexity. The filarial nematodes that cause elephantiasis are transmitted during a blood meal taken by a mosquito. The infective larvae migrate into the nearby lymphatics and take up residence where they mature, mate, and produce microfilaria that circulate in the bloodstream. Many infected individuals appear to be in a state of immunological tolerance and remain relatively asymptomatic. A proportion, however, make vigorous immunological responses to the nematode, resulting in damage to lymphatic vessels. Infection by schistosome parasites occurs following exposure to fresh water containing infected snails. The infectious cercariae literally burrow through the skin, usually through a hair follicle, enter the bloodstream, and then are swept into the lungs. After a maturation stage in the lung, parasites take up residence in the blood vessels of the gut or bladder depending on species. Each adult pair of parasites produces hundreds to thousands of eggs per day.

HELMINTHS PREFERENTIALLY TRIGGER TH2-ASSOCIATED IMMUNE RESPONSES

Despite their diversity, helminth parasites share a common feature: the propensity to activate the Th2 arm of immunity (Jankovic et al., 2006; Thomas & Harn, 2004). Historically, before the knowledge that CD41 T-helper cells could be polarized into lineages with distinct cytokine secretion profiles, helminth infection was associated with elevated eosinophils, mast cells, and immunoglobulin E (IgE). We now know that all of these features rely on cytokines produced by Th2 cells, which include interleukin 4 (IL-4), IL-5, IL-9, IL-10, IL-13, IL-21, and IL-25, to name just a few. For example, eosinophils require IL-5 for their development and maturation, while mast cell development is regulated by IL-3 and IL-9. B-cell isotype switching toward IgE production requires IL-4 and IL-13 and is antagonized by IL-21 (Pesce et al., 2006). Dendritic cells (DCs) have long been thought to play a key role in the initiation and expansion of most antigenspecific T-cell responses; however, the relative importance of DCs and Toll-like receptor (TLR) signaling in the development of TH2 effector responses is unclear. Numerous studies have suggested that DCs adopt a fairly limited activation profile when exposed to TH2-inducing helminth parasites (Pearce et al., 2004; Perona-Wright et al., 2006).

Thomas A. Wynn, Immunopathogenesis Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892-8003. Judith E. Allen, Institutes of Evolution, Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3JT, UK.

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Although some helminth-derived antigens have been shown to activate DCs (van der Kleij et al., 2002), they are inefficient producers of IL-4, the key driver of CD41 TH2-cell responses. Therefore, attention has focused on identifying accessory cells that provide an early source of IL-4 as well as additional soluble mediators that instruct DC-mediated TH2-cell differentiation. Proposed sources of IL-4 have included eosinophils, mast cells, basophils, natural killer T (NKT) cells, as well as autocrine IL-4 derived from CD41 T cells. Additional secreted cofactors have also been identified, including IL-21, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), which augments the development of TH2 responses by modulating the activation status of antigen-presenting cells (APCs). Surprisingly, it was recently suggested that DCs are not strictly required for the generation of CD41 TH2 responses. Instead, major histocompatibility complex (MHC) class II1 IL-4-producing basophils can serve as the dominant professional APC for both allergen and helminth-induced Th2 responses (Perrigoue et al., 2009; Sokol et al., 2009). Although the role of Th2 immunity in helminth infection has not always been apparent, the evidence from animal models strongly suggests that Th2 responses mediate worm killing or expulsion (Urban et al., 1992). It has recently been appreciated that, to avoid destruction, helminths are particularly adept at deactivating the immune response by stimulating various inhibitory or down-regulatory mechanisms (Maizels et al., 2004). Unrestricted Th2 immunity is damaging to most host tissues and is associated with conditions such as asthma and fibrosis (see below). Acute helminth infections in inappropriate hosts can also cause severe febrile and debilitating illnesses (such as Katayama fever, induced by schistosomes). Thus, it is to both the parasite’s and the host’s benefit to rapidly attenuate the immune response, and indeed most chronic helminth infections are characterized by what has been termed a “modified Th2 response,” in which Th2 immune responses are down regulated by a host of regulatory mechanisms, including regulatory T cells, decoy receptors, myeloid-derived suppressor cells, and crossregulatory Th1 responses (Maizels & Yazdanbakhsh, 2003). This ability of helminths to hijack regulatory pathways is currently being exploited for the development of novel treatments for a variety of autoimmune and inflammatory diseases, in which an overactive immune system is responsible for the disease. Thus, understanding the mechanistic details of how worms modulate immunity is of enormous therapeutic potential. However, the regulatory mechanisms and pathways induced by helminths are vast and likely to differ with each host–parasite combination.

GRANULOMATOUS INFLAMMATION: PROTECTION VERSUS PATHOLOGY

One of the remarkable aspects of studying helminth immunity is that despite detailed knowledge of the cytokine requirements, we have only limited understanding of how worms that reside in the tissues are killed by the host immune system. In many cases, it may be that killing the worm proves too great a task and the “sensible” approach of the immune system is to simply wall it off. This approach is certainly not unique to large multicellular parasites because mycobacterial and other microbial infections can be contained by a granuloma rather than by actual destruction of the pathogen. When encapsulation results in death, it is difficult to determine whether death occurs before or after granuloma formation (Wertheim et al., 1987). The granulomatous structure may simply be a clearance mechanism for a parasite too big to be phagocytosed, or the encapsulation

process may deprive the parasite of needed resources and lead to its death. However, most data would suggest that the worm has at least been weakened prior to encapsulation, suggesting that other effector mechanisms are contributing, including antibodies and eosinophils (Chandrashekar et al., 1985; Hoffmann et al., 1999; James & Colley, 1978). Granulomas are a feature of many diverse helminth infections, ranging across parasite phyla and anatomical location. These include nodules in the skin of people and animals with onchocerciasis (river blindness) (Henson et al., 1979; Mackenzie et al., 1985), the granuloma around the egg lodged in the tissues of individuals with schistosomiasis (Sandor et al., 2003), and the cyst encapsulating the larval stages of the tapeworm Taenia solium that leads to irreversible brain damage (Restrepo et al., 2001). In all these cases, granuloma formation is (at least in part) driven by Th2 cytokines, particularly IL-4 and IL-13 (Chiaramonte et al., 1999a; Chiaramonte et al., 1999b). An intriguing feature of macrophages involved in granuloma formation is their propensity for cell fusion leading to multinucleate cells. This fusion is an IL-4-dependent process (Helming & Gordon, 2007) and is characteristic of all macrophages that are deposited on large foreign bodies. This suggests that IL-4-dependent immunity and the associated macrophage activation process is an ancient one akin to that described by Metchnikoff himself when he stuck a thorn into the starfish larvae and made the first discovery of macrophages.

THE CENTRAL ROLE FOR MACROPHAGES IN ANTIHELMINTH IMMUNITY AND PATHOGENESIS

Until recently, the importance and role of macrophages in type 2 immunity has been largely ignored. Even today, most textbooks will show the effector arm of Th2 immunity as mediated by IgE, eosinophils, mast cells, and goblet cells, with little mention, if any, of macrophages. Not surprisingly considering their central role in granuloma formation and protection against microbial pathogens, we have a much greater understanding of macrophages activated by Th1 cytokines (gamma interferon [IFN-g] and tumor necrosis factor alpha [TNF-a]) and other proinflammatory mediators. In 1992, Siamon Gordon and colleagues noted that treatment of macrophages in vitro with IL-4 led to an alternative activation state distinct from “classical” macrophage activation. Subsequently, IL-13 was shown to have the same effects as IL-4 (Doyle et al., 1994; Stein et al., 1992). This in vitro macrophage phenotype was characterized by elevated MHC class II, mannose receptor (CD206), and arginase activity (Doyle et al., 1994; Goerdt & Orfanos, 1999; Gordon, 1999; Modolell et al., 1995; Schebesch et al., 1997; Stein et al., 1992). These cells are now frequently called alternatively activated macrophages (AAMw). In the context of helminth infection, macrophages are exposed to many signals beyond IL-4 and IL-13 that can modulate or enhance the alternatively activated phenotype. IL-10 (Modolell et al., 1995), GM-CSF, glucocorticoids (Heasman et al., 2004), IL-21 (Pesce et al., 2006), and immune complexes (Mosser, 2003), among others, are likely to be important additional signals to the macrophage during chronic Th2-mediated inflammation. These ligands may act synergistically with IL-4 to mediate anti-inflammatory functions of AAMw, consistent with the ability of IL-4 to up regulate PPARg and the glucocorticoid receptor (GR) (Henson, 2003). IL-10, which typically “deactivates” macrophages (Gordon, 2003), can synergize with IL-4 for enhanced levels of arginase production (Kropf et al., 2005). Although macrophages have been usefully classified as

28. Pathogenesis of Helminth Infections

CAMw and AAMw (also called M1 and M2 macrophages in some publications), the range of actual phenotypes is likely to be much broader, with these designations representing two ends of a wide spectrum. Indeed, these cells display a remarkable level of plasticity and can readily shift from an alternative activation state to a more classical profile in response to Th1 and/or microbial signals (Mylonas et al., 2009). In the early 2000s, two groups looked at gene expression profiles of macrophages recruited to Th2-dominated infection sites to assess the phenotype of these cells in vivo. These studies supported the in vitro work by noting significant up regulation of arginase and also identified two new highly abundant gene products that were expressed by macrophages in an IL-4-dependent manner (Loke et al., 2002; Raes et al., 2001). It appeared that, in vivo, AAMw represented a truly novel phenotype characterized by the production of two secreted proteins: a chitinase-like molecule, Ym1 (Chi3l3) and a small cysteine-rich molecule now known as Relma or FIZZ1 (Retnla). The closely related proteins, Ym2 (Chi3l4), acidic mammalian chitinase (Chia) (Boot et al., 2001), and Relmb/FIZZ2 (Artis et al., 2004), were subsequently identified, and these families of chitinase-like molecules and fizz proteins (ChaFFs) were found to be a typical feature of nematode and schistosome infections (Nair et al., 2009; Nair et al., 2005; Pesce et al., 2009b), although not all are produced by macrophages. It soon became evident that, along with arginase, Ym1 and Relma were reliable markers for alternative activation in a variety of noninfectious settings (Liu et al., 2004b; Misson et al., 2004; Sandler et al., 2003; Webb et al., 2001; Welch et al., 2002; Zimmermann et al., 2003) as well as virtually all helminth infections studied to date (Reyes & Terrazas, 2007). Although first identified in vivo, Ym1 and the closely related Ym2, as well as RELMa,b have been shown to be directly induced by IL-4 and IL-13 in vitro (Nair et al., 2003; Raes et al., 2001), and several studies have defined their dependence on the IL-4 receptor and Stat-6 (Liu et al., 2004b; Webb et al., 2001; Welch et al., 2002) along with arginase 1 (Rutschman et al., 2001). In addition to the expression of Ym1/2, RELMa,b, and arg1, AAMw express a range of other proteins (Noel et al., 2003; Rutschman et al., 2001) and exhibit several IL-4/ IL-13-dependent features that are providing valuable insight into their activities in vivo. These include the ability of IL-4/ IL-13-activated macrophages to block the proliferation of cells in cocultures (Loke et al., 2000), suppress production of proinflammatory chemokines (Loke et al., 2002), and prostaglandins such as prostaglandin E2 (Mosca et al., 2007), and regulate antitumor immunity (Van Ginderachter et al., 2006), nutrient homeostasis (Odegaard et al., 2007), and nitric oxide (NO) production (Rutschman et al., 2001). Recent studies with macrophage-specific Arg1-deficient mice suggested a critical role for arginase-1 in many of the functions that have been attributed to AAMw (Pesce et al., 2009a). The human in vivo equivalents of these divergent phenotypes discovered in mice have yet to be fully elucidated. However, AAMw frequently represent the most abundant cell type in many if not most immune responses to metazoan parasites (MacDonald et al., 2003; Ramesh et al., 2007). Although direct parallels in terms of specific gene products have yet to be made between mice and humans, there is little question that human macrophages exhibit distinct properties on exposure to Th2-type cytokines (Gordon, 2003) and, as in the mouse models, macrophages are abundant at the site of helminth infection (Edgeworth et al., 1993). The picture is complicated by an absence of human Ym1/2 genes, but several proteins of unknown function have the potential to be the functional homologs of murine chilectins

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(Elias et al., 2005; Sutherland et al., 2009). Most of the human molecules have yet to be investigated in the context of Th2 cytokine regulation, although a recent study identified a critical role for brp39 (Chi3l1), also called YKL-40 in humans, in Th2 and IL-13 induced tissue responses and apoptosis (Lee et al., 2009). Because IL-13 is known to play a critical role in the pathogenesis of many helminth infections, the IL-13-inducible gene, Chi3l3, may exhibit similar activity in these diseases. Recently Kzhyshkowska identified a human gene expressed in macrophages, SI-CLP, which has sequence similarity with Ym1 and is up regulated by IL-4 (Kzhyshkowska et al., 2005). The controversy about the human AAMw equivalent is further complicated by the apparent lack of arginase in some human macrophages, although expression in neutrophils has been identified (Munder et al., 2005) and arginase expression in macrophages can be induced by IL-4 under the right conditions (Erdely et al., 2006). Most recently, arginase-producing monocytes have been observed in individuals with filarial infection (Babu et al., 2009). This suggests that the failure to detect arginase may be due to technical limitations on the sources of human cells, as seen with the controversy over inducible nitric oxide synthase (iNOS) (Fang & Nathan, 2007).

THE PATHOGENESIS OF SCHISTOSOMIASIS IS REGULATED BY NOS2 AND ARG1

In the murine model of Schistosoma mansoni, parasites reside in mesenteric veins where they lay hundreds of eggs per day 4 to 5 weeks postinfection. Some eggs are trapped in the microvasculature of the liver and gut, where they induce a vigorous granulomatous response (Cheever & Andrade, 1967). Subsequently, fibrosis and portal hypertension develop. Consequently, much of the symptomatology of schistosomiasis is attributed to the egg-induced granulomatous response. Studies aimed at dissecting the respective roles of Th1 and Th2 CD41 T-cell-associated cytokines in granuloma formation showed that the granulomatous response evolves from an early Th1 to a sustained and dominant Th2-type cytokine response (Grzych et al., 1991; Pearce et al., 1991; Vella & Pearce, 1992; Wynn et al., 1993), with 20% to 30% of the granulomatous lesions composed of AAMw. The importance of Th2 cells to the pathogenesis of schistosomiasis was first demonstrated in experiments in which mice vaccinated with egg antigen plus IL-12 to induce an egg-specific CD41 Th1 response upon subsequent infection developed smaller lesions and less severe fibrosis than did nonsensitized Th2-polarized controls (Boros & Whitfield, 1999; Hoffmann et al., 1998; Wynn et al., 1995; Wynn et al., 1994). The decreased pathology was associated with a reduced Th2 response, markedly fewer AAMw, reduced Arg1, and increased iNOS-2 expression by macrophages. It was hypothesized that the efficacy of IL-12 as a Th2reducing agent might be improved if iNOS-2 activity was neutralized, because the antiproliferative effects of NO on Th1 cells would be eliminated as well as its potentially tissue-destructive and proinflammatory activities (Hesse et al., 2000). However, experiments with iNOS-2-deficient mice showed that while relatively normal CD41 Th1 cell responses could be established in egg/IL-12-sensitized iNOS-22/2 mice, a more than an eightfold increase in granuloma size was displayed and liver fibrosis was exacerbated. Thus iNOS-2 rather than inhibiting the protective effects of IL-12 was actually responsible for the downstream antiinflammatory and antifibrotic effects of the egg-specific Th1 response. As such, these data were the first to suggest a critical host-protective function for iNOS-2 expressing CAMw in schistosomiasis (Fig. 1).

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FIGURE 1 Distinct forms of lethal pathology develop when Th1/Th17 or Th2 responses dominate during Schistosoma mansoni infection. In murine schistosomiasis, when the immune response to the egg deposited in tissues (e.g., gut, liver, bladder) is skewed to Th1, Th17, or Th2-type cytokine responses, distinct forms of lethal immunopathology develop. When Th1/Th17 responses predominant, particularly in the acute stage of the disease or in animals genetically predisposed to develop stronger Th1/Th17 responses, S. mansoni infected mice suffer from severe acute morbidity and mortality, which is associated with the development of severe inflammation in the gut and liver, hepatotoxicity, and endotoxemia with minimal liver fibrosis. In contrast, when Th2 responses prevail, mice survive acute infection, but the persistent Th2 response contributes to the development of liver fibrosis, portal hypertension, development of collateral blood vessels, hematemesis, and death in the chronic stages of infection.

The protective role of iNOS-2 in hepatic fibrosis is supported by studies with mice deficient in the l-arginine transporter, CAT-2, which is required for sustained NO production (Nicholson et al., 2001). When CAT22/2 mice were infected with S. mansoni (Thompson et al., 2008), they developed granulomas that were three to four times larger than in the wild type, and hepatic fibrosis was significantly exacerbated, indicating a general worsening of Th2-associated pathology in the absence of CAT2. The pathological changes in the CAT22/2 mice were also associated with increased arginase activity in fibroblasts and AAMw, suggesting that CAT2 functions as an important regulator of Arg1 activity. Thus, CAT2 appears to promote iNOS2 function while antagonizing arginase activity.

A ROLE FOR ARGININE METABOLISM IN ANTIHELMINTH IMMUNITY

Previously, it was generally assumed that the large numbers of macrophages associated with dying worms were killing the parasites via the release of oxygen or nitrogen radicals (Allen & Loke, 2001; James et al., 1982; Mackenzie et al., 1985; Oswald et al., 1992; Taylor et al., 1996; Thomas et al., 1997). Indeed, in vitro studies demonstrated that thioglycollate-elicited murine peritoneal macrophages kill S. mansoni schistosomula by an l-arginine-dependent mechanism that involves the production of reactive nitrogen intermediates (Oswald et al., 1992). Similarly, J774 cells classically activated by lipopolysaccharide (LPS) and IFN-g were shown to kill larval forms of the filarial nematode Brugia malayi (Thomas et al., 1997). However, with our new understanding that AAMw are the predominant cell type associated with helminth infection, we have to ask whether AAMw (or CAMw) and/ or their associated products have the capacity to kill large

extracellular parasites in vivo (Coulson et al., 1998; James et al., 1998) or whether they play more indirect functions in this regard (e.g., parasite disposal rather than destruction). The most direct evidence to date that AAMw have antiworm effector function comes from the study by Anthony and colleagues (2006), who demonstrated that the depletion of AAMw from the intestines of mice abrogated protective immunity seen during secondary infection with the gastrointestinal nematode parasite Heligmosomoides polygyrus. Arginase inhibitors suggested that AAMw-derived Arg1 was responsible for expulsion of H. polygyrus (Anthony et al., 2006). However, the mechanism by which arginase is acting against the parasite is currently unclear. One possibility might be that arginase is depriving the worm of a needed resource, such as l-arginine. Perhaps more likely, interference with the arginine metabolism regulates the activation status of both immune and nonimmune cells. For example, inhibition of arginase leads to increased NO production that may modulate antiworm effector mechanisms as has been shown recently for protozoan parasites (El Kasmi et al., 2008). Early studies also suggested that macrophage-derived arginase can facilitate the killing of schistosomula in vitro (Olds et al., 1980). Future research needs to focus on the specific mechanism of arginase activity against helminths, as this may have broad implications in the development of vaccines and antihelminthic drugs.

ARGINASE-1 IS A CRITICAL REGULATOR OF HELMINTH-INDUCED TH2-TYPE IMMUNITY

The failure to suppress granuloma formation and hepatic fibrosis in egg/IL-12-sensitized NOS-2-deficient mice revealed a previously unrecognized protective function for nitric oxide and CAMw. This unexpected finding suggested that disturbances in the urea/l-arginine biosynthetic


pathway might regulate the pathogenesis of schistosomiasis and other Th2-associated diseases. Numerous studies have suggested that macrophages and fibroblasts are key effector cells in the pathogenesis of fibrosis. Thus, although the phenotype of CD41 T cells is clearly important (Chiaramonte et al., 1999b; Wynn et al., 1995), their primary function may be to control the activation and recruitment of inflammatory macrophages, fibroblasts, and other NOS-2-expressing cells (Hesse et al., 2000; Hesse et al., 2001). Th1 cytokines activate NOS-2 expression in CAMw, whereas the Th2-associated cytokines IL-4, IL-10, IL-13, and IL-21 preferentially stimulate arginase-1 (Arg-1) activity in AAMw (Gordon, 2003; Munder et al., 1998). Interestingly, DCs (Munder et al., 1999) and some fibroblast populations (Witte et al., 2002) show a similar differential pattern of iNOS2 and Arg1 expression when stimulated with Th1 and Th2 cytokines. l-arginine serves as the substrate for both enzymes, with iNOS-2 generating l-hydroxyarginine, l-citrulline, and NO, and arginase-1 promoting urea and l-ornithine production. l-Ornithine serves as the substrate for two additional enzymes, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT), which generate polyamines and l-proline, respectively. Because polyamines are critical for cell growth and proline serves as a substrate for collagen synthesis, ODC and OAT are both believed to be important regulators of tissue repair processes (Hesse et al., 2001). Studies in the schistosomiasis model showed that the development of hepatic fibrosis is highly dependent on the conversion of l-ornithine to proline (the basic building block of collagen), a process regulated by arginase. Therefore, the preferential activation of Arg1 versus iNOS2 in macrophages and fibroblasts has been proposed as a possible explanation for the potent profibrotic activity of Th2 cytokines (Chiaramonte et al., 1999a) and antifibrotic activity of IFN-g (Hesse et al., 2000; Wynn et al., 1995). Proline production is induced by IL-13 and tightly regulated by the activity of Arg1 in macrophages (Hesse et al., 2001). Therefore, it was thought that the profibrotic activity of IL-13 might be dependent on the activation of Arg1 in macrophages. To formally elucidate the function of macrophage-specific Arg1 mice expressing a macrophage/ neutrophil specific deletion of Arg1 (Arg1flox/flox;lysMcre) were investigated (Pesce et al., 2009a). Although susceptibility to infection was not affected by the conditional deletion of Arg1 in macrophages (Arg1flox/flox;lysMcre), chronically infected Arg1flox/flox;lysMcre mice died at an accelerated rate. The increased mortality was not due to excess NO production. However, liver sections from chronically infected Arg1flox/flox;lysMcre mice showed a significant increase in granulomatous inflammation, liver fibrosis, and portal hypertension. Blood was also frequently found in the intestines of the KO mice, suggesting that bleeding from collateral vessels was the primary cause of death. Thus, while macrophage-specific Arg1 was originally hypothesized to be an important inducer of fibrosis and portal hypertension, studies conducted with Arg1flox/flox;lysMcre mice suggested that instead of promoting disease (inflammation and fibrosis), macrophage/neutrophil-specific Arg1 was required for the resolution of disease. In the case of S. mansoni infection, the macrophage specific deletion of Arg1 led to the rapid development of the severe hepatosplenic form of schistosomiasis (Fig. 1). Thus, while numerous investigators have suggested that inhibiting arginase activity might represent a viable therapeutic strategy for a variety of Th2-associated diseases including asthma, these results suggest that arginase inhibition might actually exacerbate disease (Pesce et al., 2009a).

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FIZZ/RELM FAMILY MEMBERS DURING HELMINTH INFECTION

The conflicting roles of arginase in different contexts is also reflected in our emerging understanding of Relma. Relma is a member of the resistin-like family also called “found in inflammatory zone,” which consist of four members, Retnla/Relma/ FIZZ1, relmb/FIZZ2, Resistin/FIZZ3, and Relmg/FIZZ4. Resistin is expressed by adipocytes and has been implicated in the regulation of obesity and type-2 diabetes (Steppan et al., 2001). Relma is expressed in AAMw as described above and also in hypertrophic and hyperplastic bronchial epithelium, while Relmb is predominantly expressed in gut epithelium. Relma increases significantly during allergic responses in the lung and expression is IL-4/IL-13 and Stat6-dependent (Loke et al., 2002; Nair et al., 2005; Sandler et al., 2003). Recent studies suggested that Relma is possibly involved in the induction of fibrosis by promoting the differentiation of myofibroblasts that mediate collagen deposition (Liu et al., 2004a). Consistent with a role in tissue remodeling, another study shows that Relma can increase the proliferation of smooth muscle in vitro and has angiogenic properties in vivo (Teng et al., 2003). Relma is markedly induced in the granulomatous tissues of S. mansoni infected and egg-challenged mice and in the lungs of Nippostrongylus brasiliensis infected mice. A recent study showed that the related family member, Relmb, could impair the chemosensory activity of the parasite Strongyloides stercoralis in vitro. As such, these authors suggested that Relmb serves as a Th2-dependent effector molecule during GI (gastrointestinal) nematode infection (Artis et al., 2004). Subsequent studies with Relmb-deficient mice, however, demonstrated that resistance to tissue invasive GI nematodes is not dependent on Relmb (Nair et al., 2008). Instead, Relmb augmented the production of IFN-g by CD41 T cells and increased infectioninduced intestinal inflammation (Nair et al., 2008). Similar studies conducted with Retnla2/2 mice also found that Relma was not required for the development of helminth-induced Th2 responses in the lung, liver, or gut (Nair et al., 2009; Pesce et al., 2009b). Instead, Th2-dependent pulmonary inflammation, S. mansoni-induced liver fibrosis, and GI nematode expulsion of N. brasiliensis were all significantly enhanced in the absence of Relma. These studies suggested that Relma primarily functions as a feedback mechanism to suppress Th2 responses. In the case of schistosomiasis, Relma deficiency triggered severe inflammation in the lung and liver, leading to the accelerated development of hepatosplenic disease following infection, whereas in the case of N. brasiliensis infection, Retnla2/2 mice expelled their parasites more rapidly from the gut. Thus, Relma exhibits both protective and disease exacerbating activities by functioning as a negative regulator of Th2 responses (Pesce et al., 2009b). Interestingly however, tissue localization and cell isolation experiments have revealed that while macrophages may be the predominant source of Relma in the body cavities, gut, and skin (Anthony et al., 2006; Eming et al., 2007b; Mylonas et al., 2009; Nair et al., 2005), eosinophils and epithelial cells, rather than macrophages, are the major players in the lung and liver (Pesce et al., 2009b). Thus, while Relm family members clearly exhibit important immunoregulatory activity during helminth infection, the specific role of AAMw-derived Relma remains unclear. As with arginase, it is possible that depending on the source and timing, Relm proteins may act both as effectors and regulators of Th2 immunity.

CHITINASE FAMILY MEMBERS AND OTHER LECTINS IN IMMUNITY AND PATHOGENESIS

Relm family members and arginase are emerging as critical Th2-dependent regulators and effectors of helminth immunity and pathology. Yet the most abundant Th2-induced

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protein produced during helminth infection is the lectin Ym1 (Loke et al., 2002). Ym1 is a member of a family of chitinases, some of which have chitin-cleaving ability, whereas some do not. Ym1 lacks this enzymatic activity but presumably can still bind chitin. Some but not all helminths contain chitin and not necessarily at every stage of development (Foster et al., 2005). Whether this potential to bind the parasite translates into an antiparasite effector function has yet to be demonstrated. Ym1 has a remarkable capacity to form crystals under appropriate pH conditions (Guo et al., 2000; Harbord et al., 2001), and chitin can be found in the gut lining of some nematodes. One possible mechanism by which Ym1 may act is to form damaging crystals within the parasite. Consistent with this, the ability of Ym1 to induce cellular damage has been observed during fungal infections of the lung (Huffnagle et al., 1998). The ability of Ym1 to interact with a variety of host sugars (Chang et al., 2001) raises the possibility that Ym1 may promote the deposition of host matrix components interfering with parasite function or “trapping” the parasite in a matrix that limits mobility or normal function. Recent studies with the related chitinase member Brp-39 (Chi3l1) revealed potent immunoregulatory activity for the protein in asthma models. Similarly, Ym1/2 has been demonstrated to potentiate Th2 cytokine production (Cai et al., 2009). Thus, these proteins may exhibit similar activity during helminth infection. In addition to Ym1, Ym2, and Brp-39, the mannose receptor is also a characteristic feature of most AAMw (Noel et al., 2003). In fact, the mannose receptor was the first identified marker of alternative activation (Stein et al., 1992) and yet our understanding of its function in Th2 immunity or helminth infection has yet to be unraveled (deSchoolmeester et al., 2009; Gazi & Martinez-Pomares, 2009). Raes and colleagues (2003) also identified IL-4/IL13-dependent up regulation of two additional members of the galactose-type C-type lectin gene family, mMGL1 and mMGL2, during Taenia crassiceps infection. With the exception of a study providing evidence that the mannose receptor can bind schistosome egg components (Linehan et al., 2003), studies to identify whether these lectins bind specific helminth carbohydrates and regulate disease have yet to be been performed. Even if these interactions are identified, they may not reveal their exact function. Lectin binding could promote cell recruitment and matrix deposition on the worm, or bind to parasite regulatory molecules and thus prevent down regulation of host responses. Considerably more research is needed on both the identification of helminth carbohydrates and the function of the mammalian lectins before these questions can be answered.

TH1/TH17 RESPONSES ALSO EXHIBIT PROTECTIVE AND PATHOGENIC ACTIVITY

Survival of mice acutely infected with S. mansoni infection is dependent on the rapid development of a Th2 response. In the absence of IL-4/IL-13 signaling, mice develop hepatotoxicity, endotoxemia, and severe cachexia, which contribute to their rapid demise (Pearce & MacDonald, 2002). In most cases, when the Th2 response is inadequate, the immune response defaults to a more proinflammatory Th1/Th17-type response. Indeed, some strains of mice such as CBA are prone to developing Th1/17 responses during acute infections, and these mice develop severe pathology in response to S. mansoni infection (Fig. 1). Blocking studies have suggested that IL-17 and IL-23 are, in large part, responsible for the exacerbated pathology (Rutitzky et al., 2008; Rutitzky &

Stadecker, 2006; Shainheit et al., 2008). In the majority of cases, however, these highly pathogenic Th1/Th17 responses quickly wane and are replaced by highly protective Th2 responses (Wilson et al., 2007). While this transition is initially beneficial to the host, the persistent Th2 response itself ultimately becomes highly pathogenic by contributing to the development of hepatic fibrosis, portal hypertension, and severe hepatosplenic disease as the infection becomes increasingly chronic (Wilson et al., 2007). The ability of Th1, Th2, and Th17-derived cytokines to contribute to pathology (and protection) is also illustrated by studies of inflammation caused by nematode infection of the GI tract. Th2 immunity is absolutely required for nematode expulsion from the GI tract, but it also causes edema, cellular infiltration, mucosal mastocytosis, and crypt hyperplasia (Pennock & Grencis, 2006). However, in the absence of sufficient Th2 immunity, persistent infection leads to the development of severe intestinal inflammation, which is again mediated by Th1 and/ or Th17 cells (Schopf et al., 2002; Taylor et al., 2009; Zaph et al., 2008). Thus, as discussed below, Th2 immunity may not only have evolved to expel the parasite but also to limit inflammation associated with colonization. Whether the inflammatory effect of helminth infection on the GI tract is direct or indirect via changes to gut bacterial flora (Macpherson & Harris, 2004) has yet to be fully determined although broad-spectrum antibiotics, which eliminate gut flora, have been shown to improve outcomes in IL-102/2 mice infected with Trichuris muris (Schopf et al., 2002). The Litomosoides sigmoiditis infected mouse model has allowed a detailed investigation of the parameters required for control of filarial nematodes, parasites that live in the tissue rather than the gut (Allen et al., 2008). These studies found that although killing of the larval stages requires Th2 immunity, destruction of the adult stage is enhanced by Th1 immunity, which can act synergistically with type 2 cytokines in this setting (Saeftel et al., 2003). Similar findings have also been reported for the adult and larval forms of Trichinella spiralis, where expulsion of adults is mediated by a Th2 response whereas immunity to the muscle larvae may be more dependent on a Th1/NOS2dependent response (Fabre et al., 2009). Another exception to the Th2 worm-killing paradigm is the tapeworm, Taenia crassiceps, a model of the life-threatening helminth infection neurocysticercosis. These cestode parasites induce Th2 immunity, but this appears to confer susceptibility rather than resistance (Rodriguez-Sosa et al., 2004). Using inhibitors of NO, these investigators have shown that NO is the critical macrophage-derived factor needed to kill these parasites (Alonso-Trujillo et al., 2007). Finally, in the case of schistosomes, the parasites appear to be resistant to both arms of the immune response and can successfully establish infections in animals that develop highly polarized Th1 and Th2-type responses (Hoffmann et al., 2000). These exceptions raise important questions regarding the relative importance of Th2 responses and AAMw in antihelminth immunity, as there are clear cases where Th1 responses and CAMw are required for parasite control. However, the latter mechanism does not appear to be a typical pathway induced in response to most helminth infections, perhaps due to the potential for extensive host damage with chronic infections. Indeed, limiting proinflammatory Th1/Th17 responses may be an essential host protective response to helminth infection, especially in the GI tract.


REPAIR OF GUT AND LUNG DAMAGE DURING HELMINTH INFECTION IS ESSENTIAL FOR SURVIVAL

The association of helminths with Th2 responses and anti-inflammatory cytokines such as IL-10 may, in part, be explained by the requirement for rapid tissue repair in many infection settings. The propensity of helminth parasites to induce potentially lethal tissue damage may have provided sufficient evolutionary pressure for the development of a worm-specific tissue repair process (Graham et al., 2005). Hookworm parasites penetrate the gut wall to feed, while schistosomes use proteolytic enzymes to enter the skin and pass through the gut wall, and larval forms of both parasites traverse the vasculature of the lungs. Lesions in the skin or gut could lead to sepsis unless repair is rapid and effective. Consistent with this hypothesis, AAMw -deficient mice die of fatal sepsis after S. mansoni egg deposition (Herbert et al., 2004). Similarly, while lung-migratory phases of many different helminth parasites can lead to hemorrhage and loss of tissue integrity, repair is rapid and long-term sequelae are rare in animals or humans. A dramatic example of repair is seen during murine N.brasiliensis infection. Infective larvae migrate from the skin via the vasculature to the lung, and most larvae pass through the lungs in 48 to 72 hours. Larval migration causes severe pulmonary damage with leakage of serum proteins into the lavage fluid. This is associated with a profound inflammatory response that begins to resolve by 8 days, and by 2 weeks the lung has undergone a remarkable level of repair (P. A. Keir, D. M. Brown, A. Clouter-Baker, D. P. Knox, and L. Proudfoot, unpublished). Maximal Th2 responses occur well after the parasite has left the lung (Harris et al., 1999; Tomita et al., 2000; Voehringer et al., 2004), and Ym1/Relma levels in the lungs of N. brasiliensis-infected mice continue to increase for over 2 weeks (Nair et al., 2005; Reece et al., 2006; Reese et al., 2007). This suggests that Th2 immune responses in the lung have as much to do with repair or “cleaning up” as with antiparasitic effector function. Despite elegant studies of Th2-mediated inflammation in the early lung migratory phase (Shinkai et al., 2002; Voehringer et al., 2004), few studies have focused on aspects of tissue repair, a process that typically requires macrophages (Duffield et al., 2005). Although N. brasiliensis expulsion from the gut requires IL-13, it does not depend on AAMw (Herbert et al., 2004), suggesting these cells have other roles and raising the possibility that AAMw are required for effective lung repair following N. brasiliensis migration. The evolutionary role of AAMw in appropriate wound healing is likely to explain the association of Th2 immunity with fibrotic damage. Ironically, the specific AAMw-associated molecules previously implicated in fibrosis, arginase and RELMa, may actually regulate the overzealous Th2 processes as described above.

“ANTI-INFLAMMATORY” MEDIATORS INDUCED BY HELMINTH-INDUCED TH2 RESPONSES

AAMw are often described as anti-inflammatory despite their presence in patently “inflammatory” situations, such as the schistosome granuloma (Sandor et al., 2003), body cavities containing filarial parasites (MacDonald et al., 2003), or following nematode migration in the lung (Ramaswamy et al., 1991). Despite this apparent

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contradiction, AAMw do exhibit many anti-inflammatory characteristics. For example, synthesis by macrophages of inflammatory prostaglandins such as prostaglandin E2 is inhibited by IL-4 and IL-13 (Mosca et al., 2007), as are proinflammatory chemokines (Loke et al., 2002). Overall, in terms of cytokine secretion, chemokine profiles, and prostaglandin synthesis, macrophages activated in a Th2 environment exhibit what could be described as a noninflammatory profile. How does one reconcile this with the role of AAMw as potential effector cells recruited to the site of infection and their role as mediators of repair and fibrosis, a process that has many inflammatory components and which itself needs to be regulated? It is in the analysis of the wound-healing response that an answer can be found. For effective tissue repair to take place, inflammatory processes mediated by classically activated macrophages must end or extensive tissue destruction may result (Ashcroft et al., 2003; Eming et al., 2007a). Thus, although macrophages during helminth infection are actively recruited in an inflammatory context, the nature of this inflammation differs from that seen during classical macrophage activation by microbial products and IFN-g. Indeed, Th1 and Th2 responses are not only cross-regulated at the T-cell differentiation level, but also at the macrophage activation level. This is best illustrated with the arginase metabolism pathway described above in which macrophages must compete for arginine (Fig. 2). The induction of arginase by IL-4/IL-13 will reduce NO levels (Hesse et al., 2001; Modolell et al., 1995), thus acting to reduce one of the main mediators of “classical inflammation.” In addition to cross-regulating CAMs (El Kasmi et al., 2008), Arg1-expressing macrophages can also compete with T cells for the available stores of l-arginine, which dampens the ability of T cells to proliferate and expand, providing another mechanism for AAMw to attenuate inflammation (Pesce et al., 2009a). Arg1-expressing AAMw may also regulate the activity of important nonhematopoietic cells like fibroblasts, which also rely on l-arginine for efficient collagen synthesis. Consistent with their roles as immune regulators, in addition to expressing high levels of Arg1 and Relma, AAMw recruited to helminth infection also produce high levels of the down regulatory cytokines IL-10 and TGF (transforming growth factor)-b1. This has been seen in both animal models and analysis of macrophages from helminthinfected humans. Further, helminth-induced macrophages have a well-documented ability to block cellular proliferation (Brys et al., 2005; Loke et al., 2000; Mylonas et al., 2009; Pesce et al., 2006; Taylor et al., 2006; Terrazas et al., 2005). TGF-b beautifully illustrates the anti-inflammatory/ wound-healing association. TGF-b is absolutely essential for tissue repair and remodeling and yet is one of the most potent anti-inflammatory cytokines with critical roles in promoting regulatory pathways. The recent data with arginase and RELMa described above suggest they may have similar dual roles.

CONCLUSIONS

Until recently, macrophages have not been considered as pivotal players in helminth immunity. In the past decade, however, macrophages have emerged as central to all aspects of antihelminth immunity, including pathology, protection, and regulation. In terms of effector roles against parasites, we know that macrophages are critical as their depletion prevents killing or expulsion (Nakanishi et al., 1989; Rao

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FIGURE 2 The role of l-arginine, cationic amino acid transporters, and alternatively activated macrophages in the pathogenesis of helminth infection. Alternatively activated macrophages expressing arginase-1 suppress the Th2 response in helminth infection by competing with CD41 T cells and fibroblasts for l-arginine. In contrast, classically activated macrophages express iNOS, which is important for the development of nitric oxide that participates in the killing on intracellular pathogens. The iNOS and Arg1 pathways also cross-regulate each other.

et al., 1992). However, the specific mechanisms remain to be elucidated and definitive or direct roles for the key activation markers Ym1, MR, Relma, Relmb, and arginase have not been elucidated. Indeed, high levels of these markers are associated with parasite survival and chronic infection. Our attention has thus turned to their roles in wound healing and immune regulation, which, as described above for TGFb, Relma, and arginase, may indeed be a tightly connected processes. An emerging theme is that each of these proteins may have distinct or even contradictory functions that are context dependent. Importantly, although these proteins were first described in macrophages, their functions may differ when produced by other cell types. For example, arginase produced by AAMw appears to have protective effects against fibrotic damage, while fibroblast-derived arginase promotes it. The story may be similar with Relma and YM1/2 where expression by macrophages, eosinophils, epithelial cells, and others differs depending on disease context and tissue localization. Here, mice with cell-specific deletions, such as the newly generated macrophage specific Arg-1 deficient mice (El Kasmi et al., 2008), should help unravel the role of these proteins and AAMw in antihelminth immunity. This will be greatly facilitated by the rapid expansion in the availability of reporter mice (Reese et al., 2007) and other mice with cell-type-specific gene deletion (Herbert et al., 2004; Siewe et al., 2006) as well as new

technologies for cell-specific depletion and replacement (Cailhier et al., 2006). However, the greatest challenge lies with translating this work to humans infected with helminths. To apply our new understanding of alternative macrophage activation to people or animals infected with helminths, we urgently need to characterize and identify the cells and the relevant homologs of the key AAMw gene products in humans (Elias et al., 2005) or animals of veterinary importance (Knight et al., 2007). For example, identifying whether chitinaselike molecules (or active chitinases) play protective or regulatory roles may offer opportunities for intervention or therapy as specific inhibitors exist or are being developed (Rao et al., 2005). The findings that AAMw can positively affect fat regulation (Odegaard et al., 2007) and negatively affect cancer outcome (Van Ginderachter et al., 2006) further highlight the major implications of work on helminth infections that induce these cells in such dramatic numbers. Finally, by increasing our understanding of the “normal” functions of AAMw during helminth infection, we can better understand the circumstances in which they act inappropriately, such as with allergic asthma, fibrosis, and some forms of cancer. This will hopefully lead to the development of strategies to control the negative consequences of Th2-mediated immune dysregulation with implications for chronic noninfectious diseases, as well as helminthinduced pathology.


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29 Pathology and Pathogenesis of Malaria CHANAKI AMARATUNGA, TATIANA M. LOPERA-MESA, JEANETTE G. TSE, NEIDA K. MITA-MENDOZA, AND RICK M. FAIRHURST

PLASMODIUM AND ITS LIFE CYCLE

Only rings (Color Plate 5A) and mature gametocytes (Color Plate 5B) are typically observed in peripheral blood smears from P. falciparum-infected individuals. This is because RBCs containing trophozoites and schizonts “sequester” in the microvessels of virtually every organ in the body by binding to microvascular endothelial cells (MVECs). Sequestration is believed to enable P. falciparum-infected RBCs to avoid removal from the bloodstream by the spleen, to develop more robustly in the low oxygen tension environment of postcapillary venules, or both. In peripheral blood smears from P. vivax-infected individuals, rings (Color Plate 5C), trophozoites (Color Plate 5D), schizonts (Color Plate 5E), and gametocytes (Color Plate 5F) are all observed. This finding indicates either that P. vivax-infected RBCs do not sequester at all or that they sequester very differently than P. falciparuminfected RBCs. The symptoms of malaria are caused by asexual bloodstage parasites; neither liver-stage parasites (liver schizonts, hypnozoites), nor gametocytes contribute directly to the pathogenesis of malaria. Whether parasitemic individuals develop malaria symptoms and whether they progress to develop the severe, life-threatening manifestations of malaria is a complex process involving both parasite and human factors. This chapter aims to integrate these factors into coherent models of falciparum and vivax malaria pathogenesis, synthesizing data from epidemiological, in vivo physiological and in vitro experimental studies.

Members of the Apicomplexa family of protozoa are characterized by an apical complex of organelles that play various roles in host cell invasion. Four apicomplexan parasites, Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, cause human malaria. P. knowlesi, a parasite of macaque monkeys, causes a significant amount of malaria in humans in Malaysia (Cox-Singh et al., 2008) and is increasingly recognized as a cause of malaria in other areas of Southeast Asia (Baird, 2009; Van den Eede et al., 2009). To understand the pathogenesis of human malaria, it is helpful to appreciate some basic aspects of the Plasmodium life cycle (Fig. 1). Parasite infection of humans is initiated by the bite of a female Anopheles mosquito, which inoculates sporozoites into the dermis. After migrating through the dermis, sporozoites are carried by venous blood and lymph to the liver, where they invade and develop into schizonts within hepatocytes. Once schizonts mature in the liver, they release thousands of merozoites into the bloodstream. Shortly after merozoites invade red blood cells (RBCs), they develop into “rings” that then mature into trophozoites and schizonts, which eventually release another brood of merozoites into the bloodstream. Asexual cycles of RBC invasion and merozoite release from schizonts continue approximately every 48 hours (P. falciparum and P. vivax) until the host is either treated effectively with antimalarial drugs, clears the infection by immune mechanisms, or both. P. vivax, but not P. falciparum, can also produce dormant hypnozoites in the liver. Once the “primary” blood-stage infection is cleared, P. vivax hypnozoites can lie dormant for weeks to decades. Once reactivated, hypnozoites develop into liver schizonts from which thousands of merozoites emerge to establish a “secondary” bloodstage infection. During replication, a small minority of rings differentiates into male and female gametocytes, which may be acquired by another female Anopheles mosquito. After several stages of sexual development in the mosquito’s midgut, sporozoites form and migrate to the salivary glands, where they transmit infection to another human.

WHAT IS MALARIA?

The morbidity and mortality of malaria is truly astounding (World Health Organization, 2008). Together, P. falciparum and P. vivax parasites cause an estimated 650 million episodes of malaria in tropical countries worldwide (Snow et al., 2005). In sub-Saharan Africa alone, P. falciparum kills an estimated 1 million children each year (Rowe et al., 2006). In this chapter, we focus on the pathogenesis of malaria caused by P. falciparum and P. vivax, two parasites that are responsible for more than 90% of the world’s malaria burden. Information on the pathogenesis and clinical presentation of human malaria caused by P. malariae, P. ovale, and P. knowlesi can be found elsewhere (Collins & Jeffery, 2007; Daneshvar et al., 2009; Mueller et al., 2007).

Chanaki Amaratunga, Tatiana M. Lopera-Mesa, Jeanette G. Tse, Neida K. Mita-Mendoza, and Rick M. Fairhurst, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852.

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FIGURE 1 Life cycle of Plasmodium spp. Sporozoites injected into the dermis by female anopheline mosquitoes are carried through the blood and lymph to the liver, where they invade hepatocytes and form liver schizonts. After an incubation period of 7 to 14 days, each liver schizont releases tens of thousands of merozoites, which invade red blood cells (RBCs) and form ring-stage parasites. These forms are found freely circulating in the bloodstream until they mature to trophozoites, which sequester in microvessels while developing into schizonts. Merozoites released from schizonts establish rounds of asexual replication every 48 hours. Some ring-stage parasites develop into sexual-stage gametocytes, which circulate in the bloodstream and are taken up by female mosquitoes during a blood meal. After a cycle of sexual development in the mosquito, sporozoites enter the mosquito’s salivary gland and are injected into another host at the next blood meal. Image kindly reproduced with permission from Cell Press.

Uncomplicated Malaria

P. falciparum and P. vivax most commonly cause uncomplicated malaria, an undifferentiated febrile illness frequently accompanied by headache, body aches, and malaise. In very young children who cannot verbalize complaints such as a headache, a fever may be accompanied by irritability or poor feeding. Anemia and thrombocytopenia are commonly associated with uncomplicated malaria but often do not produce symptoms. Individuals with vivax malaria may experience a paroxysm, a dis-

tinct experience of fever, shaking chills (rigors), and drenching sweats, which coincides with the synchronous rupture of bloodstage schizonts every 48 hours. Individuals with falciparum malaria are less likely to experience periodic fever patterns.

Severe Malaria

Some individuals with falciparum malaria may develop severe, life-threatening complications (Table 1) that require parenteral (instead of oral) antimalarial therapy and

29. Pathology and Pathogenesis of Malaria TABLE 1

Criteria for the diagnosis of severe malaria

Cerebral malaria (diminished consciousness, convulsions) Respiratory distress Prostration Hyperparasitemia Severe anemia Hypoglycemia Jaundice/icterus Renal insufficiency Hemoglobinuria Shock Cessation of eating and drinking Repetitive vomiting Hyperpyrexia

aggressive clinical management (World Health Organization, 2000). These include cerebral malaria (CM; coma with or without convulsions), severe malarial anemia (SMA; hemoglobin concentration ,5 g/dL), and respiratory distress (RD; acidotic deep breathing). Additional criteria for severe malaria are prostration, renal insufficiency, hemoglobinuria, hypoglycemia, repetitive vomiting, and cessation of eating and drinking. Patients with uncomplicated falciparum malaria and high parasite densities (.100,000 asexual forms per mL of whole blood) may be at risk of developing severe malaria and are often treated and managed accordingly. While nearly all episodes of severe malaria worldwide are caused by P. falciparum, a series of patients with severe vivax malaria have recently been reported from Indonesia (Tjitra et al., 2008), Papua New Guinea (Genton et al., 2008), India (Kochar et al., 2009), and Brazil (Andrade et al., 2010). While no definition of severe vivax malaria has yet been firmly established, the World Health Organization’s criteria for severe falciparum malaria (Table 1) are currently being applied.

Pregnancy-Associated Malaria

The syndrome of pregnancy-associated malaria (PAM) is associated with P. falciparum. In areas where P. falciparum transmission is high, adults eventually develop diseasecontrolling immunity, which prevents them from developing malaria symptoms, and parasite-controlling immunity, which keeps parasite densities low in the bloodstream. Adult women, however, may become susceptible to PAM during their first pregnancy because P. falciparum parasites are able to sequester in placentae. First-time mothers with PAM may develop symptoms of uncomplicated malaria and peripheral blood parasitemia. More importantly, however, are the effects of placental parasitemia on the health of first-time mothers and their children. Women with PAM may harbor large numbers of parasites in their placentae and develop various degrees of anemia, including SMA, which afflicts 400,000 women and causes 10,000 maternal deaths each year (Desai et al., 2007). Recent evidence also suggests that PAM may be involved in the development of preeclampsia, a hypertensive syndrome that contributes to the mortality of first-time mothers (Duffy, 2007). PAM is also associated with low birth weight from the combined effects of fetal growth restriction and preterm delivery. Spontaneous abortion and stillbirth may also occur. Together, these outcomes result in the death of 100,000 to 200,000 infants each year (Desai et al., 2007). As women gain immunity to PAM during the

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first and successive pregnancies, they are more likely to have low peripheral blood parasitemias (often detectable only by PCR methods) and less likely to have fever. In low transmission areas where immunity is absent or partially effective, pregnant women are susceptible not only to PAM but also to uncomplicated and severe falciparum malaria. While P. vivax is not commonly associated with PAM, it can also cause maternal anemia and low birth weight.

Asymptomatic Parasitemia

In areas where P. falciparum and P. vivax are highly transmitted, permanent residents eventually acquire a diseasecontrolling immunity to malaria. This immunity enables them to tolerate parasitemias without developing the symptoms of malaria. Such individuals are said to have asymptomatic parasitemia but not malaria. It is important to keep this distinction in mind when reading literature on malaria, which is fraught with confusing terms such as “clinical malaria,” “asymptomatic malaria,” and “malaria infection.” In some malaria-endemic areas, the cumulative prevalence of asymptomatic parasitemia during a single transmission season can be as high as 100%. This means that individuals diagnosed with other febrile illnesses (e.g., upper respiratory tract illnesses, diarrheal diseases) can be found to have incidental parasitemia.

PATHOGENESIS OF FALCIPARUM MALARIA

To understand how individuals infected with P. falciparum develop uncomplicated or severe malaria, it is helpful to consider two well-established observations. First, parasite densities generally correlate with the severity of P. falciparum infections. That is, individuals with severe malaria tend to have higher parasite densities than those with uncomplicated malaria. Likewise, individuals with uncomplicated malaria generally have higher parasite densities than those with asymptomatic parasitemia. However, there are important exceptions to these general observations. For example, individuals living continuously in endemic areas can tolerate appreciably high parasite densities without developing symptoms of malaria. On the other hand, individuals from nonendemic areas (e.g., tourists) may develop severe malaria at relatively low parasite densities. The second observation to keep in mind is that levels of host inflammatory mediators positively correlate with the severity of P. falciparum infections. For example, levels of endothelial cellderived and monocyte-derived inflammatory mediators are higher in patients with severe malaria than in those with uncomplicated malaria (Clark et al., 2008; Kwiatkowski, et al., 1997). High P. falciparum densities are commonly seen in patients with severe malaria and contribute directly to the development of severe disease in several ways. First, parasites consume large amounts of plasma glucose during their development in the bloodstream. This contributes to hypoglycemia, which may induce coma and/or convulsions. Second, in metabolizing glucose, parasites produce large amounts of lactic acid, which drive deep breathing states that contribute to respiratory failure and death. Third, parasites produce large amounts of reactive oxygen species that are believed to oxidatively damage nonparasitized RBCs and platelets, accelerate their senescence and removal from the bloodstream by the spleen, and thus contribute to the development of anemia and thrombocytopenia. Finally, parasites are believed to secrete proinflammatory factors that may impair normal erythropoiesis and cause anemia. One example is uric acid, which derives from hypoxanthine accumulated in P. falciparuminfected RBCs and potently induces the production of IL

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(interleukin)-1, IL-6, and TNF (tumor necrosis factor) from human peripheral blood mononuclear cells (PBMCs) in vitro (Orengo et al., 2009). Simply stated, parasites that are able to achieve high densities and activate inflammatory cascades in their host have a greater propensity for causing the symptoms and severe manifestations of malaria. We now focus on two factors, parasite multiplication rate and cytoadherence of parasitized RBCs, both of which enable parasites to achieve high densities and induce microvascular inflammation.

Parasite Multiplication Rate

Robust parasite multiplication in vivo requires that merozoites efficiently invade RBCs and multiply to high density quickly ahead of the host immune responses that clear parasites from the bloodstream. To accomplish this feat, P. falciparum has evolved multiple, functionally redundant families of ligands that mediate RBC invasion (Cowman et al., 2006). Polymorphisms in these ligands are believed to promote parasite survival by evading host antibody responses that block merozoite-RBC interactions (Persson et al., 2008). While it is often stated that P. falciparum invades RBCs of all ages, P. falciparum isolates nevertheless differ in their selectivity for RBCs. Evidence of RBC selectivity comes from observations of multiple-infected RBCs in patients with falciparum malaria. That is, while most infected RBCs contain one ring form, others may contain two or more ring forms. The presence of multiple-infected RBCs indicates that different merozoites have selectively invaded the same RBC at higher frequency than would be expected by chance. To study the RBC selectivity of P. falciparum, Simpson and colleagues (1999) defined the selectivity index (SI) as the ratio of the observed number of multiple-infected RBCs to that expected from a random process (i.e., Poisson distribution). Compared to P. falciparum isolates with high SI, those with lower SI are hypothesized to be less selective for host RBCs and to have a greater propensity to rapid multiplication in vivo. Consistent with this proposal, investigators found that in patients with falciparum malaria, parasite SI decreased with increasing parasitemia and correlated inversely with disease severity. Low RBC selectivity is thus proposed to be an intrinsic parasite characteristic that enables some P. falciparum isolates to rapidly achieve high parasite densities in vivo. The ability to replicate quickly in advance of an oncoming immune response can thus be considered a parasite virulence trait that contributes to hyperparasitemia-associated complications of falciparum malaria. In addition to using efficient RBC invasion pathways, parasites may also achieve higher multiplication rates by forming rosettes. A rosette is formed by the binding of a parasitized RBC to multiple nonparasitized RBCs (Color plate 6). For P. falciparum, rosette formation brings the maturing parasite into close proximity to multiple nonparasitized RBCs by the time of merozoite release. This mechanism may enable the liberated merozoites, which are short-lived outside RBCs, to more efficiently invade a suitable RBC before they die or are cleared from the bloodstream. Evidence that rosette formation contributes to high parasite multiplication rates in vivo has come from studies in Saimiri monkeys (Le Scanf et al., 2008). Positive correlations between rosetting and P. falciparum parasitemia in African children with malaria has also been reported (Rowe et al., 2002).

Cytoadherence of Parasitized Red Blood Cells

As rings mature to trophozoites and schizonts, P. falciparuminfected RBCs become less deformable and increasingly more likely to be removed by mechanical processes in the spleen. To

avoid the spleen, parasitized RBCs sequester in the microvessels of virtually every critical organ (Color Plate 6), as well as in dermal, muscle, and adipose tissue. This phenomenon explains why only the ring form of asexual-stage parasites is observed in the peripheral blood of P. falciparum-infected individuals. Mature parasites achieve sequestration principally by expressing the cytoadherence ligand P. falciparum erythrocyte membrane protein-1 (PfEMP-1; see later) on the surface of their host RBCs. By this mechanism, most parasites are able to complete their maturation to schizonts in microvessels and release their merozoites into circulation. Those parasites that fail to cytoadhere efficiently are carried away to the spleen and destroyed. The importance of sequestration in parasite survival in vivo was beautifully illustrated in studies by David and colleagues (1983). These investigators compared the ability of P. falciparum-infected RBCs to sequester in spleen-intact and splenectomized monkeys. In spleen-intact monkeys, ringstage parasitized RBCs successively appeared and disappeared from the peripheral circulation, indicating that they were undergoing cycles of sequestration. In contrast, parasitized RBCs showed reduced sequestration in splenectomized monkeys, indicated by the appearance of mature parasite forms in the peripheral circulation. These same parasitized RBCs also showed less cytoadherence in an ex vivo cellular binding assay, indicating that the spleen modulates the ability of parasitized RBCs to sequester. The importance of the spleen in parasite clearance has also been illustrated in humans. Chotivanich and colleagues (2002) identified five splenectomized humans with falciparum malaria and high parasitemia and treated them with artesunate, an effective antimalarial drug. During follow-up for resolution of parasitemia, all five patients harbored dead circulating parasites for extended periods of time—in one case up to 63 days. In an ex vivo perfusion model of intact human spleen, Buffet and colleagues (2006) found that the spleen efficiently retained artesunate-treated ring-infected RBCs. In this same ex vivo model, these investigators also found that the spleen not only efficiently removes trophozoite-infected RBCs but also a fraction of ring-infected RBCs (Safeukui et al., 2008). These data indicate that sequestration enables parasites to evade destruction in the spleen. By promoting parasite survival, sequestration affects the overall parasite multiplication rate and thus contributes to the development of high parasite densities. To evade antibody responses that block sequestration and diminish parasite survival, P. falciparum has evolved a process of antigenic variation (Scherf et al., 2008) that produces a vast array of PfEMP-1 cytoadherence ligands. This investment in antigenic variation further supports a significant role for cytoadherence in parasite survival and malaria pathogenesis. In autopsy specimens of individuals who have died of severe P. falciparum malaria, large numbers of parasitized RBCs are seen in intimate contact with MVECs. Sequestered parasitized RBCs can be seen in virtually every organ of the body (brain, lung, kidney, heart, liver, intestine, and dermis) (Color Plate 6) (Luse & Miller, 1971; MacPherson et al., 1985; Oo et al., 1987; Pongponratn et al., 1991; Prommano et al., 2005). Parasite sequestration is not exclusively found in severe and fatal cases of malaria, but occurs in individuals with uncomplicated malaria and asymptomatic parasitemia as well. While the biomass of sequestered parasitized RBCs positively correlates with malaria severity (Dondorp et al., 2005), some individuals are nevertheless able to tolerate large numbers of sequestered parasites without developing symptoms. This indicates that the mere presence of parasites in microvessels may, in some cases, be insufficient to cause the symptoms and severe manifestations of malaria, and that additional pathogenic processes related to cytoadherence may

29. Pathology and Pathogenesis of Malaria

be contributory. We now consider several cytoadherence interactions between parasitized RBCs and host cells to better understand how parasites cause microvascular obstruction, inflammation, and dysfunction in vivo.

Microvascular Obstruction, Inflammation, and Dysfunction

Microvascular obstruction has been visualized in autopsy specimens, which reveal microvessels packed not only with parasitized RBCs, but also platelets, nonparasitized RBCs, leukocytes, and fibrin. Retinal vessels in live children with malaria have also been observed by ophthalmoscopy to be packed with parasitized RBCs (White et al., 2009). In live Thai adults with malaria, microvascular obstruction has been directly visualized in the microvessels of rectal mucosa (Dondorp et al., 2008). Such obstruction causes impaired perfusion and tissue hypoxia that contribute to microvascular inflammation. Rosettes formed by the binding of parasitized RBCs to nonparasitized RBCs may also impair tissue perfusion by enhancing microvascular obstruction (Kaul et al., 1991). In vivo evidence for MVEC activation and microvascular inflammation has also been obtained from live patients with malaria. One study showed that levels of various soluble factors (sICAM-1, sVCAM-1, and sE-selectin) of endothelial cell origin were elevated in Vietnamese adults with malaria compared to nonparasitized adults, and that these levels correlated with disease severity (Turner et al., 1998). These data and immunohistochemical evidence of MVEC activation in dermal biopsies (Turner et al., 1998) indicate that systemic MVEC activation occurs in malaria. Immunohistochemical evidence of MVEC activation has also been obtained from skeletal muscle biopsies from live patients with malaria, which showed increases in levels of ICAM-1, P-selectin, and CD36 that correlated with disease severity. Evidence for MVEC activation has also been obtained from autopsy studies. For example, some studies (Armah et al., 2005b; Turner et al., 1994) have found that sequestered parasitized RBCs colocalize with MVEC expression of ICAM-1, VCAM-1, and E-selection in the brains of patients with cerebral malaria. By immunohistochemistry, Armah and colleagues (2005a) have also found evidence of prominent expression of TNF and IL-1 in the brains of children with fatal cerebral malaria. Activation of platelets (Faille et al., 2009), the coagulation cascade (Francischetti, 2008), and the fibrinolytic system has also been observed in autopsy studies of severe malaria. Francischetti and colleagues (2008) have recently proposed that tissue factor (TF) is centrally involved in these processes. These investigators found that parasitized RBCs induce the expression of functional TF on MVECs in vitro and found evidence of TF expression in the MVECs of brain autopsy specimens from children who died from cerebral malaria (Francischetti et al., 2007). In their model of malaria pathogenesis, the sequestration of parasitized RBCs induces expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin), which promote the retention of leukocytes, as well as TF, which activates the coagulation cascade and thus promotes thrombocytopenia. Coagulation factors also induce the secretion of proinflammatory cytokines (TNF, IL-1, and IL-6), which may interact with leukocytes to further induce TF expression in MVECs and thus drive and sustain cycles of coagulation and inflammation in microvessels. In vivo evidence of endothelial dysfunction has been obtained in African children and Asian adults with malaria. In a study of Indonesian adults, the reactive hyperemia peripheral artery tonometry (RH-PAT) index, a measure of endothelial function, was found to be lower in patients with severe malaria than in patients with moderately severe malaria (Yeo et al., 2007). Endothelial dysfunction was

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associated in this study with increased parasite biomass and various measures of hemolysis (plasma hemoglobin, LDH, and arginase levels). In those patients with severe malaria, levels of nitric oxide (NO) and arginine were low. Additional evidence for endothelial dysfunction in severe malaria has now been reported from a study of Indonesian adults (Yeo et al., 2009). In this study, cell-free hemoglobin and arginase levels were elevated in patients with severe malaria, compared to those with moderately severe malaria or those who were healthy. These perturbations were not only associated with endothelial dysfunction (reduced RH-PAT index), but also with endothelial activation (elevated plasma levels of sICAM-1, sE-selectin, and angiopoietin), proinflammatory cytokinemia (elevated plasma levels of TNF and IL-6), and impaired tissue perfusion (elevated plasma levels of lactate). These data support a novel model of severe malaria pathogenesis, which can be summarized as follows. The lysis of parasitized and nonparasitized RBCs releases cell-free hemoglobin and arginase into the plasma. Hemoglobin and parasite-induced superoxides quench NO bioactivity, while arginase destroys arginine—the substrate for NO synthase. The combined effects of decreased NO bioavailability and decreased NO production exacerbate microvascular obstruction and inflammation. This is because NO maintains endothelial function, as assessed by reactive vasodilatation following ischemic stress, and exerts anti-inflammatory effects on host cells. These include reducing the levels of TF and the adhesion receptors ICAM-1 and VCAM-1 on MVECs and impairing the production of TNF and other proinflammatory cytokines from leukocytes. When NO bioavailability is reduced, the adverse effects of parasite sequestration proceed unchecked. The increased expression of ICAM-1, VCAM-1 and TF on endothelial cells and increased production of proinflammatory cytokines further activates the endothelium and exacerbates inflammation. Further, ICAM-1 up regulation promotes the adherence of parasitized RBCs, setting up a positive feedback loop of cytoadherence and inflammation.

PfEMP-1 Proteins and var Genes

PfEMP-1 is a family of parasite-encoded, antigenically variant proteins that mediate cytoadherence and thus act as important virulence factors. PfEMP-1 proteins are trafficked out of the parasite, through the RBC cytoplasm, and to the external surface of the RBC membrane, where they are incorporated into protrusions termed knobs (Color Plate 6). In microvessels, PfEMP-1 mediates the binding of parasitized RBCs not only to endothelial cells, but also to monocytes, platelets, and nonparasitized RBCs. Each PfEMP-1 protein contains multiple domains (Color Plate 7A), which can be classified as either DBL (Duffy binding-like) domains or CIDR (cysteine-rich interdomain region) domains. Most PfEMP-1 variants contain only four domains (short variants), whereas others contain five to nine domains (long variants). Each domain is believed to bind to a different host receptor, some of which have been identified (Color Plate 7B). For example, DBL1a1 domains bind to complement receptor 1 (CR1) on nonparasitized RBCs and mediate rosetting. CIDR1a domains (when in tandem with DBL1a domains) bind to CD36 on MVECs and mediate sequestration in multiple organs (except in the brain, where levels of CD36 expression are low). The expression of PfEMP-1 proteins containing particular domains can also mediate organ-specific sequestration of parasitized RBCs. For example, DBL2b domains (when in tandem with C2 domains) bind to ICAM-1 on cerebral MVECs and thus mediate parasite sequestration in the brain. Another example is the DBL3X domain, which binds to chondroitin sulphate A (CSA)

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(Singh et al., 2008) in the intervillous matrix of the placenta and thus mediates parasite sequestration in this organ. By these and other cytoadherence interactions, PfEMP-1 not only activates host inflammatory cascades, but also promotes parasite survival. Specifically, PfEMP-1 mediates the sequestration of parasitized RBCs away from the spleen and the rosetting of parasitized RBCs, from which released merozoites efficiently invade fresh RBCs. The importance of these cytoadherence interactions to P. falciparum survival is evident in the considerable intraclonal and interclonal diversity of DBL and CIDR domains in parasite populations (Kraemer & Smith, 2006; Kraemer et al., 2007). This diversity enables parasites to survive by evading PfEMP-1 variant-specific antibody responses that impair cytoadherence and rosetting, and mediate opsonization, complement activation, and antibody-dependent cytotoxicity. PfEMP-1 proteins are encoded by var genes (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995), which have a 2-exon structure. Exon 1 encodes a series of DBL and CIDR domains (Color Plate 7A), and exon 2 encodes a transmembrane domain and a short cytoplasmic domain. The haploid genome of each parasite contains approximately 60 var genes, which are distributed in various positions over all 14 chromosomes. By allelic exclusion of its var genes, each parasite clonally expresses a single PfEMP-1 variant on the surface of its host RBC. How entire populations of P. falciparum parasites switch their expression from one var gene to another is believed to be under epigenetic control and is the subject of intense research efforts (Scherf et al., 2008). Homologous recombination between var genes during meiosis in the mosquito has resulted in a bewildering diversity of var genes. Based on 5 UTR (untranslated region) sequences; however, almost all var genes can be classified into three groups, upsA, upsB, and upsC. These var gene groups differ in their chromosomal location and transcriptional orientation. For example, upsA var genes are located subtelomerically and are transcribed toward the telomere. UpsB var genes are also located subtelomerically but are transcribed toward the centromere. UpsC var genes are centrally located in the chromosome. The “ups” group designation is useful for predicting the characteristics of the PfEMP-1 molecule that the var gene encodes. For example, upsA var genes encode long PfEMP-1 variants containing multiple domains that may bind multiple cellular receptors at once. UpsA var genes, but not upsB or upsC var genes, encode DBL1a1 domains capable of binding to CR1 on nonparasitized RBCs and mediating rosetting (Color Plate 7A). UpsB and upsC var genes, but not upsA var genes, encode CIDR1 domains capable of binding CD36 on MVECs, monocytes, and platelets (Color Plate 7A). Ups classifications have recently been used to explore whether particular PfEMP-1 variants are associated with severe malaria syndromes. Preliminary studies suggest that upsA var genes are associated with malaria severity in African children (Kyriacou et al., 2006; Rottmann et al., 2006) and with noncerebral severe malaria in Brazilian adults (Kirchgatter et al., 2002). Since upsA var genes encode PfEMP-1 variants that bind CR1, rosette formation may in part be responsible for their association with severe disease. UpsA var genes tend to encode long PfEMP-1 variants (Color Plate 7A) that might mediate high-avidity binding to particular host cells, or the binding of parasitized RBCs to the microvessels of multiple critical organs.

Severe Falciparum Malaria Syndromes

Severe falciparum malaria can manifest as one or more syndromes. Here we discuss the pathogenesis of three of these syndromes, CM, severe malaria anemia (SMA), and

respiratory distress (RD), which are responsible for much of the mortality attributed to severe falciparum malaria. Both overlapping and distinct pathological processes contribute to the development of these syndromes. The pathogenesis of other life-threatening syndromes including pulmonary edema (Taylor & White, 2002) and renal failure (Das, 2008) are reviewed elsewhere.

Cerebral Malaria

Cerebral malaria is defined as unarousable coma not attributable to convulsions, hypoglycemia, or meningitis in a patient with P. falciparum parasitemia. In autopsy specimens of individuals who died from cerebral malaria, the sequestration of parasitized RBCs in cerebral microvessels can be prominent (MacPherson et al., 1985), indicating a high degree of microvascular obstruction occurs in vivo. Parasite sequestration and evidence of blood-brain barrier compromise (e.g., hemorrhages) have been observed by fundoscopy in live children with cerebral malaria (White et al., 2009). Dondorp (2008) recently observed evidence of microcirculatory obstruction in microvessels in the rectal mucosa of patients with malaria and found that the degree of microvessel obstruction correlated with disease severity. In addition to parasitized RBCs, other factors may contribute significantly to microcirculatory obstruction. For example, the reduced deformability of nonparasitized RBCs may worsen the degree of obstruction by increasing the viscosity of blood (Dondorp et al., 2002). Rosette formation may also contribute to obstruction (Kaul et al., 1991) and has been associated with cerebral malaria in epidemiological studies of African children (Rowe et al., 2009a). A role for rosetting in the pathogenesis of cerebral malaria is further strengthened by data showing that rosette-disrupting antibodies protect against cerebral malaria (Treutiger et al., 1992), and that genetic polymorphisms (CR1 polymorphisms, type O blood group), which reduce rosetting, confer protection against cerebral malaria (see later). The presence of mononuclear leukocytes and platelets (Faille et al., 2009) in microvessels may not only contribute to obstruction but also induce inflammation. For example, some parasite antigens (e.g., GPI, hemozoin) bind specifically to TLRs and other pathogen recognition receptors on the surface of monocytes. Activation of monocytes results in their secretion of proinflammatory cytokines such as TNF, which is believed to up regulate ICAM-1 on brain microvessels during episodes of cerebral malaria (Chakravorty et al., 2008). Further parasite sequestration via ICAM-1 enhances the local inflammatory response and is believed to induce proapoptotic signaling in endothelial cells. These responses may culminate in damage to tight junctions and disruption of the blood brain barrier (Medana et al., 2006). Tissue factor exposure may contribute to this pathogenetic mechanism by initiating the clotting cascade, which consumes anticoagulation factors and sets up a local procoagulant state (Francischetti et al., 2008). Consistent with these models of pathogenesis, intravascular and extravascular pathologies have been observed in autopsied brain specimens and include thrombi and hemorrhages (Taylor et al., 2004). Parasite antigens are also believed to activate platelets by binding to TLRs, which drives inflammation and up regulates the expression of ICAM-1 and VCAM-1 on endothelial cells. Since these receptors are also involved in the adhesion of platelets and leukocytes, cumulative binding of these various blood elements are believed to further activate host endothelium. Additionally, platelets have been proposed to have a special cytoadherence function. Since little CD36 is expressed constitutively in the brain, parasitized RBCs that bind CD36 may not adhere efficiently to cerebral microvessels. However, activated platelets that


express CD36 may bind to P-selectin and ICAM-1 on brain endothelial cells. In this manner, platelets may form a bridge that enables CD36-binding parasitized RBCs to sequester in brain microvessels and contribute to the pathogenesis of cerebral malaria. Parasitized RBCs may also bind directly to ICAM-1 (Chakravorty & Craig, 2005) or the globular head of the C1q receptor (gC1qR) (Biswas et al., 2007) expressed on brain endothelial cells. In addition to microvascular obstruction and endothelial cell activation, other factors associated with severe malaria may contribute to the clinical presentation of cerebral malaria. These include hypoglycemia, which may cause coma and convulsions, and acidosis-driven respiratory failure, which may produce CO2 narcosis. The contribution of these factors to the clinical presentation of cerebral malaria may be substantial, as some children may “wake up” and recover shortly after treatment. Indeed, P. falciparum parasitemia in a child clinically diagnosed with cerebral malaria (unarousable coma, with or without convulsions) does not rule out other causes of coma. In a study of 31 Malawian preschool children who were diagnosed with cerebral malaria at hospitalization but then died, 23% (7 out of 31) of them had various nonmalaria causes of coma and death identified only at autopsy, such as Reye syndrome, ruptured arteriovenous malformation, hepatic necrosis, or multiple factors (severe anemia, pneumonia, and meningitis) (Taylor et al., 2004).

Severe Malarial Anemia

Anemia frequently develops in patients with malaria. The clinical features of malarial anemia include malaise, fatigue, and dyspnea (which may be complicated by acidosis-driven respiratory distress). Before considering the pathogenesis of malaria-associated anemia, it is important to recognize that an individual’s baseline hemoglobin concentration, and thus their ability to tolerate anemia, is determined by several factors. These include age, sex, nutritional state (e.g., iron and micronutrient deficiencies), genetic polymorphisms affecting RBCs (e.g., sickle hemoglobin, thalassemia), and chronic infection with HIV and parasites (e.g., hookworm, schistosomes). From these factors alone, some individuals may meet any given criterion for anemia at baseline (e.g., hemoglobin concentration ,8.5 g/ dL). In addition, micronutrient deficiencies and HIV infection can blunt the reticulocytosis response to dropping hemoglobin levels, making it more likely that a person will develop anemia as parasitemias increase and a malaria episode runs its course. Parasite and host factors causing anemia can also predispose one to the development of severe malarial anemia (SMA), defined as a hemoglobin concentration ,5 g/dL that is attributable to P. falciparum infection. Some investigators have defined SMA as a hemoglobin concentration ,5 g/dL that is complicated by respiratory distress (Akech et al., 2008). This is because low hemoglobin levels in the absence of respiratory distress are not associated with high mortality rates in some settings. A parasite density of .10,000 per mL is sometimes used as an additional criterion for SMA. This is because SMA can occur by other mechanisms in children that are incidentally found to have relatively low parasitemias. For this reason, P. falciparum parasitemia in a child with SMA does not rule out other causes of severe anemia. In a case-control study of Malawian preschool children (Calis et al., 2008), for example, several independent yet overlapping conditions were associated with severe anemia: bacteremia, hookworm infection, HIV infection, G6PD deficiency, vitamin A deficiency, and vitamin B12 deficiency. We now discuss two major pathogenetic mechanisms of malaria-associated anemia, one that removes RBCs from the bloodstream, and another that fails to replace those RBCs that are lost. In nonimmune or semi-immune individuals with high parasite densities, the acute removal of parasitized

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and nonparasitized RBCs from the circulation may be relatively more important to the development of anemia. Indeed, such infections can rapidly drop hemoglobin levels from high baseline values to levels ,5 g/dL associated with SMA. In immune individuals with relatively lower parasite densities, chronic parasitemia may develop and cause progressive reductions in hemoglobin to levels ,5 g/dL. A low baseline hemoglobin level at the start of a malaria transmission season is a risk factor for the development of SMA. Antimalarial drug resistance can contribute to this decline in hemoglobin levels because of inadequate elimination of parasitemias. In cases of chronic parasitemia, chronic bone marrow dysfunction may play a relatively greater role in the development of anemia. Persistent and exacerbated episodes of ineffective erythropoiesis can also produce hemoglobin levels ,5 g/dL; however, some individuals may better tolerate low hemoglobin levels if the rate of RBC loss is sufficiently slow to allow time for adequate homeostatic compensation.

Removal of Nonparasitized Red Blood Cells

The pathogenesis of anemia due to P. falciparum infection is complex and no predominant pathogenetic mechanism has been identified. The removal of parasitized RBCs is affected by several mechanisms. As rings develop into trophozoites, parasite proteins are inserted in the RBC membrane and associate with cytoskeletal elements. This process reduces the deformability of parasitized RBCs, which then become trapped in the spleen and phagocytosed by macrophages. Parasitized RBCs may also be opsonized with immunoglobulins and complement, which enhances their phagocytosis. The destruction of parasitized RBCs by these processes and upon schizont rupture, however, seems to contribute relatively little to the overall loss of RBCs. This is because the amount of RBC loss far exceeds that which can be accounted for by the destruction of parasitized RBCs. Data from a study of Thai individuals with acute malaria suggest that as much as 90% of RBC loss is due to the removal of nonparasitized RBCs (Looareesuwan et al., 1987, 1991; Price et al., 2001). Data from nonimmune American adults infected with P. falciparum showed that for every parasitized RBC lost, an average of 8.5 nonparasitized RBCs were lost in addition (Jakeman et al., 1999). Indeed, erythrophagocytosis has been directly visualized in the bone marrow of patients with falciparum malaria. Further declines in hemoglobin levels occur even after elimination of parasites with antimalarial drugs, consistent with ongoing removal of nonparasitized RBCs in the spleen. In a rodent model of severe malarial anemia, Evans and colleagues (2006) showed that RBC loss was predominantly due to the uptake of nonparasitized RBCs by monocytes and macrophages. Several mechanisms have been suggested for the removal of nonparasitized RBCs. Some of these mechanisms may be initiated by an imbalance in levels of proinflammatory and antiinflammatory cytokines, for example, high TNF levels in the setting of low IL-10 levels (i.e., high TNF/IL-10 ratio), which develops in response to parasite infection. Children with malaria have high levels of plasma TNF and IFNg, a combination of cytokines that stimulates erythrophagocytosis by macrophages and activates monocytes. Monocytes respond to these cytokines by producing oxidants, which damage RBC membranes, reduce RBC deformability, and thus lead to the trapping and eventual phagocytosis of RBCs in the spleen. Oxidative stress also accelerates the senescence of RBCs. Oxidation may induce the externalization of membrane phosphatidylserine to the RBC surface, which is proposed as an antibody-independent mechanism of senescent RBC clearance. Oxidative stress can also drive antibody-dependent and complement-dependent mechanisms of RBC removal. Specifically, hemoglobin molecules oxidatively denature and bind the cytoplasmic tail of band 3 proteins in the RBC

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membrane. This process aggregates and may alter the conformation of band 3. Autoantibodies recognize this altered band 3 and activate complement on the RBC surface, thereby promoting Fc receptor-mediated and complementreceptor-mediated phagocytosis of RBCs by splenic macrophages. Complement deposition may also cause RBC lysis. Evidence that complement-mediated processes are important in the removal of RBCs is the finding that children with SMA acquire a deficiency in two complement regulatory proteins—complement receptor 1 (CD35) and decay accelerating factor (CD55) (Odhiambo et al., 2008). Reduced CD35 levels may impair the ability of RBCs to bind immune complexes and inactivate their complement component, while reduced CD55 levels may impair the ability of RBCs to inactivate C3b convertase, leading to excessive C3b deposition.

Suppression of Hematopoiesis

Hemoglobin levels are determined by both the destruction of RBCs and their replenishment by bone marrow erythroid precursors. In some studies, bone marrow suppression with reduced erythropoiesis has been observed in children with acute malaria. In children who are chronically infected with P. falciparum and experience repeated bouts of malaria, the bone marrow has been observed to be hyperactive. However, ineffective erythropoiesis is suggested by morphological evidence of dyserythropoiesis and retention of reticulocytes. In most cases, ineffective erythropoiesis is not due to reduced erythropoietin levels, which are found to be appropriately elevated for the corresponding degree of anemia (Verhoef et al., 2002). The same parasite-induced host inflammatory mediators that are believed to cause destruction of RBCs have also been implicated in bone marrow dysfunction. These include proinflammatory cytokines (TNF, IFNg) and oxidants that interfere with normal hematopoiesis and may render RBC precursors relatively unresponsive to erythropoietin. This proinflammatory cytokine profile may be enhanced by relatively low levels of IL-10 (which inhibits the production of TNF) and low levels of IL-12 (a hematopoietic growth factor). It is conceivable that parasite-derived proteins and other products might be directly toxic to developing RBCs.

Metabolic Acidosis and Respiratory Distress

Respiratory distress (RD) is a symptom of metabolic acidosis (Maitland & Marsh, 2004). When severe and prolonged, respiratory distress can cause children to tire of breathing, leading to respiratory failure and death. RD frequently accompanies CM and SMA and increases the mortality of both conditions. While decreased oxygen delivery due to anemia, and especially SMA, causes some degree of metabolic acidosis, other pathogenetic mechanisms occur. These include hypovolemia, hepatic insufficiency, and parasite metabolism. In one study of Ghanaian children, an imbalance of TNF and IL-10 (high TNF/IL-10 ratio) production has been associated with RD, as well as elevated levels of neopterin, a marker of inflammation and immune activation produced by activated macrophages (Awandare et al., 2006). Neopterin has been proposed to indirectly contribute to the development of metabolic acidosis by increasing the formation of free radicals and cooperating with IFNg to sustain TNF production. Interestingly, levels of neopterin were not elevated in children with CM or SMA compared to children with uncomplicated malaria. This finding suggests not only that this protein may play a unique role in the pathogenesis of RD, but also that the pathogenesis of RD may be very different from the pathogenesis of CM and SMA. It has been proposed that inflammatory mediators, by inducing production of

peroxynitrite radicals, might inhibit enzymes involved in oxidative phosphorylation, thereby shifting host cells to the anaerobic generation of ATP and consequent production of lactic acid (Clark & Cowden, 2003).

Pregnancy-Associated Malaria

Pregnancy-associated malaria (PAM) mainly occurs in high-transmission areas and is caused by P. falciparum (Duffy 2007; Rogerson & Boeuf, 2007; Rogerson et al., 2007a, 2007b). Women with PAM develop various degrees of anemia, which may be life threatening or fatal. Low birth weight (, 2,500 g) due to fetal growth restriction and preterm delivery may also occur. These adverse outcomes are associated with parasite sequestration in the placenta. P. falciparum isolates obtained from the placenta bind chondroitin sulphate A (CSA). This sulphated glycosaminoglycan is present throughout the intervillous space (a collection of lake-like structures through which maternal blood circulates). Parasitized RBCs are not observed to be in intimate contact with the surface of syncytiotrophoblasts, which line the intervillous space, but rather accumulate in the intervillous space by binding secreted CSA. These parasite isolates predominantly express VAR2CSA, a PfEMP-1 variant that binds CSA, but not other major host cytoadherence receptors such as CD36 or ICAM-1. It is not known whether parasites switch their PfEMP-1 expression spontaneously to VAR2CSA and then “clone out” in the presence of a placenta or whether parasites “sense” that they are in a pregnant woman and actively switch to VAR2CSA (Nunes & Scherf, 2007). The retention of parasitized RBCs in the intervillous space enables parasites to multiply to much higher densities than in peripheral blood and is associated with monocyte infiltration. Placentas from women with PAM may show various degrees of monocyte ingestion of hemozoin crystals (a by-product of parasite hemoglobin degradation) and deposition of fibrin, which may also contain hemozoin (Brabin et al., 2004). Primigravid mothers with PAM harbor much higher densities of parasitized RBCs and monocytes in their placentas and for longer durations than multigravid mothers, suggesting that the pathogenesis of PAM relates to the intensity and duration of parasite sequestration and monocyte infiltration. The predominance of monocytes over other leukocytes may be due to the local secretion of cytokines (MIP1a, MIP1b, MCP1, IP-10, and MIF) that promote the chemotaxis, activation, and retention of monocytes. High levels of proinflammatory cytokines (e.g., TNF, IFNg, and IL-1b) have been found in women with PAM. These cytokines likely have an antiparasitic role in enhancing the activation of macrophages, but excessively high levels of these cytokines are hypothesized to contribute to maternal anemia and fetal growth restriction. This is because, in normal pregnancies, the placenta has a Th2-shifted cytokine profile, and strong Th1 responses have been associated with maternal anemia and preterm delivery. The parasite products that activate monocytes have not been defined, but some candidates include hemozoin, GPI, and fibrinogen, all of which induce TNF production by monocytes. The pathogenetic mechanisms of fetal growth restriction and preterm delivery seem to be somewhat different. Fetal growth restriction is associated with chronic placental infection and is likely due to placental insufficiency, that is, compromised uteroplacental blood flow and impaired nutrient acquisition. Possible mechanisms of reduced blood flow and nutrient delivery include mechanical obstruction of the intervillous space by parasitized RBCs, monocytes, and fibrin, and inflammation-mediated effects on placental blood flow with impaired angiogenesis and reduced placental growth.


Preterm delivery has been associated with high placental parasite densities, maternal anemia, and high levels of TNF and IL-10, but the roles of these factors in initiating the birth process are not known. The same factors that cause anemia in nonpregnant adults may also contribute to maternal anemia during pregnancy. Placental monocytes may play an important role in ingesting parasitized and nonparasitized RBCs and by producing cytokines (e.g., TNF) that suppress erythropoiesis. Maternal hemoglobin levels can be chronically low due to iron deficiency, HIV infection, and other factors, which can predispose women who develop PAM to become even more anemic. The pathogenesis of PAM can be modulated by host immunity and comorbidities. Thus, placental pathology and the adverse outcomes of PAM can be mitigated over successive pregnancies, as women develop and subsequently boost antibody responses directed at the various CSA-binding domains of VAR2CSA (Hviid & Salanti, 2007). These antibody responses may inhibit parasite sequestration and opsonize parasitized RBCs for phagocytosis by macrophages. By these mechanisms, multigravid women who develop PAM are better able to control placental parasitemia and shorten the duration of placental inflammation, resulting in less maternal anemia and higher newborn birth weights.

HUMAN GENETIC RESISTANCE TO FALCIPARUM MALARIA

The life-threatening manifestations of falciparum malaria have exerted tremendous evolutionary pressure on the human genome. In 1949, J. B. S. Haldane put forth the idea that b-thalassemia was a balanced polymorphism (Haldane, 1949). Specifically, he proposed that the reduced fitness of b-thalassemia disease due to severe anemia was balanced by the increased fitness of the b-thalassemia trait due to protection against falciparum malaria. This hypothesis was extended in 1954 by A. S. Allison (Allison, 1954) to include sickle cell hemoglobin (HbS), which, in Africa, causes fatal sickle cell anemia in its SS homozygous state but protects against severe falciparum malaria in its AS heterozygous state. Today, numerous RBC polymorphisms have been described and shown in epidemiological studies to confer protection against falciparum malaria (Williams, 2006). For some of these polymorphisms (e.g., HbE, bthalassemia), robust epidemiological data associating them with protection against severe falciparum malaria are lacking and so they will not be considered here. For others (e.g., HbS, HbC, a-thalassemia, and G6PD deficiency), some proposed mechanisms of protection are difficult to reconcile with well-accepted epidemiological observations. Evidence is now emerging that many of these RBC polymorphisms impair the adherence of parasitized RBCs to host cells, including MVECs and nonparasitized RBCs. These reductions in cytoadherence and rosetting have been proposed to ameliorate microvascular inflammation in vivo and prevent the progression from uncomplicated to severe disease. Collectively, these investigations suggest that PfEMP-1, the parasite’s main cytoadherence and rosetting ligand, plays a significant role in malaria pathogenesis.

Hemoglobinopathies

The hemoglobinopathies can be divided into disorders of hemoglobin structure or production. Adult hemoglobin is a tetramer of two a-globin and two b-globin chains. Single amino acid substitutions in the b-globin chain produce the three major hemoglobin (Hb) variants: HbS, HbC, and HbE. Various deletions and mutations that reduce the amount of a-globin or b-globin chains produce a-thalassemias and

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b-thalassemias, respectively. Here we present summaries of epidemiological and experimental investigations into the malaria-protective effects of HbS, HbC, and a-thalassemia.

Hemoglobin S

Sickle hemoglobin S (HbS) is produced by a valine-for-glutamate substitution at the sixth amino acid position of b-globin. HbS arose independently on several haplotypes in sub-Saharan Africa, Arabia, and India, suggesting this polymorphism has been naturally selected for increasing fitness rather than by gene flow between human populations. SS homozygotes have a severe chronic hemolytic anemia that usually results in death in the first few years of life. AS heterozygotes, on the other hand, are healthy and experience fewer episodes of both uncomplicated and severe falciparum malaria compared to normal AA homozygotes (Aidoo et al., 2002; Hill et al., 1991; Williams et al., 2005a, 2005b). In most studies, parasite densities are lower in AS children with uncomplicated and severe malaria than their AA counterparts. This has led to the interpretation that AS confers malaria protection through mechanisms that reduce parasite multiplication in vivo. In vitro experimental support has been obtained for a variety of mechanisms, including (i) impaired merozoite invasion of RBCs, (ii) impaired intraerythrocytic parasite development at low oxygen tension (as is present in postcapillary venules), and (iii) enhanced phagocytosis of ring-stage parasitized RBCs. Despite these data, the hypothesis that HbS reduces the incidence of falciparum malaria by reducing parasite densities does not explain several observations. First, it is not known how P. falciparum achieves equivalent densities in AS and AA children in some studies. Secondly, in those studies (Williams et al., 2005b) that have found reduced parasite densities in AS children with uncomplicated and severe malaria, it is not known how fewer parasites are able to cause the symptoms of malaria in AS children compared to AA children. This observation seems to paradoxically suggest that parasites causing uncomplicated or severe malaria in AS children are more virulent than those causing disease in AA children. Third, it is not known how HbS enhances naturally acquired immunity to malaria, as suggested by an epidemiological study (Williams et al., 2005a) showing the degree of malaria protection by AS increases with age, a surrogate of disease-controlling immunity in endemic areas. To explore the basis for these various observations, we hypothesized that parasitized AS RBCs bind MVECs with lower avidity than parasitized AA RBCs. Using naturally circulating P. falciparum isolates from Malian children with malaria, we found that parasitized AS RBCs are 50% impaired in their binding to MVECs compared to parasitized AA RBCs (Cholera et al., 2008). Parasitized AS RBCs also showed commensurate reductions in the surface levels of PfEMP-1 and the density of knobs on which PfEMP-1 is concentrated. It is not known how HbS impairs the trafficking of PfEMP-1 to the surface of parasitized RBCs. One possible mechanism involves the instability of HbS. Compared to HbA, HbS undergoes accelerated denaturation to hemichromes, forms of hemoglobin that bind the inner leaflet of the RBC membrane via the cytoplasmic tail of band 3. These hemichromes may impair the docking of Maurer’s clefts (vesicles that transport PfEMP-1 through the RBC cytoplasm) at the inner leaflet of the RBC membrane or block the translocation of PfEMP-1 and other knob components to the external surface of the RBC. Another possibility is that hemichrome-associated ferric iron oxidizes RBC membrane proteins and lipids, which may compromise the parasite’s remodeling of its host cell surface. This model of protection by HbS helps to explain three puzzling observations. First, the degree of protection by sickle cell trait may be enhanced with increasing age—a

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surrogate for disease-controlling immunity. One possible mechanism for this observation is that PfEMP-1-specific antibody responses, which impair cytoadherence, are rapidly acquired in early childhood. PfEMP-1-specific IgG may thus work cooperatively with HbS at the level of PfEMP-1 to achieve even greater reductions in cytoadherence than with HbA. In support of this possibility, we have found that purified IgG from pooled Malian adult plasma abolishes the adherence of parasitized RBCs to MVECs (C. Amaratunga and R. Fairhurst, unpublished data). Second, some studies report lower parasite densities in AS children compared to AA children with uncomplicated and severe malaria. In areas where high levels of immunity are rapidly acquired and sustained (i.e., areas with intense, year-round transmission), high-titer PfEMP-1-specific IgG may greatly impair the ability of some parasitized RBCs to sequester from the spleen, thereby achieving actual reductions in parasite density in all children. However, reductions in sequestration would be greater in AS children than in AA children, which may explain the reduced parasite densities observed in AS children in some studies. Third, in studies showing reduced parasite densities in AS children, it would seem that it takes fewer parasites to cause uncomplicated or severe malaria in AS children than in AA children. To explain this, we hypothesize that P. falciparum isolates overcome abnormal PfEMP-1 display by switching to PfEMP-1 variants that encode greater avidity for MVECs. In other words, although an AS child might carry a lower parasite burden, those parasites that do sequester must cause greater levels of PfEMP-1-mediated host cell activation and microvascular inflammation.

Hemoglobin C

Hemoglobin C (HbC) is produced by a lysine-for-glutamate substitution at the sixth amino acid position of b-globin. This polymorphism arose on a single haplotype originating in present-day northern Ghana and has since spread concentrically throughout much of West Africa. AC heterozygotes have a normal phenotype and CC homozygotes have a mild, chronic hemolytic anemia and splenomegaly of little clinical consequence. Thus, HbC is not considered a balanced polymorphism, but rather a polymorphism that can conceivably reach genetic fixation in human populations if it were under net-positive selection. Only recently has HbC been associated with reduced risk of severe falciparum malaria in Mali, Burkina Faso, and Ghana (Agarwal et al., 2000; Mockenhaupt et al., 2004; Modiano et al., 2001). Unlike HbS, most studies have not associated malaria protection by HbC with reduced parasite densities in vivo. Indeed, parasite densities can reach levels as high as 200,000 per mL in AC and CC children. These observations suggest that parasites have no problem invading and developing in HbC RBCs. How might HbC protect against severe falciparum malaria without reducing parasite density? In investigating possible mechanisms or protection, we found that parasites were able to invade and develop normally in AC and CC RBCs in vitro. As for HbS, we found that HbC impairs the adherence of parasitized RBCs to MVECs and is associated with abnormal PfEMP-1 display (Fairhurst et al., 2005). In this model of malaria protection, the binding avidity of parasitized HbC RBCs for MVECs is sufficiently strong to mediate sequestration from the spleen, but not avid enough to maximally activate host endothelium. This enables parasites to multiply to equivalent densities in HbC and HbA children and cause uncomplicated malaria, but not to cause severe malaria. Like HbS RBCs, HbC RBCs contain elevated levels of membrane-bound hemichromes (Fairhurst et al., 2005), which may impair the trafficking of PfEMP-1 to the RBC surface.

a-Thalassemia

a-Thalassemia is an inherited disorder of hemoglobin synthesis, in which reduced production of a-globin chains leads to decreased amounts of normal a2b2 tetramers and increased amounts of unpaired b-globin chains. In sub-Saharan Africa, a-thalassemia states are produced by a 3.7-kb deletion that leaves one functional copy of duplicated a-globin genes. Heterozygotes (2a/aa) have an essentially normal phenotype while homozygotes (2a/2a) have mild microcytic anemia. Epidemiological studies in sub-Saharan Africa (May et al., 2007; Williams et al., 2005c) and Papua New Guinea (Allen et al., 1997) have associated a-thalassemia with reduced risk of severe falciparum malaria, particularly SMA. While the mechanism of this protection has not been established, it will need to be reconciled with a well-established observation made in numerous epidemiological settings; a-thalassemia is not associated with reduced parasite densities in children with uncomplicated or severe malaria. These observations indicate that a-thalassemia does not protect against severe malaria by impairing the ability of parasites to invade or develop within RBCs, or promoting the removal of parasitized RBCs from the bloodstream. Microcytosis has recently been implicated as a mechanism of protection against SMA by homozygous athalassemia in Papua New Guinea (Fowkes et al., 2008). According to this model, increased RBC counts protect homozygotes against SMA by reducing the amount of hemoglobin loss at any given parasitemia. Whether a-thalassemia states might contribute to malaria protection by impairing cytoadherence has not been determined. Such a mechanism seems plausible as a-thalassemic RBCs contain excess unpaired b-globins, which are unstable like HbS and HbC.

G6PD Deficiency

Glucose-6-phosphate dehydrogenase (G6PD) is important in the generation of reduced glutathione, a major antioxidant in RBCs. Many genetic mutations produce unstable G6PD enzymes with partially deficient activity (Nkhoma et al., 2009). Some of these mutations have achieved polymorphic frequencies in malaria-endemic areas worldwide. In sub-Saharan Africa, the G6PD*A2 allele is common and has been associated with protection against severe falciparum malaria (Guindo et al., 2007; Ruwende et al., 1995). Conclusions from epidemiological studies and in vitro parasite culture experiments, however, have been conflicting and have not been reconciled satisfactorily with the differential expression of X-linked G6PD*A2 in males and females. For example, one study (Ruwende et al., 1995) found that heterozygous females with mosaic populations of normal and deficient RBCs (due to random X chromosome inactivation) were resistant to severe falciparum malaria to the same degree as hemizygous males with populations of uniformly deficient RBCs. Nearequivalent protection against life-threatening malaria in both males and females is difficult to reconcile with the differential expression of G6PD deficiency between the sexes. This is because hemizygous males would be expected to show an advantage over heterozygous females under mechanisms of protection that invoke reduced merozoite invasion, impaired parasite development, and enhanced removal of ring-stage parasitized RBCs (Cappadoro et al., 1998). A recent case-control study of severe malaria in Mali showed that the uniform state of G6PD deficiency in hemizygous male children conferred significant protection against severe malaria (Guindo et al., 2007). In this study,


no such protection was evident from the mosaic state of G6PD deficiency in heterozygous females. The parasite densities of males and females with differences in G6PD status were not found to be significantly different from each other. Pooled odds ratios from a meta-analysis of these data (Guindo et al., 2007) and data from a previous study (Ruwende et al., 1995) confirmed highly significant protection against severe malaria in hemizygous males but not in heterozygous females. This finding suggests that the A2 form of G6PD deficiency is under strong natural selection in Africa because of the preferential protection it provides to hemizygous males against severe falciparum malaria. One possible mechanism for this protection is that in hemizygous G6PD-deficient males, HbA forms increased amounts of hemichromes that cause abnormal PfEMP-1 display, impair cytoadherence, and ameliorate the severity of malaria. In mosaic females, however, merozoites may preferentially invade and develop within the G6PD-normal subset of the mosaic RBC population. In this case, trophozoite-infected RBCs may cytoadhere normally, providing no protection against severe disease. This latter mechanism is consistent with initial observations of Luzzatto and colleagues (1969) who found that ring-stage parasites were more likely to be found in the G6PD-normal subset of RBCs of naturally infected heterozygous females. Whether G6PD deficiency states might contribute to malaria protection by impairing cytoadherence has not been determined. In the setting of hemizygous G6PD deficiency, it seems possible that normal HbA becomes unstable like HbS and HbC and causes abnormal PfEMP-1 display.

Type O Blood Group Antigen

Type A and B blood group antigens are produced by glycosyltransferases. A frame-shift mutation that destroys the activity of these enzymes leads to the production of type O blood group antigen. The ancestral A and B antigens were lost to varying degrees in human populations emerging from Africa, suggesting that the type O antigen was under positive selection 50,000 to 100,000 years ago on that continent. While the distribution of ABO blood group antigens varies worldwide, the type O antigen remains relatively common in sub-Saharan Africa and has long been suspected of being naturally selected by malaria (Cserti & Dzik, 2007). Only recently, however, have in vitro experimental data and epidemiological data been obtained to support the hypothesis that type O antigen confers protection against severe falciparum malaria by impairing rosetting. The rosetting phenotype (the ability of a parasitized RBC to bind multiple nonparasitized RBCs) is encoded by particular PfEMP-1 variants (Rowe et al., 1997) and has been associated with severe malaria (Doumbo et al., 2009). Because rosettes are believed to contribute to microvascular obstruction and enhanced parasite multiplication rates in vivo, rosetting is considered a virulent parasite trait (Rowe et al., 2009a). Initial work demonstrated that parasitized RBCs expressing type A and/or B antigens show enhanced rosetting compared to those expressing type O antigens (Rowe et al., 2009a). This finding generated the hypothesis that type O antigen confers protection against severe malaria by impairing rosetting. Three epidemiological studies have recently established that type O antigen confers resistance to (or, alternatively, that “non-O” antigens [i.e., A, B, and AB, taken collectively]) increase susceptibility to severe malaria in Africa (Rowe et al., 2009b). In a collection of African countries and in The Gambia, non-O antigens were found to increase the risk of severe malaria by 18% and 26%, respectively (Fry et al., 2008). In Mali, type O antigen was

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associated with a 65% reduction in the risk of severe malaria when compared to non-O antigens (Rowe et al., 2007). In this study, protection was associated with impaired rosetting of parasitized RBCs expressing type O antigen compared to those expressing non-O type antigens. These findings help to implicate rosetting in the pathogenesis of severe falciparum malaria.

Complement Receptor 1 Polymorphism

Complement receptor 1 (CR1) is a glycoprotein expressed on the surface of RBCs, which helps to remove immune complexes containing activated complement components C3b and C4b. Parasites that express particular PfEMP-1 domains (i.e., DBL1a1) are able to rosette by binding CR1 (Rowe et al., 1997) on the surface of nonparasitized RBCs. CR1 contains nine antigenic determinants that are components of the Knops blood group system (Rowe et al., 2009b). The absence of all nine antigenic determinants from CR1 molecules constitutes the Knops null phenotype, which is associated with reduced expression of CR1 on the RBC surface and markedly reduced rosetting. In Papua New Guineans and other populations, SNPs located in intron 27 and exon 22 of the cr1 gene are associated with low (L) and high (H) levels of CR1 expression, respectively. These SNPs were used to determine that the L CR1 phenotype occurs at very high prevalence (80%) in Papua New Guinea, where it has been associated with a 70% reduced risk of severe falciparum malaria in a case-control study (Cockburn et al., 2004). As for type O antigen, these findings for CR1 polymorphism help to implicate rosetting in the pathogenesis of severe falciparum malaria.

PATHOGENESIS OF vIvAX MALARIA: LOTS OF QUESTIONS

P. vivax causes an estimated 72 to 390 million episodes of malaria (Mendis et al., 2001), about half of the total malaria burden outside Africa. While this parasite kills very infrequently, it is responsible for untold suffering and debilitation. The vast majority of vivax malaria episodes are uncomplicated, causing paroxysms of fever and chronic and acute anemia. In Indonesia, vivax malaria is now recognized as a major cause of morbidity in early infancy (Poespoprodjo et al., 2009). Case series of severe vivax malaria have recently been reported from Indonesia (Tjitra et al., 2008), Papua New Guinea (Genton et al., 2008), India (Kochar et al., 2009), and Brazil (Andrade et al., 2010). In this section, we will present what little is known about the pathogenesis of vivax malaria. While no definitive answer exists for any of the questions below, recent appreciation of P. vivax as a dangerous pathogen (Baird, 2007; Price et al., 2007b) and the identification of key gaps in our knowledge of basic P. vivax biology (Mueller et al., 2009), promises to spur significant research efforts in the near future.

How Does P. vivax Preferentially Infect Reticulocytes?

P. vivax merozoites seem to have a preferential tropism for reticulocytes compared to more mature RBCs. While two reticulocyte binding proteins (PvRBP-1 and PvRBP-2) have been partially characterized (Galinski et al., 1992), and six others have been annotated in the P. vivax genome (Carlton et al., 2008), the precise roles of these proteins in merozoite invasion and reticulocyte tropism have not yet been identified. While the putative reticulocyte receptor(s) has not yet been identified, the invasion of RBCs by P. vivax

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merozoites is a multistep process involving more than one receptor-ligand interaction. The best characterized of these is the interaction of the Duffy antigen receptor for chemokines (DARC) on the RBC surface and P. vivax Duffy binding protein (PvDBP), which is discharged from one of the merozoite’s apical organelles during the invasion process. This receptor-ligand interaction was previously believed to be absolutely crucial for invasion, as P. vivax merozoites are completely unable to invade RBCs lacking DARC expression (see later). More recently in Madagascar, however, P. vivax has been found to be naturally circulating in humans who lack DARC expression on their RBCs (Ménard et al., 2010). This finding suggests that some P. vivax isolates have evolved to invade DARC-negative RBCs by an alternative route.

DNA (compared to P. falciparum DNA, which is rich in AT) results in more efficient activation of TLR-9 by CpG DNA motifs. In plasma obtained from nonimmune vivax malaria patients during a paroxysm, Karunaweera and colleagues (Karunaweera et al., 2007) identified a lipid (putatively of parasite origin) in the cholesterol-triglyceride fraction, which may be unique to P. vivax. This lipid was found to be more potent in aggregating leukocytes than GPI-linked phospholipids and may represent a novel malaria toxin. While in vitro aggregation might be linked to in vivo leukocyte activation or other host inflammatory responses, the role of this lipid in the pathogenesis of vivax malaria awaits further study.

How Does P. vivax Cause the Malaria Paroxysm?

Acute P. vivax infection can cause profound degrees of anemia. While high parasitemias in the peripheral blood are believed to contribute prominently to the pathogenesis of anemia in individuals infected with P. falciparum, this is not the case with P. vivax. As reticulocytes typically represent about 1% of circulating RBCs, the tropism of P. vivax merozoites for these cells severely restricts the peripheral parasitemia that P. vivax can achieve. Typical parasitemias of 1% indicate that destruction of P. vivax-infected RBCs (whether by lysis or immune-mediated destruction in the spleen) cannot account for much of the total RBC loss and thus the degree of anemia. Like falciparum malaria, vivax malaria is associated with the loss of nonparasitized RBCs, but this loss seems to be greater. Malariotherapy studies (in which nonimmune individuals were experimentally infected with Plasmodium for the treatment of neurosyphilis) have shown that for every P. vivax-infected RBC destroyed, 32 nonparasitized RBCs are destroyed during a bout of vivax malaria (Collins et al., 2003). This degree of nonparasitized RBC loss is 4-times greater than that estimated in bouts of falciparum malaria. The reasons for this difference in RBC loss are not known. P. vivax can invade erythroblasts in vitro (Udomsangpetch et al., 2008), but due to the paucity of pathology data from live patients with vivax malaria, we do not know the extent of hematopoietic pathology, which could contribute significantly to the development of anemia. Indeed, P. vivaxinfected erythroblasts have only recently been observed in the bone marrow (Ru et al., 2009). If future studies find that P. vivax infection of erythroblasts is extensive, they may help to explain the considerable degree of RBC loss in patients with vivax malaria. This is because the destruction of replicating erythroid precursors and reticulocytes, with unopposed clearance of parasitized and nonparasitized RBCs by the spleen, would predictably cause rapid and profound decreases in hemoglobin levels. Cytokine-mediated dyserythropoiesis is also believed to contribute to reduced production of RBCs. These processes are expected to cause even greater RBC losses in individuals who suffer repeated episodes of vivax malaria in areas where (i) relapse is frequent or treatment unavailable, (ii) individuals experience chronic asymptomatic P. vivax parasitemia due to disease-controlling immunity and do not seek treatment, (iii) individuals harbor chloroquine-resistant P. vivax that is not eradicated with chloroquine, (iv) receive treatment with hemolytic drugs (e.g., primaquine) for elimination of hypnozoites, and/or (v) become coinfected with P. falciparum. Depending on the vivax malaria-endemic area, these scenarios can be very common. P. vivax causes maternal anemia during pregnancy, which results in low birth weight and associated increases in infant mortality. While there is no evidence that P. vivax-infected RBCs accumulate in the placenta, very few

The most classical presentation of vivax malaria is described as a malaria paroxysm: a cycle of fever, drenching sweats, and shaking chills that repeats every 48 hours, with relatively asymptomatic periods intervening. These malaria paroxysms are readily observed in P. vivax-infected nonimmune individuals. In individuals with varying degrees of immunity (semi-immune to hyperimmune), vivax malaria can also present simply as fevers of various periodicities accompanied by headaches, body aches, and malaise. The malaria paroxysm coincides with the periodic rupture of schizonts at the end of each parasite life cycle and large amounts of TNF and other cytokines in the circulation (Karunaweera et al., 2003). These observations suggest that parasite products released at the time of merozoite release directly activate host cells. For example, lysates of P. vivax schizont-infected RBCs have been shown to stimulate the production of TNF from PBMCs in vitro (Karunaweera et al., 2003). Convalescent serum from individuals with vivax malaria blocks this TNF production, suggesting that antibody responses to P. vivax antigens play a role in the acquisition of diseasecontrolling immunity. This possibility is further suggested by observations of relatively lower TNF levels in semi-immune individuals during their vivax malaria episode, compared to nonimmune individuals. Compared to P. falciparum, P. vivax has a lower pyrogenic threshold, defined as the parasitemia associated with the onset of fever. Symptomatically, bouts of vivax malaria rival those of uncomplicated falciparum malaria in the degree of fever and intensity of symptoms. Even though P. vivax parasitemias are relatively low, TNF levels, especially during vivax malaria paroxysms, can be as high (or even higher) than the levels of TNF during episodes of falciparum malaria. One study found that the ratios of TNF and other host inflammatory markers per parasitized RBC were greater during episodes of vivax malaria compared to episodes of uncomplicated falciparum malaria (Hemmer et al., 2006). These findings suggest that P. vivax provokes a far greater inflammatory response than does P. falciparum. Additional evidence for this possibility was obtained by Anstey et al. (Anstey et al., 2007), who found that there is a greater inflammatory response in the lung in patients with vivax malaria than in patients with falciparum malaria who had a similar or greater parasitemia. The basis for this difference is not known, but some have hypothesized that the chemical nature of two P. falciparum pyrogens—glycosylphosphatidylinositol (GPI) and hemozoin-associated CpG DNA—might be different in P. vivax. For example, Anstey et al. (Anstey et al., 2007) proposed that P. vivax GPI activates its TLR-9 receptor more potently than does P. falciparum GPI, thereby stimulating excessive cytokine production from monocytes and macrophages. Another possibility is that the greater GC content of P. vivax

How Does P. vivax Cause Anemia and Thrombocytopenia?


pathological studies of placentae have been conducted. The finding of hemozoin in placentae suggest the prior presence and clearance of parasitized RBCs from this organ, but gross evidence of histologically significant inflammation or chronic pathological changes have not been observed. Whether the mother’s systemic inflammatory response to parasitemia compromises placental or fetal tissue perfusion is not known. Thrombocytopenia is very commonly associated with vivax malaria. Possible causes include bone marrow suppression due to inflammatory cytokines or P. vivax infection of erythroblasts in the marrow space, oxidative damage to platelets, and sequestration of activated platelets in the vascular beds of some organs.

Do P. vivax-Infected Red Blood Cells Sequester?

In the peripheral blood of patients infected with P. vivax, all blood-stage parasite forms can be seen. These include rings, trophozoites, schizonts, and gametocytes. Unlike P. falciparum, P. vivax increases the size and deformability of its host RBC (Handayani et al., 2009), which may enable P. vivax-infected RBCs to pass through 2-mm endothelial cell slits in splenic sinusoids and return to the peripheral circulation. Due to the paucity of pathology data from patients with vivax malaria, it has been difficult to determine whether P. vivax-infected RBCs are able to sequester in vivo. Just recently, a histopathological study of fatal respiratory distress in a woman with vivax malaria showed evidence of heavy monocyte infiltration of pulmonary capillaries and damage to alveolar membranes consistent with acute respiratory distress syndrome (ARDS) (Valecha et al., 2009). Monocyte accumulation has also been observed in intestine and brain microvessels, but the pathological significance of these findings has not been determined. Virtually nothing is known about possible interactions between P. vivax-infected RBCs and monocytes in the liver, spleen, and bone marrow. Such interactions may promote anemia by stimulating monocyte secretion of TNF and other inflammatory mediators or by adversely affecting the erythroblast-nurturing function of monocytes in the bone marrow. Additional autopsy studies, as well as bone marrow biopsies from live patients, should enable us to more accurately assess the size of the parasite biomass in P. vivax-infected individuals, to determine whether P. vivax-infected RBCs adhere to the microvessels of some organs, and whether P. vivax develops within erythroblasts and effectively sequesters in the bone marrow. To date, there is no direct evidence that P. vivax-infected RBCs sequester in microvessels or other circulatory conduits through the liver, spleen, or bone marrow. However, the possibility that some fraction of P. vivax-infected RBCs does cytoadhere requires some measure of exploration for the following reasons. First, P. vivax-infected RBCs frequently form rosettes in vitro (Udomsanpetch et al., 1995); that is, they possess the ability to bind to a host cell type. The function of rosetting is not known but one intriguing possibility is that P. vivax parasites developing within RBCs display rosetting ligands to capture passing reticulocytes in the bloodstream. This interaction might ensure that shortlived merozoites will be better able to find their rare host reticulocyte upon schizont rupture. Second, there is some evidence of a relative paucity of very mature schizonts in the circulation of some patients with vivax malaria, which has led some to speculate that these forms might sequester as they complete their maturation and release merozoites (Field & Shute, 1956). Third, in Indonesian adults with vivax malaria and lung injury, Anstey et al. (Anstey et al., 2007) found evidence for reduced pulmonary capillary volume and impaired alveolar-capillary function, suggesting

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the possibility that P. vivax-infected RBCs sequester in the pulmonary vasculature. Whether platelet sequestration occurs in some microvascular beds is not known, but such a process could contribute to microvascular obstruction and inflammation. It is also possible that rosettes contribute to alveolar-capillary dysfunction by causing microvascular obstruction in the lungs.

What Are the Functions of Caveolae-vesicle Complexes on the Surface of P. vivax-Infected Red Blood Cells?

Like P. falciparum, P. vivax remodels its host RBC extensively by generating clefts and vesicles within the RBC cytoplasm. Unlike P. falciparum, however, P. vivax forms caveolae-vesicle complexes (CVCs) in its host membrane. CVCs are caveolae to which multiple cytoplasmic vesicles are associated in an alveolar fashion (Atkinson & Aikawa, 1990). Monoclonal antibodies have localized several P. vivax proteins to cytoplasmic clefts, vesicles, and CVCs (Barnwell et al., 1990; Matsumoto et al., 1988; Sanchez et al., 1994), suggesting that these structures are part of a trafficking pathway from parasites to caveolae, where proteins either associate with the RBC membrane or are secreted from the parasitized RBC. While the functions of these trafficked proteins are unknown, they are conceivably involved in the pathogenesis of P. vivax malaria, for example, by displaying cytoadherence and rosetting ligands, by presenting variant antigens involved in immune evasion (see later), or by releasing soluble activators of monocytes and other host cells.

What Are the Functions of vir Gene Products?

The P. vivax genome contains a variant subtelomeric gene family called vir (del Portillo et al., 2001; Fernandez-Becerra et al., 2009). The complete sequence of the P. vivax Salvador I strain (Carlton et al., 2008) revealed 346 vir genes, including 80 fragments and/or pseudogenes, most of which can be clustered into subfamilies (A-L) encoding conserved protein motifs. The subtelomeric location of vir genes, the presence of extracellular trafficking motifs in some vir genes, and the detection of some VIR proteins on the surface of P. vivax-infected reticulocytes suggest that VIR proteins may play a role in antigenic variation and thus in the maintenance of chronic parasite infection. That VIR proteins are variably expressed in vivo is indicated by serological studies showing that recombinant VIR proteins are recognized by serum samples from some individuals with a history of vivax malaria but not others. Unlike var genes, however, vir genes are neither subject to allelic exclusion nor clonally expressed in individual P. vivax-infected reticulocytes. Also, some vir genes contain SURFIN-like or Pfmc-TM2-like sequences that suggest some of them may be expressed on the surface of merozoites or another subcellular location. It has been suggested that VIR proteins expressed on the surface of P. vivax-infected reticulocytes may play a role in immune evasion by a mechanism different from classical antigenic variation. For example, del Portillo and colleagues (Fernandez-Becerra et al., 2009) have hypothesized that VIR proteins bind to putative receptors on spleen barrier cells and thus mediate the sequestration of P. vivax-infected reticulocytes in this organ. This adherence is proposed to physically impede macrophage access to parasitized RBCs, resulting in immune evasion and the establishment of chronic parasite infection. This mechanism may enable P. vivax-infected RBCs to enter the spleen without being efficiently removed by mechanical processes or phagocytosis.

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What Causes P. vivax Hypnozoites to Reactivate in the Liver?

P. vivax causes relapsing malaria, which is defined as the reappearance of P. vivax parasitemia in a sporozoite-induced infection following adequate blood schizonticidal therapy (Cogswell, 1992). Relapses are caused by latent P. vivax hypnozoites that reside in the liver. Hypnozoites can become reactivated to develop into liver schizonts, which produce merozoites capable of infecting RBCs. How P. vivax parasites emerge from the liver at various intervals after blood schizonticidal therapy is not known, although several possibilities have been proposed. One possibility is that different strains are genetically programmed to relapse at certain intervals; that is, latency periods are an intrinsic characteristic of a P. vivax strain. In support of this, temperate strains from China and North Korea exhibit latency periods of about 1 year, while a tropical strain from New Guinea exhibits a much shorter latency period of a few months. P. vivax may have evolved different latency periods to ensure successful transmission from one human host to another. In temperate areas, where the mosquito breeding season is relatively short, parasites may have evolved to relapse annually. In some tropical areas where mosquito breeding occurs continuously year-round, parasites may have evolved to relapse every few weeks to months. In areas where P. falciparum is also transmitted, another possible trigger of relapsing malaria may be fever due to falciparum malaria or other febrile illnesses. These and other possible triggers have been speculated, but evidence for their role is lacking.

Why Do Some Individuals Develop Severe vivax Malaria?

While cases of vivax malaria complications (e.g., splenic rupture, acute respiratory distress syndrome) have been reported for decades (Imbert et al., 2009; Price et al., 2007a, 2007b), recent case series from Southeast Asia, India, and Brazil suggest that severe vivax malaria constitutes a specific clinical syndrome that may be more common in some epidemiologic settings than previously appreciated. In particular, two large prospective cohort studies from Southeast Asia (Genton et al., 2008; Tjitra et al., 2008) have helped to describe the incidence and clinical manifestations of severe vivax malaria. In these studies, severe vivax malaria was defined as coma, severe malarial anemia (SMA, Hb ,5 g/dL), and/or respiratory distress—some of the same criteria used to define severe falciparum malaria (Table 1). Over a 4-year period in Papua, Indonesia, Tjitra and colleagues (Tjitra et al., 2008) found that approximately 22% of 2,634 patients admitted to hospital for malaria had severe manifestations of the disease and that roughly half of these episodes were due to P. vivax. While most of these patients had SMA, CM and respiratory distress were also observed. The high proportion of SMA among severe vivax cases may be due to the extremely high prevalence (65% to 95%) of chloroquineresistant P. vivax infections, which might go chronically untreated and result in progressively lower hemoglobin levels. Severe vivax malaria was also associated with younger age (i.e., children) as well as coincident P. falciparum infection and falciparum malaria. Over an 8-year period in Papua New Guinea, Genton and colleagues (Genton et al., 2008) found that in children under the age of 5 years, 8.8% of severe malaria cases were associated with P. vivax monoinfection. Compared to simultaneously studied cases of severe falciparum malaria, severe vivax malaria was much less likely to involve SMA than respiratory distress. This may be due to the extremely low prevalence of drug-resistant P. vivax in this area during the study period. As found in

Papua, Indonesia (Tjitra et al., 2008), risk factors for severe vivax malaria were young age and coincident infection with P. falciparum. The level of parasitemia, on the other hand, did not seem to increase the risk of severe vivax malaria, as patients with uncomplicated or severe vivax malaria did not differ significantly in P. vivax density. It is not clear whether severe vivax malaria episodes were previously underreported and thus unappreciated, or whether they are now appearing with greater frequency due to some new parasite or host factor (Anstey et al., 2009; Price et al., 2009; Price et al., 2007b). For example, highgrade resistance to chloroquine might predispose a P. vivaxinfected individual to lower baseline hemoglobin levels from which additional P. vivax or P. falciparum infections might result in SMA. Another possibility is that waning maternal immunity—due to changes in transmission intensity—might result in the transfer of a lower titer of protective antibodies, rendering children in their first year of life more susceptible to severe vivax malaria. Yet another possibility is that severe vivax malaria cases are complicated by undiagnosed comorbidities such as bacterial pneumonia and sepsis. Studies in which occult infection occurs with P. falciparum or other Plasmodium species are now appearing in the literature. In a recent study from the Brazilian Amazon, Andrade and colleagues (Andrade et al., 2010) described a series of patients with vivax malaria complicated by severe anemia, respiratory failure, convulsions, renal failure, or jaundice, many of whom also presented with hypotension and/or splenomegaly. These patients were shown by microscopy and nested PCR protocols to be infected with neither P. falciparum nor P. malariae. Compared to patients with uncomplicated vivax malaria, those with severe vivax malaria were younger, reported fewer previous malaria episodes, reported fewer years of residence in the area, and had higher parasitemias. These observations suggest that these patients may have, in part, developed severe complications due to their relatively nonimmune or semi-immune status. These investigators have begun to explore whether there might be an inflammatory basis for these severe vivax malaria syndromes. By comparing levels of proinflammatory and anti-inflammatory cytokines in individuals with asymptomatic parasitemia, uncomplicated or severe vivax malaria, they found significant increases in levels of CRP, TNF and IFNg, as well as elevated ratios of INFg/IL-10, which correlated with disease severity. Elucidating a role for these cytokines in the pathogenesis of severe vivax malaria awaits further study. The lack of basic information on the pathogenesis of uncomplicated P. vivax malaria makes it difficult to imagine how P. vivax causes specific severe malaria syndromes such as CM, SMA, and respiratory distress. Coma and convulsions might be due to rare cerebral microvascular pathologies that do not involve the sequestration of P. vivax-infected RBCs. It is also possible that fever and anemia may exacerbate tissue hypoxia sufficiently to produce manifestations of cerebral malaria in some individuals. Severe anemia might result from repeated bouts of hemolysis due not only to chronic parasitemia but also to inadequate treatment of repeated episodes of vivax malaria. The increased fragility of nonparasitized RBCs that develops during vivax malaria episodes may be confounded by intermittent P. falciparum infections or the presence of hemoglobinopathies or G6PD deficiency, which are associated with accelerated RBC senescence in vivo. Recent evidence that P. vivax infects erythroblasts in the bone marrow (Ru et al., 2009) may explain in part the development of SMA. Individuals who cannot make reticulocytes


and at the same time are removing large fractions of nonparasitized RBCs would be at much greater risk of dropping their steady-state hemoglobin levels and developing SMA. Respiratory distress may be due to acute lung injury, which may develop from cytokine-associated increases in alveolar-capillary membrane permeability (Anstey et al., 2007). One possibility is that some individuals mount a more intense inflammatory response to the contents of ruptured or dying P. vivax parasites in the lung. Another possibility is that P. vivax-infected RBCs sequester in alveolar capillary beds, where they cause pathologic endothelial activation and dysfunction. Whether acidosis or bacterial/viral pneumonias contribute significantly to P. vivax-associated respiratory distress has yet to be investigated.

HUMAN GENETIC RESISTANCE TO vIvAX MALARIA DARC Negativity

The Duffy blood group antigens Fya and Fyb are encoded by the codominant alleles FY*A and FY*B, respectively. These antigens were found to bind IL-8 and other chemokines and were recently renamed Duffy antigen receptors for chemokines (DARC). A GATA-1 mutation in the promoters of these genes results in loss of either Fya or Fyb or both antigens from the surface of RBCs. This mutation is associated with the FY*B allele in Africa and has approached genetic fixation in vast areas of the continent, suggesting that it confers some selective advantage to humans. The absence of vivax malaria in much of sub-Saharan Africa (Culleton et al., 2008) and the finding that P. vivax is unable to infect DARC-negative individuals (Miller et al., 1976), supports the conclusion that DARC-negativity in Africa was naturally selected by vivax malaria. Recent reports of severe and fatal vivax malaria in Southeast Asia, India, and Brazil strengthen earlier speculations that vivax malaria could have once been fatal in parts of Africa and thus able to exert significant selective pressure on human populations living there. The same SNP that produces DARC-negative RBCs in Africans has been recently identified in the Wosera of Papua New Guinea (PNG), where the SNP is associated with the FY*A allele. This SNP has thus far only been observed in the heterozygous state (Fya/2) and is associated with a 50% reduction in the amount of DARC on the RBC surface. Kasehagen and colleagues (Kasehagen et al., 2007) hypothesized that this SNP was naturally selected for its protection against P. vivax malaria in PNG, where cases of severe vivax malaria occur and where DARC negativity might thus exert a selective fitness advantage. To test this hypothesis, they created cohorts of children ,15 years old and periodically examined their blood for P. vivax parasitemia. They found that Fya/2 children were less likely than wild-type Fya/a children to test positive for P. vivax parasitemia. The P. vivax parasitemia was significantly lower in Fya/2 children than in Fya/a children, indicating that merozoites do not invade Fya/2 RBCs as efficiently as Fya/a RBCs. These associations were not seen in individuals older than 15 years of age, suggesting that some elements of naturally acquired immunity may supplant the protective mechanism of DARC negativity. The natural selection of the Fya/2 and Fy2/2 phenotypes suggests that interventions that impair DBP-DARC interactions may be of clinical benefit. One such intervention being investigated is vaccination with DBP, which has been shown to elicit antibodies that partially block merozoite invasion of reticulocytes (Grimberg et al., 2007).

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Other Red Blood Cell Polymorphisms

With the notable exception of DARC negativity in Africans and in the Wosera of PNG, genetic resistance to P. vivax malaria has received little attention. This is surprising since proposed mechanisms of resistance to falciparum malaria (reduced merozoite invasion of RBCs, impaired intraerythrocytic trophozoite development, and enhanced phagocytic removal of ring stage-infected RBCs) could theoretically be implicated in genetic resistance to P. vivax malaria. Numerous RBC polymorphisms are prevalent in areas where both P. vivax and P. falciparum are transmitted and where severe disease due to both Plasmodium species has been documented. One example is Southeast Asian ovalocytosis (SAO), which is caused by a 9-amino acid deletion in the cytoplasmic tail of band 3, the major anion exchanger in RBC membranes. This polymorphism causes the RBC membrane to become more rigid and the RBC to assume an elliptical shape. Older studies (Cattani et al., 1987) suggested that SAO reduces the susceptibility of Papua New Guineans to vivax malaria, but this protection has not yet been confirmed by other studies, nor has the mechanism of this protection been defined. One possibility is that the increased membrane rigidity of SAO RBCs compromises the ability of P. vivax to increase the deformability of its host reticulocyte. This effect would likely enhance the removal of P. vivax-infected reticulocytes by the spleen. Data supporting a protective role for other RBC polymorphisms are now emerging. Louicharoen and colleagues (Louicharoen et al., 2009) recently found that a G6PD deficiency allele (Mahidol variant) common in Thailand is associated with reduced P. vivax parasitemia in vivo, which raises the possibility that this polymorphism ameliorates the severity of vivax malaria. Whether other RBC polymorphisms (e.g., hemoglobin E, a-thalassemia, Gerbich antigen negativity, and CR1 polymorphisms) have been partially selected by vivax malaria will require additional studies in Southeast Asia.

PERSPECTIvE: AN INTEGRATED vIEW OF MALARIA PATHOGENESIS

Here we briefly describe working models of malaria pathogenesis which we believe provide useful frameworks for future studies in this area. Specifically, we propose that the levels of parasitemia and the strength of pathogen-host cell interactions are critically involved in the pathogenesis of malaria, and that host factors (e.g., antibodies, RBC polymorphisms) which interfere with parasite multiplication and sequestration are centrally important in ameliorating the severity of malaria.

Falciparum Malaria

We propose that the avidity of known host-pathogen interactions is critically important to the pathogenesis of falciparum malaria. In general, disease severity is positively correlated with parasite biomass. This is because large numbers of parasites are sequestering in the microvessels of critical organs, where they activate host cells to produce microvascular inflammation, obstruction, and dysfunction. In cases where large parasite biomasses are not producing symptoms or severe manifestations of disease, this is because the parasitized RBCs are sequestering with low avidity, such that host cell activation remains below the threshold required to produce uncomplicated or severe malaria. Factors that reduce the avidity of cytoadherence interactions will ameliorate the severity of malaria and, if further reduction in avidity is attained, will reduce parasite densities in vivo by preventing the cytoadherence of some parasitized

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FIGURE 2 A new model of falciparum malaria pathogenesis and protection. The sequestration of P. falciparum-infected red blood cells (RBCs) in microvessels is believed to activate microvascular endothelial cells (MVECs) and monocytes, which contribute to the microvessel inflammation associated with severe malaria. Hemoglobin (Hb) C and sickle HbS reduce the risk of severe malaria in sub-Saharan Africa. HbC and HbS are associated with abnormal display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), a family of antigenically variant cytoadherence ligands that serves as the parasite’s main virulence factor on the surface of parasitized RBCs. Reduced levels and abnormal distributions of PfEMP-1 are associated with impaired adherence of parasitized HbC and HbS RBCs to MVECs and monocytes (this is shown schematically at right as less intimate interactions between these parasitized RBCs, MVECs, and monocytes). By lowering the avidity of cytoadherence interactions, HbC and HbS may reduce the level of MVEC and monocyte activation in vivo and prevent the progression from uncomplicated to severe malaria. PfEMP-1-specific antibodies that reduce the avidity of these interactions may work in concert with Hb variants to dampen inflammation and ameliorate disease severity.

RBCs altogether. Such factors include PfEMP-1-specific antibodies that block cytoadherence and RBC polymorphisms that impair cytoadherence by causing abnormal display of PfEMP-1. Simply stated, we propose that in highly endemic areas, naturally acquired PfEMP-1-specific antibodies work together with RBC polymorphisms at the level of PfEMP-1 to reduce the incidence of severe and uncomplicated malaria, and eventually, the density of parasitemia (Fig. 2).

vivax Malaria

Vivax malaria is traditionally distinguished from falciparum malaria in several ways, but we emphasize these two differences in summary: (i) P. vivax densities are lower than P. falciparum densities in vivo and (ii) P. vivax-infected RBCs do not seem to sequester as do P. falciparum-infected RBCs. These and other observations have been difficult to understand, especially now that cases of severe vivax malaria are being described. For example, how can P. vivax cause pulmonary symptoms when it does not sequester in the lung? How can P. vivax make individuals so ill and anemic at such low parasitemias? To explain these and other observations, we hypothesize that host–pathogen interactions may play some role in the pathogenesis of vivax malaria. We have observed that P. vivax-infected RBCs bind non-parasitized RBCs to form rosettes, but we do not know whether they adhere to the microvascular endothelial cells of specific organs. Some evidence of microvascular obstruction has been inferred from data obtained from in vivo studies of lung physiology, suggesting the possibility that P. vivax-infected RBCs bind endothelial cells and non-parasitized RBCs in

lung microvessels. The distribution of caveolae-vesicle complexes (CVCs) over the surface of P. vivax-infected RBCs is reminiscent of the distribution of PfEMP-1-laden knobs on the surface of P. falciparum-infected RBCs. Although very speculative, we hypothesize that cytoadherence and rosetting ligands are expressed in caveolae and contribute to malaria pathogenesis. The occurrence of asymptomatic P. vivax parasitemia suggests the possibility that naturally acquired antibodies ameliorate disease severity by disrupting cytoadherence and rosetting. The contribution of parasite density to the severity of vivax malaria is difficult to determine at this time. Some contribution is suggested by the finding that Duffy-negative heterozygotes in Papua New Guinea have lower parasite densities and reduced incidence of uncomplicated malaria. However, we are presently unable to accurately measure P. vivax biomass. The ability of parasites to infect erythroblasts suggests that significant numbers of P. vivax-infected RBCs might sequester in the bone marrow, where they might impair the normal development of RBCs and thus contribute to anemia. We suggest that RBC polymorphisms other than Duffy-negativity may reduce P. vivax infection of RBCs or impair cytoadherence interactions between P. vivax-infected RBCs and host cells. Like Duffy negativity, RBC polymorphisms that are found to protect against P. vivax may teach us important aspects of the pathogenesis of this fascinating and sometimes deadly parasite. This work was supported by the Intramural Research Program of the NIH, NIAID.


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30 Pathology and Pathogenesis of Virus Infections CARMEN BACA JONES AND MATTHIAS VON HERRATH

INTRODUCTION TO VIRUS-INDUCED TISSUE DAMAGE

the human cytomegalovirus genome encodes a vast number of viral gene products known to disrupt both the adaptive and innate immune response to viral infection and, as such, is able to maintain a lifelong association with the host. Likewise, a number of different host factors, which contribute to inadequate viral clearance, include deficits in antigen presentation or immune effector cell trafficking and activation. Manipulation of the aforementioned effector mechanisms, as seen in the Clone 13 strain of LCMV (lymphocytic choriomeningitis virus), contributes significantly to the persistence of a multitude of viruses. In addition, feedback loops that are in place to dampen the acute immune response to minimize tissue damage by antiviral effector cells, can allow virus to escape eradication from the host if they shut off the immune response prematurely, resulting in the establishment of a persistent or latent viral infection. Virus-induced pathology can thus be the direct result of a lytic viral infection or a consequence of immune activation, and therefore is the result of both host and viral contributions. Viruses whose replication cycle can culminate in cell lysis are not always lytic for all infected cell types. Additionally, host components may also determine the degree of virus-induced apoptotic cell death. Nonlytic viruses can be well adapted to their natural host, for example by becoming latent or by evading the host’s immune response (herpes viruses are a good example for this). Lesser symptoms during the acute infection allow the host to better tolerate the infection, thus providing greater opportunity to spread to reservoirs within the host and to spread to a new host. While some of these “smoldering” infections can be benign in the healthy immunocompetent host, the constant or serial activation of the immune system to such infections can present with significant pathology in the immunocompromised host. Lastly, differences in the pathogenicity of viruses are often determined by the cells or tissues that are permissive for virus replication. Targeting of more sensitive organ systems, as is the case for influenza (lungs) and hepatitis (liver) viruses, can lead to serious disruption of normal cell and system function, resulting in pathology associated with diminished organ function. Overall, all forms of virus-induced tissue damage involve virus factors as well as host factors and therefore, understanding the relationship between the virus and its host is essential to the development of effective interventions (Table 1).

Viruses, as obligate intracellular parasites, pose a unique challenge to the host immune system, as a balance between clearance of the invader and collateral damage to host tissues must be met to ensure survival of the host as a whole. While overall, the immune response to viral infection is protective and balance is achieved, both inadequate and overactive immune responses can result in pathology. An overly intense immune response may clear the virus at the expense of the tissues surrounding the site of replication, resulting in damage and lesions caused by immunopathology; a prime example of this (discussed later in the chapter) is the inflammatory response induced by respiratory syncytial virus in the lungs. In contrast, insufficient responses to a viral infection can permit the virus to induce excessive direct tissue damage upon acute or persistent infection (as in herpes viral infections). Persistent infections can cause pathology resulting from chronic direct effects of the virus on the infected cell (many times this can be in the development of cancers, for example, in HCV infection) as well as the burden of persistent activation on the immune system (for example, in HIV infection). A multitude of factors contribute to inadequate clearance of viral invaders resulting in persistent or chronic infections. Diverse cell type and tissue tropism can lead to the widespread distribution of virus within the host. Such seeding can provide an endless number of reservoirs for the virus, making clearance a futile task (for example, HIV can hide in antigen-presenting cells and in the central nervous system). In addition, the ability of a virus to go latent or to spread directly from cell to cell, avoiding a prolonged extracellular or viremic phase of infection, can serve as an effective immune evasion strategy, thus limiting exposure of the virus to immune effectors. Cell-to-cell spread and latency are two strategies employed widely by herpesviruses to evade direct detection. Virus-induced immunomodulation (both disruption of the innate and adaptive arms) represents yet another mechanism by which some virus families are able to promote a long-term association with the host. For example, Carmen Baca Jones and Matthias von Herrath, Center for Type 1 Diabetes Research, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037.

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TABLE 1

Examples of host and viral factors contributing to virus induced pathology

Type of pathology

Host factors contributinga

Viral factors contributing

Associated virus(es)

Cytolysis

Inadequate IFN response; robust CTL response; susceptible cell type

Lytic viral egress

Common to many viruses including Epstein-Barr virus (B cells), human papillomavirus

Apoptosis

Cytokines can induce bystander apoptosis as well as infected-cell apoptosis

Presence of dsRNA; viral blockade of host protein synthesis

Common to many viruses including bunyaviruses, flaviviruses, adenoviruses, Sindbis virus

Allergic response

Development of IgE; Th2-biased response to virus; possible genetic predisposition has been suggested

High infectivity, cytopathology; viral inhibition of interferon response

Respiratory syncytial virus

Th17

Elevated IL-17 production; TNFa/ IFNg imbalance; disruption of Fas-FasL signaling

Unknown

Theiler murine encephalitis virus

Cytokine storm

Excessive inflammatory cytokine production, particularly TNFa

Down regulation of early type I IFN

Sin nombre virus, dengue virus, poxviruses

Immune complex formation

Induction of complement cascade and incomplete clearance of virus-Ab complexes

Viremic, extracellular stage of life cycle; prolonged association with host

Lymphocytic choriomeningitis virus, hepatitis B virus

Antibody dependent enhancement of infection

Generation of nonneutralizing Ab; susceptibility of Fc receptorbearing cells to infection

Secondary infection with heterologous serotype (DHV)

Dengue hemorrhagic fever virus, human immunodeficiency virus, Ebola virus

a

IFN, interferon; CTL, cytotoxic T lymphocyte; IL, interleukin; TNF, tumor necrosis factor.

VIRUS-INDUCED IMMUNOPATHOLOGY

In addition to the direct effects of virus infection on the host cell, the potential of the immune system to respond to the invasion in itself is often the source of pathology (immunopathology). While the term immunopathology was originally used to describe an aberrant immune response to autoantigen resulting in pathology, more recent developments in our understanding of virus-induced immunopathology have revealed that this can be linked to both an aberrant immune response to the virus itself as well as unfavorable virus-induced changes to the immune response as a whole; for example, lymphopenia following certain types of infections as well as the specific loss of CD4 T cells in HIV infection. Today, advancement in available tools and techniques allows us to more precisely distinguish between pathology directly linked to virus infection of the target cell or, alternatively, as a consequence of the response to the infection by the host immune system. Viruses have been demonstrated to induce immune cell dysfunction through a variety of mechanisms, including direct infection of immune effector cells and indirect means, which impact immune cell function. Direct effects of virus infection include situations where infection ultimately leads to the death of the cell be it through the induction of apoptosis, killing by immune effectors, or cytolytic infection. Additionally, lymphotropic viruses can directly induce immunopathology through the infection of immune effectors and virally encoded immunomodulatory proteins. Herpesviruses are classic examples of virus-induced immunomodulation and represent a vast area of research, and, as such, this topic will be addressed in a separate chapter. Additional examples of virus-induced pathology include the induction of apoptosis, tissue damage resulting from oxidative burst, complement cascade, and a host of other mechanisms.

T-CELL MEDIATED CYTOLYSIS AND INDUCTION OF APOPTOSIS

Perhaps the most common virus-induced pathology is T-cell-induced destruction of host tissues. T-cell mediated

pathologies occur through both direct and indirect mechanisms including the targeted cytolysis of the infected cell by effector T cells, the induction of apoptosis in infected cells and generalized tissue damage due to bystander effects of T-cell activation. Overt pathology associated with T-cell effector functions is seen primarily in cases in which the offending pathogen is noncytolytic and where the T-cell response, not the direct effects of viral infection, induces the majority of the damage, as is the case with chronic herpes simplex virus-induced stromal keratitis (HSK). More than 400,000 individuals in the United States suffer from ocular herpes simplex infections, one of the leading causes of infectious blindness (Pepose, 1996). HSV-1 infection of the corneal epithelium leads to the infiltration of large numbers of inflammatory cells (including neutrophils, CD4 and CD8 T cells, macrophages, dendritic cells, and natural killer cells) into the cornea and the secretion of proinflammatory cytokines and chemokines, all culminating in tissue damage (Banerjee et al., 2004; Deshpande et al., 2004; Deshpande et al., 2001; Pepose et al., 1985; Thomas et al., 1997; Youinou et al., 1986; Youinou et al., 1985). It is estimated that 20% of acutely infected individuals go on to develop HSK, a chronic lesion of the corneal stroma, characterized by corneal ulceration and necrosis, neovascularization, and stromal edema (Liesegang, 2001; Streilein et al., 1997). Scarring of the cornea leads to opacity and can culminate in blindness. Two forms of HSV-induced stromal keratitis have been described in the literature: necrotizing stromal keratitis (NSK) and immune stromal keratitis (ISK). NSK is commonly associated with acute viral infection and characterized by the presence of viral antigen and intact virions in the corneal keratocytes, epithelial cells, and endothelial cells (Deshpande et al., 2004; Metcalf & Kaufman, 1976). The finding of intact virions in the cornea suggests that an active infection may directly mediate the damage to the cornea seen in NSK. Further evidence in support of the direct effects of virus in acute epithelial keratitis NSK can be found in the efficacy of antiviral treatment in alleviating

30. Pathology and Pathogenesis of Virus Infections

the symptoms of NSK (Deshpande et al., 2004; Kaufman, 2002). This is in contrast to immune stromal keratitis where viral antigen is no longer detected in the chronic lesion and disease is largely attributed to immunopathology. Mounting evidence supports an immunopathological origin in chronic HSK pathogenesis. While viral loads tend to be elevated in primary acute infection, HSK pathogenesis is relatively rare in previously naïve patients, but more commonly seen in secondary infection/reactivation from latency (Pepose, 1996). The observation that secondary infection is more commonly associated with HSK suggests that a primed preexisting adaptive immune response likely plays a role in disease. Furthermore, the use of immunosuppressive steroid therapy to treat chronic HSK, coupled with the exceptionally low incidence of HSK in immunodeficient patients as compared to immunocompetent individuals all implicate the immune response in disease progression (Liesegang, 1999). A role for T-cell mediated pathogenesis is specifically supported by a series of elegant experiments utilizing adoptive transfer of either HSV-reactive CD8 or CD4 T cells into infected mice (Banerjee et al., 2005; Metcalf & Kaufman, 1976; Shimeld et al., 1989). These studies demonstrated that while HSV reactive CD8 T cells enter the cornea during the acute phase of infection, it is the CD4 T cells that predominate in the chronic phase. Further investigation into the contribution of both CD4 and CD8 T cells demonstrated that the early infiltration of HSVreactive CD8 T cells into the cornea mediated protective clearance of the virus and was not involved in the development of corneal lesions. Later infiltration of HSV-reactive CD4 T cells, during the chronic phase where viral antigen is undetectable by current methods, was found to mediate HSK pathogenesis. The observation that HSK progresses in the absence of detectable viral antigen is addressed in the bystander activation hypothesis, which proposes that initial recognition of viral antigen by CD4 T cells results in a downstream cascade of inflammatory events culminating in the subsequent TCR independent activation of memory T cells (Banerjee et al., 2002; Deshpande et al., 2001; Gangappa et al., 1998). While T cells have been widely implicated in disease progression, HSK pathogenesis involves a complex cascade of events and, as such, there are likely multiple mechanisms of virus-induced immunopathology at play in HSK.

VIRUS-INDUCED ALLERGIC RESPONSE

Early childhood infection with respiratory syncytial virus (RSV), a noncytopathic virus associated with lower respiratory tract disease in infants, is a widely recognized risk factor for the development of childhood asthma and wheezing (Martinez, 2003; Perez-Yarza et al., 2007). RSV induced respiratory distress bears a number of similarities to allergic airway hypersensitivity response (AHR) including excessive mucus secretion, elevated levels of IgE antibody, and a sizeable infiltration of eosinophils into the lungs. While a direct cause-and-effect relationship between RSV infection and the development of subsequent AHR has yet to be demonstrated, several observations strongly suggest a link between the two. In particular, recent studies implicate the propensity of the host to initiate a Th2 biased response to the primary RSV infection as a factor in the development of an allergic-type reaction to subsequent exposures to the virus. In a mouse model of RSV induced AHR, Th2 cytokines, IL4 and IL13, were found to be required for virus-specific IgE production and subsequent AHR development. In the context of allergic responses to foreign antigen in the airway, IgE bound to its cognate antigen

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binds to FceRIs on the surface of mast cells, triggering the degranulation of mast cells. While the role of allergic IgE in the alteration of airway function is well understood, relatively little is known about the role of virus-specific IgE in airway remodeling and anti-viral host defense. A recent study found that while neutralization of anti-RSV IgE antibodies blocked the development of AHR, neutralization did not impact viral titers in the lungs, suggesting that one, viral replication alone does not cause disease and two, the IgE response is not effective in controlling viral clearance and thus may prove to be useful as a therapeutic target (Dakhama et al., 2009).

VIRALLY INDUCED TH17 MEDIATED PATHOLOGY

In addition to pathology mediated by Th1 and Th2 responses, other effector cytokines, such as IL-17 (interleukin-17) is secreted by so-called Th17 cells, although it has yet to be shown that these constitute a separate lineage, have also been implicated in virus-induced immune mediated tissue damage. Th17 cells are a subset of CD4 T cells, which express IL-17. Although they have been demonstrated to play a protective role in defending against extracellular bacterial and fungal infections, they have also been associated with inflammatory tissue damage and several autoimmune disorders (Bettelli et al., 2008, 2007; Dong, 2008; Park et al., 2005). While up regulation of IL-17 has been described for several viruses including HIV, HSV, and RSV only recently have studies utilizing the Theiler’s murine encephalomyelitis virus (TMEV) multiple sclerosis model revealed a novel role for Th17 cells in the promotion of chronic virus infection (Hou et al., 2009). Multiple sclerosis (MS) is a disease characterized by the destruction of the protective myelin sheath enveloping the neuronal axons of afflicted persons. Although generally believed to be an autoimmune disorder, the factors contributing to the initiation of MS remain an area of hot debate. Murine infection with TMEV has been used to model the hypothesis that viral infection may provide the trigger initiating an autoimmune cascade. Th17 polarization and subsequent IL-17 production in the TMEV infection model was found to promote the survival of infected cells by both inhibiting apoptosis of infected cells and rendering target cells resistant to killing by effector T cells. While IL-17 treatment of CD8 T cells did not significantly alter the frequency of granzyme B and IFN-g (interferongamma) positive cells, treatment did render the target cell resistant to Fas-FasL-mediated killing by effector T cells. Ultimately Th17 polarization promotes persistence of the virus and subsequent pathology associated with chronic demyelination.

SYSTEMIC INFLAMMATORY RESPONSE SYNDROME

Systemic inflammatory response syndrome, commonly referred to as a cytokine storm, results from an overproduction or systemic release of inflammatory cytokines (Flint, 2004). Under normal circumstances, the inflammatory response aids in the mobilization of primary effector cells of both the innate and adaptive arms of the immune response. Increased vascular barrier permeability and the localized release of cytokines direct immune cell traffic toward the site of primary infection and facilitate pathogen clearance. However, virus-induced cytokine dysregulation resulting in an overabundance of inflammatory cytokines can induce a shocklike syndrome where excessive edema, as well as cytokine and effector-cell-induced tissue damage, can lead to overall tissue destruction and subsequent organ failure. While the

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precise events triggering the disproportionate inflammatory response to viral infection are not well understood, both host and pathogen factors are believed to play a role in this potentially fatal immune response. Virus-induced cytokine storms have been implicated as the primary cause of disease in a number of viral infections, including certain types of hantaviruses. The first documented cases of hantavirus pulmonary syndrome (HPS) occurred in 1993 in the Four Corners region of the southwestern United States and resulted in a 40% case fatality rate, primarily afflicting young and otherwise healthy individuals. HPS is characterized by capillary leakage in the lungs, resulting in an acute respiratory distress syndrome, thrombocytopenia, and cardiac shock. The causative agent was later identified as a newly emergent hantavirus, later named sin nombre virus (SNV). Some years later another hantavirus, the Andes virus, inducing an HPS-like syndrome, emerged in South America. Difficulties in establishing an animal model of HPS had greatly hampered progress in understanding the factors involved in pathogenesis. However, the emergence of Andes virus opened up new avenues of research as infection of the Syrian hamster, a small animal model previously used for hantavirus vaccine strategies, for the first time reproduced a Hanta-induced HPS-like pathology, facilitating the study not only of infection, but pathogenesis as well. HPS appears to have both viral and host genetic components as illustrated in the Syrian hamster model of Hanta-induced HPS. Both SNV and Andes virus are able to productively infect both humans and Syrian hamsters. However, while both are capable of inducing HPS in humans, only Andes virus can trigger HPS in Syrian hamsters (Abel Borges & Figueiredo, 2008; Saggioro et al., 2007). Although to date a specific host factor has yet to be identified, one potential virion component of HPS has; sequence differences in the G1 viral glycoprotein lead to proteasomal degradation in pathogenic strains, whereas nonpathogenic strains remain stable (Sen et al., 2007). This difference in viral glycoprotein tail stability is hypothesized to lead to increased antigen presentation and CTL activation thus contributing to Hanta-induced immunopathology.

IMMUNE COMPLEX FORMATION

Antiviral antibodies can bind whole virus or processed viral antigen on the surface of infected cells, resulting in virus-antibody (V-Ab) immune complex formation. The generation of V-Ab complexes triggers a cascade of downstream events with both protective and pathological potential. Circulating V-Ab immune complex can be cleared through the activation of the complement cascade, culminating in the binding of complement components on the surface of erythrocytes (Janeway, 2001). V-Ab complexes bound to erythrocytes are then transported to the spleen and liver where macrophages remove the V-Ab complex from the surface of the erythrocyte and subsequently degrade the virus-Ab-complement complex, thus clearing the pathogen. However, effective clearance of immune complexes is not always achieved, particularly in the case of chronic viral infection. Immune complexes that are not cleared by phagocytes are deposited in the basement membranes of small blood vessels. Extensive tissue damage due to the deposition of V-Ab-complement complexes is a primary etiology for renal dysfunction associated with viral infection. Such cases of disease associated with immune complex deposition were first described with chronic LCMV infection, and later identified in hepatitis B virus infections of humans (Buchmeier & Oldstone, 1978; Oldstone & Dixon, 1969).

ANTIBODY-DEPENDENT ENHANCEMENT OF INFECTION

In addition to tissue damage arising from immune complex deposition in the small vessels, the formation of antibodyvirus complexes can augment viral infection of Fc receptor bearing cells. While Fc receptors expressed on cells such as macrophages, dendritic cells, B cells, mast cells, and natural killer cells cannot substitute for the endogenous viral receptor; they have been demonstrated to act as coreceptors, capable of enhancing infection of antigen presenting cells (APCs). While the precise mechanism of action is still unknown, antiviral antibody has been postulated to bring the virus in close proximity to the plasma membrane, thus increasing the likelihood of a receptor interaction. As well, some have argued in favor of a role for Fc receptor interaction induced phagocytosis in antibody dependent enhancement (ADE) of infection. Regardless of the precise mechanism(s), several viral infections appear to be enhanced by the presence of antiviral antibodies including West Nile virus, HIV, and Ebola virus (Buchmeier & Oldstone, 1978; Gubler et al., 2007; Robinson et al., 1988; Takada et al., 2003). Perhaps the most widely examined example of ADE can be found in dengue virus infections. Dengue virus, a member of the Flaviviridae family of viruses with worldwide distribution, is estimated to infect 50 to 100 million people annually (Gubler, 1997). Primary infection is typically asymptomatic, but can result in a minor febrile illness termed dengue fever (DF), however it is estimated that 500,000 people are afflicted with more severe clinical manifestations of dengue viral infection, known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Capillary leakage, increased vascular permeability, hemorrhage, and thrombocytopenia distinguish DHF from DF. DHF, where plasma leakage is so severe as to induce shock, is referred to as DSS a severe and potentially fatal outcome of dengue infection. The observation that severe adverse outcomes to dengue virus infection are almost exclusively the result of secondary infection with a heterotypic serotype of DV has lead many to postulate that DV induced tissue damage is immune mediated. Three theories dominate the literature to explain this unique finding (reviewed in Kurane, 2007; Mathew & Rothman, 2008). The first hypothesis proposes that the more severe clinical manifestations of DHF and DSS are due to the presence of preexisting antibodies to a heterologous serotype of dengue virus, which facilitate antibody dependent enhancement of infection (Littaua et al., 1990). The second theory, for which there is much support, suggests that both qualitative and quantitative differences in the T-cell response to a heterologous serotype are responsible for the clinical manifestations of DHV. Cross-reactive T cells in the memory compartment from the primary infection are selectively expanded; these T cells have lower avidity for the secondary strain of virus, are unable to control viral replication and lead to altered IFNg/TNFa (tumor necrosis factor alpha) balance contributing to tissue damage (Beaumier & Rothman, 2009; Mangada & Rothman, 2005). The more recent third hypothesis suggests that DHF/DSS are a consequence of infection with more virulent dengue viral strains, while less virulent strains lead to DF. Mounting evidence for all three hypotheses strongly suggest that multiple factors likely contribute to dengue virus pathogenesis. Epidemiological data in support of a role for heterotypic antibody in exacerbating disease severity upon secondary infection can be found in multiple settings. First, the presence of nonneutralizing antibody to a heterotypic serotype at the time of secondary infection strongly correlates with DHF progression. Second, infants enduring a primary infection

30. Pathology and Pathogenesis of Virus Infections

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with dengue virus who have passively acquired maternal antibody to a heterotypic strain of dengue virus have been documented as developing DHF. Furthermore, in vitro data has demonstrated ADE of DV in infected cultures. More convincingly, the recent development of an animal model of DHF supports a role for preexisting antibody in DV pathogenesis (Shresta et al., 2006; Zellweger et al., 2010). The study revealed that antibody can be sufficient to turn a nonlethal disease into a lethal one. Interestingly, it was found that even neutralizing antibody can enhance pathogenesis if present at subneutralizing concentrations (Fig. 1). While previous studies have centered on in vitro models of ADE, the Shresta model is the first to demonstrate an in vivo pathogenic role for antibody in DV pathogenesis.

VIRUS-INDUCED AUTOIMMUNITY

Autoreactive T cells are normally eliminated by clonal deletion in the thymus. However, T cells with minimal affinity for their cognate self-peptide can escape deletion. Additionally, not all self-antigens are available for display during this critical phase of the developing TCR repertoire. Furthermore, the inherent degeneracy of the TCR means that small sequence variation between self and non-self-peptide antigen may lead to the recognition of autoantigen as foreign by an otherwise normal TCR. Activation of T cells generally requires presentation of antigen in the context of an activated APC. Viral infection may provide the needed second stimulus for activation of autoreactive T cells. The process by which viruses may induce or enhance autoimmunity is hypothesized to occur by a number of mechanisms (reviewed in Filippi & von Herrath, 2008; Olson et al., 2001; von Herrath & Oldstone, 1996). One model of virusinduced autoimmune disease proposes that autoimmunity initiates when previously sequestered tissue antigens become exposed in an inflammatory environment. A second model promotes bystander activation where autoreactive T cells are activated by the cytokine milieu released during a normal adaptive immune response to viral invasion. A third model suggests that autoimmunity may be initiated by the release of sequestered tissue antigens as a result of virus-induced cytotoxicity, thus leading to the activation of autoreactive T cells. A fourth model supports the idea that induction of autoimmunity can be attributed to molecular mimicry, where viral encoded antigen bearing a striking sequence similarity to a host cell peptide, leads to host tissue destruction by an antiviral reactive (not autoreactive) T-cell response. FIGURE 1 Model of antibody-dependent enhancement of viral infection. In the absence of antibody, receptors on the surface of the virion attach to the cell via interactions with the cognate receptor on the surface of the cell, followed by adsorption of the virus into the host cell (A). In the presence of sufficient concentration of neutralizing antibody, viral adsorption or attachment of the virus to the cell is blocked (B). The presence of subneutralizing (either heterotypic or insufficient titer of neutralizing) preexisting antibody may enhance viral attachment or adsorption to the surface of Fc-receptor bearing cells. Alternatively, the simultaneous engagement Fc receptors on the surface of an infected cell may enhance the ability of the virus to replicate in Fc receptor bearing cells downstream of attachment and adsorption or alter the function of the infected APC (C).

IMPLICATIONS OF VIRUS INDUCED IMMUNOPATHOLOGY IN ANTIVIRAL VACCINATION STRATEGIES

In light of recent advancements in our understanding of virusinduced pathology, it is clear that each virus poses a different set of challenges; a protective immune response for one virus can prove to be pathological in another. Recent clinical trials for several different vaccines, where vaccinated groups had elevated risk for infection and or disease than their naïve cohorts, have highlighted the necessity for continued basic science research into the precise mechanisms of virus-induced immunopathology. One example of this can be found in the vastly different outcomes achieved with different strategies in RSV vaccination. Whereas vaccination of children with live attenuated virus provided protection, 69% of infants from 6 to 23 months in age vaccinated with formalin-inactivated virus suffered from acute febrile pneumonia (requiring hospitalization, as compared to 9% of unvaccinated children the same age) upon subsequent natural infection with RSV

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(Kapikian et al., 1969). Mouse models showed that adoptive transfer of CD4 T cells from mice experiencing an immunopathological response could transfer disease; depletion studies further confirmed these results. These findings suggest that CD4 T cells are acting as effectors, and not helpers, in the context of RSV pathology. This is in contrast to recent studies of hantavirus infection of its natural host, the deer mouse, where elevated levels of CD41 regulatory T cells were found (Schountz et al., 2007). Deer mice do not develop an HPSlike response, despite being potent reservoirs for viral replication; this has lead to much interest into whether the elevated Treg response observed in the natural host may be mediating protection against pathology by subduing the effector T-cell response. The observation that Tregs may alleviate downstream pathology due to an overly activated immune response opens up a new paradigm in the development of Treg therapeutics to treat virus-induced pathology. Additionally, while the development of antiviral antibody in the vaccinated host has long been a goal of vaccine design, studies in dengue virus pathogenesis, induced by the presence of heterotypic antibody, caution against generalized assumptions on the correlates of protection. Custom vaccine design tailored to the virus in question and the pathology induced should provide protection against either infection or pathology through the induction of the most effective arm of the immune response to a particular pathogen.

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EVASION AND SUPPRESSION OF THE ANTIMICROBIAL HOST RESPONSE

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31 Viral Immune Evasion LILA FARRINGTON, GABRIELA O’NEILL, AND ANN B. HILL

INTRODUCTION

and chemokines, which in turn recruit natural killer (NK) cells. Later, through highly sensitive, specific targeting, T cells and antibodies provide potent antiviral defenses. All of these defenses are targeted by different viruses in diverse and often fascinating ways. We can assume that different viruses’ replication strategies determine the host defenses that they most need to inactivate (e.g., acute viruses may not need to avoid CD8 T cells), although the data are as yet too incomplete to safely make generalizations. It is beyond the scope of this chapter, and perhaps not very helpful, to attempt a comprehensive list of mechanisms. Instead, we shall provide illustrative examples, and highlight some interesting biology that has been revealed by the study of viral immune evasion.

All living organisms are selected based on their ability to propagate their genomes. Propagation of genomes necessitates a concentration of resources that can be attractive to other organisms seeking to propagate their own genomes. All successful life forms need to be able to protect their resources from such invaders. Host defense mechanisms are probably as old as life itself; over time, increasingly, intricate mechanisms have evolved, especially in complex multicellular organisms such as vertebrates. These mechanisms, which we know as innate and adaptive immunity, are the subject of this book. Obligate parasites, such as viruses, must be able to propagate their genomes in the presence of host immunity, and so, have evolved countermeasures to facilitate their survival in this environment. Most pathogens undergo many genome replications for each one that is undergone by their host, and the evolution of successful countermeasures— immune evasion—is inevitable. Stephen Hedrick has discussed the coevolution of the immune system and parasites as an example of the Red Queen hypothesis: running as fast as they can, the immune system and its attendant parasites end up (in terms of the host–parasite balance) exactly where they started. “Incremental immune evolution surely confers a selective advantage, but such an advantage is quickly countered by the more facile evolution of thousands of parasitic agents” (Hedrick, 2004). In this view, the parasites have the upper hand, and the evolutionarily selected outcome is the one that allows propagation of the genomes of both the parasite and its obligate food source, the host. The study of viral immune evasion is the study, in a snapshot of evolutionary time, of this evolutionary race. Most viruses spread from one host to the next via secreted bodily fluids; to gain access to their target cells for replication they need to cross epithelial barriers, which can be considered the first line of host defense. When the virus reaches and enters the cell in which it will replicate, another series of hostile events ensues. Host cell apoptosis is triggered by cell stress signals responding to the virus’ attempt to co-opt cellular machinery. Pattern recognition receptors (PRRs) recognize viral components and elicit cytokines

APOPTOSIS

Apoptosis—programmed cell death—is critical for the success of complex organisms. Cells that are no longer needed and cells that have begun to malfunction are programmed to die in an orderly fashion and are promptly removed by phagocytes. Intracellular sensors recognize aberrant function and trigger apoptosis. Viruses depend upon the working transcriptional and translational machinery of their host cells to replicate; in co-opting this machinery, they trigger cellular stress responses that should result in apoptosis. In addition, several antiviral immune mechanisms trigger the apoptotic pathway as a way of clearing out infected cells and limiting viral propagation. But the deftly evolving virus has, of course, engineered an answer to these attacks in the form of virally encoded antiapoptotic proteins that function to elude or delay cell death (Fig. 1). Apoptosis is the regulated destruction of cellular machinery. Cell death follows the disassembly of the cytoskeleton, the loss of mitochondrial membrane integrity, and the fragmentation of the genome. These events are predominantly orchestrated by a family of 11 proteins known as Caspases (cysteine-dependent aspartate-specific proteases), which are involved in the initiation, execution, and regulatory phases of the apoptotic pathway. Vertebrates have evolved two main mechanisms to initiate apoptosis—from outside the cell (extrinsic) and from inside the cell (intrinsic). The intrinsic pathway begins when the cell senses stresses such as viral infection and proteins (e.g., p53) activate BH3-domain-only members of the Bcl-2

Lila Farrington, Gabriela O’Neill, and Ann B. Hill, Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239.

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FIGURE 1 The apoptotic pathway and its inhibition by viruses. The intrinsic cell death pathway is initiated when internal sensors (for example p53) activate BH3-domain only members of the Bcl-2 family. These sensors are inactivated by viral p53 inhibitors like adenovirus E1B-55K. BH3-domainonly proteins mediate assembly of pro-apoptotic Bcl-2 family members (for example Bid, Bax, Bok) into pores in the outer mitochondrial membrane, actions that are antagonized by Bcl-2 orthologs produced by viruses like adenovirus, KSHV, EBV, and HCMV. Cytochrome c and other factors are released into the cytoplasm, promoting formation of a complex containing Apaf-I and pro-caspase 9 called the apoptosome. Caspase-9 is activated, triggering executioner caspases 2, 3, 6, and 7. vIAPs (encoded by baculoviruses and African swine fever virus) as well as many non-BIR containing viral proteins such as p35 (baculoviruses) and the serpin CrmA (poxviruses) inhibit caspases. The extrinsic cell death pathway is initiated by TNF-family death receptors (e.g., FAS, TRAIL, TNFR) binding to their cognate ligand and facilitating the binding of adaptor proteins to pro-caspase-8 and/ or 10 to form the DISC. Inactive caspases (pro-caspases) are cleaved to their active forms, triggering caspase-3 and initiating the mitochondrial cell death pathway via activation of Bid. Herpesviruses and orthopox viruses produce soluble decoy receptors to block death-receptor signaling. The adenovirus E3 protein targets TNF-family receptors for degradation. The HCMV protein vICA inhibits pro-caspase 8 activation and vFLIPs prevent the formation of the DISC complex.

family like Bid. These proteins translocate to the mitochondria where they form hetero-oligomeric pores with other Bcl-2 family members Bax and Bak in the outer mitochondrial membrane. As a result, Cytochrome c is released. Cytochrome c binds to Apaf-1 (apoptosis protease-activating factor) to induce the formation of the apoptosome, an oligomeric assembly platform. Caspase-9 is recruited to the apoptosome and is activated. The active form of Caspase 9 then targets executioner caspases 3 and 7, which, once active, perform key cleavage reactions that lead to the cell’s demise. Apoptosis via the extrinsic pathway is initiated through ligation of the tumor necrosis factor (TNF) superfamily of receptors by death receptor ligands. These ligands are up regulated on the surface of immune effector cells like NK cells and dendritic cells in response to the detection of viral products by pattern recognition receptors (PRRs). When members of this superfamily (e.g., TNF, Fas, and TRAIL) are bound by their respective ligands, adaptor proteins and initiator caspases are recruited to form the death-inducing signaling complex (DISC). Initiator Caspases 8 and 10 directly convert pro-caspase-3 to its active form, which initiates cell death. Caspase 8 can also cleave Bid, leading to the induction of the mitochondrial

cell death pathway described above. Granzymes, delivered by Cytolytic T cells and NK cells, can activate caspase 3 directly or indirectly through either of the pathways described. Viruses target four main steps in the cell death pathway: the expression of internal sensors, the release of cytochrome-c by Bcl-2 family members, caspase activation, and death receptor signaling (Benedict et al., 2002). P53 is an example of an internal sensor responsible for detecting cellular stresses and up regulating death receptors and proapoptotic Bcl-2 family members. Many viruses inactivate this protein in an attempt to thwart their host’s self-destruct mechanism. SV40 large T-antigen complexes with p53 and renders it inactive (Lane & Crawford, 1979). The papillomavirus E6 protein and adenovirus E1B-55K promote degradation of p53 via ubiquitination (Steegenga et al., 1998). Hepatitis B virus encodes the pX protein, which binds to p53 and inhibits p53 mediated transcriptional activation (Wang et al., 1995). As a way to block the release of cytochrome c from the mitochondria, many viruses encode proteins that mimic the antiapoptotic regulatory protein Bcl-2. For example, adenovirus protein E1B-19K is similar in both sequence and function

31. Viral Immune Evasion

to Bcl-2 and blocks a conformational change in Bax required for Bax-Bak oligomerization at the mitochondrial membrane (White et al., 1991). Human herpesviruses also use Bcl-2 orthologs. Epstein-Barr virus (EBV) encodes BHRF-1 and BALF-1, while Kaposi’s sarcoma herpesvirus (KSHV) expresses KSbcl-2, all of which mimic cellular Bcl-2 (White et al., 1991). Human cytomegalovirus (HCMV) employs another strategy. UL37, which encodes the protein vMIA, does not share sequence homology with Bcl-2 but does reside in the mitochondria where it inhibits Fas-mediated apoptosis (Goldmacher et al., 1999). Other strategies include modulating Bcl-2 family members at the transcriptional or posttranslational level. The human T-cell leukemia virus type-1 (HTLV-1) tax protein promotes transcription of the antiapoptotic Bcl-xl while repressing transcription of Bax (Goldmacher et al., 1999). HIV-1 Nef suppresses apoptosis of T cells by phosphorylating and thereby attenuating the activity of Bad, a proapoptotic protein (Wolf et al., 2001). HSV-1 encodes US3, which also targets Bad via posttranslational modifications that block its cleavage and subsequent activation (Munger & Roizman, 2001). The third step in the apoptotic pathway that is routinely targeted by viruses is the activation of caspases. Caspases are inhibited by a family of cellular proteins known as inhibitor of apoptosis proteins (IAPs). Eight cellular IAPs have been identified that regulate both initiator and executioner caspases. They are characterized by their inclusion of zincfinger binding motifs called baculoviral IAP repeat (BIR) domains that bind caspases directly. IAP antagonists also exist to modulate the activity of antiapoptotic IAPs. Viral IAPs are expressed by baculoviruses, entomopoxviruses, iridoviruses, and African swine fever virus. Rather than binding to caspases, vIAPs are thought to act as decoys for the IAP antagonists (Munger & Roizman, 2001). Non-vIAP proteins are also encoded by viruses to inhibit caspases. Crm-A, which is derived from cowpox and is present in most poxviruses, is a member of the Serpin superfamily of serine protease inhibitors and inhibits several caspases by binding to their active sites. p35 is another caspase inhibitor derived from baculovirus that binds to directly to caspase active sites (Best, 2008). Lastly, viruses regulate death receptor signaling by a number of different methods, including the neutralization of TNF by soluble decoy receptors. An example is the secreted TNFR2 ortholog expressed by the rabbit poxvirus, myxomatosis virus (MYXV) (Smith et al., 1990). Interestingly, the TNF decoy receptors expressed by vaccinia, which is not highly pathogenic, are mutated (Benedict et al., 2002). A different strategy is employed by HSV-1, which actually uses its envelope glycoprotein D to access the lymphatic compartment via a TNFR family member, herpesvirus entry mediator (HVEM), and induce the apoptosis of T cells (Raftery et al., 2001). The adenovirus E3 region encodes proteins that clear the cell-surface of death receptors, effectively desensitizing infected cells to death-receptor-mediated killing (Benedict et al., 2002). Several viruses block death receptor signaling at the level of DISC assembly. Viral FLICE (caspase-8) inhibitory proteins (vFLIPs) are homologous to cellular FLIPs. Both are recruited to the DISC and inhibit proteolytic activation of caspase-8 (Benedict et al., 2002). The HCMV UL36 gene product vICA shows no sequence similarity to the cFLIPS, but is also able to bind and block activation of caspase-8 (Skaletskaya et al., 2001). Additionally, vFLIPs interact with adaptor proteins that regulate the activation of transcription factors important for inhibiting apoptosis. Viral interference with apoptosis is probably the most common form of immune evasion. It may be that all viruses need to prevent or delay intrinsic apoptosis in order to maintain cell viability long enough to reproduce.

INNATE CYTOKINES

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Membrane bound and cytosolic pattern recognition receptors (PRRs) in host cells recognize viral molecules and trigger the release of antiviral cytokines and chemokines to attract cellular host defense. Interferons (IFNs) and TNF are particularly important and rapidly set off a series of signaling events that create a hostile environment for viral infection and replication. This is a powerful system and viruses must have many measures for circumventing its inhibitory effects. Often, the specific interactions of viral proteins and antiviral signaling pathways are so important that they dictate tissue and species tropism. IFNs are grouped into three classes (I, II, and III) according to amino acid sequence, chromosomal location, and receptor specificity. Type I IFN production (IFNs a and b) is stimulated by cellular detection of pathogen-associated molecular patterns (PAMPs), most notably dsRNA, by Toll-like receptors (TLRs) and the molecules RIG-I and MDA-5. Type II IFN, IFN-g, is produced predominantly by T cells and NK cells upon activation. Once expressed, IFNs bind to ubiquitously expressed cellular receptors and set off signaling events, the details of which depend upon the type of IFN involved. In general, signaling begins with the phosphorylation of janus family kinases (JAKs) and tyrosine family kinases (TYKs). These molecules then promote the phosphorylation and activation of signal transducer and activator of transcription family proteins (STATs). Phosphorylated STATs translocate to the nucleus and induce the expression of interferon-stimulated genes (ISGs), which have effects ranging from degradation of viral and host RNA transcripts, inhibition of cellular and viral mRNA translation, and interference with virus assembly and trafficking. Each class of IFN is necessary for antiviral defense; experiments with knockout mice have shown that they are not functionally redundant (McFadden et al., 2009). TNF signaling can lead to caspase-dependent cell death or the induction of an antiviral state similar to that induced by IFNs. Binding of TNF to its cognate receptors TNFR1 and 2 leads to receptor phosphorylation and recruitment of signal transduction molecules that can activate the proinflammatory transcription factor NF-kB. TNF signaling can also alter the expression levels of cell surface receptors used by viruses for entry. Depending on the direction of regulation, this effect can either increase or decrease the efficiency of virus infection (McFadden et al., 2009). Five ways through which viruses hijack the IFN response have been described (Randall & Goodbourn, 2008). These include (i) globally inhibiting host-cell transcription and translation, (ii) minimizing IFN induction by limiting viral detection or by targeting IFN-induction cascades, (iii) inhibiting IFN signaling, (iv) blocking the action of IFNinduced enzymes, and (v) having a replication strategy that is unaffected by the actions of IFNs. Viruses that engage in the first tactic include Bunyamwera virus (BUNV), which produces the NSs protein that blocks activity of RNA pol II, thereby inhibiting cellular mRNA. Without this protein, BUNV grows to high titers only in cells that do not produce IFN (Randall & Goodbourn, 2008). Foot and mouth disease virus (FMDV) produces L proteinase, which abrogates host-cell protein synthesis. A recombinant virus lacking this protein induces elevated levels of IFN-a/b upon infection (Chinsangaram et al., 1999). While this strategy prevents production of antiviral IFNs, it also has the effect of limiting the replication of the virus. Many viruses specifically minimize IFN production. One way that this is done is by limiting the detection of PAMPs such as dsRNA. Negative-strand RNA viruses encapsulate both genomic and antigenomic RNA, paramyxoviruses cap

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the 5 end of their mRNA to avoid recognition by RIG-I, influenza viruses steal caps from their host for this purpose, picornaviruses attach a protein to the 5 end of their RNA, and retroviruses integrate their genomes into that of their host and use cellular machinery for replication thereafter. Many other viruses produce dsRNA-binding proteins that sequester and protect dsRNA from detection (Randall & Goodbourn, 2008). Additionally, viral proteins have been identified that block the activities of MDA5 and RIG-I. If IFNs are produced, many viruses still have a means of interrupting their downstream effects. IFNs themselves can be bound and neutralized, as in the case of ectromelia virus, cowpox, and camelpox, which encode proteins that bind IFN-g. Most orthopoxviruses encode soluble IFN-a/b binding proteins and Yaba-like disease virus expresses a related protein that is able to inhibit both type I and type III IFNs. By inhibiting IFN signaling components common to type I, II, and III IFN signaling cascades, viruses can block the induction of cellular antiviral enzymes and stop the up regulation of MHC class I, rendering invaded cells poor targets for CTL. There are examples of viral proteins that interrupt all aspects of these pathways, from receptor signaling to the activity of IFN-induced transcription factors. The T antigen of murine polyomavirus binds to and inactivates JAK1. Most herpesviruses, as well as human papillomavirus 18 (HPV18) and measles virus (MeV), down regulate IFN receptors. HCMV’s immediate early 1 protein sequesters STAT1 and STAT2. MeV, vaccinia, and adenovirus are known to interfere with STAT activation and/or alter STAT phosphorylation. HPV16, HSV, and hepatitis C virus block the activity of IFN-induced transcription factors (Randall & Goodbourn, 2008). Many more examples could be listed. Additionally, there are many examples of viral proteins that inhibit or promote degradation of IFN-induced antiviral enzymes (Randall & Goodbourn, 2008). dsRNA-dependent protein kinase R (PKR) is activated by dsRNA produced during viral replication and targets the translation initiation factor eIF2a. Additionally, PKR has a role in the induction of apoptosis and cell-cycle arrest. dsRNA is also a cofactor for 25-oligoadenylate synthetase (OAS), which activates RNase L, leading to the degradation of cellular and viral RNAs. Pox, herpes, influenza, and reoviruses sequester dsRNA to minimize activation of PKR and OAS. Other viruses encode proteins and highly structured RNA molecules that bind to and inactivate these enzymes. Furthermore, viral molecules have been identified that serve as competitive inhibitors, promote degradation of, or up regulate proteins that inhibit PKR and OAS (Langland et al., 2006). HIV as well as other lentiviruses target APOBEC3, a protein shown to restrict viral replication by mutation of template nucleotides and inhibition of reverse transcription. Lentiviral protein Vif complexes with cellular E3 to target APOBEC3 proteins for ubiquitination and degradation (Malim, 2006). This litany of targets and mechanisms of evasion bears witness to type I IFN’s potent ability to interrupt viral replication.

INNATE IMMUNE EVASION AS A DETERMINANT OF HOST RANGE RESTRICTION

Most viruses are limited in the cell types and species that they can infect, a phenomenon known as viral tropism or host range restriction. Immune evasion of apoptosis and cytokines can determine host cell tropism (Werden et al., 2008). For example, the rabbit orthopox virus, myxomatosis virus (MYXV), infects the skin and causes disseminated disease by infecting lymphocytes and spreading throughout the body. At least four

MYXV genes have been identified which inhibit different stages of apoptosis, without which lymphocytes undergo rapid apoptosis upon infection. Viruses in which these genes have been deleted can replicate in fibroblasts but not lymphocytes in vitro, and are markedly attenuated in vivo. Evasion of apoptosis can also determine tropism at the species level. Cytomegaloviruses are highly species specific and can only replicate in cells of their native host or closely related species. Murine CMV (MCMV) is unable to replicate in human fibroblasts because it triggers the intrinsic pathway of apoptosis. However, providing MCMV with the human CMV gene that inhibits mitochondrial apoptosis enables MCMV to replicate in human cells, thus breaking the species barrier (Jurak & Brune, 2006). Cytokine evasion can also affect the viral host range (McFadden et al., 2009). MYXV is a highly species-specific virus, which, as previously stated, can only infect rabbits. The species barrier is determined by the ability to prevent a type I interferon response (Wang et al., 2004). In mouse but not rabbit cells, MYXV activates erk and IRF3, leading to type I IFN synthesis. Consequently, MYXV cannot establish infection in wild-type mice; however, it is lethal in mice deficient in STAT-1, which is necessary for type I IFN signaling. Similarly, infection of mice with a number of human viruses, including Edmonston measles virus, influenza A, poliovirus, and West Nile virus, results in mild, tissue-specific infections unless the IFN response is disabled, in which case highly disseminated systemic infections develop. The fact that immune evasion genes can determine species specificity has opened intriguing possibilities in the field of oncolytic virus therapy (Stanford & McFadden, 2007). Many cancer cells are deficient in type I IFN signaling, and, in consequence, human tumors such as glioblastoma and pancreatic cancer are susceptible to MYXV infection. In animal models of cancer, MYXV infected and cleared tumors, but did not infect normal tissue and caused little inflammation. While it remains to be seen whether MYXV and other oncolytic viruses can be developed into useful therapeutics in humans, this field is an unexpected, exciting spin off from the study of viral immune evasion.

EVADING NATURAL KILLER CELLS

Natural killer (NK) cells are an essential component of the first line of defense against viruses. NK cells have two antiviral mechanisms, cytotoxicity and cytokine secretion, and they are the main source of INF-g early in infection. NK cell activation occurs when the balance of signaling by stimulatory and inhibitory receptors is tipped to favor activation. Some NK receptors (both stimulatory and inhibitory) bind to polymorphic regions on MHC class I molecules; these receptors are highly polymorphic and are rapidly evolving. In fact, different gene families serve this function for mammals (KIRs) and mice (Ly49 genes). The pressure during rapid NK receptor evolution is unclear, but it may be due to the role of NK cells in maintaining tolerance during pregnancy, as well as the rapid coevolution of viral immune evasion mechanisms. NK evasion mechanisms include direct effects on NK cells by the virus, interference with NK cell cytokine/chemokine pathways, expression of viral MHC class I-homologs, modulation of MHC class I molecules by viral proteins, and inhibition of NK cell activation, all of which have one common goal: to oppose NK function in order to establish a productive infection (Fig. 2). The most straightforward way for a virus to evade the NK cell response is to inhibit NK cells through infection or direct contact (Orange et al., 2002). NK cells cultured in vitro are susceptible to infection with HIV-1 resulting in decreased viability of NK cells. Direct inhibition but not


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FIGURE 2 Mechanisms of viral inhibition of NK cells. Viruses have developed a variety of mechanisms to oppose NK function including: (i) direct effects on NK cells by the virus, (ii) expression of viral MHC class I-homologs, (iii) modulation of MHC class I molecules by viral proteins, (iv) inhibition of NK cell activation, and (v) interference with NK cell cytokine/chemokine pathways. HCV envelope protein E2 binds and cross-links NK cell surface protein CD81 resulting in inhibition of NK cell cytotoxicity, proliferation, and IFN-g production. HCMV encodes UL18, an MHC class I homolog, which is able to inhibit NK cells via inhibitory receptor ILT2. HIV selectively down modulates HLA-A and HLA-B but not HLA-C or HLA-E via viral protein nef. MCMV prevents NK cell stimulation by decreasing cell surfaces levels of RAE-1, MULT1, and H60, NKG2D receptor ligands in mice, via viral proteins m152, m145 and m155, respectively. HCMV also down modulates MICB and MICA, human ligands for NKG2D, via UL16, UL142, and miRNA miR-UL112. Finally, KSHV encodes for a broad chemokine antagonist, vMIP-II, which binds CC and CXC chemokine receptors and prevents immune cell chemotaxis.

infection of NK cells is best illustrated with hepatitis C virus (HCV). HCV envelope protein E2 binds CD81 on NK cells and inhibits NK cell cytotoxicity, proliferation, and IFN-g production. Another way viruses have managed to undermine NK cell responses is by encoding viral homologs of cytokines or chemokines that can then antagonize the interactions with their cognate receptors (Orange et al., 2002). KSHV protein vMIP-II is a broad chemokine antagonist capable of binding CC and CXC chemokine receptors (Kledal et al., 1997), thus preventing chemotaxis of important immune cells. Both HCMV and EBV encode viral homologs of IL-10 (interleukin-10), which inhibits the activity of NK cells as well as the production of proinflammatory cytokines. Many viruses down regulate MHCI, which engages inhibitory receptors on NK cells (Jonjic et al., 2008; Orange et al., 2002). Some viruses encode MHCI homologs, which have been presumed to engage NK inhibitory receptors to prevent NK activation. HCMV encodes UL18, an MHC class I homolog, which can inhibit NK cells via inhibitory receptor ILT2 (immunoglobulin-like transcript 2; also known as LIR1 and CD85j). ILT2 is expressed on monocytes, B cells, and some CD8 T cells, but only on a subset of NK cells. Interestingly, UL18 has been found to activate a subset of NK cells that lack ILT2, making the role of UL18 immune evasion quite controversial. MCMV also encodes an MHCI

homolog, m144, whose receptor has not yet been identified and whose function is also controversial. Many viruses that down regulate MHCI selectively spare MHCI molecules that engage inhibitory NK receptors (Orange et al., 2002). For example, human MHCI molecules, HLA-C and HLA-E interact with NK cell inhibitory receptors, KIR2DL1 and CD94:NKG2A, respectively, and in typical fashion viruses have “learned” to exploit this. HCMV proteins US2 and US11 cause selective degradation of HLA molecules. HLA-C and HLA-E are resistant to the effects of US2 and US11 and are still able to interact with their respective inhibitory receptors. Lentiviruses such as HIV and SIV are able to selectively down modulate HLA-A and HLA-B but not HLA-C or HLA-E via viral protein Nef. Similarly, KSHV encodes a protein, K5, which down regulates only HLA-A and HLA-B. Viral inhibition of the expression of activating NK receptors has produced the strongest in vivo phenotypes (Jonjic et al., 2008). Viral infection triggers the expression of stress-induced ligands in both human and mouse, although, interestingly, quite different molecules are induced in each species: RAE-1, MULT-1, and H-60 in mice, and MICA, MICB, and ULBPs in humans. These diverse ligands all engage NKG2D, a stimulatory NK receptor expressed on all NK cells. MCMV encodes three genes, m152, m145, and m155, that down regulate RAE-1, MULT-1, and H60,

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respectively. Deletion of any one of these three genes results in the rescue of their respective NKG2D ligands at the cell surface, resulting in NK cell activation and reduced virus titers in vivo. HCMV encodes different genes, UL16 and UL142, to down regulate the human NKG2D ligands ULBP 1, ULBP2, MICA, and MICB. In fact, UL16 led to the discovery of the ULBP (UL16 binding protein) family. More recently, herpesviruses have been discovered to encode microRNAs that target MICB mRNA for destruction. Since MICB is only synthesized in response to virus infection in most cell types, this is an effective evasion mechanism. The fact that HCMV, KSHV, and EBV each encode unrelated microRNAs that perform this function provides further evidence for its importance (Nachmani et al., 2009).

VIRAL GENES THAT TRIGGER NATURAL KILLER ACTIVATION

Investigation of a difference in susceptibility to MCMV between mouse strains yielded the surprising finding that MCMV encodes a gene that specifically activates NK cells (Pyzik et al., 2008). MCMV m157 encodes an MHCI-like glycoprotein that is the only known ligand for the NK cell activating receptor, Ly49H. This receptor is expressed in approximately 50% of NK cells in C57BL/6 mice. M157 potently activates Ly49H1 NK cells, resulting in efficient control of MCMV infection. Conversely, BALB/c mice lack the ly49h gene, and develop 1 to 2 log higher titers during acute infection. Evolutionarily, it does not seem advantageous for MCMV to activate NK cells. However, m157 has also been

found to interact with the NK cell inhibitory receptor Ly49I, which is expressed on a subset of NK cells in 129/J mice, and it has been speculated that m157 evolved to engage this inhibitory receptor. Most wild isolates of MCMV do not encode an m157 that binds to Ly49H. Furthermore, when MCMV is placed under intense selective pressure by NK cells (by repeated passage in C57BL/6 mice, or by infection of B6-SCID mice, which have NK cells but no adaptive immunity), mutant viruses that lack functional m157 rapidly emerge. Thus, it has been tentatively concluded that m157 activation of Ly49H is an experimental artifact of laboratory mouse and viral strains. Intriguingly, however, a second occurrence of an MCMV gene involved in the activation of NK cells has been recently discovered (Pyzik et al., 2008). Similar to C57BL/6, MA/My mice display an NK-cell-dependent resistance to MCMV. MA/My mice, however, lack the ly49h gene. Instead, resistance is due to a different gene in the ly49 complex, ly49p, which encodes the activating NK receptor, Ly49P. Ly49P activation requires the MHCI molecule H-2Dk and the MCMV gene m4, plus some other as yet unidentified viral factor. It may be that this specific activation of NK cells by MCMV is another laboratory “accident.” It is also possible that a more complex game is being played here by the virus than we have yet appreciated.

CD8 T-CELL IMMUNE EVASION

Viral genes that interfere with the MHCI pathway of antigen presentation to CD8 T cells were the first viral immune evasion genes to be described (Fig. 3). Given the important

FIGURE 3 Mechanisms of viral inhibition of the MHC class I antigen presentation pathway. Proteins in the cytosol are degraded by the proteasome, transported into the ER by TAP, and loaded onto MHCI, which then traffics to the cell surface as indicated by dashed arrows. Many viruses inhibit the TAP transporter by a variety of methods, from both cytosolic and lumenal sides. HCMV US3, Ad5E3/19K, CPXV203, and MCMVm152 retain MHCI in pre-Golgi compartments. HCMV US2 and US11 target MHCI for retrotranslocation and proteasomal degradation. MCMV m6 directs MHCI to the lysosome for degradation. HIV nef reduces MHCI at the plasma membrane by a complex set of reactions. KSHV and MHV-68 K3 and K5 proteins ubiquitinate the cytosolic tail of MHCI and target it to the lysosome. This list is not exhaustive: MHCI synthesis is also targeted, and other new mechanisms are being discovered all the time.


role of CD8 T cells in antiviral immunity, it was assumed that these genes would play a critical role for viral fitness, particularly for viruses that establish chronic infection and need to survive in the face of a fully primed immune response. However, experimental results have not routinely confirmed this assumption. MHCI immune evasion genes appear to have been strongly favored evolutionarily; in particular, every member of the herpesvirus family encodes MHCI evasion genes, many with exquisite specificity for this pathway, and with such disparate mechanisms that they must have evolved independently (Hansen & Bouvier, 2009). The HSV protein ICP47 blocks the transport by TAP of peptides from the cytosol to the ER (endoplasmic reticulum) and profoundly inhibits peptide loading and antigen presentation. Cytomegaloviruses are highly species specific, and each encodes multiple genes that interfere with the MHC class I pathway, although there is no homology between the genes encoded by human and mouse CMVs. The CMV genes are all glycoproteins that attack MHCI or TAP from the lumenal side, causing its degradation and/or intracellular retention (Pinto & Hill 2005; Powers et al., 2008). The gamma 2 herpesviruses KSHV, RRV, and MHV68 encode MARCH family ubiquitin ligases that target MHCI for degradation from the plasma or ER membranes (Stevenson, 2004). In its lytic replication cycle, EBV also inhibits TAPI to down regulate MHCI (Croft et al., 2009). During latency, expression of EBNA1 is essential to maintain the EBV genome. EBNA1 contains an amino acid sequence that renders it resistant to antigen processing; a similar defect occurs in the analogous protein during MHV-68 infection (Bennett et al., 2005). MHC I evasion has also been described in nonherpesviruses. For example, type 5 adenoviruses encode the E3/19K protein, which retains MHCI in the ER (Paabo et al., 1987). The lentivirus HIV and SIV nef protein degrades MHCI from the cell surface (Collins et al., 1998). The orthopox virus cowpox has two genes to impair MHCI expression; one retains MHCI in the ER while another inhibits TAP (Alzhanova et al., 2009). Finally, of course, viruses can escape specific CD8 T-cell recognition by mutation of the recognized epitope; this occurs mostly with RNA viruses such as HIV and HCV, because of their error-prone genome replication. Some of these genes have been shown to play a role in vivo. There is ample evidence for selection of epitope escape variants from CTL recognition for HIV and SIV infection (Goulder & Watkins, 2004). Similarly, when the ability of SIV nef to down regulate MHCI was abolished by a single codon mutation, there was such strong selective pressure for this function that the mutation had reverted to wild type within 2 weeks of infection (Munch et al., 2001). The gammaherpesvirus MHV-68 lacking the MHCI down regulating gene K3 is impaired in the transient mononucleosis-like expansion of latently infected B cells, although acute infection is unimpaired and long-term latency is still established (Stevenson et al., 2000). Resistance to antigen processing by the MHV68 gene needed for latent genome maintenance seems to be necessary for successful gammaherpesvirus latency (Bennett et al., 2005). Murine cytomegalovirus lacking MHCI immune evasion has lower salivary gland titers during chronic infection, suggesting that these genes may facilitate transmission to the next host (Lu et al., 2006). These findings probably indicate a real selective advantage for the MHCI immune evasion genes and could explain their evolutionary conservation, although the observed impacts seem paltry when compared to the dramatic phenotypes for apoptosis and cytokine evasion genes described above.

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The real surprise from studying the impact of MHCI evasion in vivo is the recognition of just how well a virus can do without it. This has been studied most thoroughly in the MCMV model of infection, which is a natural hostpathogen pairing. MCMV has three genes that attack the MHC class I pathway: m4, m6, and m152 (Pinto & Hill, 2005). Wild-type MCMV-infected cells are not lysed by MCMV-specific T cells in vitro, whereas cells infected with a virus lacking these three genes can be killed. These genes have also been shown to impair CD8 T-cell lysis in vivo; in a lethal irradiation/bone marrow transplantation model, adoptively transferred CD8 T cells control immune evasiondeficient virus much more efficiently than wild-type virus (Holtappels et al., 2004). Thus, the genes do indeed function in vivo as predicted by the in vitro studies. However, studies in immunodeficient animals cannot address the evolutionary purpose of these genes, and the results of experiments in normal hosts have been less impressive. The first surprising result was that these three genes have absolutely no impact on the priming of the CD8 T-cell response (Munks et al., 2007). The response to MCMV in C57BL/6 mice is very broad; 26 epitopes have been identified. CD8 T cells specific for 17 of these were tested for recognition of virus-infected cells in vitro: all lysed cells were infected with virus lacking m4, m6, and m152, but failed to lyse wild-type virus-infected cells. However, when mice were infected with one or the other virus, the rank ordering of the response to these 26 epitopes—the immunodominance hierarchy—was identical. Similar results were obtained in BALB/c mice. The best explanation for this result is that the CD8 T-cell response to both mutant and wild-type virus is primed by cross-presented antigen rather than by directly infected cells, perhaps because other immune evasion genes that impact dendritic cell function are still able to prevent direct priming. Even if T cells could be primed by cross-priming, it was expected that MHC I evasion genes would have a decisive impact on the ability of T cells to control infection. However, these genes had little or no impact on the titer of virus nor on the resolution of acute infection. Further, they did not prevent the establishment of latency, and (based on the T-cell response) both viruses remained equally active for the life of the animal. These initial findings have been probed further by attempting to mimic natural routes of infection and viral inocula with the same outcome: the immune evasion mutant infected and persisted as robustly as the wild-type virus (Doom & Hill, 2008). As mentioned above, immune evasion genes did impact salivary gland titers, but the impact was modest (about a 1 log reduction); and the immune-evasion-deficient virus was still replicating in the salivary gland weeks later. The lack of impact cannot be explained by T-cell dysfunction because, in contrast to chronic virus infections such as LCMV, CD8 T cells in MCMV-infected mice remain fully functional. Viral evasion of apoptosis and IFN frequently has a dramatic impact on infectious outcome, even to the extent of conferring species specificity. In contrast, these studies of MHCI evasion have provided little explanation for the extraordinary evolutionary imperative for herpesviruses to encode these genes.

EVOLUTIONARY CONSIDERATIONS

The particular immune evasion mechanisms evolved by each virus presumably reflect the host defenses that most impact the ability of that virus to propagate its genome, spreading from one host to the next. MHCI evasion mechanisms are not unique to herpesviruses. However, the ubiquity of these genes amongst herpesviruses, the variety

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of mechanisms that are encoded by unrelated genes, and the fact that some herpesviruses encode multiple MHCI evasion genes implies that this mechanism of immune evasion is particularly important for a critical aspect of the herpesvirus lifestyle. Since lifetime latency or persistence is a hallmark of herpesvirus infection, the automatic conclusion was that these genes would be necessary for robust persistence once the CD8 T-cell response was primed. However, notwithstanding a modest impact in some model systems, it is perfectly clear that these genes are not needed for lifelong latency. Is there another common characteristic of herpesvirus infections for which these genes could provide a critical selective advantage? A striking feature of cytomegalovirus infection is the coexistence of multiple viral strains in the same host, which can result from simultaneous primary infection or from subsequent superinfection (Gorman et al., 2006). Infection with multiple strains also occurs with HSV and EBV, suggesting that competition between herpesvirus strains within an immune host may be the norm. Evolutionary theory tells us that the most intense selective pressure driving changes to genomes comes from competition within a species (intraspecific), rather than between species. If multiple strains of CMV within a host are competing for limited host resources, genes that provide a competitive edge are likely to be strongly selected. Even with the immune evasion genes removed, CD8 T cells may not be able to eradicate herpesvirus infections. However, if CD8 T cells can detect the infected cells, they will have a significant impact on the critical reproductive ratio, the average number of infectious virions produced per infected cell. After several rounds of replication in the presence of CD8 T cells, a virus that could avoid CD8 T-cell recognition would have produced far more progeny than a competing virus that could not, and thus have a decisive competitive advantage. If intraspecific competition drives selection of immune evasion genes, these genes should favor the strain that encodes them and not provide an advantage to the strain infecting a neighboring cell. The MHCI immune evasion genes of herpesviruses, which inhibit antigen presentation, all act in cis; they protect only the cell infected with the virus that encodes them. It is interesting to note that secreted immune evasion molecules, which offer no selective advantage to the individual infected cell, are rare among herpesviruses. In contrast, they are common in poxviruses, which cause only acute infections. Even CD8 T-cell evasion by poxviruses, when it occurs, may not act to the exclusive advantage of the infected cell; monkeypox causes a generalized paralysis of CD4 and CD8 T cells (Hammarlund et al., 2008). It is tempting to speculate that competition between different strains of a poxvirus within an individual host is uncommon.

CONCLUSIONS

Viral evolutionary success requires propagating the viral genome within a host and spreading to new hosts to repeat the process. Long-term evolutionary survival requires a continued supply of susceptible hosts, which are engaged in their own evolutionary survival battles. There are multiple viral strategies for achieving this feat. For example, influenza viruses may exhaust the available human host supply within a season, but utilizing a second (avian) host and mutation of the targets of neutralizing antibodies enables them to reinfect their human hosts the next year. Herpesviruses establish lifelong latent infection and can wait patiently for the next host generation to pass on their genomes. Competition between viral strains within a host will favor virulence, but a strain that kills its host before it can spread does not survive. Strains that allow the host to survive but significantly

impair its biological fitness will also fail over time, as the host species or community fails to compete for resources with fitter individuals. Within these constraints, there can be intense competition between viral strains for access to host resources, a phenomenon that is very evident in RNA virus quasispecies infection. The ability to access host resources requires molecules that enable the virus to enter the host cell and specifically co-opt the host cell resources it needs for replication (enzymes, transcription factors, etc.). Access also requires adaptation to the host environment, such as the pH of different intracellular compartments and bodily fluids, which can be as extreme as gastric acids encountered by enteroviruses. Immune defense mechanisms are part of the host environment for the virus, and they offer varying degrees of impediment to the ability of a virus to propagate its genome and spread to a new host. The facile evolution of viruses compared to their hosts probably means, as Hedrick has argued (Hedrick, 2004), that any immune mechanism can be countered, constrained only by the long-term selective advantage of maintaining a stable host population. Specific deletion of viral immune evasion genes has revealed the extraordinary potency of innate immune responses compared to the modest impact of the sophisticated adaptive immune system. However, all viral immune evasion genes, whether they are absolutely required for host species infection or confer a barely perceptible benefit, have been selected by the same balancing evolutionary pressures: the ability of a virus to propagate its genome within a host, the ability to spread to a new host, and the need to maintain a supply of new hosts.

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32 Growing Old and Immunity to Viruses JANKO NIKOLICH-ŽUGICH AND MARCIA A. BLACKMAN

INTRODUCTION

some of these changes remains incomplete. We will briefly review known and well-described changes in the immune system with aging (the reader is encouraged to supplement this by liberally using the recent review articles of Cambier, 2005; Gruver et al., 2007; Nikolich-Zugich, 2008; Wagner et al., 2004), and will focus upon those whose relevance and reproducibility is firmly established.

Resistance to infections is a major driver of natural selection. Only the successful host, the one that survives infections by environmental pathogens up to and including the reproductive age, will transfer its genes to the next generation. It is often assumed that the coevolution of hosts and pathogens is an arms race, where the host is evolving increasingly potent immune defenses, while pathogens follow in lockstep, by evolving novel mechanisms to evade host immunity. The extremely short reproduction span and rapid mutation rate allows microbial pathogens to evolve for hundreds of thousands of generations over the lifetime of a single longlived host. Despite this apparent advantage to the pathogens, multicellular organisms have found ways to survive against these odds and to evolve in size, complexity, and life span; while the life spans of most evolutionary ancient animals is measured in hours to weeks, many more recently evolved vertebrates, including humans, enjoy life spans of several decades. Surviving after the initial reproductive age poses interesting questions for infectiologists, evolutionary biologists, and biologists of aging—how did the success of immunity carry over to protect vertebrates into senescence, and why does immunity wane at these late stages of life? Although this chapter will not answer such questions, it is useful for the reader to bear them in mind while considering interactions of microbial pathogens with aging hosts. This chapter deals with immunity to viruses in old age. Biology of prominent viral pathogens mentioned in this chapter will not be discussed in detail here, but the reader is referred to other chapters in this volume for detailed virological aspects. Here, we have merely described broad features of each infection from the standpoint of general host–pathogen interaction.

Innate Immunity

The impact of aging on innate immunity remains incompletely understood, and some of the effects are still controversial. Age-related changes have been described both in cells, including neutrophils, dendritic cells, macrophages and NK (natural killer) cells, and in soluble factors (cytokines, chemokines, and acute phase reactants) (Kovacs et al., 2009; Lord et al., 2001; Solana et al., 2006). It is unfortunate, however, that many of these changes are not consistently observed and that many studies themselves are less than rigorous, and it remains difficult to reconcile the contrary results obtained in some of them. Thus, no change or age-related declines were reported on granulocyte and macrophage (Mf) function (Solana et al., 2006). Numbers and representation of granulocytes were reported to increase in the mouse spleen (Linton & Dorshkind, 2004), which could have origins in the observed shift in hematopoietic stem cell commitment away from the lymphoid and towards the myeloid lineage in older mice (Min et al., 2004). It seems clear that there is an impact of aging on NK cells; the more mature subset accumulates with age, but there is also a decline in cytotoxic function. NK T cells may accumulate or decrease with age and exhibit functional changes as well, but the impact and extent thereof are incompletely understood (Solana et al., 2006). Dendritic cells (DCs) are key antigen-presenting cells that function at the interface between innate and adaptive immunity. They not only respond to microbial pathogens by immediate cytokine secretion, but also capture antigens and initiate the adaptive immune response by presenting them to T and B cells. Some reports suggest that numbers and function of DCs remain normal in aging mice, but that their migratory properties degrade (Agrawal et al., 2007; Linton & Dorshkind, 2004). It is unclear, however, whether this is reproducible, and whether this is due to intrinsic defects in DCs (e.g., expression of receptors for chemokines that guide DC migration) or alterations in microenvironment (e.g., dysregulated production of

AGE-ASSOCIATED CHANGES IN IMMUNITY

Age-related changes have been described for nearly all aspects of immunity. However, it is fair to say that at the present, our understanding of the precise importance of Janko Nikolich-Žugich, Department of Immunobiology and the Arizona Center on Aging, University of Arizona College of Medicine, Tucson, AZ 85718. Marcia A. Blackman, The Trudeau Institute, Saranac Lake, NY 12983.

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chemokines and cytokines in the milieu). Moreover, DCs are heterogenous and studies of DC subsets with aging, which are likely to be most informative, have not been reproducibly performed so far. Often, changes in innate immunity are collectively described as resulting in a “proinflammatory” state, and, indeed, many proinflammatory cytokines are somewhat elevated from the baseline seen in adult humans or animals, particularly IL-6 (interleukin-6), TNF-a (tumor necrosis factor a), and type I IFNs (interferons). In fact, it was proposed that a trade off exists between adaptive and innate immune systems so that the decline of the former is followed by an increase in the activity of the latter (Franceschi et al., 2000). While this idea is intellectually intriguing, hard experimental data are still lacking to support it, and it is more likely that dysregulation, rather than a simple decline, exists in both arms of immunity. Whether this dysregulation is extrinsic, mediated by the aged microenvironment, rather than intrinsic, with defects residing in the cells themselves, remains to be delineated; data from rodents suggest the former, while some data in humans point to the latter. With that in mind, it is important to note that we know little about the age-related changes in extracellular matrix, blood, and lymph endothelium, and secondary and tertiary lymphoid site architecture, and such knowledge will be essential to properly understand changes in innate, as well as in adaptive, immune responses with aging (Aw et al., 2007; Murasko & Jiang, 2005; Panda et al., 2009). Overall, it will be critical to conduct standardized and rigorous studies to decisively answer what structural and functional changes develop in innate immunity with aging and what is their biological meaning.

Adaptive Immunity

Age-related defects in CD4 and CD8 T cells and B cells have been known for decades, and these are linked to impaired antibody production and cellular immunity (reviewed in Cambier, 2005; Gruver et al., 2007; Maue et al., 2009; Nikolich-Zugich, 2008; Wagner et al., 2004). Several features are shared between the three main classes of adaptive immune cells during aging. Thus, total numbers of lymphocytes are either unchanged or somewhat reduced with aging, based upon measurements of lymphocytes in blood (in humans) or blood and the spleen (in mice), although other sites need to be surveyed. A highly consistent finding is the shift in population balance between antigen-experienced, or memory lymphocytes, and virgin, antigen-naïve lymphocytes, in favor of the former (reviewed in Goronzy & Weyand, 2003; Miller, 1996), largely due to decline in production of new naïve lymphocytes by primary lymphoid organs (bone marrow for B cells, and, even more markedly, the thymus for T cells, and utilization of the existing naïve lymphocytes due to life-long exposure and immune responses to pathogens). Reduced lymphocyte proliferative responses have been observed across a diverse spectrum of stimuli, including mitogens, phorbol ester, and calcium ionophore combinations and anti-TCR and anti-BCR agonistic antibody (Ab), with or without costimulatory signals (reviewed in Miller, 1996). Further, declines were found in proximal T-cell receptor signaling, cytokine secretion (in particular that of IL-2) (reviewed in Miller, 1996), and cytotoxicity (Effros & Walford, 1983) and B-cell proliferation, somatic hypermutation and isotype switching (reviewed in Cambier, 2005), as well as in the response to homeostatic stimuli (Nikolich-Zugich, 2008). Fortunately, single-cell analysis studies (Garcia & Miller, 2001) (rather than bulk population studies, which are often clouded by altered naïve:memory composition of lymphocytes found in old mice) have corroborated the notion

that there are cell-autonomous age-related changes that impair response to stimulation. Therefore, it has been firmly established that formation of the immunological synapse is delayed and the concomitant assembly and phosphorylation of critical downstream substrates, such as the linker of activated T cells (LAT) and the protein tyrosine kinase Zap-70, which then propagate to the MAPK (mitogen-activated protein kinase) and other downstream pathways, are diminished in old naïve CD4 T cells (Garcia & Miller, 2001). Finally, there is consensus that the overall diversity of aging lymphocytes is further reduced by expansions of clonally identical lymphocyte populations (T-cell and B-cell clonal expansions, TCE and BCE respectively) (reviewed in Clambey et al., 2005; Nikolich-Zugich, 2008). These cells are likely to be of two different and distinct origins, driven by (i) homeostatic proliferation—presumably in response to a likely (but not formally substantiated) relative surplus of homeostatic cytokines (chiefly, IL-7), some central memory CD8 T cells give rise to “spontaneous” TCE, very often seen in laboratory mice older than 18 to 20 months; and (ii) virus, mostly CMV (cytomegalovirus), resulting in accumulation of effector memory cell expansions, which is seen in the vast majority of CMV-infected humans (Pawelec et al., 2004) and also in mice infected with latent persistent viruses (NikolichZugich, 2008). Significance of the two categories of TCE remains under investigation, but in human octogenarians they were found to inversely correlate with residual life span and there is some evidence that they have the potential to impair functional immunity to unrelated infection with previously not encountered microbial pathogens (Messaoudi et al., 2004). TCEs that are related to viral infections will be addressed in more detail later in the chapter.

ACUTE VIRUSES Respiratory Viruses/Influenza

The elderly are highly susceptible to respiratory viruses, and tend to develop secondary bacterial infections, resulting in increased frequencies of hospitalization and death. In addition, current influenza virus vaccines are less effective in the elderly. It is a high priority for human wellness to develop better vaccine strategies targeting the elderly, particularly given the demographic trends, predicting an explosion of population over 65 years of age. Thus, there has been intense interest in defining the impact of aging on the development of acute and memory responses to respiratory infections, and much of this work has been carried out in mouse models. Influenza virus is initially controlled by cytotoxic CD8 T cells, which can recognize components of the virus that are shared among serologically distinct viruses. In addition, infection results in the development of neutralizing antibodies specific for the highly variable surface proteins, hemagglutinin and neuraminidase. The initial infection generates longlived humoral and cellular immunity. Neutralizing antibody thus provides protection against subsequent infection with the homologous virus, whereas cellular immunity provides limited protection against heterosubtypic infection. Although cellular immunity is not able to prevent reinfection, it can reduce the maximal viral load, mediate faster viral clearance, and prevent death from challenges with lethal doses in animal models. Vaccines elicit neutralizing antibodies that will protect against the particular variant of virus currently circulating. Unfortunately, the majority of the elderly (by some estimates, up to 65%) do not mount a good antibody response to new viruses or the current vaccines because of ageassociated immune defects in generating humoral immunity. In contrast, a T-cell response is often generated, and it has

32. Growing Old and Immunity to Viruses

been suggested that cellular immunity is a better correlate of immune protection following vaccination in the elderly. However, this may not be a completely de novo response, but is likely strongly influenced by cross-reactive T cells generated by previous infections (McElhaney et al., 2006). Studies in mice have provided details on three phases of the cellular immune response in which aging may have an impact. These include (i) the generation of the primary response in aged individuals that have not previously encountered the virus, (ii) the subsequent generation of longlived memory after primary infection of aged mice, and (iii) the impact of aging on maintenance of memory generated earlier, following influenza virus infection of young mice.

Impact of Aging on the Acute Response to New Respiratory Infections

It is well established that intranasal infection of aged mice with sublethal doses of respiratory viruses such as influenza virus results in delayed viral clearance compared to young mice. The underlying mechanism has been extensively investigated (reviewed in Murasko & Jiang, 2005). There is evidence to suggest that poor antigen presentation function in aged mice is a major contributing factor to poor responsiveness (Katz et al., 2004). Another potential defect is impaired CD8 T-cell cytotoxicity. However, most studies show that cytotoxic activity of the CD8 T cells on a per cell basis is not impaired by aging, but that fewer virus-specific CD8 T cells are elicited following infection. When CD8 T cells specific for an immunodominant epitope, NP, were enumerated after infection of aged mice, it was observed that the peak response was delayed and the maximal number of NPspecific CD8 T cells was highly variable, although the numbers of CD8 T cells in the lungs of young and aged mice were comparable (Murasko & Jiang, 2005; Yager et al., 2008). The ability to generate a vigorous response to newly encountered infections is dependent on a diverse T-cell repertoire. It is well established that the ratio of naïve to memory CD8 T cells declines dramatically with age, as a consequence of reduced thymic output of new T cells, the increasing antigen experience of the host leading to more memory cells, and the development of clonal expansions of memory CD8 T cells. The overall effect is a dramatically reduced precursor number and diversity of naïve CD8 T cells, predicted to result in narrowing of the epitope-specific CD8 T-cell repertoire in aged mice compared with young mice. This hypothesis has recently been experimentally tested. The results confirmed that the age-associated reduction of diversity in the naïve T-cell repertoire has a profound impact on the generation of an effective primary and recall response to de novo influenza virus infection in aged mice (Yager et al., 2008). Aged mice showed delayed clearance of influenza virus compared to young mice. Analysis of the CD8 T-cell response in the lung showed that comparable numbers of CD8 T cells were found in the lungs of both young and aged mice following infection, but the epitope immunodominance was profoundly shifted, and there were dramatic effects on the Tcell receptor repertoire of responding T cells. Following infection of H-2b C57BL/6 (B6) mice with influenza A virus, over 50% of the virus-specific CD8 T cells in the lung were specific for three dominant epitopes, NP, PA, and PB1, derived from the nucleoprotein, and acidic and basic polymerases, respectively. In young mice, T cells specific for NP, PA, and PB1 were essentially equidominant in the primary response, whereas NP dominated the memory response. The response of individual aged mice was very heterogeneous, which was not completely unexpected because the repertoire in each mouse is unique, but there was an overall reduction in the NP component of the response, in some cases so severe as to be essentially missing. In addition, in many cases where the

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aged mice did mount an NP response, the T-cell receptor Vb repertoire of NP-specific T cells was skewed away from the dominant Vb8.3 usage of the young mice, suggesting that the preferred T-cell receptors were no longer available to these old mice. As preferential loss of an immunodominant epitope was unexpected, an in vivo limiting dilution assay was used to determine the functional precursor frequency of cells specific for each of the three epitopes in young mice. Interestingly, the ratio of precursors for NP:PA:PB1 was 1:10:30. The lower precursor frequency of NP-specific cells provided an explanation for why age-associated reductions in the T-cell repertoire would preferentially impact the response to NP, as specificities of low precursor frequency would be most profoundly impacted by a decline in repertoire diversity. Thus, these data formally demonstrated that age-associated reductions in repertoire diversity can lead to diminished responses to a normally immunodominant epitope and may lead to compromised protective immunity. Importantly, the failure to develop a response to NP during the primary infection severely compromised the ability of mice to mount a secondary protective cellular immune response. Thus, there was a correlation between an inability to develop an NP-specific response in the primary infection and accelerated viral clearance characteristic of a secondary response. These data confirm that the age-associated decline in the CD8 T-cell repertoire can profoundly impact the primary and recall response to influenza virus. Virus-specific CD4 T cells are also impacted by aging, showing reduced intensity of T-cell receptor signaling; defects in activation, differentiation, and expansion upon stimulation; reduced IL-2 production; and impaired cognate help for B cells (reviewed in Maue et al., 2009). Aging thus impacts the ability to generate strong antiviral humoral immunity to influenza and other respiratory viruses, largely the consequence of defective CD4 T-cell help for B cells. This is likely the major reason for the failure of infection and vaccination to generate a robust, class-switched antibody response in the elderly. It is also likely that impaired CD4 help impacts the failure of de novo infection of aged mice to generate effective memory (see below).

Impact of Aging on Generation of CD8 T-Cell Memory

A hallmark of the immune response is that after generation of the primary effector CD8 T-cell response, long-term memory is developed. This generates an accelerated response upon secondary viral challenge, which, although it does not prevent reinfection, does allow accelerated viral clearance and reduced viral load after challenge with the homologous pathogen. It has been shown that CD8 T-cell memory to respiratory virus infections established in aged individuals is inferior to memory established in the young, although the underlying mechanisms are not well understood. The data suggest that memory cells can develop in aged individuals, but that their proliferative capacity during the recall response is impaired. Thus, more nonresponsive memory cells are generated in aged mice. In order to determine whether this was an inherent defect of the memory cells (intrinsic effect) or a consequence of the aged environment (extrinsic effect), dual adoptive transfer studies in which memory cells generated in young and aged donors were transferred into the same naïve (young) host. The results showed that the aging effect was intrinsic to the CD8 T cells (Ely et al., 2007b; Kohlmeier et al., 2009; Roberts et al., 2005). A likely explanation is that the CD8 T cells receive inadequate CD4 T-cell help during their generation, a consequence of the well-characterized effects of aging on CD4 T-cell function, and thus are now intrinsically defective (Haynes & Eaton, 2005; Kovaiou & Grubeck-Loebenstein, 2006). Thus, overcoming the defect in CD4 T-cell help during vaccination

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may result in enhanced efficacy of vaccines for the elderly, not only in terms of a better antibody response, but also in terms of cellular immunity.

The Impact of Aging on Peripheral Memory T Cells Generated in Young Mice

Aging is generally not associated with loss of immunity previously established when young, whether elicited by infection or vaccination (a notable exception is the infection with the varicella-zoster virus, a latent persistent infection often followed by the waning of T-cell memory, accompanied by painful outbreaks of shingles [Oxman & Levin, 2008], through incompletely understood mechanisms). This is because humoral immunity is long lived and does not wane substantially with age, and because memory CD8 T cells that are generated when young maintain their function. In fact, in certain cases, CD8 memory may actually improve with age. Central memory cells progressively accumulate with time after infection. Dual adoptive transfer studies, which allowed direct comparison of the function of young and aged memory cells in the same young host, were carried out to compare the capacity of long-term and short-term memory cells to proliferate. The results showed that aged (12 month) memory cells proliferated more strongly than young (1 month) memory cells, suggesting that T-cell memory improves over time on a per cell basis. Further studies showed that both central and effector memory cells improved in terms of proliferative capacity over time. Analysis of two phenotypic markers, KLRG1 (killer cell lectin-like receptor G1), a marker associated with senescence and poor proliferative capacity, and PD-1 (programmed death-1), a marker of T-cell exhaustion, showed that KLRG11/PD-12 cells declined over time, leading to increasing frequencies of highly proliferative memory cells (reviewed in Kohlmeier et al., 2009). In conclusion, there are both detrimental and beneficial effects of aging on CD8 T-cell memory.

Repertoire Effects of Aging have Implications for the Ability of the Elderly to Respond to De Novo Infections

There are two major manifestations of aging on the T-cell repertoire. The first major effect is a dramatic reduction of the number of naïve T cells, leading to reduced repertoire diversity among the naïve T-cell population. Largely as a consequence of thymic involution and reduced output of new thymic immigrants, and the life-long utilization of naïve T-cells to contain invading microbial pathogens, there is a dramatic shift in the ratio of naïve to memory T cells in the periphery. In young mice, there is usually a ratio of 80:20 naïve:memory T cells, whereas this is dramatically and heterogeneously shifted in old mice to range between 50:50 and 5:95. A direct consequence of reduced numbers of naïve T cells is reduced repertoire diversity. We recently directly demonstrated this by analyzing repertoire diversity in naïve T cells from young and aged mice using DNA spectratype and sequence analysis (Ahmed et al., 2009). Reduced repertoire diversity of naïve T cells has the potential to generally compromise the ability to respond to new infections. As dramatically illustrated above for influenza virus in B6 mice (Yager et al., 2008), reduced naïve T-cell repertoire diversity in aged mice can have profound effects on the response to epitopes with a low precursor frequency, and can lead to “holes in the T cell repertoire.” We have proposed that a reduced naïve T-cell repertoire suggests that the response of the elderly to de novo infections may be heavily dependent on fortuitously cross-reactive memory T cells (Woodland & Blackman, 2006). The consequences of heavy dependence of de novo responses on crossreactive memory T cells rather than a diverse repertoire of

naïve T cells may include highly stochastic responses with extreme variation between individuals, reflecting the individual’s previous antigen exposure. The responses may be of reduced avidity, or may be pathologic, as has been shown for some defined cross-reactivities. A second major effect of aging on the T-cell repertoire is the development of CD8 T-cell clonal expansions in the memory pool of peripheral CD8 T cells. Clonal expansions are commonly associated with persistent infections (see detailed discussion below). However, we have also shown that clonal expansions can develop in the memory population of CD8 T cells specific for acute respiratory viruses (Ely et al., 2007a). Kinetic analyses of mice that had recovered from infection revealed that antigen-specific T-cell clonal expansions developed over time, and by 20 months postinfection, a significant percentage of mice demonstrated the presence of clonal expansions within tetramer-positive populations of memory CD8 T cells. These clonally expanded populations were both from the central and effector memory pools, did not require antigen for their maintenance, and maintained function in terms of proliferation and cytokine secretion. A similar scenario was observed with a different acute virus, the West Nile virus (Lang et al., 2008), leading us to conclude that, in general, memory T-cell pools can serve as recruiting grounds for these TCEs that do not require antigen but rather cytokines for their long-term maintenance. Experiments are underway to determine the impact of antigen-specific clonal expansions on the recall response to challenge infection.

West Nile Virus

The West Nile virus (WNV) is a flavivirus that inflicts a disproportionally heavy toll on the elderly, and more than 95% of the mortality from this virus is confined to those over the age of 65 years. The virus has spread throughout the northern hemisphere, and has increased in virulence, particularly in North America, where strain I (clade a) had dominated all clinical cases. In addition to the fatal or heavily debilitating meningoencephalitis, the disease presents in a minority of cases as peripheral polyneuritis. Correlates of protection in adult humans are not well established; however, in experimental adult animals, it is clear that numerous innate (including IFN-I, complement, NK cells, and TCRgd (T-cell receptor gd) cells) and adaptive (B cells, CD4, and CD8 T cells) cells all play a role in defending against this virus, and that IFNg and the lytic granule pathway (perforin) are involved in a nonredundant manner, presumably in T-cell-mediated and NK-cell-mediated defense (Diamond, 2005). It remains incompletely understood whether and which of the above protection mechanisms may be redundant, but it is clear that each of them contributes to viral control. We found that WNV reaches the brain and spinal cord in all infected animals, old or adult, regardless of the outcome of the infection; therefore, differences between old and adult mice did not occur at the level of early viral control (confirmed by viral load determination) nor of neuroinvasion, but rather at the level of neurovirulence/ neuropathology. Consistent with that, our work in old C57BL/6 (B6) and BALB/c mice bore out few age-related differences in innate immunity, however, these experiments were not exhaustive, and we cannot rule out more subtle differences. By contrast, we observed substantial agerelated differences in CD4, CD8, and Ab (B cell) responses, where neither the magnitude nor the protective ability of either of the three adaptive immunity mechanisms reached the levels achieved in the adult (Brien et al., 2009). Both T-cell subsets responding to WNV in old mice were reduced numerically and relatively, as a percentage of total T cells;


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moreover, fractions of CD8 and CD4 T cells capable of mounting polyfunctional (IFN-g, TNF-a, and granzyme B) responses were also significantly reduced. Finally, on a per cell basis, all three molecules were produced at significantly lower levels by old T cells, and ex vivo lytic capacity of old T cells against WNV peptide-coated targets was either extremely low or absent. Importantly, examining these same parameters in the brains of infected old and adult animals revealed that there is a cumulative effect of several sequential defects in aged mice; lower overall T-cell numbers in the old brains were followed by reduced numbers of WNV-specific T cells. Furthermore, of these cells, fewer were able to produce effector molecules, and production of effector molecules per cell was further reduced. In the end, we estimate that old brains contained 15 to 20 times fewer differentiated, fully armed T cells ready to tackle the virus, resulting in the loss of viral control at the level of neurovirulence (Brien et al., 2009). Memory responses against WNV have not been fully studied in old animals, but tend to be more robust than primary responses in these same animals. In fact, restimulation of primary T cells, obtained by vaccination or by natural infection, holds hope that full differentiation and protective immunity can be induced in old animals.

This disproportionate expansion of one population, perhaps at the expense of T cells specific for other pathogens could directly or indirectly contribute to increased vulnerability of the old immune system. This possibility is discussed further below, particularly as it pertains to CMV. When considering the two main classes of persistent viruses, latent persistent and chronic persistent viruses, it is important to make a sharp distinction between the two, as discussed in greater detail recently (Nikolich-Zugich, 2008). Latent persistent infections rarely make healthy hosts sick, and these viruses have coevolved with hosts to persist for life. Their subclinical reactivation(s) likely play a part of what is considered normal, physiological aging—the host is not sick, but the virus could be playing an important role in what is perceived as aging of the immune system. Chronic persistent viruses, by contrast, are never controlled and therefore the host is sick or becoming sick, with pathophysiological processes being active, which cannot be considered to be part of “normal” aging. While the degree of reactivation can be different, and therefore quantitative and qualitative manifestations of latent persistent infection can vary, the lack of or the presence of the disease ultimately makes the most important difference between the two types of infection and their roles in aging.

LCMV

Latent Persistent Viruses

Immunity to LCMV in old animals (Kapasi et al., 2002) bears some striking parallels to the situation described above for WNV. Briefly, it was found that old LCMV-specific CD8 T cells were inferior to adult cells with regard to proliferative expansion, cytokine secretion, and cytotoxic activity. That was accompanied by delayed virus clearance in old mice. Once memory cells were formed, they were apparently maintained in a similar manner in old and adult mice, with somewhat larger responses present for both virus-specific CD8 and CD4 T cells, consistent with larger primary responses. Reexposure of these mice to secondary challenge resulted in slightly weaker initial viral control by old memory cells compared to adult ones, but differences were lost after the early time points. This study reaffirms that repeated rounds of stimulation may be a viable strategy to improve immunity in old animals and, perhaps, in humans.

Other Viruses

Few other acute viruses have been studied in depth (or at all, for that matter) to follow upon the observed age-related increase in morbidity and mortality. Clinically, it is know that the epidemics of the SARS (severe acute respiratory syndrome)-causing coronavirus disproportionally affected older adults so that deaths were tens of times more frequent in those over the age of 65, and epidemiological evidence suggests that prior outbreaks of variola had a pronounced age-related mortality. These and other models await experimental investigation in the future to elucidate whether and which host:pathogen interactions promote vulnerability of the older population.

PERSISTENT VIRUSES

Because the persistent viruses are not eliminated from the host, constant immune surveillance is necessary for their control. As immune function declines with age, this raises the possibility of viral reactivation in old age, with pathological consequences. However, another intriguing scenario was borne out of the human studies (reviewed in Pawelec et al., 2004), namely that subclinical activation of these latent persistent viruses may contribute to dysregulation of the immune system with aging, by repeatedly stimulating and expanding, those T cells specific for the virus itself.

Varicella-Zoster Virus

As a typical representative of herpesviruses, VZV causes a primary infection (e.g., chickenpox, varicella), and then becomes latent in sensory ganglia. Herpesviruses have a propensity to reactivate from sites of latency, and it is not clear whether latent, subclinical reactivations occur with VZV, as they seem to occur with HSV (herpes simplex virus) and CMV. However, late in life in a subset of human subjects, the virus reactivates and causes clinical disease, with painful outbreaks of shingles. Decline in cellular immunity invariably marks these outbreaks and boost vaccination has been shown to be effective in reducing frequency and severity of shingles. The elderly have the highest rate of recurrence and clinical disease, and the disease is highly debilitating. Lack of a suitable animal model, precipitated by the high species specificity of VZV, has rendered studies of VZV reactivation, immunity, and its waning in old age very difficult, and the studies using the simian varicella virus (SVV) in various primate models have fallen somewhat short of a reproducible and faithful animal model (reviewed in White et al., 2001). However, most recent studies in the rhesus macaque model suggest that this may become a very promising model, because most of the essential features with regard to immunological control and the establishment of latency appear to mirror the situation in humans. What remains to be seen, however, is whether natural reactivation can be achieved, particularly in old monkeys. Otherwise, the main immunological information on the age-related changes in viral control is that there is a decline in cell-mediated immunity, and clinical studies suggest that revaccination with varicella virus vaccine live (ZostaVax) vaccine is beneficial in reducing the incidence of shingles (Levin et al., 2008). Memory cells, therefore, fail in VZV infection, and it is likely that this occurs by mechanism(s) unique to this virus. Indeed, while children need only one shot of the vaccine, there is a need for boosters during primary vaccination in adults (Levin, 2008) for reasons that remain to be elucidated.

The g-Herpesviruses

The human g-herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are important oncogenic viruses that establish lifelong latency in

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infected individuals and are associated with a wide variety of malignancies, including Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, Kaposi’s sarcoma, and B-cell lymphoproliferative syndromes. Most of the malignancies develop after years of viral dormancy and are associated with viral reactivation. An important role for immune control in preventing the development of malignancies is illustrated by the fact that immunosuppression, as a consequence of disease or suppressive immunotherapy, leads to the development of EBV-associated lymphoproliferative syndromes and lymphomas, and KSHV-associated Kaposi’s sarcoma. A key question is whether declining immunity associated with aging has consequences for immune control of latency and increased frequencies of malignancies. There have been sporadic reports of reactivation of both EBV and CMV in aged individuals (Stowe et al., 2007), and enhanced numbers but impaired responsiveness has been described for EBVspecific CD8 T cells in elderly individuals (Pawelec et al., 2004). However, a strong correlation between aging and the development of g-herpesvirus-associated malignancies has not been observed. Despite this, there are a few reports suggesting that increasing age may enhance susceptibility. First, there is an age-associated development of non-HIV-associated Kaposi’s sarcoma (Sarid et al., 1999). This classic form of Kaposi’s sarcoma is endemic to the Mediterranean Basin and Africa and is present most often in elderly males. Second, there is a bimodal age distribution of EBV-associated Hodgkin’s disease with an increased frequency among the elderly (Mueller, 1987). However, this may have more to do with the timing of the initial infection and whether there was an associated onset of infectious mononucleosis rather than a declining of immunity with aging. More recently, a large study concluded that impaired immune status may contribute to the development of EBV-positive Hodgkin’s lymphoma in older patients (Jarrett et al., 2005). Finally, there is a recently described, age-related EBV-associated Bcell lymphoproliferative disorder (Shimoyama et al., 2008). Taken together, considering that more than 90% of individuals worldwide harbor the virus, it would be expected that if age-associated declining immunity allowed viral recrudescence, there would be more frequent incidence of malignancies in the elderly. However, it is clear that the virus is only one predisposing factor for oncogenesis, so that an age-associated loss of immune control of the virus would not be expected to translate directly into increased incidence of g-herpesvirus-associated malignancies in the elderly.

The Cytomegalovirus

CMV, a b-herpesvirus, holds a special place among the latent persistent viruses (and all microbial pathogens as a whole) with regard to immunosenescence. CMV is a highly species-specific latent persistent virus that persists in several cell types including endothelial cells, macrophages, glandular epithelia, and others, and which has evolved a vast array of immune evasion mechanisms. For those reasons, after the primary infection (typically in childhood), it is very difficult to detect replicating CMV in the latent phase. Immunological signs of viral reactivation, however, clearly exist and are robust. Unlike with acute viruses—where virus-specific T cells increase from very low levels in primary infection, reach the peak at or just after the virus is controlled, and then contract so that only a few percent convert into stable, long-lived memory cells—CMV produces a state christened “memory inflation.” This state is characterized by initial expansion of CMV-specific T cells, contraction and low levels of memory cells for several months (in mice) or years (in humans), but with subsequent accumulation or inflation of

the CMV-specific CD8 T-cell pool for months, years, and even decades (reviewed by Nikolich-Zugich, 2008). The accumulation can reach up to 30% of total CD8 T cells in the blood of both mice and humans of advanced age, and the response targets a broad array of viral open reading frame products (reviewed in Nikolich-Zugich, 2008). Nonetheless, T-cell accumulation reaches a plateau, suggesting that there are limits to the accumulation of these populations of virusspecific cells. Concomitantly, studies in humans in the last 10 years have shown that many signs of T-cell aging correspond to those seen in cells responding to CMV. This includes the loss of CD28, a major costimulatory molecule on T cells, and acquisition of the effector/effector memory phenotype, with low proliferative potential in response to Ag or agonist anti-TCR mAb stimulation, high immediate cytokine production and cytotoxicity, and an increased expression of inhibitory NK cell receptors (Pawelec et al., 2004). Moreover, in longitudinal studies, most but not all people with CMV infection lost diversity of that response with aging, and overall were more likely to die sooner than their counterparts with no or smaller responses to CMV (Hadrup et al., 2006). There is also anecdotal evidence at the present that people who are CMV-positive or CMV-exposed but who do not accumulate large populations of CMV-specific T cells may be healthier and even live longer than those who do, and it was proposed that these individuals may be elite controllers of CMV, that either resist or control the virus without having to mobilize enormous immunological resources. Based on these findings, it was suggested that CMV could be one of the main drivers of immune senescence. CMV could potentially do this by several mechanisms. CMV is one of the most potent inducers of type I interferons (IFN-I, IFNa, and IFNb), and repeated waves of frequent production of this proinflammatory cytokine can adversely impact many of the age-related pathologies. Second, by virtue of stimulating a growing pool of T cells, CMV could shift the balance further in favor of memory (effector memory) cells (L. Cicin-Sain and J. Nikolich-Zuglich, unpublished data). At the present, it is not clear whether that has further impact upon naïve T cells themselves in the absolute sense (e.g., whether this contributes to the decline of naïve T-cell numbers and/or diversity over and above what aging does), although these experiments are in progress in our labs. Preliminary data we have generated suggest that CMV has a detrimental effect upon resistance to unrelated infection in old age, but not in adult age, presumably by constricting T-cell repertoire. This is contrary to the protective effect that CMV and other herpesvirus infections have in adult mice upon unrelated secondary infections by activation of the innate immune system (Barton et al., 2007). Therefore, it remains to be seen whether CMV will have a net protective or a net deleterious effect in aged mammals, particularly in the long-lived ones. Another question related to CMV is whether this virus impairs the very T cells that are supposed to defend against it. Indeed, there have been several descriptions of “anergic” or paralyzed T cells that accumulate in older adults in response to CMV (reviewed in Pawelec et al., 2004). Some of these cells are believed to be terminally differentiated (in humans often called TEMRA cells [T-cell effector memory cells that reexpress differentiation marker CD45RA]). Are these really not protective and does CMV lead to attrition of virus-specific T-cells? Data from rhesus macaques, obtained in collaboration between Nikolich-Zugich and Dr. Louis Picker’s group (L. Cicin-Sain, A. Sylvestre, S. Hagen, J. Nikolich-Zuglich, and L. J. Picker, unpublished data) strongly suggest that this is not the case, and that by any and all measures, monkeys retain fully functional, robust responses to CMV deep into senescence. It is more likely


that some of the CMV-specific T cells stop proliferating (but not necessarily stop functioning) in order to prevent an even larger accumulation of CMV-specific lymphocytes. However, cell transfer studies will need to be done to directly answer this question. Overall, CMV remains a strong candidate to drive at least some aspects of immune senescence, but physiological consequences of this interaction of the virus with an aging immune system remain incompletely understood and will require further study.

Herpes Simplex Virus

HSV is a latent persistent a-herpesvirus that, after an acute infection controlled by CD8 T cells, becomes latent in sensory ganglia, from where it reactivates, subclinically, and in a subset of people, clinically. In human immune aging, HSV does not play as prominent role as CMV as it does not draw large T-cell populations to itself, most likely due to its limited and sequestered site of latency. In its natural route of infection, via mucosal or skin surfaces, HSV does not produce memory inflation in mice (Lang et al., 2008), and the only mechanism by which it can participate in clonal expansion is by providing a memory T-cell pool from which cytokines can expand “spontaneous” TCE in an Ag-independent manner (Lang et al., 2008), much like the Sendai virus (Ely et al., 2007a), WNV (Lang et al., 2008), and probably any other acute microbial infection. In a systemic (intraperitoneal) experimental infection, HSV produces robust memory inflation, akin to CMV. This inflation is completely Ag-dependent, relying upon establishment of viral latency and reactivation for its maintenance and perpetration (Lang et al., 2009). Antiviral drugs reduce memory inflation in this model, and, if administered before the virus, can completely prevent it. Therefore, this remains a good and tractable animal model to learn more about longterm latent virus/aging-host interactions.

Chronic Persistent Viral Infections Human Immunodeficiency Virus

A hallmark of human immunodeficiency virus (HIV) infection is immunodeficiency resulting not only in the impaired ability to control other infections, but also with manifestation of AIDS-defining malignancies, including Kaposi’s sarcoma, non-Hodgkin’s lymphoma, and cervical cancer. With improved therapies, there is a dramatically increased life expectancy among patients, raising the question of how age-associated immune decline impacts the course of HIV infection as well as the development of AIDS-associated manifestations. There are striking similarities between the immune consequences associated with HIV infection and some aspects of immune aging, and it has been suggested that HIV compresses the aging process. HIV leads to a huge overstimulation of the immune system, both directly (because it is constantly present and it floods CD8 T cells and B cells with its antigens in a relative or absolute dearth of CD4 cells), and also indirectly. This indirect action can, in principle, be at least twofold. First, as HIV depletes CD4 T cells, the remaining cells attempt to compensate for that loss and proliferate homeostatically. Second, and probably more importantly, the massive depletion of memory T cells from the gut by the virus seems to irreversibly open the floodgates for microbial translocation in the gut (Brenchley et al., 2006), leading to systemic immune activation. This provides a basis for potential replicative exhaustion and “replicative senescence” of cells specific for HIV and for associated opportunistic microbes, and the accumulation of cells demonstrating replicative senescence with age is indeed

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accelerated during chronic HIV infection (Effros, 2007). It is therefore obvious that the mechanisms operating during natural aging and HIV infection are quite different, quantitatively and probably qualitatively. The consequences of these mechanisms in HIV lead to a loss of control of previously contained persistent infections including CMV, EBV, and VZV. In addition, there are more frequent manifestations of non-AIDS-defining cancers, although the specific types of cancer and their frequencies vary widely, and the role of immunosuppression has been controversial. However, meta-analysis of the incidence of cancers in patients with HIV compared with immunosuppressed transplant recipients showed similar patterns, prompting the conclusion that immune deficiency rather than other risk factors for cancer is responsible for the increased incidence. Thus, it is likely that the immunosuppressive consequences of HIV infection in a population of increasingly aging individuals may have important clinical complications. This remains an issue in need of more thorough investigation, and as the HIV patients that control the virus with the advent of new antiviral therapies become old, opportunities will present themselves for further incisive studies. At least two scenarios will need to be investigated: (i) for patients with HIV that become old, what is the impact of the virus upon the process of immune dysregulation with advanced age; and (ii) for patients that may get infected in later years (e.g., above the age of 50), what is the impact of aging upon control of HIV?

Hepatitis C Virus

Hepatitis C virus (HCV) is another chronic persistent virus that causes major mortality and morbidity in humans, and which has the potential to alter and exhaust the parts of the immune system designed to control it. Like HIV, HCV floods the immune system with its antigens and there is a high dose of viremia that makes virus-specific T and B cells swim in the ocean of antigens, providing incessant stimulation and eventual exhaustion of most of these cells. Other nonspecific stimulatory mechanisms via elevated inflammation are also likely at work. Like HIV, HCV is infecting an increasing segment of the older adult population, and questions of its relationship to the aging immune system will need to be answered. At the present, however, we have no good datasets to provide insight into this interplay, and the only animal model (using great apes), has been withdrawn from research for humane reasons, making this line of research even more difficult.

VACCINE STRATEGIES FOR THE ELDERLY

Despite the fact that vaccination efficacy wanes with aging, to this day there is not a single vaccine tailored towards addressing the specific needs of the elderly population. Some vaccines utilized in the general population remain advised and, to some extent, efficacious in older adults. Influenza vaccination, for example, is effective in reducing hospitalizations and deaths resulting from complications in the elderly (reviewed in Monto et al., 2009). But that efficacy often may be occurring via improved herd immunity, and studies exist showing that vaccination of schoolchildren in Japan was the single most effective measure to reduce morbidity and mortality in older adults (Reichert et al., 2001). In older adults, estimates of annual influenza vaccination efficacy ranged from 30% to 60% (determined by the fourfold increase of neutralizing antibody titers) (reviewed in McElhaney, 2009), making it clear that we have a long way to go to protect this population. Moreover, serum antibody titers are not a good correlate of immune protection and vaccination efficacy in the elderly. Rather, correlates of

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protection that evaluate T-cell responses seem to be helpful and therefore must be developed, and, not surprisingly, the cellular immune response to inactivated influenza virus vaccines is diminished in older adults. Approaches to improve vaccines for the elderly will depend on better understanding of the molecular and cellular immune defects in older animals and humans, but time is now ripe to utilize the current knowledge and begin designing rational second and third generation vaccines for this population. Several strategies are inviting, and supplementing vaccines with targeted immunomodulatory agents and optimized adjuvants to support robust initiation of immunity is one of the most prominent ones (Katz et al., 2004). Also, the development of mucosal vaccinations for the elderly has been suggested, but we still understand very little about regional immunity, migration, and cross-talk between different players that initiate a response to the vaccine. Another possibility is to enhance antigen presentation to improve immune responses, and also to target stimulation of T-cell responses that are cross-reactive to serologically distinct strains to enhance protection. Reduced repertoire because of clonal expansions and reduced numbers of naïve T and B cells has implications for vaccination strategies. Here, several issues are critical. We must understand the limits of T-cell repertoire and T-cell numbers needed to provide a response to vaccination (or a microbial pathogen) in an older adult. That knowledge will then inform us on whether we have B/T cells (even the sluggish ones) to work with, which can be then stimulated to work better by new generation of vaccines, or whether we have no or few naïve cells that have no realistic hope to provide protection. That latter scenario calls for modes of immune rejuvenation and reconstitution (reviewed in Nikolich-Zugich, 2007), which would be critical to extend protection to those individuals whose lymphocytes have been depleted and/or burned out. Stem cell therapies, thymic stromal rejuvenation therapies, and bone marrow stromal rejuvenation therapies have been tested in mice with several promising leads and are being done in primates and humans at the present. Finally, in some of the cases we may be able to boost preexisting or even heterologous immunity using memory cells. Timing issues are crucial here as well, because a window in late adulthood when immune system still remains vital could be one of the best vehicles to protect individuals as they grow older by using primary, as well as memory-boosting, vaccination. All these possibilities provide exciting avenues to be pursued in basic and translational research in upcoming years.

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33 Growing Old and Immunity to Bacteria JOANNE TURNER

INFECTIOUS CHALLENGES OF AN AGING GLOBAL POPULATION

components of innate immunity, including poor neutrophil and macrophage phagocytic function, cytokine secretion, and expression of pathogen associated molecular patterns such as Toll-like receptors (TLRs). Such defects lead to poor killing and clearance of pathogens. Immunity to bacterial pathogens requires a broad array of physical, chemical, and cellular mediators that either independently or combined can control an infectious agent. Deficiencies in complement, antibody, and innate or adaptive cellular function can account for an increased susceptibility to bacterial pathogens, and the specific immune responses that are required for control can also differ depending on the local site of infection.

By 2050, one in every five individuals in the world will be older than 60 years of age (United Nations, 2009). As the world’s population expands, we are faced with an important challenge: how to provide appropriate healthcare for the elderly. To achieve this, we need a firm understanding of how life span regulates our physiology and how this makes an older individual more prone to specific diseases. In addition to heart disease and cancer, and chronic illness such as COPD and diabetes, the elderly are at increased risk of developing disease following exposure to infectious agents (Heron, 2007). Increased susceptibility to infection in the elderly is a result of both altered physiology in old age and decreased immune function. The primary goal of this chapter is to discuss immune function and its association with increased susceptibility of the elderly to bacterial infections.

COMMON BACTERIAL PATHOGENS OF THE ELDERLY

The focus of this chapter is on immunity to bacterial pathogens in the elderly. Bacterial infections are a significant cause of morbidity and mortality in older individuals, but are surpassed in attention by influenza, which causes substantial death in older individuals (Table 1). Pneumonia (due to influenza and/or bacterial infection) is the 7th leading cause of death in the elderly, and septicemia (systemic bacterial infection) is listed as the 10th (Heron, 2007). These infectious diseases are preceded only by cardiac failure, cancer, diabetes, and accidental death. Other common bacterial diseases in the elderly include urinary tract infections (UTI), soft tissue infections, endocarditis, tuberculosis, and a higher incidence of foodborne infection (Cristofaro, 2004). For simplicity, this chapter will be divided based upon common sites of infection, with an emphasis on pulmonary and gastrointestinal infections. Within each section, the prevalence of infection in the elderly population will be discussed, followed by our current understanding of immunity in the elderly.

IMMUNE FUNCTION IN THE ELDERLY

It has long been accepted that increasing age results in changes in immune function that can lead to increased susceptibility to infectious diseases. Such changes have been discussed in detail elsewhere (Globerson & Effros, 2000) and will only briefly be reviewed here. As we age, a loss of thymic output results in a reduced naïve T-cell output and an abundance of T cells in the periphery that have an effector/memory phenotype. This imbalance leads to a net loss of naïve T cells that can respond to new infectious challenge, and an accumulation of antigen experienced cells that reach immune senescence and no longer function optimally when they reencounter a pathogen. Altered T-cell phenotype and function is believed to directly contribute to the increased susceptibility of the elderly to many infectious diseases, and is associated with reduced cytotoxic T-cell activity and altered secretion of cytokines. Furthermore, B-cell function, both T-cell dependent and independent, is reduced in old age. Pathogen specific antibody production is reduced or of poor affinity in old age, leading to inefficient binding and neutralization of infectious agents. Alterations are also reported in

PULMONARY INFECTIONS OF THE ELDERLY

Pneumonia can be defined broadly as a respiratory disease characterized by inflammation of the lung parenchyma that can be caused by either viral or bacterial infection. Amongst the extracellular bacterial pathogens that are implicated in bacterial pneumonia, Streptococcus pneumoniae is the causative agent of almost half of all cases, with Pseudomonas

Joanne Turner, Center for Microbial Interface Biology, and Department of Internal Medicine, Division of Infectious Diseases, The Ohio State University, 460 West 12th Ave., Columbus, OH 43210.

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EVASION AND SUPPRESSION OF THE ANTIMICROBIAL HOST RESPONSE TABLE 1 Common bacterial infections of the elderly Bacterial infectious disease

Causative bacterial agents

Pneumonia

S. pneumoniae, H. influenzae, various others

Septicemia

S. aureus, various others

Tuberculosis

M. tuberculosis

Endocarditis

S. aureus, various others

Urinary tract infection

E. coli, various others

Skin infection

S. aureus, S. pyogenes, various others

Foodborne infection

L. monocytogenes

Salmonella species

Shigella species

Ocular keratitis

P. aeruginosa, S. aureus

aeruginosa, Chlamydia pneumoniae, and Haemophilus influenzae causing most other cases (Janssens, 2005; Vila-Corcoles et al., 2009). Intracellular pathogens that cause pulmonary infection in the elderly include Mycobacterium tuberculosis, Mycobacterium avium/intracellulare complex, and Legionella pneumophila (Cristofaro, 2004). The bacterial pathogens S. pneumoniae and M. tuberculosis have been studied in detail and will therefore be the primary focus of this chapter. The respiratory tract is one of the most significant routes of infection, confirmed by current reports that pneumonia is the leading cause of death from an infectious disease in the elderly. Respiratory pathogens have evolved to utilize this easily accessible portal into our bodies, however, in the elderly, this is facilitated further by physiological and social changes that occur with increasing age. As we age, we lose pulmonary elasticity, which results in a reduced and less effective cough reflex. Combined with an increased risk of food aspiration, this allows for more opportunity of bacterial

Disease incidence/prevalence in elderly persons In the United States, 7th leading cause of death in individuals over the age of 60 (combined with influenza); 50% of all pneumonia cases and 90% of all pneumonia deaths are in individuals over the age of 60 In the United States, 10th leading cause of death in individuals over the age of 60 Up to 20% of all TB cases in developed countries are in individuals over the age of 65; the elderly have increased case rate when adjusted by age 4.6 times greater risk in individuals over the age of 65 versus general population Cumulative prevalence is 30% in elderly women and 10% in elderly men Approximately 5% to 10% of all dermatologic problems in elderly are bacterial infection In the United States, 86% of hospitalizations, with 19% case fatality rate, were in individuals over the age of 50 In the United States, 40% of hospitalizations, with 1.3% case fatality rate, were in individuals over the age of 50 In the United States, 27% of hospitalizations, with 0.4% case fatality rate, were in individuals over the age of 50 In a U.S. study, 38% of conjunctivitis cases were in individuals over the age of 50, with 54% due to S. aureus, 14% due to P. aeruginosa

colonization in the lung. Furthermore, respiratory infections are transmitted most effectively in close quarters, and social change over the last decade has led to an increase in community housing of our elders, increasing their risk of infection. Once within the lung, bacterial pathogens encounter cells and molecules of the innate and adaptive immune response that seek to eradicate the pathogen.

Streptococcus pneumoniae

Protective immunity to extracellular pathogens that cause pneumonia are dependent on the generation of T-cell dependent and T-cell independent antibodies, driven by a robust innate immune response. Generation of antibodies to the polysaccharide capsule is particularly important for protection. This is the basis for the current S. pneumoniae vaccine for the elderly that targets capsular polysaccharides from the 23 most common strains of S. pneumoniae. This vaccine has been used to study immune function in the elderly.

33. Growing Old and Immunity to Bacteria

In humans, studies have primarily focused on antibody production in response to pneumococcal vaccination. Studies have shown that antibody titers, in response to pneumococcal vaccine, are either equivalent or slightly reduced in older individuals relative to young, however, the functional activity of antibodies is reduced with regard to the capacity of antibody to opsonize pneumococci for phagocytosis by granulocytes. Furthermore, antibody avidity and the ability of specific antibody to protect mice against pneumococcal challenge is reduced (Romero-Steiner et al., 1999; Schenkein et al., 2008). These studies demonstrate that the elderly are fully capable of producing pneumococcal-specific antibodies in response to vaccination, however, the antibody that is produced is less effective at clearing the infectious agent. Investigators have also reported a skewing of the antibody variable heavy chain (Kolibab et al., 2005) and light chain (Smithson et al., 2005) gene usage in the elderly in response to vaccination, as well as reduced somatic hypermutation and a loss of antibody oligoclonality with increasing age. All of these changes may impact antibody affinity and function. The exact mechanism that leads to these changes is unclear but may reflect the accumulation of memory B cells in older individuals that are specific to other antigens, or the reported decrease in IgM memory B cells that are known to generate antibodies against polysaccharides (Shi et al., 2005). In old mice, the reduced antibody responses to S. pneumoniae can be linked to upstream changes in accessory cell function, most likely macrophages that act via a secreted factor (Garg et al., 1996). Chelvarajan showed that compared to young mice, splenic macrophages from old mice were less able to stimulate T-independent antibody secretion from antigen primed B cells (Chelvarajan et al., 2005). This was linked to a reduced secretion of inflammatory cytokines by splenic macrophages because B-cell stimulation could be reversed by the addition of the proinflammatory cytokines interleukin (IL)-1b and IL-6 (Chelvarajan et al., 2005). Reduced circulating inflammatory cytokines have also been reported in older individuals with S. pneumoniae infections, including IL-6 and IL-1b (Gon et al., 1996). These data support the hypothesis that macrophages from old mice secrete lower levels of proinflammatory cytokines in response to S. pneumoniae infection, reducing their capacity to drive effective antibody production from B cells. In old mice and elderly persons, TLR expression is reduced and is one potential mechanism for altered macrophage function seen in old age (Chelvarajan et al., 2005; van Duin et al., 2007). Furthermore, studies in mice have shown that adding CpG to the conjugate polysaccharide vaccine for S. pneumoniae can boost the generation of T-independent antibody production (Sen et al., 2006), supporting a role for altered accessory cell function as a correlate of increasing susceptibility to S. pneumoniae infection in old age. An alternative hypothesis for increased susceptibility to S. pneumoniae infection in the elderly is that B-cell function can be inhibited by macrophage-derived IL-10. IL-10 is secreted in abundance in macrophage cultures from old mice in response to LPS (lipopolysaccharide), and neutralization of IL-10 in vitro can improve the secretion of proinflammatory cytokines such as IL-12 and IL-6 (Chelvarajan et al., 2005). IL-10 is also increased in cell cultures from old mice (Chelvarajan et al., 2005; Sen et al., 2006) and protracted production of IL-10 has been reported in elderly individuals with S. pneumoniae infection (Bruunsgaard et al., 1999). In support of a suppressive effect of IL-10, a secondary pneumonia model following influenza infection has shown that neutralization of IL-10 in vivo can reduce the growth of S. pneumoniae and also decrease the mortality rate of young

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mice (van der Sluijs et al., 2004). In contrast, a different study reported that IL-10 can have a beneficial adjuvant effect for vaccination in old mice, with improved secretion of antigen-specific antibodies that was dependent on the presence of T cells and macrophages. This is contrary to what might be expected given the immunosuppressive properties of IL-10, however, in addition to its anti-inflammatory role, IL-10 has known B-cell stimulatory properties (Moore et al., 2001). The exact mechanism by which IL-10 impacts control of S. pneumoniae infection in old age is unclear, but the data currently available certainly indicate that the role of IL-10 warrants further investigation. For S. pneumoniae infection, the balance between proinflammatory and anti-inflammatory cytokines, secreted by accessory cells, is a good indicator of protective immunity and appears to influence the generation of T-cell independent antibody secretion. Primary defects in B-cell function, independent of macrophage responses, also cannot be ruled out. How aging impacts T-cell-dependent responses during S. pneumoniae infection are less clear, although defective CD4 T-cell help has been reported during primary infection (Sen et al., 2006) in addition to alterations in CD4 T-cell recall responses to secondary challenge (Borghesi & Nicoletti, 1995). The reduction in recall response is highly significant when we consider that most encounters with S. pneumoniae in the elderly are unlikely to be representative of a primary infection. Additional studies, either in mice or in humans, are necessary to begin to dissect the mechanisms for impaired innate or adaptive immunity in old age.

Mycobacterium tuberculosis

M. tuberculosis infection is most frequently established in the lung, after inhalation of the bacteria in droplet particles (Flynn & Chan, 2001). Pulmonary tuberculosis is the most common form of disease, although M. tuberculosis can be located at many other sites in the body. Control of M. tuberculosis infection is dependent on the phagocytosis of bacteria and the generation of TH1 cell mediated immunity, with IL-12 secretion by macrophages and activation of antigen specific CD4 and CD8 T cells that secrete interferon (IFN)-g (Flynn & Chan, 2001). In most individuals infected with M. tuberculosis, eradication of the pathogen does not occur and the host encapsulates the bacteria in a granuloma that serves to wall off the infection and prevent growth and dissemination of M. tuberculosis to other sites of the body. In the absence or loss of adequate immune function, the granuloma structure breaks down and M. tuberculosis continues to replicate freely, causing active tuberculosis disease (Flynn & Chan, 2001). Reactivation tuberculosis occurs in individuals that have successfully contained M. tuberculosis within a granuloma for many years but then subsequently develop signs of bacterial regrowth and disease. Altered immune function is considered to be the most significant factor that leads to reactivation of M. tuberculosis infection (Flynn & Chan, 2001), and the immune changes that occur with increasing age are likely to contribute to disease progression in the elderly. Reactivation tuberculosis is the most common form of tuberculosis in the elderly and waning immunity is considered to be the primary mechanism for disease, however, the association of tuberculosis with other chronic illness in the elderly (e.g., heart disease, diabetes) also indicates that general poor health is also a contributing factor (Rajagopalan, 2001). The diagnostic test for exposure to M. tuberculosis is a skin test whereby a soluble preparation of M. tuberculosis antigens is injected intradermally. This test relies on memory recall by antigen specific CD4 T cells and their localization to the skin,

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resulting in a delayed type hypersensitivity (DTH) reaction, which is indicative of previous exposure. In humans, aging is associated with a reduced DTH reaction and this loss of reactivity is a primary indicator that immune function is reduced in old age (Dorken et al., 1987). However, booster skin tests can lead to a DTH reaction in older individuals, suggesting that antigen-specific CD4 T cells are not lost in old age, but their localization to the skin may simply be delayed. This may be a consequence of poor local antigen presentation or chemokine secretion, delayed migration of dendritic cells to the draining lymph nodes, or reduced or delayed T-cell proliferation. The immunological mechanism leading to poor M. tuberculosis DTH skin test reactivity in humans is unclear, however, it is also important to recognize that physiological age-related changes in the skin could equally contribute to the apparent loss of immune reactivity. In mice, age-associated reactivation of M. tuberculosis infection has been modeled using mice that have been infected with M. tuberculosis for greater than 300 days. M. tuberculosis infection in mice is a chronic disease with a relatively high bacterial load in the lung that eventually leads to a reduced life span relative to noninfected controls (Orme, 1988). Disease progression is associated with breakdown of granuloma integrity that leads to dissemination of bacterial infection, overwhelming lung pathology, and death (Rhoades et al., 1997). Although experiments have revealed a general disorganization of granuloma integrity with increasing age— suggestive of a loss of protective immunity—little data is available to address the specific changes in immune function that contribute to increased susceptibility of older mice to reactivate an infection with M. tuberculosis. In humans and in mice, M. tuberculosis-specific CD4 T-cell responses can be detected in asymptomatic individuals, suggesting that antigen exposure and immune activation is an ongoing event even during quiescent infection (Flynn & Chan, 2001). Constant antigenic challenge may lead to persistent immune activation leading to cellular exhaustion (infectiondriven reactivation). Alternatively, the failure to contain M. tuberculosis infection may be directly linked to changes in immune function in old age (age driven reactivation). Recent evidence suggests that the failure to contain a longterm infection with M. tuberculosis may be due to rising levels of IL-4 and IL-10 in the lungs of mice as they increase in age (our unpublished observations). Although reactivation tuberculosis is the most common form of disease in the elderly, there is little research being done to determine why immune function breaks down and leads to the reactivation of M. tuberculosis in the elderly. The paucity of animal models that truly model latent M. tuberculosis infection is perhaps a factor for this deficit in information. Furthermore, very few studies of immune function are performed in elderly persons with active, latent, or cured M. tuberculosis infection even though the elderly make up one-fifth of all tuberculosis cases in the United States and Europe (Rajagopalan, 2001). Tuberculosis can also develop immediately following exposure to M. tuberculosis in individuals that have compromised immune function (primary disease). Primary tuberculosis occurs in the elderly, particularly within institutionalized settings such as nursing homes, where respiratory transmission of infectious diseases is common. The most likely cause of primary tuberculosis in the elderly is the natural accumulation of memory CD4 and CD8 T cells in old age that impedes the generation of an immune response to a newly encountered pathogen. Murine studies have confirmed that there are defects in the generation of CD4 T-cell-mediated immunity during primary infection of old mice with M. tuberculosis. Intravenous infection of old mice with M. tuberculosis showed a delayed accumulation of CD4

and CD8 T cells into the spleen relative to young mice, and a reduced capacity of CD4 T cells to secrete IFN-g in vitro upon antigen stimulation (Orme et al., 1993). In contrast, pulmonary infection with M. tuberculosis led to a delayed accumulation of CD4 T cells only, and no differences in CD8 T-cell numbers between young and old mice over a 3 week period (Turner et al., 2002). The reduction in pulmonary CD4 T-cell numbers was a consequence of reduced local proliferation leading to fewer antigen-specific IFN-g producing CD4 T cells in the lungs of old mice (our unpublished observations). Interestingly, antigen-specific CD8 T-cell numbers and responses remained intact in old mice (our unpublished observations). These studies highlight the importance of investigating immune function using the natural route of infection and analysis of cell function at the most relevant site, but they also clearly identify a specific defect in CD4 T-cell function in old mice (Chiu et al., 2008). Adoptive transfer of CD4 T cells from M. tuberculosis-infected old or young mice into young recipients followed by M. tuberculosis challenge conclusively showed that CD4 T cells from old mice were not as effective at controlling infection as CD4 T cells from young mice (Orme, 1987). The presence of functional antigen specific CD4 T cells is critical for the control of M. tuberculosis infection in young mice and in humans (Flynn & Chan, 2001) and therefore the delayed or defective CD4 T-cell responses observed in old mice likely contributes to the increased susceptibility of old mice, and elderly humans, to infection with M. tuberculosis. It is unclear exactly why CD4 T-cell function is reduced in old mice during M. tuberculosis infection but several mechanisms can be proposed. The migration of antigen presenting cells to the draining lymph nodes to prime T cells may be delayed in old mice, however, the normal generation of antigen specific IFN-g-producing CD8 T cells in the lung (Chiu et al., 2008; Turner et al., 2002) argues against this. The unique environment of the lung does appear to play a role as systemic infection alters both CD4 and CD8 T-cell accumulation in the spleen, whereas pulmonary infection only impacts CD4 T-cell accumulation. Altered T-cell migration to the lung may also be impacted in old age, although chemokine expression appears to be intact in old mice during M. tuberculosis infection (Vesosky & Turner, 2005). The expression of chemokine receptors on activated T-cell subsets may reveal differential chemokine receptor expression between CD4 and CD8 T cells in old mice. Finally, the local lung environment may negatively impact the recruitment or local stimulation of CD4 T cells in the lung. Innate immune function in old mice has been investigated using a M. tuberculosis antigen-coated bead model, which revealed that old mice recruited more mononuclear cells to the lung lesions early after infection and that these cells could secrete the immunosuppressive cytokine IL-10 (Chiu et al., 2008). Blocking the action of IL-10 restored macrophage chemokine secretion to similar levels as young mice. These studies looked extremely early after bead delivery but indicated that IL-10 may play an important role in dampening immune responses in the lungs of old mice. While antigen-specific CD4 T-cell responses are deficient or delayed in old mice during infection with M. tuberculosis, antigen-specific CD8 T cell responses remain intact, delineating specific differences between these two T-cell subsets in old age. Antigen-specific CD8 T-cell responses are unable to fully compensate for failing antigen-specific CD4 T-cell function in old mice, and old mice ultimately have reduced survival relative to young, but studies of CD8 T-cell function have revealed an additional property of CD8 T cells that develops with increasing age; the expression of properties more akin to cells of the innate immune system.


Resident CD8 T cells in the lungs of old mice are capable of responding to an early burst of IL-12, generated in response to M. tuberculosis infection, by secreting IFN-g. This early IFN-g secretion, in turn, is thought to contribute to the capacity of infected macrophages to limit bacterial growth in the lung (Vesosky et al., 2006b) leading to an early resistance to infection. CD8 T cells that mediate this mechanism reside within the lungs of naïve old mice, increase in numbers as mice age (Vesosky et al., 2006a), and express cell surface molecules associated with effector/memory T cells. It is thought that this CD8 T-cell population is derived from antigen-specific CD8 T cells that have responded to antigen/ infection throughout the life span of the mouse and migrated to the lung parenchyma to provide rapid recall responses to antigens independent of M. tuberculosis. In vitro studies have shown that CD8 T cells isolated from naïve old mice can secrete IFN-g when stimulated with M. tuberculosis infected macrophages, with supernatants from M. tuberculosisinfected macrophages or, more significantly, with IL-12p70 alone (Vesosky et al., 2009). Furthermore, IFN-g secretion could occur in the absence of MHC class I, confirming the lack of specificity for M. tuberculosis antigens in this response. Additional studies have confirmed that CD8 T cells from old mice that brightly express the activation marker CD44 are capable of responding directly to IL-12p70, driven by enhanced STAT4 dependent signaling pathways (Rottinghaus et al, 2009). Clearly, a subset of effector/memory CD8 T cells can respond in an antigen-independent manner in an environment rich in TH1 cytokines. M. tuberculosis infection, which drives the early production of IL-12 and IL-18 in old mice (Vesosky et al., 2006b), provides such an environment, leading to IFN-g production by CD8 T cells in vivo. These data provide convincing evidence for an early IL-12 mediated innate or bystander function of resident CD8 T cells within the lungs of old mice, which leads to an early resistance to infection with M. tuberculosis. Studies of primary M. tuberculosis infection using the aged mouse model clearly show that old mice possess a resident population of CD8 T cells that are capable of responding to infection in an antigen-independent manner and that these cells contribute to the early control of M. tuberculosis infection. Despite this early innate response, the capacity to control an infection with M. tuberculosis is exquisitely dependent on the generation of a robust antigen-dependent CD4 T-cell mediated immune response. Old mice have a delayed or defective functional capacity of CD4 T cells in old age, which is supported by evidence in humans of reduced reactivity to the diagnostic skin test. There remain several deficiencies in our knowledge of how old mice and elderly humans control infection with M. tuberculosis. Few studies have addressed pulmonary macrophage function in old age, even though the macrophage is the host cell for M. tuberculosis (Flynn & Chan, 2001). It will also be important to determine why CD4 T-cell function is altered in old age, and whether immunosuppressive or TH2 cytokines, or T-cell exhaustion in response to chronic antigen exposure, plays a role. It is unclear whether the early responsiveness of CD8 T cells in the lungs of old mice can influence the long-term control of infection. Finally, and perhaps most significant, is the need to investigate immune function during M. tuberculosis infection in the aging human population.

Other Pulmonary Pathogens

The elderly are also susceptible to many other bacterial pathogens of the respiratory tract, but relatively few studies have compared infection in old and young persons or mice, and even less have studied immune function. Legionella pneumophila (Fujio et al., 1995) and Chlamydophila pneumoniae

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(Little et al., 2005) infections have been performed in the aged mouse model and, for both infections, old mice showed evidence of increased susceptibility associated with increasing pulmonary bacterial loads. Immune mechanisms for increased susceptibility to C. pneumoniae were not performed, whereas in vitro studies using L. pneumophila showed that peritoneal macrophages from old naïve mice were able to kill L. pneumophila more effectively than young (Fujio et al., 1995). This early control may reflect a robust innate immune response, similar to the early control of M. tuberculosis in old mice, perhaps indicating a general enhanced innate immune function in response to intracellular bacterial pathogens in old age. Contrary to studies with M. tuberculosis, naïve spleen cell cultures from old mice secreted less IFN-g in response to formalin fixed L. pneumophila than young mice (Fujio et al., 1995), and although early production of IFN-g was found to be important for the control of L. pneumophila infection in young mice, it appeared to be redundant in old mice. These differences may simply reflect the different delivery route and source of T cells in the L. pneumophila studies, but could also highlight the fact that each human pathogen drives a unique immune profile. It may be impossible for us to extrapolate our findings from one pathogen to another to understand how changes in immune function impact the control of infections in the elderly.

FOODBORNE INFECTION

Foodborne pathogens are an important public health concern and include pathogens such as Campylobacter, Listeria, Shigella, and Salmonella. Data from the Center for Disease Control and Prevention (CDC, 2009) indicate that individuals over the age of 50 are more likely to be hospitalized due to foodborne infection and also have a higher case fatality rate than younger persons. The increased susceptibility of the elderly to foodborne pathogens can be related to several physiological changes in old age, such as low stomach acidity and reduced intestinal motility, but is also linked to decreased immune function. Gut-associated defenses rely heavily on chemical mediators and the natural barrier of the intestinal wall. Once pathogens attach and penetrate the gut wall, there is a requirement for both innate and adaptive cellular mechanisms to control infection. Although animal models have been used to study several foodborne infections in the elderly, relatively few have studied immune function using the natural route of oral infection. Despite this, relevant information on immune function in old age has been generated using the foodborne pathogen Listeria monocytogenes.

Listeria monocytogenes

The incidence of L. monocytogenes infection and the development of listeriosis in the general population is relatively low, however, listeria is certainly more common in the elderly and infection frequently leads to hospitalization and death. Compared to other foodborne infections in the United States, listeria has the highest case fatality rate in the elderly (CDC, 2009). With regard to aging, L. monocytogenes infection has been the most widely studied of all the foodborne pathogens, although relatively few studies have delivered this pathogen via the natural route, perhaps due to the availability of tractable animal models that express the appropriate form of E-cadherin, the receptor for L. monocytogenes, in the gastrointestinal tract (Lecuit & Cossart, 2002). Studies of intravenously infected old mice have shown that old mice can be both more or less susceptible to infection with L. monocytogenes (Gardner & Remington, 1977; Løvik & North, 1985), however, these conflicting

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observations appear to be related to dose and virulence of the infecting strain. Furthermore, studies indicate that old mice have either functional (Løvik & North, 1985) or defective (Patel, 1981a, 1981b) adaptive immunity in response to L. monocytogenes infection. Studies by Løvik and North clarified these conflicting results by clearly demonstrating that old mice have a more robust pre-immune or innate response to L. monocytogenes infection leading to an early reduction in the number of bacteria disseminating into the spleen and liver (Løvik & North, 1985). This marked difference in the inoculum between young and old mice led to old mice having higher LD50 and increased survival. By normalizing the inoculum, delivering up to 10-fold more L. monocytogenes to old mice, they were able to show that old mice controlled infection at an equivalent rate as young, and were equally able to generate adaptive immunity and long-term memory. This study highlights the importance of accounting for the early innate responses that can be expressed in old mice when investigating adaptive immune function in response to infection in old age. Finally, Løvik and North also emphasized the importance of correctly controlling infection studies in old mice by normalizing for age-related deaths within the uninfected control group (Løvik & North, 1985; Orme, 1988). Spontaneous deaths, unrelated to infection, often contribute to the shortened life span observed in old mice. In recent studies of aging, this issue is less significant as the majority of studies are performed on mice that are not close to their maximum life span. It is not apparent why old mice have such a robust innate immune response to L. monocytogenes infection, however, parallels with studies in young mice or other infections in old mice can be made. Young mice infected with L. monocytogenes generate a rapid IFN-g response that is mediated by CD8 CD44hi T cells (Berg et al., 2003). These CD8 T cells respond to infection in an antigen-independent manner, much like those observed during early M. tuberculosis infection in old mice (Vesosky et al., 2006b). It will be interesting to determine whether the expansion of CD8 CD44hi T cells in old age can contribute to their capacity to limit early infection with L. monocytogenes, and whether this specific T-cell population has a more generalized capacity to respond to infection in old age. Studies with M. tuberculosis have shown that the delivery route of infection is highly relevant when determining immune function (Orme et al., 1993; Turner et al., 2002). For L. monocytogenes, the primary route of infection to date has been intravenous, leading to a systemic infection. For the mouse model, delivery to the gastrointestinal tract has not been successful because mice do not express the correct form of E-cadherin, the receptor for L. monocytogenes in the gut (Lecuit & Cossart, 2002). Intragastric delivery of L. monocytogenes to guinea pigs showed that the aged guinea pig model may be a promising model for determining the relative contribution of innate and adaptive immunity to L. monocytogenes infection in old age (Pang et al., 2007).

Salmonella Species

Like listeria, salmonellosis accounts for a significant number of hospitalizations in the elderly (CDC, 2009). In elderly humans, there have been no studies of immune function in response to Salmonella infection, although comparative studies of infection in old and young animals have been performed. In response to intraperitoneal infection with S. enterica serovar Typhimurium, older rats had a delayed febrile response that was associated with a more robust early control of infection in the spleen and liver between days 1 and 5 of infection (Bradley & Kauffman, 1990). Despite this early control, older animals were subsequently found to have a higher bacterial burden than young rats. Although preliminary, these

data again have correlates with studies of M. tuberculosis infection and we can speculate that Salmonella infection also generates a robust innate immune response in old age, but the generation of adaptive immunity is delayed or defective and ultimately leads to an increased susceptibility to infection. Finding a way to diagnose and treat the elderly during the peak of innate immunity may be a potential strategy to reduce the incidence of infection in this specific population.

OCULAR INFECTION

Bacterial conjunctivitis is a common disorder that is frequently self-limiting, however, antibiotics are often prescribed to reduce the symptomatic phase of infection. In the elderly, Pseudomonas aeruginosa and Staphylococcus aureus infection are the most common causes of conjunctivitis, with a high incidence of methicillin-resistant S. aureus (MRSA) being reported in the elderly (Hautala et al., 2008). In some individuals, ocular infection can lead to bacterial keratitis, a more debilitating disease. Bacterial keratitis due to S. aureus or P. aeruginosa infection results in corneal swelling and perforation, and is more prevalent and causes greater clinical severity in the elderly compared to younger individuals. The eye is an immune-privileged site and a fine balance between the control of infection and control of local inflammation is premium. As such, immune responses in the eye can reveal more about inflammation and cell trafficking in old age than other locations. Studies of immune function and bacterial infections of the eye are sparse in the elderly, and the majority of our knowledge has been generated using animal models. The murine experimental models of P. aeruginosa and S. aureus keratitis have been extensively studied and have provided a wealth of information about how an aged immune system responds to an extracellular pathogen in an immune privileged site. Bacterial keratitis in healthy individuals and in young mice is characterized by an influx of neutrophils into the cornea that can clear the infection, thus resolving ocular pathology. In older mice (ranging from 9 to 24 months of age) neutrophil influx is delayed, corneal swelling is marked, and the anterior chamber becomes opaque, finally leading to corneal perforation (Girgis et al., 2004; Hobden et al., 1995). This heightened susceptibility of the aged mouse model to P. aeruginosa keratitis correlates with the delayed recruitment of neutrophils into the anterior chamber and subsequent escalation in bacterial numbers at the infection site (Kernacki et al., 2000). The mechanism for delayed recruitment can be linked to reduced local secretion of inflammatory cytokines (IL-1b and IFN-g) and poor up regulation of the adhesion molecule ICAM-1, which facilitates neutrophil migration to an inflammatory site (Hobden et al., 1995, 1997). Additional immune changes have been described, which may also contribute to poor outcome in old mice. These include increased production of the chemokine MIP-2 (Kernacki et al., 2000), defects in the alternative arm of the complement cascade (Hazlett et al., 1999), and reduced phagocytosis of bacteria by macrophages and neutrophils (Hazlett et al., 1990). Despite these changes, phagocytic killing of P. aeruginosa is fully functional in old mice (Hazlett et al., 1999), suggesting that if phagocytic cells were able to reach the local infection site, they would be fully capable of controlling infection. Because bacterial keratitis occurs at a site where immune cell recruitment is tightly regulated, this model of infection has been important for increasing our understanding of cell migration, which, in old age, appears to be delayed. This delay is tightly linked with the capacity of older animals to secrete inflammatory and chemotactic molecules that lead to adhesion molecule


up regulation and efficient migration of cells that can effectively eradicate the pathogen. More directed studies of cellular migration using other infection models may reveal similar findings and contribute to our understanding of defective immunity in old age.

SEPTICEMIA

Septicemia refers to systemic inflammation in response to infection, which is associated with detectable bacteria in the bloodstream and organs. The elderly are at increased risk of developing septicemia following a bacterial infection, with septicemia listed as the 10th leading cause of death in the elderly. Much of what we understand about septicemia in the elderly comes from studies using purified LPS as the stimulus, representative of infections caused by gram-negative bacteria. Using the mouse model, investigators have shown that old mice are up to 10 times more sensitive to LPS lethality than young mice (Chorinchath et al., 1996; Tateda et al., 1996) and this sensitivity is associated with increased serum levels of TNF, IL-6, and IL-1. IL-6 production is certainly associated with increased susceptibility as old IL-6 deficient mice were shown to be much more resistant to LPS shock than old wild-type mice (Gomez et al., 2006), although TNF neutralization studies TABLE 2

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demonstrate that TNF is the most significant cytokine that contributes to poor outcome (Chorinchath et al., 1996). The anti-inflammatory cytokine IL-10 is also elevated in old mice that have been given LPS although the relevance of this cytokine in vivo has yet to be tested in old mice. Furthermore, old mice given LPS challenge increase their circulating levels of corticosterone (Chorinchath et al., 1996), and experimental reduction of corticosterone led to decreased TNF production, indicating an important link between innate inflammatory responses and hormone control. Indeed, LPS delivery has been shown to directly impact the neuroendocrine system leading to depressive behavior in old mice (Godbout et al., 2008). The influence that infection driven immunity has on behavior in the elderly, and how this impacts general health and recovery from disease, is an area that has been overlooked but may significantly contribute to the increased susceptibility of the elderly to infectious diseases. Studies in humans confirm the in vivo mouse studies, with peripheral blood mononuclear cells (PBMC) from older individuals generating more TNF, IL-1, or IL-6 in response to LPS (Gabriel et al., 2002; Motegi et al., 2006). This was also observed using dendritic cells derived from human donors (Agrawal et al., 2007), which showed reduced phagocytic activity and migration. Interestingly,

Immune findings in animal models of aging

Bacterial disease

Pathogen/ agent studied

Model system

Route of infection

Pneumonia

C. pneumoniae S. pneumoniae

Aged mouse Aged mouse

Intranasal Intraperitoneal vaccination

Septicemia

S. aureus LPS

Aged mouse Aged mouse

Intravenous Intravenous

Tuberculosis

M. tuberculosis

Aged mouse

Inhalation

Legionella

L. pneumophila

Aged mouse

Intraperitoneal

Endocarditis Urinary tract infection Skin infection Foodborne infection

none none

None None

– –

none S. enterica serovar Typhimurium L. monocytogenes

None Aged rat

– Intraperitoneal

Aged guinea pig Aged rat

Intragastric Intratracheal

Aged mouse

Intravenous

Aged rabbit Aged mouse Aged mouse

Intrastromal injection Corneal scarification Corneal scarification

Ocular keratitis

S. aureus P. aeruginosa

Immune findings in models (observation in old relative to young) Increased bacterial load and dissemination Reduced macrophage function, less inflammatory cytokines, increased IL-10, decreased CD4 T-cell help Increased mortality, toxin sensitivity Increased LPS sensitivity, elevated IL-1, IL-6, TNF, and IL-10 Increased long-term susceptibility (delayed CD4 T-cell adaptive responses), poor maintenance of granuloma, increased early resistance (CD8 T-cell-mediated innate IFN- g), Increased bacterial load, decreased LD50, macrophages more effective at killing bacteria, less IFN-g from naïve splenic cells in response to formalin fixed bacteria – – – Delayed febrile response, early control, later susceptibility Susceptibility not studied Increased susceptibility, decreased NO production Decreased bacterial load, due to robust preimmune state, equivalent adaptive immune response More resistant to keratitis More susceptible to keratitis More susceptible to keratitis, delayed neutrophil influx, increased bacteria, poor ICAM-1 up regulation, good phagocytic activity

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in vitro studies using macrophages derived from old mice show opposing results to those seen in vivo, with reduced TNF, IL-1, or IL-6 in LPS-stimulated macrophage cultures relative to macrophage cultures from young mice (Boehmer et al., 2005; Vega et al., 2004). One significant difference between the in vitro studies using mouse or human cells is the common utilization of PBMC in humans versus purified macrophages in mice. Motegi and colleagues showed that LPS-induced TNF was dependent on IL-12 secretion and the stimulation of CD561 NK or T cells that could secrete IFN-g (Motegi et al., 2006), which was required to drive the production of TNF. This finding indicates that the LPS response is partially mediated by innate immune responses in vivo (such as NK-cell and T-cell derived IFN-g production), and may explain the discrepancies between in vitro and in vivo data in mice. Therefore, in old age, LPS exposure leads to a robust proinflammatory response with potential for TNF-mediated LPS shock, which is partially mediated by cells of the innate immune system that secrete IFN-g. Furthermore, LPS delivery leads to significant changes in the stress response and increased depressive behavior in old age that is likely to impact control of infection in general. One significant confounding factor in the studies of LPS responsiveness in humans is the importance of age and health of the study subject. Although cells from elderly individuals have a robust TNF response after LPS stimulation, older individuals that have indicators of frailty (a significant risk factor for poor outcome) have a dramatically reduced proinflammatory cytokine response (Leng et al., 2004; van den Biggelaar et al., 2004). How this observation correlates with susceptibility to LPS shock and infection is unknown. Finally, although systemic LPS delivery has been an important model to further our understanding of systemic bacterial infection, it cannot model responses to whole bacteria where the interplay between immune responses and pathogen adds more complexity than one single pathogen-associated virulence factor. Systemic infection with the gram-positive bacterium S. aureus leads to poor outcome in an aged murine model. Interestingly, poor outcome was defined as decreased survival in the absence of any increase in bacterial growth in the

kidney (Louria et al., 1986). Furthermore, reduced survival was not associated with early phagocytic function as mononuclear and polymorphonuclear phagocytosis of S. aureus within the peritoneal cavity was similar between young and old mice within the first hour of infection. Phagocytic ability was, however, reduced in old mice after 4 hours despite the fact that neutrophil influx in old mice was significantly elevated relative to young mice (Louria et al., 1986). Why old mice had increased mortality, even when cell recruitment and phagocytosis was intact, lies in the increased sensitivity of old mice to staphylococcal toxins, which, when injected in a purified form, led to increased mortality in old mice relative to young mice. While immune function is clearly an important correlate of susceptibility to infection in old age, these limited studies of staphylococcal infection provide an alternative mechanism for increased susceptibility of the elderly to bacterial infections. A better understanding of why the elderly are more sensitive to bacterial toxins, and how this impacts control of infection, would be an important avenue of research.

CONCLUSIONS

As we age, we become more susceptible to infection with bacterial pathogens, which is intimately linked to alterations in immune function. There are several common themes that can be found embedded within the studies of different pathogens, models, and delivery routes (Table 2). In old age, innate immune function appears to be fully functional and robust, with a developmental change in CD8 T cells that provides the aging immune system with an additional mechanism for generating IFN-g. CD4 T-cell responses are delayed or defective in old age, and this loss appears to be one of the most significant changes that leads to poor infection outcome. A lack of CD4 T cell help may also be responsible for the loss of high affinity antibody, although it is clear that there are specific intrinsic alterations in B-cell function as well. Finally, in old age, there appears to be an increased production of IL-10, which may lead to suppression of protective immunity. It is clear that immune function has a significant impact on susceptibility of the elderly to bacterial infection, yet mechanistic studies to understand

TABLE 3 Immune findings in human studies of aging Bacterial disease Pneumonia

Pathogen/agent studied

Immune findings in humans

H. influenzae vaccine

No change in antibody light chain usage after vaccination

S. pneumoniae vaccine

Similar antibody titers but reduced affinity and opsonization capacity, skewed heavy and light variable chain gene usage, reduced somatic hypermutation, loss of oligoclonality Protracted IL-10 production Elevated IL-1, IL-6, and TNF in response to LPS (except in frail elderly) Delayed DTH response None None None None None None

Septicemia

S. pneumoniae infection Undefined

Tuberculosis Legionella Endocarditis Urinary tract infection Skin infection Foodborne infection Ocular keratitis

M. tuberculosis L. pneumophila None studied None studied None studied None studied None studied


the basis for such immune-mediated susceptibility have seldom been performed. There is a pressing need to generate more information about how the immune system ages, and how these changes impact the control of infectious disease. This is not only critical for treatment of the elderly when they become infected, but is also essential for us to develop appropriate vaccines that are specifically designed to target functional components of an aged immune system. Within the last 5 years only seven articles with S. pneumoniae and four articles on M. tuberculosis have studied immune function in old age (versus 18 articles for influenza alone). There is also a distinct absence of research being performed on other significant bacterial pathogens that cause disease in the elderly (Table 3). These include bacteria that cause urinary tract infection, endocarditis, skin, and soft tissue infection (Cristofaro, 2004). There are infectious diseases that are expected to increase in the elderly in the future. The great success we have had with antiretrovirals means that a significant portion of HIV-positive individuals will successfully survive into old age. This elderly HIV-positive population will face the challenges of drug intolerance with increasing age, and there is an expectation that age-associated changes in immunity will lead to an increased incidence of opportunistic bacterial infections. Furthermore, improved quality of life for the elderly, in addition to the availability of innovative drugs and changes in social behavior, has also led to an increased incidence of sexually transmitted diseases, and, as the food chain continues to be automated and mass produced, we can anticipate that foodborne infections will continue to increase in the elderly. Global aging will also have an impact on the incidence of bacterial infection. For example, M. tuberculosis is prevalent in developing countries and, as their population ages, it is highly likely that reactivation tuberculosis will become an increasing problem in the elderly, and may also lead to an increased rate of transmission to others. We also cannot dismiss new and emerging infections. The greater incidence of hospital-acquired MRSA is just one example of a significant emerging bacterial pathogen (Klevens et al., 2007), and history has shown us that we must be vigilant for new pathogens that can cause significant disease in the population, including the elderly. Finally, the potential for bacterial pathogens to be used a biological weapons has already been realized (Jernigan et al., 2001), and our elderly population is particularly susceptible. Our ability to protect the elderly from bacterial infection is highly dependent on the generation of knowledge about how the elderly respond to infectious disease and additional research is warranted.

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34 Bacterial Strategies for Survival in the Host ANNA D. TISCHLER AND JOHN D. McKINNEY

In 1994, a 40-year-old Danish man was diagnosed with pulmonary tuberculosis (TB) based on his presentation with typical symptoms, visible tuberculous lesions on chest radiography, and isolation of Mycobacterium tuberculosis organisms from sputum cultures. The man recalled that when he was a boy, his father had also contracted TB. Hospital records confirmed that the father was treated for pulmonary TB in 1961, when his son was only 7 years old. Was this merely coincidence, or could the two cases of father and son, separated by 33 years, be connected? The question was answered by molecular subtyping of more than 2,000 clinical M. tuberculosis specimens collected in Denmark in the 1960s and 1990s, including isolates from both the father and son. In this type of analysis, isolates with identical DNA restriction fragment patterns are likely to be related epidemiologically. The study uncovered 14 distinct DNA patterns that were present in both “recent strains” from the 1990s and “historical strains” from the 1960s, suggesting that some patients recently diagnosed with TB were infected 30 years earlier (Lillebaek et al., 2003). Most remarkably, identical DNA restriction fragment patterns were observed in isolates from the father and son. These were the only Danish samples with this specific molecular signature, strongly suggesting direct transmission of M. tuberculosis from father to son (Lillebaek et al., 2002). Since transmission likely occurred in 1961, when the father was treated for active pulmonary TB, the tubercle bacilli were presumably latent in the lungs of the son for 33 years without causing signs or symptoms of infection before reactivating to cause full-blown disease. This work provided the first compelling molecular evidence for reactivation after decades-long persistence of latent M. tuberculosis infection. This anecdote highlights several features of persistent bacterial infections that are also evident in animal models of disease. First, although most bacterial pathogens cause transient infections because they are efficiently cleared by the host immune response, persistent bacterial pathogens can survive in immune competent hosts for many decades without causing disease signs and symptoms (Fig. 1). Second, for most acute bacterial infections, the mammalian immune system generates immunological memory that, in

the case of reinfection, effectively eliminates the pathogen before the onset of disease symptoms. In contrast, the immune response that is generated against persistent bacterial pathogens is often not sufficient to prevent reinfection or superinfection with other strains (Fig. 1). Third, although persistent pathogens can survive in the host for many years or decades without eliciting clinical signs and symptoms, if the immune system is suppressed, the bacteria can reactivate to cause overt disease. In this chapter, we focus on the strategies that persistent bacterial pathogens use to evade, subvert, and disarm the host immune system. For each strategy, we begin with a general description and then elaborate on specific examples. It is worth emphasizing that persistent bacterial pathogens must use several of these strategies simultaneously to thwart the immune response. In addition, bacterial pathogens that cause acute infections often use similar strategies to colonize their hosts, albeit temporarily.

STEALTH: PERSISTENCE BY MOLECULAR DISGUISE

Host cells, particularly phagocytic cells, detect and respond to infection by expressing so-called “pattern recognition receptors” (PRRs). These include both membrane-localized Toll-like receptors (TLRs) and cytoplasmic Nod-like receptors (NLRs) that are activated by microbe-specific molecules. Upon binding the cognate ligand, PRRs activate signal transduction cascades that culminate in production of proinflammatory cytokines and recruitment of additional phagocytic cells to clear the infection. Some bacterial pathogens avoid activating this inflammatory response by producing molecules that retain biological function but are not readily bound by PRRs. For example, TLR4 is bound and activated by lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria. LPS from the persistent gastric pathogen Helicobacter pylori is a poor activator of TLR4 because the lipid A moiety contains fewer fatty acid chains with longer chain lengths than usual (Algood & Cover, 2006). Similarly, TLR5 is bound and activated by flagellin, the major protein subunit of the bacterial flagellum, which many bacterial pathogens require to reach their preferred colonization niche within the host. To circumvent this defense, some pathogens, including H. pylori, express flagellin amino acid sequence variants that assemble into functional flagella but fail to activate TLR5 (Algood & Cover, 2006).

Anna D. Tischler and John D. McKinney, Global Heath Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland.

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FIGURE 1 Replication dynamics of transient and persistent bacterial pathogens in animal infection models. The transient pathogen Listeria monocytogenes (gray line) rapidly replicates to high bacterial titers in the mouse gastrointestinal tract, but the infection is quickly resolved by the adaptive immune response. The immune response also generates immunologic memory that largely prevents Listeria replication upon secondary reinfection (gray dashed line). The persistent pathogen Mycobacterium tuberculosis (black line) also replicates to high bacterial loads in the lungs of infected mice, but these bacteria persist despite inducing a robust immune response. In addition, this immune response does not generate effective protective memory, such that infected mice can be superinfected upon secondary exposure to M. tuberculosis (black dashed line).

Another persistent pathogen that uses stealth tactics to establish infection is Salmonella enterica serotype Typhi (S. enterica serotype Typhi), a strictly human pathogen that causes typhoid fever. S. Typhi is related to nontyphoidal Salmonella serotypes that cause gastroenteritis, including Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium), but these pathogens elicit dramatically different host responses (Tsolis et al., 2008). Both S. enterica serovar Typhi and S. enterica serovar Typhimurium are enteric pathogens that invade their hosts through the intestinal mucosa. After crossing the intestinal epithelium, serovar Typhimurium is readily detected by host PRRs, resulting in a massive influx of neutrophils at the infection site (Raffatellu et al., 2006). In most cases, neutrophil recruitment is sufficient to contain the infection within the intestine and local draining lymph nodes. In rare cases when the immune system is compromised, serovar Typhimurium invades systemically where it induces a strong inflammatory response that leads to septic shock (Tsolis et al., 2008). In contrast, serovar Typhi invasion of the mucosa does not trigger influx of neutrophils, allowing the bacteria to disseminate systemically to colonize the liver, spleen, and bone marrow (Tsolis et al., 2008). Serovar Typhi bacteremia only weakly stimulates production of proinflammatory cytokines such as tumor necrosis factor-a (TNF-a) and does not cause septic shock (Tsolis et al., 2008). In a minority of cases (1% to 5%), systemic spread of serovar Typhi results in persistent colonization of the gall bladder and conversion to a carrier state. Although they do not exhibit any signs or symptoms of disease, serovar Typhi carriers continuously shed serovar Typhi in their stools and contribute to the spread of infection to naïve hosts and the persistence of the pathogen within the host population (Parry et al., 2002). The differences in the host responses to S. enterica serovar Typhimurium and S. enterica serovar Typhi suggest that serovar Typhi possesses virulence mechanisms that modulate

host inflammatory responses. One possibility is that serovar Typhi, like H. pylori, expresses molecules with reduced capacity to activate PRRs. However, LPS and flagellin purified from serovar Typhi are equally potent elicitors of proinflammatory cytokines as serovar Typhimurium LPS and flagellin, suggesting that serovar Typhi uses other mechanisms to prevent inflammation (Tsolis et al., 2008). The genome sequence of serovar Typhi revealed several major genetic differences between serovar Typhi and other Salmonellae, including the presence of a unique 134 kilobase region designated Salmonella pathogenicity island 7 (SPI-7) (Baker & Dougan, 2007). SPI-7 is a genetically unstable element and is readily lost after repeated passage of serovar Typhi in the laboratory (Raffatellu et al., 2006). Recent studies of SPI-7-deficient serovar Typhi strains indicate that SPI-7 is required for inhibition of inflammatory cytokine production via interference with TLR4 and TLR5 signaling in host cells (Tsolis et al., 2008). Further studies have shown that different factors encoded by SPI-7 are required to evade recognition by these two TLRs. SPI-7 encodes genes that are required for production and export of the Vi capsular polysaccharide antigen that is expressed during human serovar Typhi infection. The Vi capsule has been shown to contribute significantly to serovar Typhi pathogenesis in human volunteers (Raffatellu et al., 2006). Ectopic expression of the Vi capsule in serovar Typhimurium results in reduced TLR-4 dependent TNF-a production both ex vivo in a macrophage infection model and in vivo in a mouse sepsis model (Wilson et al., 2008). Although the mechanism by which Vi capsule prevents TLR4 signaling is unclear, it is possible that the capsule layer shields the outer membrane LPS from detection by TLR4. This hypothesis is supported by the observation that strains expressing the Vi capsule are not agglutinated by antibody against the LPS O-antigen (Wilson et al., 2008). Alternatively, the Vi capsule might bind and sequester a soluble

34. Bacterial Strategies for Survival in the Host

component of the TLR4 signaling complex (e.g., CD14) and thus prevent active complex formation, a mechanism by which the capsule of Neisseria meningitidis blocks TLR signaling (Winter et al., 2008). The SPI-7 locus also encodes a regulatory protein TviA that inhibits flagellin expression by repressing transcription of flhC and flhD, the master positive regulators of flagellar biosynthesis (Winter et al., 2008). Deletion of TviA in serovar Typhi causes increased flagellin expression and enhanced production of the proinflammatory cytokine IL-8 (interleukin-8) by human colonic epithelial cells infected with the tviA mutant strain (Winter et al., 2008). Thus, serovar Typhi uses factors encoded by SPI-7 to conceal its identity as a gram-negative bacterium and to avoid inducing a strong inflammatory response, thereby promoting systemic invasion and persistent gall bladder colonization.

ANTIGENIC VARIATION: PERSISTENCE BY MOLECULAR EVOLUTION

To persist in the bloodstream or on mucosal surfaces, extracellular pathogens must evade the acquired antibody response. Specific antibodies contribute to elimination of bacterial pathogens by activating the complement system and by opsonizing pathogens to enhance phagocytic clearance. Bacteria can avoid these host defense mechanisms by presenting the immune system with a continuously evolving antigen repertoire. Two related processes, antigenic variation and phase variation, generate molecular variants that escape antibody detection (comprehensively reviewed in van der Woude & Bäumler, 2004). Antigenic variation is the expression of antigenically distinct but functionally conserved versions of a single protein. In each bacterial cell, the genetic information for production of all possible variants is available but at any given time only one variant is expressed. Antigenic variation is commonly achieved by homologous recombination of silent gene cassettes into a single expression locus (van der Woude & Bäumler, 2004). Phase variation is reversible on/off switching of gene expression that can occur by inversion or DNA methylation of promoter elements, or by slipped-strand mispairing of short DNA sequence repeats during DNA replication (Moxon et al., 2006; van der Woude & Bäumler, 2004). Insertion or deletion of DNA repeats can cause premature termination of transcription or translation in the resulting phase variants (Moxon et al., 2006). Both antigenic and phase variation occur with high frequency at specific “contingency loci,” and result in heritable changes to the genome that generate diversity in the bacterial population and improve the fitness of the population as a whole. Antigenic and phase variation are well characterized in Neisseria gonorrhoeae, a human pathogen that causes the sexually transmitted disease gonorrhea. To persist in the genital tract, N. gonorrhoeae express pili, filamentous surface structures that facilitate adhesion to host epithelial cells but that also elicit a strong antibody response (Craig et al., 2004). To escape detection by pilin-specific antibodies, N. gonorrhoeae has elaborate mechanisms for phase and antigenic variation of pili expression (Hill & Davies, 2009). Pili are composed primarily of the major pilin subunit PilE, which undergoes antigenic variation by recombination of sequences from silent pilS cassettes into the single expressed copy of pilE. The pilS loci are not expressed because they lack a promoter and the conserved 5 sequence of the pilE gene. Each pilS copy has variable sequences flanked by short conserved repeats; nonreciprocal recombination between these conserved sequences in pilE and pilS replaces segments

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of pilE with the corresponding sequence from pilS without altering the pilS donor locus, a process termed gene conversion (Fig. 2A) (Kline et al., 2003). These recombination events change the PilE sequence in regions exposed on the pilus surface, but do not alter the conserved N-terminus that is required for pilus assembly (Craig et al., 2004). A virtually limitless number of PilE variants can be generated since there are many pilS sequences in the genome and they can replace either in whole or in part the sequence at pilE. Pili also contain several minor subunits including PilC, which localizes to the pilus tip and is required for pilus assembly. Slipped-strand mispairing during DNA replication at a poly-G tract within the pilC coding sequence causes a translational frameshift and expression of a truncated, nonfunctional PilC protein (Jonsson et al., 1991) (Fig. 2B). Since expression of PilC is necessary for assembly of the pilus, isolates of N. gonorrhoeae that have switched pilC to the “off” phase do not express pili on their surface (Jonsson et al., 1991). Recently, antigenic variation was also described for Treponema pallidum, a spirochete that causes the sexually transmitted disease syphilis. Syphilis is acquired during direct contact with another infected individual when T. pallidum organisms from an active lesion penetrate dermal microabrasions. Typically, the infection results in a single primary lesion that heals spontaneously, but during this primary stage of infection, T. pallidum disseminates systemically and colonizes a variety of tissues where its replication can lead to secondary manifestations that commonly include skin lesions (LaFond & Lukehart, 2006). Without antibiotic treatment, T. pallidum infection can persist and cause tertiary disease symptoms including granulomatous lesions of the skin and bones, cardiovascular complications resulting from T. pallidum colonization of the aorta, and syphilitic meningitis due to colonization of the central nervous system years or even decades after the initial infection (LaFond & Lukehart, 2006). T. pallidum has been dubbed the “stealth pathogen” because it has very few integral outer membrane proteins that can be detected by the host immune system and like other spirochetes it lacks LPS (LaFond & Lukehart, 2006). These features may temporarily contribute to evasion of the innate immune response at the initial site of infection, but persistence of T. pallidum likely depends on evasion of the antibody response, because macrophages more readily phagocytose treponemes when they are opsonized by specific antibodies (LaFond & Lukehart, 2006). Indeed, after most of the treponemes have been cleared from the initial site of infection by the immune response, the remaining bacteria are resistant to phagocytosis, suggesting that the persistent population somehow avoids opsonization (LaFond & Lukehart, 2006). Antigenic variation could therefore contribute to the ability of treponemes both to persist at the primary infection site and to survive during dissemination via the bloodstream to colonize other tissues (Deitsch et al., 2009). The genome sequence of T. pallidum uncovered a 12-member family of tpr (T. pallidum repeat) genes, some of which are predicted to encode outer membrane proteins (LaFond & Lukehart, 2006). One of these genes, tprK, exhibited striking sequence heterogeneity between T. pallidum isolates in seven variable (V) regions, suggesting that it might undergo antigenic variation (LaFond & Lukehart, 2006). Extensive sequence analysis of V regions from different T. pallidum isolates revealed that the tprK gene has a complex architecture. Each V region is flanked by short conserved sequence repeats, and in some V regions there are additional internal repeats of the same

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FIGURE 2 Mechanisms of antigenic and phase variation of Neisseria gonorrhoeae pilus expression. (A) Antigenic variation. Nonreciprocal recombination of variant pilin sequences from silent pilS cassettes to the expressed pilE locus generates variant PilE pilin subunits. pilS sequences are not expressed because they lack the 5 coding sequence and promoter of pilE, but they can replace, either in whole or in part, the sequence at pilE to yield mosaic PilE variants that escape recognition by specific host antibodies. (B) Phase variation. Slipped-strand mispairing occurs during DNA replication at a poly-guanosine (poly-G) tract near the 5 end of the pilC gene, which encodes a minor pilin subunit that is required for pilus assembly. This changes the number of G residues and alters the translational reading frame such that downstream sequences are either in or out of frame for translation. Strains of N. gonorrhoeae that have switched PilC to the “off” phase do not express pili on their surface and therefore escape detection by pilin-specific antibodies.

sequence (Centurion-Lara et al., 2004). Bioinformatic analysis of the T. pallidum genome identified DNA sequences that could function as donors for recombination into the tprK V regions (Centurion-Lara et al., 2004). To demonstrate that recombination in tprK occurred during experimental infection, a clonal T. pallidum isolate was passaged in rabbits. tprK sequence diversity accumulated during successive passages, and each sequence change could be explained by a recombination event with a donor sequence (Centurion-Lara et al., 2004). No changes were observed in the donor sequences during passage of the clonal isolate, suggesting that variation in tprK occurs by a gene conversion mechanism, similar to that described for N. gonorrhoeae pilE antigenic variation (Centurion-Lara et al., 2004).

There is increasing evidence that antigenic variation of TprK occurs during infection and contributes to T. pallidum survival in the host. First, changes in the sequence of tprK are observed only during slow passage of T. pallidum in rabbits, suggesting that pressure from the acquired immune response is necessary to select for the rare TprK antigenic variants (LaFond et al., 2006a). Second, vaccination with TprK protects against subsequent infection with T. pallidum expressing the same TprK variant, but does not protect against clones expressing other versions of TprK (Morgan et al., 2003). Finally, in the rabbit infection model, T. pallidum clones expressing a single unique TprK protein elicit antibodies that are highly specific for the V regions of the infecting clone and that rarely cross-react with other V regions (LaFond et al., 2006b). Thus, it is likely that TprK antigenic


variation is important for T. pallidum to evade the host antibody response in order to establish and maintain a persistent infection.

COMPLEMENT EVASION: AVOIDING CLEARANCE FOR EXTRACELLULAR PERSISTENCE

Complement is a critical component of the innate immune system that recognizes foreign cells and marks them for destruction either by additional complement factors or by phagocytic cells that are recruited to the site of invasion (comprehensively reviewed in Walport, 2001a; Walport, 2001b). Complement is a network of circulating serum proteins that serve as enzymes and substrates in a complex hierarchical proteolytic cascade. The cascade can be activated in three ways: the classical pathway recognizes foreign cells that are marked by specific antigen-antibody complexes; the lectin pathway recognizes carbohydrate ligands specific to microbial cells; and the alternative pathway is activated by spontaneous hydrolysis of complement component C3. All three of these complement activation pathways culminate in proteolytic activation of C3. One C3 cleavage product, C3a, is released and serves as a neutrophil chemoattractant; the other, C3b, is covalently linked to the surface of the foreign cell where it fulfills three functions. First, opsonization of the foreign cell by C3b enhances uptake by phagocytic cells that express complement receptors. Second, with other complement components, C3b forms a surface-bound C3 convertase that amplifies the cleavage of C3 and deposition of C3b. Third, C3b is a component of the C5 convertase that cleaves C5 to induce formation of the membrane attack complex (MAC) and to release a second neutrophil chemoattractant, C5a. The MAC is composed of complement proteins C6-C9 and directly lyses gram-negative bacteria by forming pores in the bacterial cell membrane. Gram-positive bacteria that have a thick outer peptidoglycan cell wall and bacteria that produce a polysaccharide capsule are naturally resistant to the MAC (Lambris et al., 2008). To survive in the bloodstream or on mucosal surfaces, however, pathogenic bacteria must avoid opsonization by complement. One general strategy used by bacterial pathogens to inhibit the complement cascade is recruitment of host complement regulators (Blom et al., 2009; Lambris et al., 2008). These factors normally prevent inappropriate complement activation on host tissue, but they have been co-opted by some pathogens. Host complement regulators inhibit complement deposition by accelerating the decay of proteolytically active complexes in the complement cascade or by serving as cofactors for host factor I-mediated proteolytic degradation of complement proteins (Blom et al., 2009). Factor H, for example, is a complement regulator that binds directly to C3b to inhibit further proteolysis of C3. Factor H is also a cofactor for proteolytic cleavage of C3b by factor I (Blom et al., 2009). Factor H functions primarily on host cell surfaces because the affinity of factor H for C3b is much higher in the presence of glycosaminoglycans and sialic acid, molecules that typically coat only host cells (Blom et al., 2009). Factor H and other complement regulators including factor H-like proteins and C4 binding protein (C4BP) are present at relatively high concentrations in the fluid phase of human serum and can easily be captured by pathogens (Lambris et al., 2008). In addition to evading host antibodies by antigenic variation, N. gonorrhoeae avoids complement-mediated killing by recruitment of host complement regulators. All gonococcal strains can recruit factor H by sialylation of the outer membrane LPS (Blom et al., 2009). In addition, some strains of

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N. gonorrhoeae express an outer membrane porin, Por1A, that binds to factor H and C4BP to inhibit all three complement activation pathways (Blom et al., 2009). Binding of these complement inhibitors is related to N. gonorrhoeae host restriction. Por1A binds only to human C4BP and factor H; it cannot bind these factors from rodents or other primates (Ngampasutadol et al., 2005, 2008). Thus, N. gonorrhoeae cannot inhibit complement deposition in hosts other than humans, which may explain why it has been difficult to develop an animal model of gonococcal infection. Pathogens can also interfere with the complement system by producing proteins that degrade complement factors or inhibit formation of proteolytic complexes in the complement cascade (Blom et al., 2009; Lambris et al., 2008). Staphylococcus aureus, which persistently colonizes the nasopharynx of 20% of the human population, is a master of these forms of complement evasion (Lowy, 1998). Colonization of the nasopharynx is a risk factor for S. aureus infections, which range from skin and soft tissue infections, characterized by abscess formation, to systemic bacteremia (Lowy, 1998). In addition to these acute infections, S. aureus is associated with persistent and recurrent infections of bone tissue, implanted medical devices, and the nasopharynx (Proctor et al., 2006). Inhibition of the complement system is likely a critical factor for S. aureus survival on mucosal surfaces and in the bloodstream. Since S. aureus is a gram-positive bacterium that is naturally resistant to lysis by the MAC, its complement inhibitory factors primarily block opsonization and neutrophil recruitment (Lambris et al., 2008). S. aureus produces a vast array of proteins that target C3, the central component of the complement cascade (Fig. 3). Efb (extracellular fibrinogen binding protein) was identified first by virtue of its ability to directly bind C3 in vitro (Lee et al., 2004). Preincubation of human serum with Efb prevents C3b deposition on the S. aureus surface and inhibits opsonophagocytosis (Lee et al., 2004). Bioinformatic analysis of the S. aureus genome revealed another secreted protein, Ehp (Efb-homologous protein), that has a similar C3-binding domain (Hammel et al., 2007). Ehp also binds to C3 in vitro, and the Ehp-C3 cocrystal structure suggests that Ehp alters the conformation of native C3 to block formation of C3b (Hammel et al., 2007). Efb and Ehp can also inhibit proteolytic complexes that contain C3b, possibly by inducing this conformational change in C3 (Jongerius et al., 2007; Lambris et al., 2008). Efb and Ehp can inhibit neutrophil chemotaxis in a mouse model (Jongerius et al., 2007), but whether the C3-binding activity of these proteins is required for S. aureus virulence has not been tested. Three other orthologous S. aureus proteins, the SCINs (staphylococcal complement inhibitors), efficiently prevent phagocytosis by human neutrophils in vitro by blocking C3b deposition on the bacterial cell surface (Jongerius et al., 2007; Rooijakkers et al., 2005). SCINs bind specifically to surface-bound C3 convertases and stabilize them to prevent the formation of additional convertases, and thus block further C3 cleavage and C3b deposition (Rooijakkers et al., 2005). Since proteolytic cleavage of C3 is central to complement activation, the SCINs block all three complement activation pathways (Jongerius et al., 2007; Rooijakkers et al., 2005). Although SCINs are potent complement inhibitors, they are highly specific for the human complement system (Rooijakkers et al., 2005). Thus, standard animal models cannot be used to decipher the role of these complement inhibitory factors in S. aureus virulence. S. aureus also uses proteolysis to prevent opsonization by C3b. Sbi is a bifunctional S. aureus protein that binds antibody and activates the alternative pathway by enhancing

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FIGURE 3 Staphylococcus aureus complement inhibitors. S. aureus expresses several factors that prevent opsonization by C3b. The staphylococcal complement inhibitors (SCINs) bind to and stabilize C3 convertases to block their activity and prevent C3b deposition. Efb and Ehp bind directly to C3 to interfere with C3 cleavage. The secreted protein Sbi indirectly blocks C3b deposition on the S. aureus surface by cleaving C3 in the fluid phase. S. aureus also recruits host proteases that degrade surface associated C3b: clumping factor A (ClfA) recruits host factor I (fI) that cleaves C3b to C3d, and staphylokinase (SK) recruits host plasminogen and activates it to the protease plasmin that degrades C3b. S. aureus interferes with signaling between complement proteins and cells of the immune system. S. aureus prevents neutrophil migration to the site of infection by blocking neutrophil detection of C5a, a chemoattractant released upon complement activation. CHIPS (chemotaxis inhibitory protein of S. aureus) binds and antagonizes the neutrophil C5a receptor. The C3 binding proteins Efb and Ehp also interfere with communication between the complement system and B cells by blocking the interaction between C3d and complement receptor 2 (CR2).

cleavage of C3 to C3b (Burman et al., 2008). Although it seems counterintuitive for a bacterial pathogen to activate the complement cascade, Sbi is a secreted protein, so its proteolytic activity results in futile consumption of C3 in the fluid phase, rather than on the bacterial cell surface (Burman et al., 2008). S. aureus also recruits host proteases that inhibit the complement cascade. Clumping factor A (ClfA) is a S. aureus cell wall associated protein that binds to and serves as a cofactor for factor I, which cleaves C3b to an inactive form and thereby prevents opsonization (Hair et al., 2008). Finally, S. aureus staphylokinase recruits human plasminogen to the cell surface and converts it to the active serine protease, plasmin (Chavakis et al., 2007). Plasmin inhibits opsonization by cleaving C3b and IgG that have been deposited on the S. aureus cell surface (Chavakis et al., 2007). Finally, S. aureus interferes with the downstream activities of the complement system by blocking host cell receptors. Most S. aureus human isolates express the chemotaxis inhibitory protein of S. aureus (CHIPS) that binds to and antagonizes the neutrophil C5a receptor (Chavakis et al., 2007). This interaction presumably prevents neutrophil recruitment to the site of infection, although it has not been possible to test this idea experimentally since CHIPS is highly specific for the human C5a receptor (Chavakis et al., 2007). In addition to activating the innate immune response, the complement system communicates with cells of the adaptive immune system to enhance their response to infection (Carroll, 2004). C3d, a fragment of C3b

generated by factor I cleavage, plays a crucial role in this communication. B cells recognize C3d-tagged pathogens via complement receptor 2 (CR2). Engagement of CR2 lowers the threshold for B-cell activation and enhances B-cell survival (Carroll, 2004). The S. aureus C3 binding proteins Efb and Ehp may block this communication since purified Efb and Ehp prevent C3d from binding to CR2 in vitro (Ricklin et al., 2008). Although the S. aureus factors described clearly interfere with complement activation in vitro, it has not been established whether these proteins contribute to S. aureus colonization or virulence. In fact, for several reasons, it may be difficult to demonstrate that S. aureus requires these complement inhibitors during infection. First, given the sheer number of S. aureus complement evasion factors, there is likely to be some functional redundancy such that inactivation of a single factor may not significantly attenuate virulence. Second, since several S. aureus complement inhibitory factors, including SCIN and CHIPS, are specific for proteins of the human complement system, it is not possible to test the importance of these factors for S. aureus complement evasion, colonization, and virulence in standard animal infection models.

IMMUNE ADAPTATION: INTRACELLULAR PERSISTENCE BY STRESS RESISTANCE

As a first line of defense for the host immune system, phagocytic cells internalize and rapidly destroy most invading


microorganisms in maturing phagolysosomes. The lumen of the phagosome is acidified by vacuolar proton ATPases (V-ATPases) that utilize the energy from ATP hydrolysis to transport protons (H1) across the phagosomal membrane (Huynh & Grinstein, 2007). Acidic luminal pH can directly interfere with the metabolism and growth of some bacterial species, and enhances the activity of hydrolytic enzymes that are delivered to the phagolysosome (Huynh & Grinstein, 2007). Reactive oxygen and nitrogen species (ROS and RNS) are generated by the NADPH phagocyte oxidase (NOX2/gp91phox) and inducible nitric oxide synthase (iNOS), respectively (Fang, 2004). NOX2 is a multisubunit complex that assembles on the phagosomal membrane and, upon macrophage activation by interferon-g (IFN-g) produces superoxide anions (O22) by transfer of electrons from NADPH to oxygen. O22 can further dismutate in the phagosome to toxic hydrogen peroxide (H2O2) and hydroxyl radicals (Fang, 2004). iNOS is a cytoplasmic enzyme that is induced at the transcriptional level by IFN-g and produces nitric oxide (NO) from l-arginine. NO freely diffuses across the phagosomal membrane, where oxidation and acidic pH result in the formation of additional RNS, including nitrous acid and nitrogen dioxide (NO2) (Fang, 2004). ROS and RNS are highly toxic compounds that can react with and damage many microbial targets including protein tyrosine residues, DNA bases, lipids, thiols, and metal centers (Fang, 2004). Unless repaired, this damage can inhibit bacterial replication or bring about cell death. Phagocytic cells also produce cationic antimicrobial peptides including defensins, cathelicidins, and ubiquitin-derived peptides that permeabilize bacterial cell membranes (Flannagan et al., 2009; Purdy, 2007). Finally, hydrolytic enzymes within the phagolysosome degrade bacterial components, including proteins and lipids (Flannagan et al., 2009). Some pathogens have evolved mechanisms to inhibit the host cell phagocytic machinery and thereby avoid destruction in the phagolysosome. For example, Yersinia species utilize a type III secretion system to inject effector proteins into host cells that block actin cytoskeletal rearrangements and prevent phagocytosis (Navarro et al., 2005). Other bacterial pathogens have evolved to survive, replicate, and even persist in phagocytic cells. While this strategy can shelter bacterial pathogens from secreted antibody and complement, which extracellular pathogens must evade or counter, professional intracellular pathogens must avoid being killed in the harsh environment of the phagolysosome. Some intracellular pathogens actively manipulate the cellular machinery that drives phagosome maturation to establish residence in a unique compartment that is less hostile than the mature phagolysosome (Flannagan et al., 2009). For example, Legionella pneumophila delivers effector proteins to the host cell cytosol via a type IV secretion system. These effectors impede fusion of the phagosome with the endocytic compartment and promote fusion with membranes derived from the endoplasmic reticulum (Isberg et al., 2009). Other intracellular pathogens, such as Listeria monocytogenes and Shigella flexneri, escape from the phagosome into the cytoplasm, which is a more permissive environment for replication (Ray et al., 2009). The widespread human pathogen Mycobacterium tuberculosis has evolved stress resistance mechanisms that allow it to persist within the phagolysosome of activated macrophages. Although M. tuberculosis produces lipid and protein factors that block phagosome maturation in resting macrophages (Philips, 2008), macrophages activated with IFN-g deliver M. tuberculosis to a mature phagolysosome (Via et al., 1998). M. tuberculosis is apparently able to withstand the harsh phagolysosomal environment, since mutants that fail

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to block phagosome-lysosome fusion in resting macrophages are not deficient for intracellular survival (Ehrt & Schnappinger, 2009). Below, we survey M. tuberculosis factors that contribute to resisting stresses encountered in the phagolysosome and that likely enable its decades-long persistence in the lungs of immune-competent humans (Fig. 4). M. tuberculosis uses three general strategies to avoid the effects ROS and RNS: detoxification, scavenging, and damage repair (Ehrt & Schnappinger, 2009). Detoxification is accomplished by the action of specific enzymes that convert ROS and/or RNS to less toxic compounds. The M. tuberculosis catalase/peroxidase KatG shows a clear relationship between enzyme activity and survival of ROS generated by NOX2. KatG decomposes H2O2 to water and oxygen and is required for M. tuberculosis resistance to H2O2 in culture (Ng et al., 2004). KatG specifically detoxifies ROS generated by NOX2 since a katG null mutant is attenuated in wild-type mice but fully virulent in mice deficient for the enzymatic subunit of NOX2, gp91phox (Ng et al., 2004). Other M. tuberculosis enzymes that detoxify ROS and/or RNS include superoxide dismutases (SodA and SodC), alkyl hydroperoxide reductase (AhpC), and thiol peroxidase (TpX). SodA and SodC both convert O22 to H2O2 (Ehrt & Schnappinger, 2009), while AphC and TpX both catalyze the reduction of a broad range of hydroperoxides and peroxynitrite (Bryk et al., 2002; Jaeger et al., 2004). Each of these enzymes mediates M. tuberculosis resistance to oxidative and/or nitrosative stress in vitro (Ehrt & Schnappinger, 2009; Hu & Coates, 2009), but it is not yet clear whether each is required to counteract the ROS and RNS encountered in the phagosome. TpX is necessary for M. tuberculosis survival in IFN-g activated macrophages and in the lungs of mice, but survival of a tpx null mutant is only partially restored in IFN-g activated macrophages from iNOS2/2 mice, suggesting that TpX might be required to detoxify both ROS and RNS (Hu & Coates, 2009). An M. tuberculosis mutant with reduced sodA expression is attenuated in mice, but this may be due to a general growth defect since sodA is essential for growth in vitro (Ehrt & Schnappinger, 2009). Finally, sodC and aphC are dispensable for M. tuberculosis replication and virulence in the mouse model, suggesting that other enzymes with redundant functions might compensate for the loss of SodC or AhpC (Ehrt & Schnappinger, 2009). Scavenging of ROS and RNS is accomplished by noncatalytic antioxidants that maintain a reducing environment in the cytoplasm. The most abundant antioxidant in M. tuberculosis is mycothiol (MSH), a low-molecular weight compound composed of two sugar moieties and a reactive cysteine that forms a disulfide bond with a second MSH molecule concomitant with reduction of ROS (Newton et al., 2008). Mutations in the MSH biosynthesis pathway increase sensitivity of M. tuberculosis to oxidative stress in vitro and impair bacterial replication in macrophages, suggesting that MSH is required for growth in the oxidizing environment of the phagosome (Newton et al., 2008). There is biochemical evidence for RNS scavenging by M. tuberculosis truncated hemoglobins, which coordinate an oxygenated heme moiety that rapidly and irreversibly reacts with nitric oxide (Ascenzi et al., 2007), but a role for these proteins in M. tuberculosis virulence has not been reported. DNA and protein repair/degradation pathways that counteract nitrosative stress were identified by a genetic screen for RNS-sensitive M. tuberculosis mutants (Darwin et al., 2003). UvrB is a component of the nucleotide excision repair pathway that removes damaged nucleotides to initiate the DNA repair process. UvrB deficiency causes sensitivity to RNS in vitro and attenuation in the mouse model

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FIGURE 4 Antimicrobial factors in the macrophage phagolysosome and Mycobacterium tuberculosis resistance mechanisms. In the maturing phagosome, bacteria encounter reactive oxygen species (ROS) synthesized by the NADPH phagocyte oxidase (NOX2); reactive nitrogen species (RNS) synthesized by the inducible nitric oxide synthase (iNOS); acidic pH resulting from the action of vacuolar ATPase (V-ATPase) proton pumps; and cationic antimicrobial peptides (CAMPs). M. tuberculosis resists ROS and RNS using various detoxification enzymes (described in the text) including degradation of hydrogen peroxide (H2O2) by the catalase/peroxidase KatG. M. tuberculosis repairs oxidative and nitrosative damage to DNA by the nucleotide excision repair pathway (UvrB) and uses the proteasome to degrade proteins damaged by oxidation. The complex M. tuberculosis cell wall serves as a permeability barrier to the influx of protons and the membrane-associated protease Rv3671c maintains cytoplasmic pH homeostasis during growth in the acidified phagolysosome. M. tuberculosis resists the action of CAMPs by lysinylation of the membrane lipid phosphatidyl glycerol (PG).

of infection (Darwin & Nathan, 2005). Attenuation of uvrB deficient bacteria is partially reversed in iNOS2/2 mice and completely reversed in mice deficient for both iNOS and NOX2, suggesting that M. tuberculosis requires nucleotide excision repair to counteract DNA damage resulting from exposure to ROS and RNS in the phagosome (Darwin & Nathan, 2005). Mutations in genes encoding two putative accessory factors of the M. tuberculosis proteasome, mpa and pafA, were also found to confer RNS sensitivity (Darwin et al., 2003). The proteasome is a proteolytic complex that degrades damaged proteins targeted for degradation by a short peptide tag, the prokaryotic ubiquitin-like protein, Pup (Darwin, 2009). Proteasomal degradation of Pup-tagged proteins might be required for survival of nitrosative stress because it removes proteins that are irreversibly damaged by NO, removes NO-damaged proteins that are particularly toxic, or up regulates transcription of genes encoding antioxidants by removing a transcriptional repressor (Darwin, 2009). The marked attenuation of mpa deficient bacteria in wild-type mice is partially but not completely reversed in iNOS2/2 mice, suggesting that proteasome-mediated protein degradation is required to counteract RNS and other stresses that M. tuberculosis encounters in the host (Darwin et al., 2003). M. tuberculosis also encounters a strongly acidic environment within the mature phagolysosome (Vandal et al.,

2009a). Recent studies identified M. tuberculosis factors that contribute to acid resistance and pH homeostasis by screening for acid-sensitive mutants in vitro. Most of the mutations isolated in this screen affect genes required for biogenesis of the mycobacterial cell wall, a complex lipid-rich structure that functions as a permeability barrier (Vandal et al., 2009b). These mutations also cause hypersensitivity to other stressors, including lipophilic antibiotics and detergents, suggesting compromised cell wall integrity and permeability of the mutant cells to these compounds, as well as to protons (Vandal et al., 2009b). Attenuation of the cell wall deficient mutants in the mouse model suggests that the mycobacterial cell wall might contribute to survival of acidic phagolysosomal conditions during infection (Vandal et al., 2009b). This work also revealed that Rv3671c, a membrane-localized serine protease, is critical for intrabacterial pH homeostasis under acidic conditions. Unlike wild-type M. tuberculosis, the Rv3671c mutant cannot maintain neutral cytoplasmic pH either in vitro in acidic growth medium or in vivo in IFN-g activated macrophages (Vandal et al., 2008). Although its precise function is still a mystery, the Rv3671c protease might modify the mycobacterial cell wall or activate stressresponsive signaling pathways to influence intrabacterial pH and promote survival in the phagolysosome (Vandal et al., 2009a).


The stresses that M. tuberculosis encounters in the mature phagolysosome include the cationic antimicrobial peptides (CAMPs) cathelicidin, hepcidin, and peptides derived from ubiquitin, which kill mycobacteria by disrupting the cell wall (Alonso et al., 2007; Liu et al., 2007; Sow et al., 2007). Other bacteria avoid being targeted by CAMPs by making their cell surface structures less negatively charged, thus reducing the probability that the positively charged peptides will bind to the cell surface (Ernst et al., 2001). Recent evidence suggests that M. tuberculosis uses similar membrane modifications to resist CAMPs. The gene lysX, which encodes a putative lysine transferase, is required for the addition of positively charged lysine moieties onto phosphatidyl glycerol (PG), a lipid component of the M. tuberculosis membrane (Maloney et al., 2009). An M. tuberculosis lysX mutant is more sensitive to HNP-1, a CAMP produced by neutrophils, as well as the cationic antibiotics vancomycin and polymyxin-B (Maloney et al., 2009). Replication of lyxX deficient bacteria is impaired in animal infection models, suggesting that PG lysinylation contributes to M. tuberculosis survival in the host (Maloney et al., 2009).

BIOFILMS: PERSISTENCE BY COMMUNITY ORGANIZING

Although bacteria are typically considered unicellular organisms, they can colonize surfaces as biofilms—organized multicellular communities of aggregated cells embedded in a matrix of extracellular polymeric substances (EPS), usually polysaccharides. Growth within biofilms offers bacteria protection from a variety of environmental insults including acidic pH, reactive oxygen species, UV exposure, dehydration, metal toxicity, and antibiotics (Hall-Stoodley et al., 2004). The EPS matrix provides much of this protection by binding to or neutralizing toxic compounds (Hall-Stoodley et al., 2004). The physiology of bacteria within biofilms may also contribute to their increased resistance. Although antibiotics can diffuse freely through the EPS matrix, bacteria at the center of the biofilm may be nonreplicating or metabolically sluggish and thus recalcitrant to antimicrobials that act only on growing and metabolically active cells (Fux et al., 2005). In addition to enhancing resistance to environmental challenges, the biofilm mode of growth can protect bacteria against host immune responses. Bacteria within a biofilm resist phagocytosis; although phagocytic cells penetrate biofilms through fluid-filled channels, they fail to access bacteria embedded in the EPS matrix (Hall-Stoodley & Stoodley, 2009). The EPS matrix also protects bacteria from humoral immune responses by acting as a physical barrier to opsonization by antibodies (Hall-Stoodley & Stoodley, 2009). Bacterial biofilms are now recognized as the cause of many persistent infections that are refractory to antibiotic treatment. Some biofilm infections are associated with surfaces of medical devices such as catheters, shunts, prostheses, and mechanical heart valves (Fux et al., 2005). Staphylococcus epidermidis is one of the leading causes of this type of infection. S. epidermidis colonization of the device surface is facilitated by the polysaccharide intercellular adhesin (PIA), which protects S. epidermidis from host antimicrobial peptides and phagocytic uptake (Foster, 2005). Bacterial biofilms can also form on mucosal surfaces of the upper and lower respiratory tracts, the oral cavity, and the middle ear (Fux et al., 2005; Hall-Stoodley & Stoodley, 2009). Pseudomonas aeruginosa is an opportunistic pathogen that colonizes the lungs of cystic fibrosis (CF) patients as a biofilm (Hassett et al., 2009). The CF lung is characterized by accumulation of thick viscous mucus that prevents normal mucociliary clearance of particulates, including bacterial pathogens. Most P. aeruginosa CF

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isolates have a mucoid colony morphology caused by overproduction of the polysaccharide alginate, which increases adherence, enhances resistance to antibiotics, and protects P. aeruginosa from phagocytosis by cytokine-activated macrophages (Hassett et al., 2009). In a unique twist on the theme of persistent biofilm infections, uropathogenic Escherichia coli (UPEC) forms biofilmlike communities inside bladder epithelial cells (Anderson et al., 2003). UPEC is the most common cause of urinarytract infections (UTI), which can be recurrent, even if treated with antibiotics. Traditionally, UTIs were thought to be caused by extracellular UPEC colonization of the bladder epithelium, and were thought to recur by reinfection from the gastrointestinal tract (Anderson et al., 2004). Both of these traditional views were debunked by microscopic examination of UPEC infection in a mouse UTI model. UPEC invades epithelial cells of the bladder to initiate infection, replicates in these cells as complex intracellular bacterial communities (IBCs) with biofilm-like characteristics, and establishes persistent intracellular reservoirs that can cause recurrent infection (Anderson et al., 2003; Justice et al., 2004; Mysorekar & Hultgren, 2006). In addition, growth within the biofilm-like IBCs induces morphological changes in UPEC that contribute to evasion of host immunity (Justice et al., 2006). Biofilm formation on abiotic surfaces has been described as a developmental pathway with distinct stages of attachment, cell aggregation, maturation, and detachment with unique genetic requirements (Hall-Stoodley et al., 2004). Like biofilms that form on a surface, UPEC IBC formation in the mouse UTI model is a developmental process with four distinct phases that can be distinguished by differences in bacterial growth rate, size, motility, and organization (Justice et al., 2004). Each phase of IBC development probably requires production of specific bacterial factors, although these are just beginning to be characterized. IBC formation is initiated by attachment of individual motile rod-shaped UPEC cells to superficial umbrella cells of the bladder epithelium. Attachment is mediated by the FimH adhesin, which is localized at the distal end of UPEC type 1 pili (Anderson et al., 2004). FimH binds to mannosylated uroplakins, proteins of the bladder epithelium that serve as a permeability barrier to the toxic molecules concentrated in urine (Anderson et al., 2004). The interaction between FimH and uroplakins activates a signal transduction cascade in the superficial umbrella cell that induces localized rearrangements of the actin cytoskeleton and internalization of UPEC by a zippering mechanism (Anderson et al., 2004). Shortly after penetration of the superficial umbrella cell, UPEC escapes from the phagosome into the cytoplasm, where the bacteria replicate to form an “early IBC” (Color Plate 8A) (Justice et al., 2004). UPEC cells within the early IBC are fast-growing, rod-shaped, nonmotile, and only loosely associated with each other (Justice et al., 2004). After approximately 6 to 8 hours of growth in the early IBC, all UPEC cells within the IBC undergo a differentiation process to slow-growing coccoid cells that are densely packed in “pods” that protrude from the surface of the bladder epithelium (Anderson et al., 2003; Justice et al., 2004). UPEC differentiation to the “middle IBC” (Color Plate 8B) is probably orchestrated by a specific signal because all bacteria within the IBC undergo the process simultaneously (Justice et al., 2004). Visualization of mature middle IBCs by electron microscopy revealed that each UPEC cell is embedded in a matrix consisting of fibers that are likely type 1 pili and an electron lucent material that is probably polysaccharide (Anderson et al., 2003; Wright et al., 2007). Thus, the mature middle IBC is a

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highly organized structure of bacteria embedded in a matrix that strongly resembles a biofilm and serves as a safe haven for UPEC replication. Although infected epithelial cells release cytokines that attract phagocytic cells, like surfaceassociated biofilms, the IBC is protected from phagocytosis (Color Plate 8D). Even in rare cases when phagocytes penetrate the IBC, they fail to clear all of the bacteria (Justice et al., 2004). In the final stage of IBC development, UPEC disperses from the IBC to initiate new rounds of infection in naïve superficial umbrella cells of the bladder (Justice et al., 2004). During differentiation to the “late IBC,” UPEC at the outer edge of the IBC revert to the prototypical rod shape, become motile, and exit the infected epithelial cell, a process termed “fluxing” (Color Plate 8C) (Justice et al., 2004). A subpopulation of UPEC fluxing out of late IBCs are filamentous, a morphological change resulting from bacterial cell growth in the absence of division (Color Plate 8C). UPEC filaments are less efficiently phagocytosed, suggesting that differentiation to the filamentous form in the IBC may protect UPEC during dispersal (Justice et al., 2004, 2008). Indeed, UPEC requires the cell division inhibitor SulA to differentiate into filaments in vivo, and a DsulA mutant is able to initiate infection but fails to persist in the mouse UTI model (Justice et al., 2006). IBC development is one potential fate of UPEC that invade the bladder epithelial cells, but UPEC can also enter a nonreplicating state in these cells (Justice et al., 2004). UPEC quiescent intracellular reservoirs (QIRs) are characterized by small bacterial clusters that are sequestered in a compartment with characteristics of a late endosome or early lysosome (Mysorekar & Hultgren, 2006). If the bladder epithelium is damaged prior to infection, QIRs can also develop in the underlying transitional cells of the bladder that participate in renewal of the bladder epithelium (Mysorekar & Hultgren, 2006). If UPEC QIRs develop in this cell layer, they can contribute to recurrence of infection upon epithelial cell differentiation (Mysorekar & Hultgren, 2006). In addition, because UPEC in QIRs are nonreplicating, they likely contribute to antibiotic tolerance (Anderson et al., 2004). Although UPEC IBC development has been characterized in the mouse UTI model, there is evidence that UPEC replicates in IBCs during human infection. Superficial umbrella cell exfoliation is a host response induced by UPEC infection that causes sloughing of infected cells in the urine (Kau et al., 2005). Examination of urine from UTI patients by microscopy revealed large UPEC communities encased in uroplakin-containing membranes (Rosen et al., 2007). UPEC within these communities had similar morphology and were organized in a matrix, as in the mouse UTI model (Rosen et al., 2007). Filamentous UPEC were also found frequently in urine from UTI patients, suggesting that UPEC uses similar strategies to evade immune responses in mice and humans (Rosen et al., 2007). UPEC is not the only pathogenic bacterial species that forms biofilm-like communities inside host cells. IBCs have also been described for another UTI pathogen, Klebsiella pneumoniae (Hall-Stoodley & Stoodley, 2009), and for P. aeruginosa in cultured tracheal epithelial cells (GarciaMedina et al., 2005). Several other bacterial pathogens that colonize mucosal surfaces as biofilms may also have an intracellular phase to their life cycle, since they are able to invade epithelial cells in vitro and have been observed inside cells in patient biopsies (Hall-Stoodley & Stoodley, 2009). These include, for example, S. aureus, Streptococcus pneumoniae, and Haemophilus influenzae, all of which can cause chronic middle ear infections (Hall-Stoodley &

Stoodley, 2009). Sequestration of these bacteria inside host cells may contribute to evasion of immunity and tolerance of antibiotic therapy.

IMMUNE SUBVERSION: PERSISTENCE BY MANIPULATION OF CYTOKINE PRODUCTION

Regulatory responses that suppress the activity of immune system effector cells are a natural part of the immune response to infection. These modulatory responses are crucial for limiting damage to host tissues caused by inflammation and returning the immune system to a state of homeostasis. Usually, immune-suppressive functions are not induced until after effector cells have eliminated the invading microbe and they serve to terminate the effector response. Some bacterial pathogens, however, subvert these immune-suppressive signaling pathways to ensure their survival (Belkaid, 2007). Pathogens can suppress activity of the immune system either by inducing antigen-presenting cells to produce regulatory cytokines or by inducing differentiation of regulatory T cells (Belkaid, 2007). IL-10 is a potent anti-inflammatory and immunesuppressive cytokine that affects antigen presenting cells and T cells. IL-10 has been called the “macrophage deactivation factor” because it inhibits macrophage production of ROS, RNS, and proinflammatory cytokines, and down regulates expression of MHC class II and costimulatory molecules that are critical for antigen presentation (Redpath et al., 2001). IL-10 also inhibits proliferation of T cells and, when produced by antigen-presenting cells, can instruct naïve T cells to differentiate into an antigen-specific regulatory T-cell population, Tr1 cells, that abundantly secrete IL-10 and suppress inflammation (Belkaid, 2007). IL-10 can be secreted by most cells of the immune system, but is usually produced only late during infection (Redpath et al., 2001). Persistent pathogens may indirectly induce IL-10 production and Tr1 differentiation by interfering with the function of antigen-presenting cells. Repeated exposure of naïve T cells to the same antigen or to deactivated antigen presenting cells induces Tr1 differentiation (Belkaid, 2007). Bacterial pathogens can also directly activate IL-10 production in antigen-presenting cells by expressing specific molecules that manipulate host cell signaling pathways. Following, we describe factors produced by persistent pathogens that manipulate host cell signaling pathways to induce IL-10 production by binding to integrins, TLR2, or the dendritic cell (DC)-specific C-type lectin DC-SIGN. Stimulation of dendritic cell (DC) IL-10 production by a bacterial product was first described for Bordetella pertussis filamentous hemagglutinin (FHA) (McGuirk & Mills, 2002). FHA is an adhesin that mediates B. pertussis binding to host integrins, heterodimeric protein complexes composed of a and b subunits that bind to extracellular matrix proteins. Ligation of DC integrins by FHA activates IL-10 secretion and suppresses production of the proinflammatory cytokine IL-12 (McGuirk & Mills, 2002). DCs stimulated with FHA instruct naïve T cells to differentiate into Tr1 cells, both in vitro and in vivo; these FHA-specific Tr1 cells inhibit IFN-g secretion by Th1 cells and thereby suppress immunity to B. pertussis (McGuirk & Mills, 2002). Integrins bound by FHA include avb3 and the b2 integrin complement receptor 3. Both of these integrins facilitate uptake of antigen from apoptotic cells and induce tolerogenic DCs that secrete IL-10 to prevent activation of the immune system by “self” antigen (Mahnke et al., 2003). Thus, manipulation of integrin signaling may be a general mechanism by which pathogens can induce immune suppression.


TLR2 is a promiscuous receptor that forms heterodimers with either TLR1 or TLR6 to recognize triacylated or diacylated bacterial lipoproteins, respectively (Zähringer et al., 2008). TLR2 has been reported to have both proinflammatory and anti-inflammatory functions, producing signals that can promote either T-helper cells or Tr1 differentiation (Netea et al., 2004). Recent analysis of the Yersinia pestis protein LcrV, which activates IL-10 production in a TLR2-dependent manner, has revealed a specific role for TLR2/6 signaling in the induction of tolerogenic DCs that produce IL-10 and drive differentiation of Tr1 cells (DePaolo et al., 2008). Y. pestis causes plague, an acute infection that is transmitted by fleas. To ensure that fleas ingest bacteria when they take a blood meal, Y. pestis replicates to high titer in the blood (108 bacteria per ml) by inhibiting the inflammatory response. There is strong evidence from animal plague models that Y. pestis inhibits secretion of the proinflammatory cytokines IFN-g and TNF-a by inducing IL-10 production (Brubaker, 2003). IL-10 knockout mice are, in fact, resistant to Y. pestis because they produce a normal inflammatory response to infection (Brubaker, 2003). In vitro, the Y. pestis LcrV protein induces IL-10 and suppresses IL-12 secretion by DCs in a manner that depends on TLR2, TLR6, and the adaptor protein CD14, but not TLR1, suggesting that LcrV signals specifically through a TLR2/6 complex to activate IL-10 production (DePaolo et al., 2008). TLR6 and CD14 deficient mice are relatively resistant to Y. pestis infection, suggesting that immune suppression mediated by the LcrV-TLR2/6 interaction is critical for Y. pestis virulence (DePaolo et al., 2008). Analysis of known TLR2 ligands demonstrated that only TLR2/6 specific ligands, including the model diacylated peptide Pam2CysK4, strongly induced IL-10 production by DCs and promoted differentiation of naïve T cell to Tr1 cells (DePaolo et al., 2008). The proinflammatory effects of TLR2 signaling, in contrast, are mediated by the TLR2/1 heterodimer, since DCs stimulated with triacylated lipoproteins that are TLR2/1 ligands produced copious amounts of IL-12 (DePaolo et al., 2008). The results of this work suggest that diacylated bacterial lipoproteins can hijack the TLR2/6 signaling pathway to promote production of IL-10 by DCs and differentiation of Tr1 regulatory T cells. One pathogen that may depend on TLR2/6 activation and IL-10 production for persistence is Borrelia burgdorferi, the causative agent of Lyme disease. Like Y. pestis, B. burgdorferi is transmitted by an arthropod vector, the Ixodes tick. Although infection of humans represents a dead end for its life cycle, B. burgdorferi can persistently infect joint tissue and cause severe inflammatory arthritis (Singh & Girschick, 2004). The mouse is a natural mammalian reservoir for B. burgdorferi and has been used extensively to model infection. Of the cytokines that have been analyzed in the mouse model, only IL-10 significantly contributes to the outcome of B. burgdorferi infection. IL-10 deficient mice harbor reduced B. burgdorferi bacterial loads in chronically infected tissues, albeit at the cost of increased inflammation (Brown et al., 1999). The B. burgdorferi genome encodes an abundance of putative lipoproteins (more than 150) (Radolf & Caimano, 2008) that are presumed to be triacylated and thus proinflammatory, based on putative acyl-transferase enzymes encoded by the B. burgdorferi genome (Cullen et al., 2004). There is mounting evidence, however, that B. burgdorferi and its purified lipoproteins induce IL-10 production by antigenpresenting cells (Guillermo et al., 2002; Lazarus et al., 2008; Murthy et al., 2000). In fact, stimulation of human monocytes with B. burgdorferi makes the cells tolerant (i.e., less

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responsive) to subsequent proinflammatory stimuli, and this activity requires TLR2 and IL-10 production (Diterich et al., 2003). Since B. burgdorferi lipoproteins induce production of IL-10, perhaps some of these lipoproteins are diacylated and activate the TLR2/6 receptor complex. The B. burgdorferi lipoprotein OspC is critically important for transmission from the tick vector to the mammalian host. OspC prevents clearance of B. burgdorferi from the site of inoculation by cells of the innate immune system and promotes dissemination to other tissues (Radolf & Caimano, 2008). Recent work suggests that OspC can be functionally replaced by overexpression of other B. burgdorferi lipoproteins (Xu et al., 2008). It is tempting to speculate that the common function of B. burgdorferi lipoproteins is to interact with the TLR2/6 receptor to induce IL-10 production, and thereby suppress immunity. A third host cell receptor that can be exploited by pathogens to suppress the immune response is DC-SIGN, a C-type lectin that facilitates adhesion of DCs to endothelial cells and naïve T cells, and that functions as a pattern recognition receptor by binding to mannose-containing carbohydrates of pathogens. Interaction of DC-SIGN with specific ligands, including the M. tuberculosis cell wall component, mannosylated lipoarabinomannan (ManLAM), interferes with the response of DCs to other TLR specific ligands, such as the TLR4 ligand LPS (Geijtenbeek et al., 2003; Geijtenbeek & Gringhuis, 2009). The interaction between ManLAM and DC-SIGN stimulates production of IL-10 and prevents LPSinduced DC maturation by triggering a signal transduction cascade that leads to acetylation of the transcription factor nuclear factor-kB (NF-kB) (Geijtenbeek et al., 2003; Gringhuis et al., 2007). Acetylated NF-kB has altered properties that enhance and prolong transcriptional activation from the IL-10 promoter. Because NF-kB is maintained in an inactive state in the cytoplasm by inhibitory IkB proteins, triggering of DC-SIGN by ManLAM only influences NF-kB transcriptional activity in DCs that have already activated NF-kB, for example, by activation of signal transduction pathways downstream of TLRs (Geijtenbeek & Gringhuis, 2009). Thus, M. tuberculosis ManLAM modulates the response of DCs to produce more IL-10 and instruct naïve T cells to differentiate into regulatory T cells. Whether activation of DC-SIGN signaling by ManLAM is beneficial for M. tuberculosis or for the host is still a matter of debate. Because the interaction between ManLAM and DC-SIGN is specific for the human DC-SIGN receptor (Geijtenbeek et al., 2003), it has been difficult to examine the role of DC-SIGN in experimental animal models of infection. Recently, transgenic mice expressing the human DC-SIGN receptor were described. These mice exhibited no difference in the ability to control M. tuberculosis replication in the lung, but had reduced tissue pathology and survived longer than control mice (Schaefer et al., 2008). These results suggest that DC-SIGN-mediated host recognition of pathogens may be an appropriate host response that induces tolerance to specific pathogens to protect the host from excessive immune-mediated tissue damage.

IMMUNE SUPPRESSION: PERSISTENCE BY HOST INTOXICATION

For some bacterial pathogens, persistence in the host depends on the production of protein toxins that interfere with cellular physiology. Bacterial toxins can be delivered to host cells by several pathways. Some toxins, more commonly termed “effectors,” are directly translocated from the bacterial pathogen to the host cell cytoplasm by specialized secretion systems. We have already briefly discussed

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bacterial effectors in the context of pathogen interference with phagocytic cell functions. Translocated effectors can also induce bacterial uptake by cells that are normally nonphagocytic, modulate host cell death pathways, and interfere with innate immune signaling (Galán, 2009; Mattoo et al., 2007). Other bacterial toxins are secreted to the extracellular milieu by the general secretion machinery and can thus act at a distance from the bacterial cell, but they must bind to specific host cell receptors to exert their function. Secreted toxins can influence host cell physiology by interacting with cell surface receptors to modulate signal transduction pathways, enzymatically modifying cell surface proteins, forming permeable pores in the host cell membrane, or triggering their internalization to reach intracellular targets (Cover, 2005). Several secreted bacterial toxins have been identified that contribute to persistent infections. Cytolethal-distending toxin (CDT) is produced by many gram-negative bacterial species that colonize the gastrointestinal tract, including Campylobacter jejuni, a common cause of food-borne diarrheal illness, and Helicobacter hepaticus, a mouse pathogen that causes chronic hepatitis. The active subunit of CDT has DNase I-like activity that damages DNA in target host cells, leading to cell cycle arrest, and, in some cell types, programmed cell death. For both H. hepaticus and C. jejuni, there is evidence that persistent colonization of the mouse gastrointestinal tract requires production of CDT (Ge et al., 2008). Since CDT is cytotoxic to cells of the immune system— including antigen-presenting cells, T cells, and B cells—CDT is probably required for these bacteria to escape immune surveillance (Ge et al., 2008). Bordetella species, which cause persistent infections of the mammalian respiratory tract, manipulate the immune system using the adenylate cyclase toxin, CyaA, a bifunctional toxin that forms pores in target host cell membranes and synthesizes the second messenger signaling molecule cyclic AMP (cAMP). CyaA specifically targets antigen-presenting cells, including macrophages and DCs that express its receptor, complement receptor 3 (Vojtova et al., 2006). In these cell types, cAMP suppresses secretion of the proinflammatory cytokines IL-12 and TNF-a, and enhances LPS-induced production of anti-inflammatory IL-10 (Vojtova et al., 2006). Thus, intoxication of antigen-presenting cells by CyaA may promote the expansion of regulatory T-cell populations and suppress immunity. Helicobacter pylori is another bacterial pathogen that produces toxins to manipulate the immune system. H. pylori persistently colonizes the stomachs of about half the world’s human population and is highly adapted to growth in this acidic environment (Algood & Cover, 2006). Upon entry into the stomach, H. pylori uses flagellar motility to penetrate the gastric mucus layer and colonize the gastric epithelium (Algood & Cover, 2006). Although the epithelial surface of the gastric mucosa is significantly more neutral (pH  5.5) compared to the lumen of the stomach (pH , 2), H. pylori still requires mechanisms to resist acid shock and to grow at low pH. H. pylori acid resistance is primarily mediated by the enzymes urease and carbonic anhydrase. Urease hydrolyzes urea to generate ammonium and CO2; carbonic anhydrase subsequently converts the CO2 to bicarbonate (HCO32), which buffers the H. pylori periplasm and cytoplasm (Kusters et al., 2006). In addition to surviving stomach acid, H. pylori must obtain nutrients for replication. H. pylori uses a type IV secretion system to deliver an effector protein, CagA, to gastric epithelial cells. CagA interferes with host cell signaling pathways to disrupt epithelial cell polarity and the tight junctions between epithelial cells. These disruptions release nutrients on the apical surface of the epithelial cell that H. pylori uses for biomass growth (Backert & Selbach, 2008; Tan et al., 2009).

Although most people colonized with H. pylori do not exhibit any overt symptoms of disease, H. pylori infection induces an inflammatory response termed “chronic superficial gastritis” that is characterized by influx of macrophages, neutrophils, DCs, T cells, and B cells. This chronic inflammation is probably caused by continuous stimulation of the innate immune response and a cellular immune response dominated by IFN-g-secreting T cells, which can, over time, lead to development of gastric ulcers and gastric cancer (Algood & Cover, 2006; Kusters et al., 2006). Despite eliciting a robust inflammatory response, H. pylori persists in the gastric mucosa for the lifetime of its human host and, based on the frequency of reinfection, rarely induces protective immunity, suggesting that H. pylori directly interferes with immune system function (Algood & Cover, 2006). Since H. pylori binds to DC-SIGN and the number of regulatory T cells is increased in the gastric mucosa of H. pylori infected individuals, it is possible that H. pylori, like M. tuberculosis, subverts immunity by manipulating DCs to produce the anti-inflammatory cytokine IL-10 (van Kooyk & Geijtenbeek, 2003). Here, we elaborate on H. pylori toxins that suppress immunity by intoxicating T cells. The H. pylori vacuolating cytotoxin VacA, so named because it induces formation of large intracellular vacuoles in cultured mammalian cells, affects the function of epithelial cells, phagocytic cells, B cells, and T cells. Following endocytosis by target cells, VacA forms anion-selective channels in the endosomal membrane and conducts chloride ions through these channels to the lumen of the endosome, which triggers osmotic movement of water and vacuole swelling (Cover, 2005). In phagocytic cells and gastric epithelial cells, which H. pylori can occasionally invade (Dubois & Boren, 2007), VacA induces the formation of large vesicular compartments known as megasomes (Cover, 2005). VacA may promote survival of H. pylori in this compartment by directly interfering with phagosome maturation or by decreasing the concentrations of microbicidal factors (Cover, 2005). VacA-mediated vacuolation of antigen-processing endocytic compartments also inhibits antigen presentation by B cells (Cover, 2005). The effects of VacA on T cells, however, are more complex and involve several intoxication mechanisms. Inhibition of T cell function by VacA was first suggested by the observation that H. pylori culture supernatants suppressed proliferation of cultured T-cell lines and primary human T lymphocytes in a VacA-dependent manner (Gebert et al., 2003). VacA inhibits nuclear translocation of the transcription factor NFAT (nuclear factor of activated T cells) and thereby prevents induction of IL-2, a cytokine that promotes T-cell survival (Gebert et al., 2003). Nuclear translocation requires activation of NFAT by the calcium-responsive phosphatase calcineurin. Normally, antigen binding to the T-cell receptor triggers calcium influx leading to NFAT activation. VacA blocks calcium mobilization by forming anion-selective channels that depolarize the T-cell plasma membrane (Boncristiano et al., 2003). VacA channel forming activity has also been implicated in an IL-2-independent and NFAT-independent block in cell cycle progression that prevents T-cell proliferation, but the underlying mechanism of this cell cycle block has not been elucidated (Sundrud et al., 2004). VacA can also interfere with T-cell function by a mechanism independent of its channel-forming activity. A Cterminal fragment of VacA that does not form ion channels and therefore does not induce cellular vacuolation still binds to T cells and modulates signal transduction pathways (Boncristiano et al., 2003). Either the C-terminal fragment or full-length VacA induces phosphorylation of the guanine


nucleotide exchange factor Vav and subsequent activation of the small GTPase Rac, which is involved in actin cytoskeleton rearrangements (Boncristiano et al., 2003). VacAtriggered reorganization of the actin cytoskeleton may inhibit formation of a functional immunological synapse between intoxicated T cells and antigen-presenting cells and thereby prevent T-cell activation (Boncristiano et al., 2003). Recently, a VacA receptor specific for activated and migrating primary human T lymphocytes was identified. CD18 is a b2 integrin that mediates T-cell adherence to the vascular endothelium and interaction with antigen-presenting cells. VacA colocalizes with CD18 on activated T cells and is internalized upon T-cell migration, resulting in vacuolation and inhibition of NFAT activation and IL-2 secretion (Sewald et al., 2008). Primary murine T cells are insensitive to the VacA toxin in part because they lack a specific receptor. A “humanized” murine T cell line that expresses human CD18 bound and internalized VacA, leads to vacuolation (Sewald et al., 2008). Internalized VacA did not, however, inhibit IL-2 secretion by the CD18-humanized murine T cells, suggesting that VacA targets cellular signaling pathways that are specific to human T cells. The inability of VacA to interfere with murine T-cell function may explain why only a few H. pylori human isolates have been successfully adapted to persistently colonize the mouse stomach (Algood & Cover, 2006). Paradoxically, although VacA is a potent inhibitor of human T-cell function, a DvacA H. pylori strain still inhibits T-cell proliferation (Gebert et al., 2003). Analysis of H. pylori culture supernatants revealed that a secreted protein, distinct from VacA, arrested T cells in the G1 phase of the cell cycle and thereby blocked proliferation independent of NFAT activation and IL-2 induction (Gerhard et al., 2005). Biochemical purification of the factor identified it as the enzyme g-glutamyl transpeptidase (GGT), which catalyzes the transfer of a g-glutamyl moiety between substrate peptides (Schmees et al., 2007). Purified recombinant H. pylori GGT inhibited T-cell proliferation in a manner dependent on enzyme activity, and an H. pylori Dggt mutant failed to inhibit T-cell proliferation (Schmees et al., 2007). T cells treated with wild-type H. pylori, but not the Dggt mutant, exhibited altered Ras-dependent protein phosphorylation and changes in the levels of the cyclin proteins that control cell cycle progression, suggesting that GGT modulates T-cell signal transduction pathways (Schmees et al., 2007). Both H. pylori VacA and GGT intoxicate T cells and prevent their proliferation in response to antigen stimulation. These factors have thus far been characterized only in vitro in cell culture systems, and it is not yet clear whether these toxins are also effective in vivo. T cells are present only on the basolateral surface of the gastric epithelium, not on the apical surface that H. pylori colonizes, so it is uncertain how these secreted bacterial toxins access their T-cell targets. It is possible that breaches in the epithelial cell permeability barrier, caused by CagA and VacA intoxication of epithelial cells, are sufficient to allow penetration of H. pylori secreted proteins to the basolateral surface of the mucosa (Algood & Cover, 2006). Given the apparent host specificity of VacA, however, it will be a challenge to demonstrate interference with T-cell function in animal models of infection.

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35 Suppression of Immune Responses to Protozoan Parasites DAVID L. SACKS

INTRODUCTION

And, Toxoplasma, Leishmania, and T. cruzi remodel or escape the endocytic compartments to insulate themselves from lysosome fusion and destruction (reviewed in Sacks & Sher, 2002). The specific host-cell signaling pathways that are targeted by intracellular pathogens to suppress the antimicrobial activities of mononuclear phagocytes is extensively reviewed in chapter 36 in this volume. This discussion will focus on the manner in which protozoan pathogens have learned to condition their initial encounter with dendritic cells (DCs) as a means to suppress or delay the onset of the adaptive response. DCs represent a minor, heterogeneous population of hematopoietic cells that coevolved with the development of the adaptive immune system. DCs possess unique functional attributes that have earned them the designation “professional” antigen-presenting cells, including their ability to efficiently take up and process macromolecules and cell associated antigens, their ability to up regulate expression of MHC class II and costimulatory molecules, their ability to secrete IL-12 (interleukin-12) and other Th1 instructional cytokines, and their ability to migrate from sites of antigen uptake to sites of T-cell interactions in lymphoid tissue. One or more of these functions have been found compromised following their encounter with protozoan parasites, as summarized in Table 1. One of the first studies to look at DC interactions with malaria parasites employed an in vitro human system involving Plasmodium falciparum (Urban et al., 2001). Coculture of monocyte derived DCs (MDDCs) with P. falciparum-infected erythrocytes inhibited the maturation response, including the LPS (lipopolysaccharide)-induced up regulation of MHC class II, adhesion molecules (ICAM-1), and costimulatory molecules (CD83 and CD86). The DCs were severely impaired in their ability to induce allogeneic and antigen-specific primary and secondary immune responses. The process involved the binding of iRBC to CD36 on DCs, which may mimic the suppressive signals induced by the binding and uptake of apoptotic cells. More recent findings suggest that only very high doses of iRBCs will suppress the function MDDCs, and that an alternative receptor, chondroitin sulfate A, may be involved (Elliott et al., 2007). The parasite ligands implicated in these interactions include a conserved domain of P. falciparum erythrocyte

The hallmark of infections caused by parasitic protozoa is chronicity, designed to maintain the transmission of vector-borne (e.g., malaria, Leishmania, African trypanosomes, Trypanosoma cruzi), or food- or water-borne pathogens (e.g., Toxoplasma, Entamoeba, Giardia) that might otherwise fail to sustain their life cycle were they to be eliminated during the acute stages of infection in their mammalian hosts. For all of these organisms, the development of chronicity depends, at least in part, on finely tuned mechanisms of immunoregulation that serve both to prevent parasite elimination and to suppress host immunopathology. This chapter will focus on three protozoan parasites: malaria, Leishmania, and T. cruzi, that have in common their transmission by insect vectors, their intracellular lifestyle in the mammalian host, and the requirement for Th1 responses to control these infections. The chapter will discuss the mechanisms used by these parasites to actively suppress the development and/or expression of cell-mediated immunity that contribute to the state of equilibrium that is established between the host and the parasite in sites of chronic infection.

SUPPRESSION OF DENDRITIC CELL FUNCTION

A series of important adaptations occur during the initial establishment of infection when parasites that have gained entry into the host are confronted by the innate immune system. These innate defenses include the epithelial barrier of the skin, humoral factors such as the alternative complement system, and the toxic metabolic products and hydrolases of mononuclear phagocytes. Protozoan pathogens have evolved specific mechanisms to evade destruction by these host defenses. For example, the infective stages of T. cruzi and Leishmania prevent the stable formation and surface deposition of C3 convertases require for complement lysis. Leishmania inhibit the assembly of NADPH oxidase complex necessary for killing during phagocytosis by macrophages. David L. Sacks, Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 126, 4 Center Dr., MSC 0425, Bethesda, MD 20892-0425.

441

442


TABLE 1 Parasite Malaria

T. cruzi

Leishmania

a b

Dendric cell (DC) functional defects induced by protozoan pathogens Inhibitory signala

DCb

Response defect (Reference)

P. falciparum iRBC, PfEMP1 binding to CD36

Human MDDC

Poor maturation and APC function (Urban et al., 2001)

P. falciparum iRBC, PfEMP1 binding to CSA

Human MDDC

Poor maturation and APC function (Elliott et al., 2007)

P. falciparum hemozoin

Human MDDC

Poor maturation (Skorokhod et al., 2004)

P. chaubaudi iRBC, hemozoin

Mouse splenic DC

Poor T- and B-cell priming (Millington et al., 2006)

P. yoelli lethal strain in vivo

Mouse splenic DC

Low IL-12 and T-cell priming (Wykes et al., 2007).

P. yoelli, later stage infection with nonlethal strain in vivo

Mouse splenic DC

Low IL-12, TNF-a, increased IL-10 (Perry et al., 2005)

P. yoelli, later stage infection with nonlethal strain in vivo

Mouse splenic regulatory DC

Low CD40, increased IL-10 (Wong & Rodriguez, 2008)

P. chaubaudi, acute stage infection in vivo

Mouse splenic CD82 DC

Enhanced activation of Th2 responses (Sponaas et al., 2006)

Blood stage trypomastigotes

Mouse BMDDC

Poor maturation, increased TGFb /IL-10 (Poncini et al., 2008)

Trypomastigote GIPL- ceramide portion

Human MDDC

Poor maturation, low IL-12, TNFa (Brodskyn et al., 2002).

T. cruzi HSP70

Human MDDC

Reduced IL-12p40, enhanced IL-10 secretion (Cuellar et al., 2008)

Metacyclic promastigotes from multiple species

Human MDDC

Low CD40L induced IL-12p70 (McDowell et al., 2002)

L. donovani infection in vivo

Mouse splenic regulatory DC

Enhanced IL-10 secretion, Tr1 cell activation (Svensson et al., 2004).

Antibody opsonized L. amazonensis amastigotes

Mouse BMDDC

Enhanced IL-10 secretion, Tr1 cell activation (Wanasen et al., 2008)

L. major phosphoglycan (PG) deficient lines in vivo and in vitro

Mouse BMDDC, lymph node DC

PG-deficient lines enhanced IL-12p40 secretion and Th1 responses (Liu et al., 2009)

iRBC, infected red blood cell; CSA, chondroitin sulfate A; GIPL, glycoinositolphospholipid. MDDC, monocyte derived DC; BMDDC, bone marrow derived DC.

membrane antigen (PfEMP1), and hemozoin, a byproduct of infected erythrocytes that has been shown to directly inhibit the maturation of human DCs (Skorokhod et al., 2004). In a rodent model of malaria, hemozoin-containing DCs could be identified in T-cell areas of the spleen, and were shown to inhibit B-cell responses to heterologous antigens in vivo (Millington et al., 2006). Importantly, mice infected with a nonlethal malaria strain accumulated functional DCs in the spleen that could transfer protection that was dependent on IL-12, whereas those infected with a lethal strain lacked functional DCs (Wykes et al., 2007). Even during nonlethal infection, splenic DCs were found to become refractory to Toll-like receptor (TLR)-mediated IL-12 and TNF-a (tumor necrosis factor a) production later during infection, while increasing their ability to produce IL-10 (Perry et al., 2005). This TLR tolerance is similar to that induced by endotoxin shock, and may explain the prevalence of regulatory CDllclowCD45RBhigh DCs later in infection that were shown to induce IL-10 secreting T cells as a negative feedback mechanism to control immunopathology (Wong & Rodriguez, 2008). A regulatory CD82 DC subset supporting IL-10 and IL-4 production from MSP-1 specific CD4 T cells was shown to emerge as the predominant APC (antigen-presenting cell) population in the spleen during the later stages

of Plasmodium chabaudi infection in mice (Sponaas et al., 2006). The relevance of these findings to human disease is indicated by the observation that the percentage of HLA-DR1 DCs was significantly lower in children with severe or mild malaria compared to healthy controls, and they also had increased frequencies of DCs expressing BDCA-3, a marker that is up regulated on IL-10-treated MDDC (Urban et al., 2006). Together, the mouse and human studies are consistent with functional DCs, priming T-cell responses to blood stage antigens early in infection, followed by the emergence of regulatory DCs that modulate the effector response, limiting both immunopathology and pathogen clearance. Infection with T. cruzi parasites typically produces an acute blood and tissue parasitemia that is controlled, followed by long-term persistence of low numbers of parasites in muscle and nerve tissue, leading to chronic inflammation in these tissues and the formation of Chagas’ disease. Compromised DC function has been linked to immune suppression in chronically infected mice. T. cruzi blood stages inhibited the LPS-induced activation of mouse bone marrow derived DC, including their up regulated expression of MHC class II and costimulatory molecules, and their capacity to stimulate mixed lymphocyte reactions (Poncini et al., 2008; Van Overtvelt et al., 1999). Both IL-10 and

35. Suppression of Immune Responses to Protozoan Parasites

TGF-b (transforming growth factor b) were shown to be important in the induction of the regulatory DC phenotype. The ceramide portion of glycoinositolphospholipid (GIPL) from T. cruzi could reproduce many of the effects of the live parasite on DC function, though the concentrations used were nonphysiological (Brodskyn et al., 2002). The limited human data on APC function in Chagas’ disease patients indicates that monocytes from patients with the indeterminate or mild form of the disease displayed lower levels of HLA-DR and higher levels of IL-10, compared with monocytes from cardiac-disease patients, suggesting a role for these cells in modulating the pathologic response (Souza et al., 2004). By contrast, MDDC from chronic cardiac chagasic patients stimulated with T. cruzi HSP70 that is capable of maturing human DCs produced a higher level of IL-10 and a lower level of IL-12 compared to cells from healthy donors, suggesting that the MDDC may contribute to parasite immune evasion (Cuellar et al., 2008). In leishmaniasis, there has been a concerted effort to understand the immunologic defects controlling both the delayed onset of acquired resistance and the long-term persistence of organisms in the immune host following clinical cure. Of greatest relevance may be the fact that Leishmania appear not to express strong TLR agonists that, on their own, can fully activate DCs. In a survey of various Leishmania species for the ability of infective metacyclic stage promastigotes to activate human MDDC for IL-12p70 secretion, none of the strains was sufficient on their own despite their efficient and comparable uptake by the DCs, and only Leishmania major primed the cells for a subsequent CD40L-induced response (McDowell et al., 2002). The observations may be significant because whereas L. major produces self-limiting infections that are controlled in the skin, the other strains tested (i.e., Leishmania donovani, Leishmania tropica) typically disseminate from the skin to the viscera or to other cutaneous sites. In the mouse, while promastigotes of some Leishmania strains can induce dermal DCs and myeloid DCs to mature, on their own the parasites only transiently activate these cells to produce IL-12p40 and little or no IL-12p70 (reviewed in Soong, 2008). Infection of DCs with amastigotes from New World species that are associated with profoundly impaired immune responses in mice, failed to activate mDCs, even in conjunction CD40 agonist, and furthermore inhibited their responsiveness to LPS and IFN-g (interferon g) that was due, at least in part, to deficient STAT1 and STAT2 phosphorylation (Xin et al., 2008). DCs with clear regulatory properties have been described in the context of chronic infection with L. donovani in mice. These CD11clo CD45RB1 CD11b1 IL-10 producing cells emerge as the predominant DC subset in the VL spleen to induce antigen-specific tolerance in vivo. Importantly, spleen stromal cells from infected mice can direct the development of regulatory DCs from bone marrow progenitor cells in the absence of exogenous cytokines (Svensson et al., 2004). Evidence that Leishmania-derived molecules can directly compromise DC function was obtained from L. major mutants specifically deficient in biosynthesis of surface and secreted phosphoglycan-containing molecules that induced increased levels of IL-12p40 in BMDDC in vitro and promoted Th1 priming in vivo (Liu et al., 2009).

SUPPRESSION OF IMMUNE RESPONSES BY REGULATORY AND IL-10 PRODUCING T CELLS

The balance between proinflammatory and regulatory immune mechanisms is crucial in determining the outcome of parasitic infections. A number of regulatory T-cell

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populations have been described that have in common their ability to suppress the generation and function of effector T cells. These include (i) natural Treg (nTreg), which are generated in the thymus, constitutively express the IL-2Ra chain (CD25) and the transcription factor forkhead box P3 (Foxp3), and may or may not rely on IL-10 or TGF-b for their suppressive function in vivo; (ii) induced Treg (iTreg), which are converted from activated CD252Foxp32 CD41 T cells to CD251Foxp31 suppressor cells following an encounter with antigen and TGF-b in the periphery; (iii) adaptive Treg or Tr1 cells, also generated from naïve cells in the periphery in response to immature DCs and/or IL-10 from APCs, but which remain Foxp32 and secrete high levels of IL-10; and (iv) Foxp32T-bet1 Th1 effector cells, activated by high-dose antigen and IL-12 or IL-27 to secrete IL-10 as a mechanism of feedback control. There are data linking one or more of these populations to the suppression of immune responses to each of the protozoan pathogens considered in this review, as summarized in Fig. 1.

Malaria

In malaria, a strong proinflammatory response, characterized by IFN-g and TNF-a, contributes to the initial killing and clearance of malaria-infected RBC. High production of TGF-b and IL-10 during the first few days of infection, as occurs with the Plasmodium yoelii 17XL strain in mice, is responsible for the inhibition of proinflammatory responses and the early lethality of this strain. At the same time, a number of studies have highlighted the important roles for IL-10 and TGF-b in regulating immunopathology during malaria infection (reviewed in Riley et al., 2006). In mice, in vivo neutralization of IL-10 promoted severe disease following Plasmodium berghei infection in resistant mice, and Plasmodium chabaudi infections were universally fatal following TGF-b neutralization in BALB/c mice or in C57Bl/6 IL-102/2 mice. In humans, high ratios of inflammatory to regulatory cytokines are associated with decreased risk of malaria infection but increased risk of clinical disease in those who do become infected. Extensive literature now exists for both humans and mice that address the nature of the regulatory cells and cytokines that control malaria immunity and pathology. The studies by Hisaeda and colleagues (Hisaeda et al., 2004) were the first to suggest a role for nTreg in compromising immunity to malaria infection. Depletion of Treg prior to and at the time of challenge using anti-CD25 antibodies rescued mice from lethal infection with P. yoelli that was associated with enhancement of parasite driven T-cell responses. However, in a subsequent, more comprehensive analysis of Treg function in P. yoelli infected mice, antiCD25 treatment had no effect on the course of lethal infection, neither did adoptive transfer of CD25 depleted, naïve T cells to Rag-12/2 mice (Couper et al., 2008). Importantly, the IL-10 that was preventing wild-type mice from surviving a low-dose challenge was produced almost exclusively by CD252Foxp32 CD41 T cells (i.e., Tr1 cells). Moreover, the reason these cells failed to rescue Rag-12/2 mice from being killed by the high-dose challenge was because, in their absence, despite better control of infection, immunopathology was exacerbated. IL-10 was also found to suppress pathology in a mouse model of cerebral malaria (ECM) following infection with P. berghei ANKA (PbA) (Kossodo et al., 1997). Since mice depleted of Foxp31 Treg cells by treatment with anti-CD25 mAb prior to infection were actually protected against ECM, it is clear that these cells are not the source of disease protective IL-10 in this model (Amante et al., 2007). The role of Foxp31 Treg in promoting ECM was associated with suppression of antiparasite responses,

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FIGURE 1 Subsets of regulatory and IL-10 producing T cells implicated in the suppression of immune responses to protozoan pathogens. nTregs undergo selection in the thymus for relatively high affinity recognition of self-peptide/MHC (major histocompatibility complexes). Their expansion in response to parasitic infection can be due to parasite antigens bearing cross-reactive epitopes with the self, or to activation of self-reactive clones in inflammatory sites rich in IL-2 and activated DCs. In most experimental and clinical studies in which the expansion of Foxp31 Treg cells have been described, it is not possible to distinguish their origin from iTregs, which are generated following an encounter of conventional T cells with antigen in the periphery and in response to high levels of TGF-b. The presence of iTregs in patients with malaria is inferred from antigen specificity and correlation with elevated serum concentrations of TGF-b. Tr1 cells are peripherally differentiated IL-101Foxp32CD41 T cells generated in response to antigen presented by regulatory DCs, which typically express subimmunogenic levels of antigen-MHC and costimulatory molecules, and secrete IL-10. IL-10 Th1 cells are T-bet1IFN-g1 effector cells that simultaneously secrete IL-10. Their development can be driven by high dose or persistent antigen and IL-12 or IL-27.

since the depleted mice had fewer parasites in the brain and vasculature. The argument that the real ameliorating effect of the anti-CD25 mAb was to also deplete activated, disease-promoting CD41 and CD81 T cells, was countered by the finding that these effector responses were in fact enhanced, and that mice treated 2 weeks prior to infection were still protected against ECM. This experiment also suggests that natural Treg, but not converted Treg, cells were involved in ECM pathogenesis, since most of the antibody would have been cleared from the blood by the time any infection-induced Treg cells might appear. However, these findings must contend with similar experiments in which anti-CD25 mAb treatment at the time of infection, while protecting the mice against early mortality due to ECM, eventually led to parasitemic death a few days later, suggesting depletion of effector cells (Vigario et al., 2007). These studies point out the serious difficulties in interpreting Treg depletion experiments, particularly when disease outcomes are controlled by such a delicate balance between immunity and pathology as mediated by the same effector cells, and that may be targeted for removal along with the Treg. Using the recently described DEREG mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-enhanced GFP (eGFP) fusion protein

under the control of the foxp3 gene locus, it is now possible to specifically deplete Foxp31 Treg in vivo (Lahl et al., 2007). Importantly, when the DEREG mice were infected with PbA, no difference in the severity or incidence of ECM was observed, strongly suggesting that nTreg and/or iTreg play only a limited role in controlling this pathology (Steeg et al., 2009). Elucidating the role of Treg in human malaria infection is further challenged by the inability to experimentally address the function of Treg subsets in vivo, and by the difficulty in following the dynamics of effector and regulatory T-cells responses from an uninfected state through to acute and chronic infection or cure. Sporozoite challenge by infected mosquitoes to evaluate the efficacy of a pre-erythrocytic stage malaria vaccine in human volunteers afforded the opportunity to study immune responses during the prepatent and acute stages of malaria infection (Walther et al., 2005). The most striking finding was the marked peak of bioactive TGF-b in the plasma within 12 hours of the first detection of circulating Plasmodium falciparum DNA in individuals whose subsequent parasitemias rose significantly faster than those individuals who did not make an early burst of TGF-b. The other key observation was that the TGF-b concentration correlated with the up regulation of CD41CD25hiFoxp31


lymphocytes in peripheral blood, whose removal enhanced antigen-induced T-cell proliferation and IFN-g production in vitro. The data are consistent with infection induced, TGF-b driven conversion of conventional T cells into iTreg that enhance blood-stage parasite growth, but that may also contribute to the control of inflammatory pathologies and help to moderate clinical disease. The seasonal variation in numbers of both circulating CD41Foxp31CD27lo/2 cells, currently the most discriminating markers for classical Treg in humans, as well as Tbet1 cells in healthy, malaria-exposed individuals, provides further evidence that antigen-driven Treg and T-effector cells are coregulated to maintain control of both parasitemia and pathology during repeated exposure to malaria (Finney et al., 2009). A deficit in Foxp31 Treg numbers and function was found associated with the lower susceptibility to P. falciparum malaria of ethnic Fulani in West Africa compared with other sympatric ethnic groups (Torcia et al., 2008). Whether the deficit was due to an inherent difference in the frequency of natural Treg or antigendriven, converted Treg was not determined. Nor was there any indication that amongst the Fulani who did develop clinical malaria, their risk of severe disease was increased due to their reduced number of Treg. This issue has been specifically addressed in a recent study of Gambian children with severe versus uncomplicated P. falciparum malaria, with the result that no difference in the number or function of CD41Foxp31CD27lo/2 Treg was observed (Walther et al., 2009). Importantly, the proportion of CD41 T cells simultaneously producing IL-10 and IFN-g was threefold higher in uncomplicated cases than severe cases, suggesting that as proinflammatory responses intensify and pose a heightened risk of immune-mediated complications, the ability of Th1 cells to self-regulate by producing IL-10 provides a critical layer of homeostatic control.

Leishmaniasis

In leishmaniasis, aside from the Th2 polarizing conditions that underlie the extreme susceptibility of BALB/c mice to cutaneous strains of Leishmania, chronic forms of cutaneous or visceral disease in humans are better explained by ongoing Th1 or mixed Th1/Th2 responses compromised in intensity or function by suppressor cells, especially regulatory T-cell subsets, and the production of suppressive cytokines, IL-10 and TGF-b. The clinical data has consistently supported an association of elevated IL-10 levels and the pathogenesis of visceral leishmaniasis (VL) (reviewed in Nylen et al., 2007). IL-10 has been implicated in the suppression of antigen-specific T-cell responses in human VL based on the elevated levels of IL-10 observed in plasma and in target organs like the spleen and bone marrow, and the reconstitution of antigen-specific responses when IL-10 is neutralized in vitro. Continued production of IL-10 following a clinical cure has been strongly linked to the development of post-kala-azar dermal leishmaniasis (PKDL), which is a reactivation disease. Importantly, murine models of VL have established the potential of in vivo neutralization of IL-10 as an adjunct therapy to dramatically enhance the antileishmanial activity of pentavalent antimony, still the first-line drug for treatment of VL (Murray et al., 2002). Clinical trials involving IL-10 neutralization or IL-10 receptor blockade will ultimately be needed to validate the role of IL-10 in the pathogenesis of human VL and PKDL. In the meantime, the source of elevated IL-10 in these patients remains an important issue, especially as the murine models have implicated multiple and diverse subsets of IL-10 producing cells, including nTreg, Th1, Th2, macrophages, NK (natural killer) cells, and B cells. In Indian kala-azar patients, the high levels of splenic IL-10 mRNA was primarily

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found in the CD252Foxp32 T cells, which accumulate in the VL spleen (Nylen et al., 2007). Furthermore, natural Treg cells were not found to be elevated in the blood or accumulate in the spleen in active VL cases. Thus, parasite-driven IL-10 secreting T cells, either Tr1 cells or Th1 effector cells, appear to be more important than nTreg or iTreg in suppression of antileishmanial immunity in human VL, although the relevance of IL-10 from these cells to suppression remains to be shown. In line with these clinical findings, in L. donovani-infected mice, IL-10 production by splenic CD41CD252 T cells strongly correlates with disease progression (Stager et al., 2006), and in IL-102/2 mice in which expression of a human IL-10 transgene was restricted to myeloid cells, the absence of T cell IL-10 failed to recapitulate the parasite persistence seen in the wild-type mice (Ranatunga et al., 2009). In cutaneous leishmaniasis, elevated IL-10 in lesional tissue is again a consistent finding in the more chronic forms of disease, as well as a predictor of a poor response to treatment (Melby et al., 1994; Salhi et al., 2008). An IL-10 promoter region polymorphism associated with increased production of IL-10 by LPS-stimulated PBMCs was also found strongly associated with an increased risk of developing active lesions following infection by Leishmania braziliensis. There is some evidence that Foxp31 cells accumulate in active lesions and may be a source of localized IL-10 production in the inflammatory site (Campanelli et al., 2006). Comparing relative tissue levels of Foxp3 mRNA, recruitment of Treg cells from the blood to the lesion was implied, as was the ability of intralesional CD41CD251 T cells to suppress antigen-specific IFN-g production by responder cells (Bourreau et al., 2009). These clinical studies are unable to address whether the accumulation of Foxp31 cells in the lesion represent natural or induced Treg, and in no case has the Treg been shown to be a critical source of IL-10 (or TGF-b) that can suppress the effector response and promote infection. These questions have been more directly addressed in mouse models of cutaneous leishmanias (CL). Based on the results using IL-10 deficient or anti-IL-10R antibody treated mice, IL-10 is known to play an essential role in the evolution of nonhealing lesions and in promoting L. major persistence following clinical cure in resistant mice (reviewed in Sacks & Anderson, 2004). As the site of chronic infection in the skin contains IL-10 producing CD41 cells, the relationship of these cells to regulatory cell subsets has been carefully investigated. CD41CD251 Foxp31 regulatory T cells were found to accumulate in sites of persistent L. major infection in the skin, at least some of which were nTreg, based on adoptive transfer of positively selected CD251 T cells from naïve mice that homed to the site of L. major challenge in Rag12/2 mice. However, the main CD41 T cell source of IL-10 in chronically infected mice, and especially in mice with nonhealing lesions, appear to be Foxp32 Th1 cells, rather than Foxp31CD251 cells (Anderson et al., 2007). IL-10 production by antigen-specific CD41CD252Foxp32 T cells, the majority of which also produced IFN-g, was necessary for suppression of acquired immunity in Rag2/2 reconstituted mice. Interestingly, the elimination of nTreg from the T cells used to reconstitute the Rag2/2 mice actually resulted in exacerbated disease, due to an increase in the frequency of IL-10 producing Th1 and Th2 cells. Similar effects of Treg depletion on disease severity have been reported involving other Leishmania species and susceptible mouse strains, which point out the complex nature of a regulatory network in which activated T cells that release suppressive cytokines like IL-10 can themselves be targets of regulation.

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It is important to emphasize that in both the experimental and clinical studies, IL-10 producing cells appear to be activated in a strong proinflammatory tissue environment. Even in active VL patients in whom the parasitization process appears uncontrolled, elevated splenic levels of IL-10 mRNA are accompanied by equally elevated levels of IFN-g mRNA, as well as elevated plasma concentrations of IFN-g, IL-12p40/p70, TNF-a, and IL-1 (Nylen et al., 2007). The activation and/or accumulation of IL-10-producing cells in the liver and spleen is consistent with their suggested role in preventing collateral tissue damage in sites of strong inflammation (O’Garra et al., 2004). This might be especially important in preventing liver fibrosis in VL, as suggested in patients chronically infected with hepatitis C virus, in whom less fibrosis was detected in those areas of the liver where virus-specific IL-10-producing T cells had accumulated (Accapezzato et al., 2004). Too little IL-10 and/or IL-10 receptor expression is in fact associated with the pathogenesis of mucocutaneous leishmaniasis, which is characterized by low but persistent numbers of viable organisms, hyperresponsiveness to leishmanial antigens, and immune-mediated destruction of mucosal tissue (Gomes-Silva et al., 2007).

Chagas’ Disease

An immune response to persistent parasites is thought to be a primary cause of Chagas’ disease, best demonstrated by the strong correlation in murine models between the persistence of Trypanosoma cruzi and the presence of disease in muscle tissue (Zhang et al., 1999). Chagasic lesions are mainly concentrated in the cardiac tissue or digestive tract and clearly involve T-cell activation and release of inflammatory cytokines and chemokines, including IFN-g and MCP-1 (reviewed in Golgher & Gazzinelli, 2004). This inflammatory reaction is responsible for the local control of parasite replication, but it also appears to cause local tissue damage, best illustrated by the outcome of T. cruzi infection in IFN-g knockout mice that have increased parasitemia but attenuated cardiac inflammation (Michailowsky et al., 2004). There is clear evidence that IL-10 plays a critical role in regulating this immunopathology, at least during the acute stage of infection, since IL-10 knockout mice, while experiencing reduced parasitemia, develop lethal and systemic inflammation similar to septic shock (Hunter et al., 1997). Unfortunately, these mice do not survive long enough to determine whether, in the absence of IL-10, they would be capable of achieving a sterile cure. The source of IL-10 has not been clearly addressed in the mouse models. TGF-b has also been shown to promote parasite survival in vivo and in vitro, and can be produced by infected macrophages following their uptake of apoptotic lymphocytes. This process has been shown to fuel intracellular growth of T. cruzi, and may play an important role in parasite persistence in the host (Freire-de-Lima et al., 2000). Natural or converted Treg do not seem to be an important source of IL-10 or immunosuppression, in general, because infection of mice with the same strain of T. cruzi that produced lethal pathology in IL-10 knockout mice did not produce more acute inflammation or reduced parasitemia in mice treated with anti-CD25 antibody at the time of infection (Kotner & Tartleton, 2007). Nor did antiCD25 treatment alter the cardiomyopathy resulting from chronic infection with a strain of T. cruzi tropic for cardiac tissue (Sales et al., 2008). In another related study, treatment with anti-GITR, but not anti-CD25 antibody, markedly increased both myocarditis and parasitemia (Mariano et al., 2008). These results need to be interpreted with caution because, although GITR is highly expressed on CD41 Foxp31 Treg, it is also up regulated on conventional CD41 and CD81 T cells upon activation.

In humans, the proinflammatory nature of Chagas’ disease is supported by immunohistochemistry studies of cardiomyopathy heart tissue and analysis of heart infiltrating T cells that disclosed a predominance of IFN-g and TNF-a producing cells, and the observation that peripheral blood T cells of cardiac patients make a stronger Th1 response to various T. cruzi antigens compared to asymptomatic individuals (reviewed in Dutra, et al., 2005). Since a high proportion of asymptomatics also have a relatively high frequency of antigen-specific IFN-g producing cells (at least in their peripheral blood) the balance of inflammatory and anti-inflammatory mediators may be the more meaningful correlate or predictor of chronic disease. The evidence that IL-10 might limit the intensity of the inflammatory pathology is based on the observation that higher expression of IL-10 by monocytes and T cells was observed in patients with the asymptomatic or indeterminate form compared to patients with cardiac disease. Furthermore, a good correlation between IL-10 levels produced by antigen-stimulated PBMCs and improved cardiac function was observed in Chagas’ patients, and IL-10 gene promoter polymorphisms linked to low IL-10 expression was associated with the development of chagasic cardiomyopathy (Costa et al., 2009). There is a single report of elevated IL-10 expression associated with CD25hiFoxp3hi T cells in antigen-stimulated PBMCs from indeterminant patients compared to noninfected individuals, though not compared to cardiac patients (Araujo et al., 2007). Their production of IL-10 aside, ex vivo analysis of peripheral blood CD25hiFoxp3hi T cells revealed significantly higher frequencies in indeterminant patients as compared to cardiac patients (Vitelli-Avelar et al., 2005). In no case have the cells been demonstrated in the inflammatory lesions and their relevance to the control of morbidity is far from clear.

THE LINK BETWEEN DENDRITIC CELL FUNCTION AND Treg ACTIVATION

What are the antigen-presenting cell requirements, signaling pathways, and cytokines involved in the induction of nTreg, iTreg, Tr1, and IL-10-producing Th1 cells during chronic parasitic infection? For those studies demonstrating a role for CD41CD251Foxp31 T cells in suppression of antiparasite responses, few have clarified whether these are nTreg or extrathymically induced iTreg. This distinction is especially difficult to address in clinical studies, whereas in experimental models, the transfer of trackable nTreg from naïve mice into infected, T-cell deprived recipients can be used to confirm their activation and contribution to the regulation of immunity or immunopathology during chronic infection. This approach has only been applied to the L. major mouse infection model to demonstrate a role of nTreg in the persistence of organisms following clinical cure (Belkaid et al., 2002). And only in this model has the antigen-specificity of the infection-driven nTreg been directly addressed, with L. major infected DCs inducing the proliferation of CD41CD251Foxp31 T cells from lymph nodes draining the site of infection, and T-cell lines established from these cells able to produce IL-10 in response to antigen in vitro (Suffia et al., 2006). The specificity of nTreg for foreign epitopes has been a matter of some debate due to the fact that relatively high-affinity interactions of the abTCR with agonist ligands expressed in thymic epithelial cells are thought to be needed for the efficient generation of nTreg. Thus, L. major may possess epitopes that are cross-reactive with self-reactive nTreg. Apart from antigen specificity, the conditions that preferentially activate nTreg have been investigated with regard


to the maturation state of the DC. Immature DCs are efficient at capturing antigen but are poor at processing and presenting antigen to T cells. Immature DCs are thought to prime nTreg involved in maintaining peripheral tolerance, and have been shown to induce nTregs specific for foreign peptides. There is, however, considerable data to indicate that mature DCs are able to expand antigen-specific human and mouse nTreg that is dependent on up regulated costimulatory molecules and on provision of exogenous IL-2 (reviewed in Yamazaki & Steinman, 2009). Thus the APCs that activate nTreg may not be so different from the APCs that activate effector T cells. It is therefore likely that the expansion of CD41CD251Foxp31 T cells typically observed in inflammatory sites might be due, at least in part, to the presence of mature DCs and IL-2 provided by effector T cells that efficiently activate self-reactive nTreg, even in the absence of foreign epitopes that are cross-reactive with self-peptides. In contrast to nTreg, the antigen specificity of the de novo generated CD41CD251Foxp31 T cells from conventional Foxp32 cells is less in doubt because they are driven by encounters with foreign antigen in the periphery. There is also greater clarity as to the conditions required for their induction and expansion, with select subsets of DCs, including those from the gut, spleen, and skin, able to convert antigen-specific Treg under the influence of TGF-b and typically under conditions of low costimulation (Yamazaki & Steinman, 2009). The source of endogenous TGF-b in these tissues is not always known, though in the spleen, CD81DEC2051 DCs produce substantial levels of the latent form of TGF-b to its bioactive form, and rapid induction of TGF-b leads to impaired Th1 responses and a failure to control infection with lethal P. yoelii (Omer et al., 2003). Of note, the source of the TGF-b and the contribution of converted Tregs to the lethality was not shown in these studies. TGF-b also promotes T. cruzi infection in mouse models, and live trypomastigotes or protein extracts could activate latent TGF-b to yield a mature, bioactive homodimer that promotes invasion and intracellular survival (Waghabi et al., 2005), though again there is no direct evidence for induction of antigen-specific, converted Tregs playing a role in the pathogenesis of clinical or experimental Chagas’ disease. Finally, while TGF-b is also known to exacerbate Leishmania infections in mice, using adoptively transferred, naïve CD42CD252Foxp32 T cells from Foxp3GFP reporter mice, Treg conversion during L. major infection was directly addressed, with few iTregs detected in the skin or the draining lymph nodes during either the acute or chronic stage of infection (Anderson et al., 2007). The IL-10 producing CD4 T cells, or Tr1 cells, that function to help maintain peripheral tolerance were first shown to be induced by a distinct subset of CD11clowCD45RBhigh DCs that are present in the spleen and lymph nodes of normal mice and enriched in the spleen of IL-10 Tg mice (Wakkach et al., 2003). These DCs maintain an immature phenotype (i.e., few dendrites and low expression of costimulatory molecules) even after in vitro activation with LPS or CpG oligodeoxynucleotides (ODN). The first evidence for pathogen specific Tr1 cells was reported for Bordetella pertussis infection in mice in which the Th1 response in the lung was suppressed by IL-10-secreting CD4 cells (McGuirk et al., 2002). A molecule derived from B. pertussis, filamentous hemagglutinin, was shown to inhibit IL-12 and stimulate IL-10 production by DCs in vitro, which, in turn, were shown to induce naïve T cells to differentiate into Tr1 cells. Freshly isolated DCs in the respiratory tract and Peyer’s patches have the propensity to secrete high levels of IL-10, which may explain why Tr1 cells are induced at mucosal

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surfaces, where they function to maintain tolerance. However, liver and splenic DCs can also secrete IL-10, and may be important to the induction of Tr1 cells during malaria or visceral leishmaniasis. During P. yoelli infection in mice, DCs with a regulatory CD11clowCD45RBhigh IL-10-expressing phenotype become the predominant DC subset in the spleen, and were found to induce Tr1 cells from naïve ovalbumin (OVA)-specific T cells in vitro (Wong, et al., 2008), although a role for Tr1 cells in suppressing immunity was not established in these studies. By contrast, in the studies by Couper et al. (Couper, et al., 2008), in which a case for malaria-specific Tr1 cells preventing parasite clearance has been most convincingly made, the role of regulatory DCs in the induction of these cells was not investigated. Only in the murine model of visceral leishmaniasis, where IL-10 from Foxp32 T cells was shown to be critical for L. donovani persistence (Stager et al., 2006), has a link between suppression by Tr1 cells and their induction by regulatory DCs been directly drawn. In these studies, spleen stromal cells from infected mice, comprising a mixture of fibroblasts and macrophages, were shown to support the development of CD11clowCD45RBhigh IL-10 producing DCs, which were in turn shown to induce Leishmania-specific Tr1 cells in vitro and in vivo (Svensson et al., 2004). The ability of Leishmania to promote an IL-10 producing regulatory DC phenotypes was also demonstrated for Leishmania amazonensis, which in this case could be driven by antibody-FcgR-mediated signaling following uptake of serum opsonized parasites (Wanasen et al., 2008). It would appear somewhat paradoxical that immature, regulatory DCs that induce Tr1 cells come to predominate in the spleens of mice with malaria or visceral leishmaniasis when these same pathogens are known to drive highly proinflammatory immune responses. It is becoming increasingly apparent, however, that proinflammatory cytokines can themselves induce T cells to express IL-10, providing a classical mechanism of feedback control. These findings are highly relevant to a host of chronic infections in which the production of IL-10 from T cells participating in a Th1type response is a common finding (reviewed in Trinchieri, 2001). Specifically, IL-12 family members (IL-12, IL-23, IL-27), have each been shown to be self-regulating. Early in vitro studies revealed that IL-12 could instruct both IFN-g and IL-10 secretion by anti-CD3 or PHA-activated human T cells. Recent studies employing OVA-specific TCR transgenic T cells showed that the differentiation of Th1 cells coproducing IFN-g and IL-10 required IL-12-induced STAT4 signaling that, together with a high antigen dose, produced sustained ERK1 (extracellular signal-regulated kinase 1) and ERK2 phosphorylation (Saraiva et al., 2009). IL-12 induction of IFN-g was shown to provide an obligate signal for local reactivation of IL-10 production by effector Th1 cells in T. gondii-infected mice (Shaw et al., 2006). Other studies in T. gondii, however, observed high frequencies of antigenspecific, IL-10 producing Th1 cells in both wild-type and IL-12-deficient mice (Jankovic et al., 2002), indicating that STAT4 signaling is not required for the differentiation of T cells producing IFN-g, either alone or simultaneously with IL-10. These authors also noted that whereas IFN-g secretion was triggered with the same kinetic regardless of the state of Th1 cell activation, IL-10 secretion was more transient and confined to recently activated cells (Jankovic et al., 2007). An alternative IL-12 family member, IL-27, which activates Th1 transcription factors T-bet and STAT1, was shown to up regulate IL-10 expression by Th1 cells in T. gondii infected mice but in a STAT1 and STAT3 dependent manner (reviewed in Stumhofer & Hunter, 2008).

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Accordingly, IL-27ra2/2 mice develop lethal inflammation that is associated with defective IL-10 production by Th1 cells isolated from the brains of infected mice. IL-27ra2/2 mice infected with L. major also develop more severe pathology, which, in the case of nonhealing L. major infections, is associated with fewer numbers of T cells coexpressing IL-10 and IFN-g (Anderson et al., 2009). The absence of suppressive IL-10 from these T cells sources was not a sufficient condition to promote parasite clearance, however, because the IL-27 signaling defect also severely impaired Th1 differentiation in these mice. Recently, the Notch pathway was identified as a common signaling pathway for both IL-12 and IL-27 induced IL-10 production by Th1 cells (Rutz et al., 2008). The negative feedback loop described above, in which high dose antigen and IL-12 and/or IL-27 from innate cells act to up regulate IL-10 production from Th1 effector cells, would seem to accurately reflect the conditions established

by malaria, Leishmania, T. cruzi, and T. gondii. At least during the acute stages of infection, each of these intracellular parasites undergo rapid division and release immune stimulatory molecules, including strong TLR agonists (Gazzinelli & Denkers, 2006) that activate DCs and initiate a robust Th1 effector response. The IL-10 produced by Th1 cells in these inflammatory settings, in conjunction with the diminishing concentration of antigen available to DCs as a substantial degree of pathogen clearance is achieved, might begin to create the conditions necessary for the generation of regulatory DCs and the induction of Tr1 cells. Such a Th1 IL-10 driven, regulatory feedback loop is depicted in Fig. 2. Clearly, IL-10 from multiple sources, including Th1, Tr1, Th2, Th17, and innate cells, could help to establish a dominant regulatory or immature DC phenotype in target organs, which would limit the effector response and help to maintain the balance between immunity and pathology in sites of chronic infection.

FIGURE 2 Negative feedback loop initiated by protozoan pathogens inducing strong Th1 responses. TLR agonists and other stimulatory molecules delivered to DCs by malaria-infected RBCs (red blood cells), T. cruzi trypomastigotes or Leishmania amastigotes, during the acute stage of infection, activate Th1 effector cells that, under the influence of persistent, high dose antigen and instruction by IL-12 or IL-27, will comprise a population of cells that are transiently activated to coexpress IL-10. The IL-10 from Th1 cells will down regulate APC (antigen-presenting cell) function and, in conjunction with reduced antigen available for processing during the immune clearance phase, will establish conditions suitable for the predominance of a regulatory DC phenotype and activation of Tr1 cells that help to limit the effector response during the chronic stage of infection.


CONCLUSIONS

Collectively, the data discussed above support the notion that inflammatory cytokines and chemokines are essential for immune-mediated control of parasite growth and dissemination. On the other hand, regulatory cells or antiinflammatory cytokines are absolutely necessary to limit the tissue damage resulting from persistent infectioninduced inflammation, which may lead to the death of the host. The results from both experimental and clinical studies highlight the importance of maintaining the balance between a strong effector immune response and a regulated immune response that will help maintain tissue homeostasis. To the extent that these homeostatic control mechanisms are driven by persistent antigen stimulation, the argument that they are responsible for the chronicity of infection is obviously somewhat circular and begs the question as to why such strong effector responses fail to eliminate the pathogen in the first instance. It is more likely that immunosuppression is superimposed on or is secondary to various immune evasion strategies that prevent the complete clearance of the parasite during the acute stage of infection. For P. falciparum malaria, antigenic variation of parasite proteins expressed on infected erythrocytes is the key mechanism ensuring survival of somatic variants during antibody-mediated clearance of blood-stage parasites. For Leishmania sp., which resides normally within a macrophage phagolysosome, its intrinsic resistance to enhanced microbicidal mechanisms, as well as its ability to inhibit immune activation signals in infected cells necessitates especially strong and sustained levels of activation to achieve killing. For T. cruzi, its ability to infect any nucleated cell type (e.g., muscle cells) and to subsequently escape from endocytic vacuoles, allows it to persist in cell types and in an intracellular compartment that minimizes exposure to the toxic metabolites of activated cells. Thus even in the face of a robust, class appropriate immune response, these organism are not eliminated efficiently or quickly enough to remove them as a source of stimulation before counterregulatory mechanisms kick in to limit the response and make their ultimate clearance all the more difficult to achieve. This research was supported by the Intramural Research Program of the NIAID, NIH.

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36 Immune Evasion by Parasites JOHN M. MANSFIELD AND MARTIN OLIVIER

INTRODUCTION

mechanisms employed by African trypanosomes suggest these organisms are masters of the craft.

All successful microbial pathogens evade or alter host immune responses. The evolution of immune evasion by microbes is thought to have occurred in step with evolution of the vertebrate immune system, and is therefore intimately associated with all aspects of innate and adaptive immunity. Like other pathogenic microorganisms, parasites have evolved the means to manipulate host immune cell recognition, activation, and regulation by employing a wide range of discrete mechanisms. For parasites, the necessity of (indeed the dependence on) immunological evasion mechanisms is a direct consequence of the fact that these organisms must live for prolonged periods within host tissues during their life cycle; at a minimum this period must be sufficient for the organisms to replicate and develop into the life cycle stages and numbers of organisms necessary for successful transmission in nature. This chapter examines several mechanisms by which two well-known protozoan pathogens, one extracellular and one intracellular, successfully evade host immunity.

Practical Constraints on Antigenic Variation

There are substantial structural as well as other practical constraints on molecular variation by parasites. Antigenic variation is limited in that it must occur in concert with the maintenance of critical molecular structures that provide cellular integrity and permit functional molecular interactions within the parasite’s environment. For organisms that are extracellular at any stage of their life cycle, all surface-exposed molecules are potential targets for host B-cell-mediated immunity, and these antigens, as well as other cellular constituents, are also capable of triggering T-cell mediated immune responses that can affect parasite survival in the extracellular environment. The problem for the parasite is to express reduced numbers of surfaceexposed molecules, to permit selective or limited immune responses to target antigens that can accommodate molecular variation, and/or to couple limited antigenic variation with immune modulation that prevents immune targeting of all antigens. For parasites residing inside nucleated host cells, the release or secretion of any molecules intracellularly or extracellularly potentially can result in the immune targeting of infected cells by major histocompatibility complex (MHC) class I- and II-restricted as well as CD1-restricted effector T cells. The problem for these parasites is to control the accessibility of antigenic molecules and their peptide substituents to class I or class II antigen processing pathways, to permit antigenic variation in noncritical cellular constituents that access these pathways, and/or to modulate host cell antigen processing or immune effector cell recognition of invariant and critical parasite molecules. As noted above, the problems for both extracellular and intracellular parasites are exponentially more complex if potential immune target antigens serve as critical structural, metabolic, transport, or other essential invariant components of the microbial cell. Following, we discuss how one protozoan parasite has evolutionarily addressed some of these problems by displaying a highly ordered molecular surface coat that serves largely to protect the trypanosome plasma membrane from immunological assault.

EVASION OF IMMUNITY BY AFRICAN TRYPANOSOMES

Parasites evade host immunity by employing both “passive” and “active” mechanisms. Passive evasion in this context includes the altered display of MAMPs (microbe-associated molecular patterns) to avoid activation of host innate immune cell pattern recognition receptors, as well as classical antigenic variation in which the display of antigenic epitopes on potentially protective parasite antigens is altered to avoid recognition by T and B cells of the adaptive immune system. Active evasion of immune responses refers to alterations in post-receptor signaling pathways and gene activation profiles in which cells of the innate and adaptive immune system are prevented from generating a sufficient response to receptor-mediated stimuli. In this section we explore the multiple means by which an extracellular protozoan parasite, the African trypanosome, alters and evades host immunity by using several passive and active mechanisms. The complexity and extent of immune evasion John M. Mansfield, Department of Bacteriology, Microbial Sciences Building, 1550 Linden Drive, University of Wisconsin-Madison, Madison, WI 53716. Martin Olivier, Department of Microbiology and Immunology, Duff Medical Building, 3775 University, McGill University, Montréal, Québec, Canada.

The Trypanosome Variant Surface Coat

Many successful parasites exhibit antigenic variation to avoid immune elimination during infection. Yet arguably the most well-known example of immune evasion by parasites 453

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is antigenic variation by the African trypanosome, which for nearly a century has provided the classical paradigm for microbial antigenic variation as a means of escaping host immunity (Mansfield, 1995). African trypanosomes have evolved a structured monomolecular surface coat that covers the entire plasma membrane of the parasite, including the flagellum (Vickerman & Luckins, 1969; Cross, 1975). This surface coat consists of a densely packed array of 107 identical glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) homodimers (Color Plate 9) that both determine the antigenic phenotype of the parasite and prevent Ab and innate immune elements such as complement from binding to subsurface invariant epitopes of the coat or to any plasma membrane determinants underlying it. VSG molecules are 55-kDa to 65-kDa glycoproteins that contain internal antiparallel A- and B-alpha helices that lend rigidity to the folded structure (Metcalf et al., 1987; Freymann et al., 1990); VSG homodimers are oriented with the hydrophilic N-terminal domain of the protein partially exposed to the extracellular environment and with GPI anchors tethering the C-terminal domain to the plasma membrane (Carrington et al., 1991; Blum et al., 1993; Chattopadhyay et al., 2005). Together, these characteristics permit a highly ordered packing of VSG molecules into the surface coat structure. Despite extensive primary sequence variation among different VSGs that comprise different surface coats, secondary and tertiary structural features of these molecules (and the amino acids residues that determine such features) are highly conserved (Metcalf et al., 1987; Reinitz et al., 1992; Blum et al., 1993), perhaps ensuring that all VSG molecules are packaged similarly into a surface coat structure during the process of antigenic variation (see following). The VSG coat is capable of rapidly activating B cells to produce VSG-specific Ab that eliminates trypanosomes from the bloodstream. It is a T-cell-independent B-cell response to the multiepitope array on the VSG coat surface that represents the earliest event in the protective adaptive immune response to trypanosomes and this alone is sufficient to control parasitemia (Campbell et al., 1978; Mansfield et al., 1981; Reinitz & Mansfield, 1990; Mansfield, 1994; Schopf et al., 1998; Dubois et al., 2005; Radwanska et al., 2008). This response represents “antigen pattern” recognition and is similar to B-cell responses to viral capsid determinants; Ab is directed at exposed epitopes displayed as part of the three-dimensional architectural configuration of VSG molecules within the surface coat. Classically, pathogen molecular pattern recognition has been held to involve antigen nonspecific receptors displayed primarily by cells of the innate immune system. However, it has been proposed that B cells also have the capacity to scan architectural surface structures of microbes for polymeric epitope patterns associated with an assemblage of identical unit molecules (Mansfield, 1994; Bachmann & Zinkernagel, 1996; Fehr et al., 1998; Zinkernagel, 2000). This type of polymeric epitope recognition event is distinct from individual unit molecule epitope recognition by B-cells, which requires T-cell help in generating a significant Ab response. The ability to stimulate T-independent B-cell responses in such an antigen pattern-specific manner is dependent on the homogeneity, orientation, density, and rigidity of molecules expressed within an exposed structure such that cognate B-cell antigen receptors are extensively cross-linked in a manner sufficient to trigger cellular activation (Snapper et al., 1994; Mond et al., 1995; Snapper & Mond, 1996; Vos et al., 2000). In this regard, the trypanosome VSG coat represents an ideal T-independent B-cell-stimulatory surface structure: the dense packaging of identical VSG homodimers on the plasma membrane, the internal rigidity of VSG homodimers in the coat, and the orientation of each subunit VSG

monomer in the coat all contribute to a repetitive antigen pattern recognized by B cells.

Trypanosome Antigenic Variation at the Genetic Level

Given that the VSG coat is highly immunogenic and capable of provoking a rapid early immune response, trypanosomes have evolved a successful mechanism for coping with host immune responses by extensive antigenic variation of the VSG coat during infection (Vickerman & Luckins, 1968, 1969; Cross, 1975). This variation occurs as the result of transcriptional activation of distinct VSG genes from among a large resident VSG gene family. Stringent allelic exclusion ensures that only 1 of potentially 1,000 different VSG genes in the genome is transcribed at any given time from a chromosome telomere (Cross, 1990; Van der Ploeg et al., 1992; Borst & Rudenko 1994; Borst et al., 1996; Borst & Ulbert, 2001; Borst, 2002; Donelson, 2003). Thus, normally only one species of VSG molecule is expressed at a time and is present within the trypanosome surface coat. VSG gene switching may occur at a relatively high frequency of 1 switch in 102 to 106 cells (Turner & Barry, 1989; Turner, 1997; Vanhamme et al., 2001; Lythgoe et al., 2007). As noted above, only one VSG gene is expressed at a time, however, and expressed VSG genes are transcribed from active chromosome telomeric expression sites under strict allelic restriction mediated by a specialized extranucleolar expression site body that is a complex of Pol I polymerase and other proteins (Navarro & Gull, 2001; Landeira et al., 2009). These expression sites normally generate large polycistronic transcripts that encompass a number of upstream expression site-associated genes as well as the telomeric VSG gene (Cully et al., 1985; Borst et al., 1996; Horn & Cross, 1997; Cross et al., 1998; Navarro & Cross 1998; Hertz-Fowler et al., 2008). There are several mechanisms that serve to duplicate and transpose copies of internal chromosome basiccopy VSG genes into telomeric sites or to transcriptionally activate existing copies of silent VSG genes already present in telomeric sites (Hoeijmakers et al., 1980; Borst & Cross, 1982; Cross, 1990, 1996; Navarro & Cross, 1996; Horn & Cross 1997; Cross et al., 1998; Barry & McCulloch, 2009; Verstrepen & Fink, 2009). Therefore, during each wave of parasitemia, new antigenic variants constantly arise and it is the Ab-mediated destruction of the predominant variant antigenic type (VAT) that ultimately leads to immune selection for one or more new VATs that have arisen within the previous population. This cycle of host VSG-specific Ab production and VAT elimination, coupled with changes in trypanosome VSG expression and selection, occurs throughout infection until, at some point, the host dies without ever completely eliminating the organisms from the body.

Removal of Immune Complexes from the Coat

Trypanosomes also possess another clever immune evasion weapon in their VSG coat armamentarium. Since VSG homodimers are tethered in the membrane by the lipid soluble dimyristoylglycerol (DMG) moiety of the GPI anchor, they are able to “flow” across the membrane from and to the flagellar pocket. This movement is influenced by several factors including parasite motility and hydrodynamic forces within the vasculature (Engstler et al., 2007). Recent studies have shown quite clearly and dramatically that binding of IgM and IgG Ab to exposed VSG surface coat determinants results in their removal via a hydrodynamic drag effect in which Ab molecules serve as molecular “sails,” dragging immune complexes across the membrane and into the flagellar pocket where they are rapidly internalized (Fig. 1). Subsequently, VSG molecules may be recycled back to the membrane but

36. Immune Evasion by Parasites

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FIGURE 1 VSG-Ab complexes on the trypanosome surface coat serve as “molecular sails” that, when exposed to the hydrodynamic forces in the bloodstream and trypanosome directional motility, move towards the flagellar pocket and are internalized. Adapted from Engstler et al., 2007.

Ab molecules are degraded. Thus, even in the presence of Ab to the VSG coat, immune complexes may be removed via a mechanism dependent on parasite forward motility in an environment where hydrodynamic drag forces predominate. Presumably, however, this evasion process ceases to be effective in the presence of high Ab concentrations that can cross-link VSG molecules sufficiently to retard movement towards the flagellar pocket. At such a point, the targeted VAT would be cleared from the blood by complement and/or FcR-dependent mechanisms and replaced by the new VATs that have already appeared in the population.

Mosaic VSG Coat Switch Variants Escape Early B-Cell Detection

During the process of antigenic variation, one homogeneous surface coat is replaced over a period of time by a new homogeneous coat; this presents a potential conundrum for the parasite because switch variants appear frequently and continuously throughout infection (Esser & Schoenbechler, 1985; Baltz et al., 1986).

Transient VSG double expressers (see Color Plate 10) potentially may prime the host immune system to VSGs expressed by such emerging VATs and afford the host an immunological advantage during a long-term chronic infection. Transcription of a new VSG gene plus translation and trafficking of new mature VSG homodimers to the cell surface, coupled with residual mRNA and protein stability of the old VSG coat, result in transient expression of both the former and the nascent VSG species in a “mosaic” VSG coat on the cell surface for a period of time during the process of antigenic variation. However, the kinetics of antibody responses (appearance, Ig class, titer) to VSG surface coats displayed by trypanosomes in the first peak of parasitemia and the kinetics of antibody responses to different VSG coats subsequently displayed by variant antigenic types (VATs) in subsequent waves of parasitemia are similar, suggesting that B cells are not primed to new VSG molecules expressed in the mosaic surface coats of any preceding double expressers or switch variants (Dempsey & Mansfield, 1983; Mansfield et al., 2002).

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For this reason, the trypanosome “mosaic” VSG surface coat (see Color Plate 10) was recently examined for its ability to prime the host immune system to newly arising VSG species. Building on existing evidence that recombinant trypanosome VSG double expressers displaying a mosaic surface coat were viable and able to infect mammalian hosts (MunozJordan et al., 1996), an experimental approach was designed in which recombinant trypanosomes stably expressing two different VSG genes were constructed, resulting in a mosaic surface coat with equivalent amounts of the two VSGs (Dubois et al., 2005). When animals were infected with trypanosomes expressing a typical monotypic VSG coat, they produced a rapid protective T-independent B-cell response. However, when animals were infected with trypanosomes stably displaying a mosaic VSG surface coat, they failed to produce a significant T-independent B-cell response to either VSG species (Dubois et al., 2005). Thus, the process of antigenic variation itself produces temporal VSG mosaic surface coats that, architecturally, no longer display epitope arrays capable of directly activating B cells. These mosaic coats provide a temporal “stealth” mode during the antigenic switch process that prevents activation and outgrowth of B cells capable of responding to the newly emerging VATs. Furthermore, there is recent evidence that memory B cells are altered in function, phenotype, and tissue distribution during trypanosomiasis (Radwanska et al., 2008), which may compound the problem for infected hosts in terms of memory responsiveness to previously encountered VSGs during infection. Thus, trypanosomes evade elimination from the bloodstream of infected hosts by the process of antigenic variation in which new VSG surface coats are presented to the host immune system, and also in which the molecular process of producing new antigenic surface coats fails to prime the immune system to emerging VATs due to temporal production of mosaic VSG coats, and by significant alterations in B-cell memory. Despite the demonstrable efficiency of the VSG-specific B-cell response in destroying trypanosome VATs present in the bloodstream during infection, however, the B-cell response alone is not functionally or genetically linked to overall host resistance to infection. This is because African trypanosomes cross the vascular endothelium and invade host tissues, including the brain, where the Ab response is considerably less effective.

VSG Specific T Cells: A Central Role in Host Protection

For nearly a century, the paradigm of trypanosome antigenic variation embraced the concept that Ab-mediated control of parasite burden was the key factor in the control of the disease and in host resistance. However, a profound shift in this paradigm occurred as the result of new insights into the immunobiology of trypanosomiasis. The old central paradigm first began to unravel as the result of functional and genetic studies on the specific role of Ab in determining relative host resistance to experimental trypanosome infections. It was demonstrated using semiallogeneic bone marrow radiation chimera mice that control of parasitemia by Ab was not functionally linked to overall host resistance (De Gee & Mansfield, 1984). The central finding was that susceptible mice, which are genetically unable to make Abs to VSG surface coat determinants and to control parasitemia, were afforded a functional VSG-specific B-cell response after transplantation and reconstitution of the lymphoid system with H-2-compatible bone marrow cells from resistant mice; this response was sufficient to eliminate trypanosomes from the blood in a variant-specific manner. However, despite Ab-mediated control of trypanosomes, the radiation chimera mice lived no longer than wildtype susceptible mice that could not make VSG-specific Ab

or control the parasitemia. Subsequently, classical genetic approaches employing crosses between resistant and susceptible animals demonstrated that Ab-mediated control of parasitemia by itself was not genetically linked to the host resistance phenotype (De Gee et al., 1988). This work has been substantiated in other laboratories and by different approaches (e.g., see Seed & Sechelski, 1989). Thus, although VSG-specific Abs provide a very important mechanism for controlling trypanosome numbers in the blood (and perhaps also provide an unerringly specific and useful means to select for new VATs in the process), this event alone is insufficient to provide a significant level of host resistance. These seminal experiments laid the foundation for studies in which resistant and susceptible animals were examined for T-cell-mediated immune responses to trypanosome antigens. T-cell responses to VSG previously had not been detected or characterized in animals infected with the African trypanosomes, despite evidence that such animals made T-dependent B-cell responses to VSG molecules (Reinitz & Mansfield, 1988) and that primary sequence variation was evident in nonexposed subregions of VSG molecules making up the surface coat (see following) (Reinitz et al., 1992; Blum et al., 1993; Field & Boothroyd, 1996). The first direct evidence for VSG-specific T-cell responses came from experimental studies with Trypanosoma brucei rhodesiense infections of mice (Schleifer et al., 1993; Schleifer & Mansfield, 1993). T-cell populations derived from lymphoid tissues of infected mice were tested for activation following exposure to purified VSG in vitro. The results of these studies showed that VSG-specific T cells exhibited a degree of tissue-specific compartmentalization and did not proliferate in vitro in response to antigen but rather produced substantial cytokine responses when stimulated; the principal cytokines produced were gamma interferon (IFN-g) and interleukin-2 (IL-2) but not IL-4 or IL-5. The cellular phenotype of VSG-responsive T cells was that of classical Th cells that were CD4 positive, expressed the CD3 a/b T-cell receptor membrane complex, were APC (antigen-presenting cell)-dependent and MHC-II restricted (Schleifer et al., 1993; Hertz et al., 1998; Schopf et al., 1998; Dagenais et al., 2009a). Thus, VSG appeared to preferentially drive a polarized Th1 cell cytokine response during Trypanosoma brucei subspecies infection. Intrinsic molecular characteristics of the VSG did not induce mice to make this response, however, since VSG-specific T-cell lines derived from VSG-immunized, but not infected, mice displayed cytokine profiles characteristic of both Thl and Th2 cells (Schleifer et al., 1993). Subsequently, it was demonstrated that IL-12 production by dendritic cells and macrophages, in conjunction with infection-associated inhibition of Th2-cell outgrowth, were responsible for induction of this polarized Thl-cell response (see additional discussion following) (Magez et al., 1998; Kaushik et al., 2000; Drennan et al., 2005; Barkhuizen et al., 2008; Dagenais et al., 2009b). Parasite antigen specific B-cell responses were also affected by Th1 cell cytokines in terms of immunoglobulin Ig isotype switch events of T-dependent B-cell responses to VSG (Schopf et al., 1998). Th1 cells appear to serve as a major source of IFN-g, a cytokine that is linked to host resistance against T. brucei trypanosomes through macrophage activation events (De Gee et al., 1985; Hertz et al., 1998; Hertz & Mansfield 1999; Drennan et al., 2005; Dagenais et al., 2009a). VSG-specific Th1-cell responses result in IFN-g-mediated activation of macrophages, leading to the release of factors such as TNF-a (tumor necrosis factor a), ROI, and NO (nitric oxide) (that are known to be cytotoxic for trypanosomes) (Vincendeau et al., 1989; Vincendeau et al., 1992; Mnaimneh et al., 1997; Beschin et al., 1998; Daulouede et al., 2001; Lucas et al.,


1993, 1994; Magez et al., 1997, 1999, 2006). Infection of relatively resistant mice lacking the IFN-g gene was shown to result in susceptibility similar to that of scid mice, which have no adaptive immune system, despite the fact that such IFN-g knockout animals made Ab responses that controlled trypanosomes in the bloodstream (Hertz et al., 1998; Schopf et al., 1998) (also, see previous discussion regarding genetic and functional linkage of adaptive immune responses to relative resistance). Thus, the current presumption is that IFN-g activation of macrophages with concomitant production of microbicidal factors is responsible for the control of parasite burden within the extravascular tissues where Ab is relatively inefficient in killing trypanosomes (n.b., an exception exists with T. congolense infections of animals; this parasite of veterinary interest is limited to the vasculature where B-cell responses appear to be the primary factor in controlling infection). Indeed, several studies have demonstrated that treatment of animals with microbial products capable of activating macrophages resulted in enhanced control of infection and prolonged host survival (Murray & Morrison 1979; Harris et al., 2007). Thus, these types of results support the contention that Th1-cell and IFN-g cytokine responses to VSG regulate a major component of host resistance to African trypanosomes. However, like B-cell responses to VSG, Th1-cell responses alone are insufficient to provide full resistance, and there is evidence that IFN-g may contribute to some pathological changes during infection (Shi et al., 2006).

Evasion of Host T-Cell Responses

Antigenic variation in trypanosomiasis also results in evasion of VSG-specific T-cell responses. Early studies demonstrated that protective B-cell responses were directed at exposed epitopes of VSGs in the surface coat, but the specific submolecular sites that trigger T-cell responses were not known. Sequence and structural analyses of VSG molecules revealed variable subsequences that were predicted to be buried in the surface coat structure. The formal hypothesis was made that VSG-specific T-cell responses were generated to peptides derived from these buried variable region sites (Blum et al., 1993). A follow-up study was conducted in which the VSGs of a single VSG gene family were compared at the primary sequence level in the context of a conserved tertiary structure for these molecules. The analyses suggested that there were three hypervariable subregions detectable in trypanosome VSGs: HV-2 and -3 that were predicted to be exposed on the trypanosome surface coat structure and that may have evolved under B-cell selective pressure, and HV-1 that was clearly not exposed in the coat and that may have evolved under T-cell selective pressure (Field & Boothroyd, 1996). A recent test of these predictions yielded interesting results. An analysis of infected mouse T responses to epitopes displayed by the T. brucei rhodesiense LouTat 1 VSG clearly demonstrated that T-cell reactive sites were distributed throughout the N-terminal domain in multiple microvariable sites that, while overlapping with all HV subregions, were not constrained exclusively to any one HV region (Fig. 2) (Dagenais et al., 2009a). Thus, Th cells appear to recognize peptides distributed throughout the N-terminal (two-thirds of the VSG) without selective recognition of HV-1 epitopes. An unexpected finding from this study was that T cells failed to recognize potential antigenic peptides derived from within the C-terminal domain of VSG, which contains subsequences that were conserved among different VSGs. While it was clear from this and previous studies that there was no demonstrable VSG cross-reactivity by VATspecific Th cells (Schleifer et al., 1993; Schopf et al., 1998; Dagenais et al., 2009a, 2009b), the finding that T cells

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were not activated during infection that were capable of recognizing these conserved sequences suggested that the processing of peptides from the C-terminal subregion may be stringently regulated by trypanosomes. This finding also opens the door to using contemporary vaccine methodology to perhaps “force” recognition of conserved VSG subregions by Th cells with the goal of providing cross-protective immunity at the T-cell level.

Alterations in Antigen-Presenting Cell Function

There may be biochemical or structural features associated with the C-terminal domain of VSGs (including the presence of GPI anchor residues; see discussion following), that may prevent proper unfolding, access to proteolytic cleavage sites, or degradation in appropriate compartments of the MHC-II processing pathway of APCs. If true, this would be an additional passive evasion mechanism associated with VSG expression and it is easy to see how such structural features would have been selected. But the processing and presentation of parasite antigens is also actively modulated by trypanosome infection. In addition to an apparent absence of VSG C-terminal peptide processing by APCs following primary exposure, there is evidence that the processing of parasite antigens may be more broadly regulated during progressive infection. Generalized immunosuppression of T-dependent immune responses is a key feature of African trypanosomiasis (see discussion following), and, as part of this suppression, the ability of APCs to process and present nonparasite protein antigens to T cells is dramatically altered by infection (Paulnock et al., 1989; Namangala et al., 2000), with the result that new MHC-II antigenic peptide complexes are not displayed on APCs. Recent studies found that exposure to trypanosomes during infection rendered DCs (dendric cells) and microphages unable to process and present new variant VSG molecules to VSG-specific Th cells as well as to generate peptides from nonparasite exogenous protein antigens (Dagenais et al., 2009b). While the mechanism underlying these events is unclear, there are a few clues from preliminary studies. For example, endocytic uptake and intracellular trafficking of VSG and other proteins by APCs and the membrane display of MHC II-peptide complexes appear relatively normal only in the earliest stage of trypanosomiasis. However, if APCs are taken from animals after a few days of infection, during which time they already are displaying VSG peptides and activating VSG-specific Th cells, the APCs are unable to display additional MHC-II peptide complexes derived from exposure to other antigens or to newly arising VSGs (Dagenais et al., 2009b). These cells exhibit normal uptake and trafficking of antigen but with no recycling of existing MHC-II peptide complexes from the membrane, and newly formed intracellular MHC-II peptide complexes are either unstable or fail to properly assemble intracellularly (Freeman et al., manuscript in preparation). Thus, infection with African trypanosomes seems to prevent Th responses both to conserved elements of variant surface antigens as well as general Th cell responses to exogenous protein antigens, including newly expressed VSG molecules as infection progresses. Thus, African trypanosomes use antigenic variation and other unique features of the surface coat to passively evade immune detection and elimination during infection. Potentially active alterations in antigen processing further may not only regulate Th cell responses to exogenous protein antigens but also to invariant subsequences of the VSG. Clearly, however, the trypanosome has evolved additional mechanisms to help evade host immunity after triggering an adaptive immune response to its antigens. These mechanisms are all of

458 EVASION AND SUPPRESSION OF THE ANTIMICROBIAL HOST RESPONSE

FIGURE 2 Sequence comparisons among VSGs related by type and class showing composite and overlapping T-cell reactive sites identified using VSG-specific T cells. The data reveal that T cells preferentially recognize epitopes within the N-terminus of the molecule but not the more conserved C-terminal subregion. Adapted from Dagenais et al., 2009.


an “active” nature and involve both up and down regulation of postreceptor cellular activation.

Modulation of Cellular Activation by GPI Substituents

The extravascular survival of trypanosomes is affected by activation of macrophages for the production of microbicidal factors such as RNI, ROI, and TNF-a, as noted previously. This important aspect of host resistance to trypanosomes is largely Th1 cell-dependent and IFN-g-dependent and is ultimately shaped by exposure of macrophages to parasite factors early in infection. The degree of antigen-specific Th cell polarization seen in T. brucei group trypanosomiasis is unusual in that it appears absolute (Schleifer et al., 1993; Hertz et al., 1998; Schopf et al., 1998; Drennan et al., 2005; Dagenais et al., 2009a), unlike other infectious diseases in which only a Th1 or Th2 predominance occurs. In trypanosomiasis, a specific parasite molecule is linked to early polarization of the Th1 cell response; this is the glycosylinositolphosphate (GIP) substituent of GPI molecules that anchor VSGs to the plasma membrane (see previous). During infection, GIP-sVSG is released from the membrane by activation of an endogenous GPI-phospholipase C that is localized to an external linear array along the flagellum (Mensa Wilmot et al., 1990; Field et al., 1991; Carrington et al., 1998; Hanrahan et al., 2009). When GPI-anchored VSG flows across the membrane into this site, the activated enzyme cleaves the anchor and releases VSG from the membrane with the residual GIP residue attached and the DMG lipid soluble portion in the membrane (Hanrahan et al., 2009). The GIP-sVSG residues (and potentially residual DMG substituents in the trypanosome membrane) activate cells of the innate immune system early in infection (Tachado et al., 1997; Paulnock & Coller, 2001; Magez et al., 2001, 2002; Mansfield & Paulnock, 2005). GIP-sVSG binds to type A scavenger receptor (SR-A) on the macrophage membrane (Fig. 3) and triggers internalization that helps initiate a cascade of subcellular signaling events leading to activation of NF-kB and MAPK (mitogenactivated protein kinase) signaling pathways and the expression of a subset of proinflammatory genes (Leppert et al., 2007; Lopez et al., 2008). These events may be modulated by additional factors, including: a TRAF6- and TLR-9-dependent down-regulatory effect on the GPI-induced NF-kB response

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(Leppert et al., 2007), augmentation of the response in the presence of IFN-g (Coller et al., 2003; Leppert et al., 2007; Lopez et al., 2008), and by CpG DNA potentially released from senescent or damaged trypanosomes (Harris et al., 2006; Harris et al., 2007). One of the genes activated by GIP-sVSG exposure is IL-12p40, with the result that biologically active IL-12p70 is released very early during infection, as noted previously. Early polarization of the nascent VSG-specific Th cell IFN-g response is clearly IL-12 dependent (Barkhuizen et al., 2007; Dagenais et al., 2009b). However, there are other factors that may stabilize or contribute to polarization since a Th2 phenotype does not emerge in the absence of IL-12; rather, in the absence of IL-12, the Th1 cell phenotype simply emerges and becomes established later in infection (Dagenais et al., 2009b). This delay in polarization and, ultimately, IFN-g production by Th1 cells is sufficient to promote susceptibility of otherwise relatively resistant animals. An additional key component in macrophage activation is interaction of IFN-g with the IFN-gR that leads to the formation and phosphorylation of STAT-1 homodimers and activation of downstream proinflammatory genes. However, the relative timing of macrophage exposure to GPI substituents released by trypanosomes and to IFN-g is critical for the ability of macrophages to kill the parasites. If cells are simultaneously exposed to significant levels of both IFN-g and GPI, there is augmentation of macrophage activation and the activation of additional proinflammatory genes (Fig. 3). This event may happen in early infection with IFN-g derived from T cells (and, potentially, NK [natural killer] cells) (Demick et al., 2010, manuscript in preparation) and may help to control the early explosive growth of trypanosomes within host tissues so the host survives the first wave of infection. However, if macrophages are exposed to GPI substituents prior to sufficient levels of IFN-g, STAT-1 fails to phosphorylate in response to IFN-g and the production of trypanocidal factors is inhibited (Coller et al., 2003). These events probably occur as the infection progresses, when increasing numbers of parasites release GIP-sVSG prior to sufficient activation of new T-cell responses for production of IFN-g. Thus the interplay and timing of parasite and host factors are critical for control of the parasite burden in the extravascular tissues. The ability of residual GPI residues to block additional parasite killing by interfering with IFN-g-dependent

FIGURE 3 GIP-sVSG binds to the macrophage membrane, is internalized, and triggers activation of the NF-kB and MAPK signaling pathways with subsequent activation of proinflammatory genes. This response is TRAF6-dependent, augmented in the presence of IFN-g and down regulated by a TLR-9-dependent pathway. Adapted from Leppert et al., 2007. Note, GPI-mediated membrane binding and cell activation are dependent on scavenger receptor interaction(s).

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activation of macrophages may be central to downstream parasite evasion of host adaptive immunity. Furthermore, this ability of GPI substituents to alter macrophage function may extend to the observations cited previously in which APC function is significantly altered as the infection progresses. There are also additional elements of GPI-induced cellular activation that impact host resistance. For example, GIPsVSG induces a wide range of IFN-related genes, including IRF-7 (interferon regulatory factor 7), and subsequent production of the type I interferons, IFN-ab (De Gee et al., 1985; Lopez et al., 2008) may set in motion the recently described IRF-7-dependent and IFN-ab-dependent modulation of host resistance during late stage infection that inhibits IFN-g production by Th1 cells (Lopez et al., 2008).

Suppressor Macrophages

A side note on immunoregulation is that activation of macrophages during infection impacts not only trypanosomes but also nascent Th-cell responses to VSG and other antigens. One of the earliest findings regarding alterations of host immunity in trypanosomiasis was that animals were immunosuppressed with respect to T-cell-dependent responses to unrelated foreign antigens and perhaps also to self-antigens (Seed & Gam, 1967; Mansfield & Kreier, 1972; Mansfield & Wallace, 1974; Mansfield & Bagasra, 1978; Mackenzie et al., 1979). Th cell responses to exogenous foreign antigens were dramatically depressed by infection and, while early speculation was that Ts cells were involved (Jayawardena & Waksman, 1977; Jayawardena et al., 1978), it subsequently and clearly was shown that macrophages exhibited suppressor cell effects on host immunity during infection (Wellhausen & Mansfield, 1979, 1980a, 1980b; Mansfield et al., 1981). The suppressive effect primarily was directed at T-dependent responses including VSG-specific responses with a predominant effect on Th cell clonal expansion rather than cytokine production since proliferative but not cytokine responses to VSG largely were affected (Sternberg & McGuigan, 1992; Schleifer et al., 1993; Sternberg & Mabbott, 1996; Millar et al., 1999). Ironically, it was the IFN-g-dependent activation of macrophages that produce NO (and other factors such as TNF-a and prostaglandins) that had a direct suppressive effect on T-cell proliferative responses, but minimal effects on the cytokine responses of T cells (Schleifer & Mansfield, 1993; Hertz & Mansfield, 1999). Thus, by provoking an early Th1 response during infection, trypanosomes both sow the NO-dependent seeds of their own destruction as well as trigger an NO-dependent suppressor mechanism in macrophages that limits clonal expansion of parasite antigen-activated Th1 cells that produce IFN-g.

purposes of parasite growth and transmission, there appears to be expressed a genetically based program of ever increasing trypanosome virulence. This pattern of virulence expression, recently termed “virulence rheostat” (Mansfield & Paulnock, 2005, 2008), is inherent to each trypanosome cell and is expressed by daughter cells arising by binary fission from less virulent parental cells (Inverso & Mansfield, 1983; Inverso et al., 1988). The consequence of elevated virulence expression is that, following a useful period of infection in which a host serves as a suitable reservoir for transmission of disease, no resistant hosts survive to pass their genetic information on to their offspring. This mechanism may have been selected for in order to provide trypanosomes, which possess a limited genome, the ability to ultimately escape destruction no matter the spectrum of genetic resistance mechanisms displayed by the animal or human hosts upon which infected tsetse flies feed. An analysis of low and high virulence trypanosomes within a genetically homogenous parasite model system has revealed several important features. First, virulence is not linked to the VSG coat, the mechanism of VSG gene expression, or the specific chromosome telomeric VSG gene expression site (Inverso et al., 1988; Inverso et al., 2010). Second, virulence expression results in the abrogation of measurable host innate and adaptive resistance (Fig. 4) (Mansfield & Paulnock, manuscript in preparation). For example, while a spectrum of relative resistance is observable when genetically different mouse strains are infected with low virulence organisms, infection of these animals with high virulence trypanosomes derived from the low virulence parental cells eliminates this spectrum and results in rapid death. Similarly, infection of scid mice, which

Trypanosome Virulence: The Ultimate Trump Card

It should be clear from the previous sections that African trypanosomes regulate the host’s ability to recognize parasite antigens, activate cells for parasite killing, and regulate the quality and magnitude of the adaptive immune response by various means. However, there is another weapon in the parasite’s armory that trumps all of the immune evasion mechanisms displayed during infection: Trypanosomes express the ability to progressively up regulate their virulence (Inverso & Mansfield, 1983; Inverso et al., 1988; Mansfield, 2006). The evolutionary pressure to survive in the mammalian host for a period of time (i.e., the time sufficient for trypanosomes to increase their numbers so that natural transmission by the tsetse fly is successful), has resulted in the complex evasion mechanisms noted previously that alter host innate and adaptive immunity. The end result is that parasite numbers remain episodically elevated but not to the point of terminally and prematurely overwhelming the host. However, coincident with preserving the infected host for

FIGURE 4 Virulent trypanosomes overcome genetically based differences in adaptive immune resistance as well as common elements of innate resistance. T. brucei rhodesiense LouTat 1 is a low virulence parasite while T. brucei rhodesiense LouTat 1A is a genetically related high virulence organism that arose from LouTat 1. Adapted from Mansfield & Paulnock, manuscript in preparation.


have no adaptive immune system with low virulence trypanosomes, reveals a level of innate resistance and this resistance is also ablated when such animals are infected with more virulent trypanosomes. Third, measurable and repeatable differences in the pattern of intraclonal parasite gene and protein expression occur as trypanosomes become more virulent over the period of infection, and some genes and proteins are predicted to impact on host immunity (Inverso et al., 2010). This pattern is, however, not reversible in infection of the mammalian host but must, somehow, be reset to a low level of virulence in nature, perhaps in the intermediate host and vector, the tsetse fly. Fourth, trypanosomes displaying an elevated virulence phenotype do not induce the same pattern of early gene expression in the infected host as do low virulence trypanosomes. A microarray analysis of immune system related genes activated within 72 hours of infection revealed that whereas the virulent organisms activate some of the same genes as less virulent organisms (Lopez et al., 2008), it was a very small subset of the total array of genes activated (J. M. Mansfield & D. M. Paulnock, unpublished). Thus, virulent trypanosomes do not trigger the same danger signals and genetic events as those triggered by less virulent organisms. This is not related to the GPI residues expressed by high and low virulence trypanosomes, which appear to be identical in composition and activity in vitro (J. M. Mansfield, unpublished data).

Summary

Trypanosomes utilize a passive form of immune evasion, antigenic variation of the VSG surface coat, and modulations in the coat structure to avoid immune detection and elimination during infection. This affords the parasite ample time to replicate in host tissues and for the infected host to serve as a reservoir of parasites so that natural transmission of the disease by tsetse flies occurs. Clearly however, as previously detailed, the process of antigenic variation is supplanted by other, more active forms of immune evasion that permit the parasite to persist and which affects every parameter of postreceptor signaling, gene transcription, cell activation, and, ultimately, all expression of antiparasite effector mechanisms during infection. Because of the complete and varied manner of immune evasion displayed by African trypanosomes, these protozoan pathogens readily deserve the moniker of “master assassins.” In the next section, the complex array of immune evasion mechanisms exhibited by the African trypanosomes is contrasted with types of immune evasions displayed by another trypanosomatid. The Leishmania spp. reside intracellularly for most of their life cycle and, as a result, are not exposed to the broader array of innate and adaptive immune defense mechanisms by the host relative to African trypanosomes. As a result, the evolution of and selection for immune evasion mechanisms that are highly specific for the intracellular survival and propagation of these parasites are evident.

IMMUNE EVASION BY LEISHMANIA

Some parasites reside intracellularly during infection and do not exhibit substantial antigenic variation. For these organisms, some of which reside in antigen-processing cells capable of triggering protective host T-cell responses to antigenic peptides displayed by MHC molecules, other active mechanisms are employed to evade host immunity. In this section, the novel means by which Leishmania modulates intracellular signaling pathways of the infected cell in order to evade immunity are explored and evaluated. Leishmania parasites infect host macrophage cells during their life cycle. Development of effective cell-mediated immune responses to these organisms requires that infected macrophages induce significant Th1 cell activation against leishmanial antigens. The subsequent type 1 cytokine response

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activates macrophages to become microbicidal effector cells (reviewed in Olivier et al., 2005). In this regard, IFN-g has been recognized as the most potent activator of macrophage functions needed for parasite control. IFN-g ligation with the IFN-g receptor leads to rapid activation of the JAK-STAT-1a signaling pathway (Leonard & O’Shea, 1998), which regulates the expression of key macrophage genes and proteins. The integrity of this type of signaling pathway is of paramount importance for host protection against Leishmania.

General Mechanisms of Immune Modulation

However, infection of macrophages with Leishmania donovani leads to the inhibition of many cellular functions including phagocytosis, IFN-g-inducible MHC class II expression, IL-1 production, lipopolysaccharide (LPS)-mediated and phorbol ester-mediated c-fos gene expression, and generation of oxygen radicals in response to the chemotactic peptide fMetLeu-Phe (fMLP) or phorbol myristate acetate (Olivier et al., 2005). It has been demonstrated that Ca21-dependent and protein kinase C (PKC)-dependent signaling is deficient in L. donovani-infected human monocytes (Olivier et al., 1992; Olivier et al., 1992), and this was directly responsible for inhibition of several macrophage functions (see Fig. 5). One of the consequences of L. donovani infection was the induction of abnormal macrophage plasma membrane permeability to Ca21, leading to rapid and sustained elevation of the intracellular Ca21 concentration ([Ca21]i) (Olivier et al., 1992), possibly involving a capacitative mechanism maintained by persistent depletion of intracellular Ca21 stores. The most abundant parasite surface molecule, lipophosphoglycan (LPG), and its structural substituents may be involved to some extent in this cellular process. Such Ca21 mobilization may also have led to the activation of an inositol triphosphate (IP3) phosphatase that can dephosphorylate and degrade the inositol phosphates (Kukita et al., 1986), thus explaining in part the low fMLP-stimulated IP3 activity measured in L. donovani to infected macrophages (Olivier et al., 1992). On the other hand, such [Ca21]i elevation may have promoted the activation of Ca21-sensitive phosphoprotein phosphatases, such as the Ser/Thr phosphatase calcineurin, leading to the dephosphorylation and inactivation of several critical cellular proteins. In addition, it should be noted that other phosphatases, such as phosphotyrosine phosphatases (PTP), endogenously produced by host cells in response to Leishmania infection or produced by various pathogens—as reported for Yersinia infection—in which the PTP YopH is inducible by (Ca21)i present in infected cells, may cause dephosphorylation of tyrosyl residues of host cell proteins and interfere with signaling cascades (Bliska et al., 1993; Olivier et al., 2005).

PTP Regulation

Phosphorylation of tyrosyl residues by phosphotyrosine kinases (PTK) is a primordial step in the regulation of many cellular events. A number of growth factors have been demonstrated to use tyrosine phosphorylation as a mechanism for transduction of an extracellular signal via their specific receptors (Hunter & Cooper, 1985). Tyrosine phosphorylation is a common event in the initiation of cell proliferation, but its role in signal transduction regulating the cellular functions of nonproliferative hematopoietic cells is also well documented (Olivier et al., 2005). With regard to PTP, it has been estimated that there are more than 500 genes coding for this phosphatase, and studies of the role of PTP and PTK in the regulation of various cellular functions are of great interest. Activation of macrophage functions such as generation of a respiratory burst in response to zymosan, phagocytosis via Fc receptors, tumoricidal activity induced by LPS and IFN-g, NO production in response to LPS and/or IFN-g, induction of IL-12 and TNF-a by LPS (reviewed in

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FIGURE 5 Leishmania-induced macrophage signaling alteration. Binding of Leishmania to host cell receptors is potentially responsible for the induction of deactivating events involving proteasome and SHP-1 activation. SHP-1 negatively affects JAK2 kinase and Erk1/Erk2 MAPK conducting to the inhibition of IFN-g-inducible macrophage functions. Proteolysis of signaling molecules such as STAT-1 contributes to this inactivation process. Other phosphatases (e.g., IP3 phosphatase and calcineurin) and surface parasite molecules (i.e., GP63 and LPG) are recognized for their role in the alteration of various second messengers, acting directly (e.g., LPG-mediated PKC inactivation) or indirectly (e.g., GP63-mediated SHP-1 activation and concurring to kinase inactivation), signaling alteration that ultimately are reflected by abolition of agonist-induced macrophage functions.

Olivier et al., 2005; Abu-Dayyeh et al., 2008), regulation of eicosanoid biosynthesis, and MHC class II expression following IFN-g stimulation of 2C4 cells in somatic-cell genetic experiments have all been shown to involve, at least in part, tyrosine phosphorylation-mediated signaling events. The importance of macrophage PTP in NO down regulation has been demonstrated (Olivier et al., 1998); inhibition of PTP with peroxavanadium (pV) compounds led to increased responsiveness to IFN-g stimulation, which was reflected in increased NO production. A correlation between PTP inhibition in macrophages and enhancement of NO production was further supported by an increase in PTK activity and tyrosyl residue hyperphosphorylation (Olivier et al., 1998). It is clear that IFN-g induces tyrosine phosphorylation-mediated cellular functions (Olivier et al., 1998), and there is strong evidence that this cytokine can specifically activate JAK2 kinase, a PTK, to achieve this effect (Hunter, 1993). JAK2 kinase becomes rapidly phosphorylated following IFN-g activation and subsequently induces tyrosine phosphorylation of the

latent cytoplasmic transcriptional activator STAT proteins. This signaling pathway is altered in L. donovani-infected macrophages due to immediate PTP SHP-1 activation following the initial parasite–macrophage interaction (Blanchette et al., 1999; Olivier et al., 2005) (Fig. 5). Importantly, it has been recently observed that activation of macrophage PTPs SHP-1 and PTP1B by several Leishmania species is mediated by the Leishmania surface metalloprotease GP63 and that parasites lacking this protease are affected in their capacity to induce host PTPs concurring to abolish LPS and/or IFN-g-induced macrophage functions (Gomez et al., 2009) (see Fig. 6). Recent findings revealed that SHP-1 can rapidly inactivate IRAK-1, a critical kinase of TLR-mediated signaling, by interacting with an evolutionarily conserved kinase tyrosyl-based inhibitory motif (KTIM) (Abu-Dayyeh et al., 2008) and therefore concurring to inhibit several LPS-mediated macrophage functions (e.g., IL-12, NO, TNF-a). Those findings permit a better understanding of how LPS and/or IFN-g-inducible phagocyte functions (e.g., MHC class II expression and IL-12


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FIGURE 6 Rapid inactivation of IRAK-1 kinase by PTP upon Leishmania infection concur to tame down macrophage innate immune response. IRAK-1 is a critical kinase-regulating majority of TLR family members at the exception of TLR-3. The mechanisms whereby the Leishmania parasite can avoid the induction of the macrophage innate immune response involve the activation of PTPs by the metalloprotease GP63 and the rapid inactivation of IRAK-1 by SHP-1 recognizing a KTIM motif found in the kinase domain of this pivotal second messenger. This inactivation concurs to avoid activation of TLR signaling as also reflected by the complete inhibition of LPS-mediated functions.

generation) are inhibited by Leishmania. In regard to IFN-g, it is possible that the IFN-g receptor may be differentially expressed in Leishmania-infected macrophages, and this might explain the cellular unresponsiveness to IFN-g stimulation. However, contradictory observations, potentially due to the different types of macrophages studied or the use of inadequately differentiated cells (Reiner et al., 1990; Ray et al., 2000), render this interpretation uncertain. In fact, whereas IFN-g-induced IL-12, inducible NO synthase (iNOS), and MHC class II expression are affected by Leishmania infection, the induction of MHC class I antigen presentation (Kima et al., 1997) and the expression of immunoproteasome subunits LMP-2, LMP-7, and MECL-1 mRNA in response to this cytokine are normal (M. Olivier, unpublished data). These observations suggest that the IFN-g receptor generally is not affected in Leishmania-infected cells and further reinforces the notion that other signaling pathways are triggered in response to IFN-g stimulation. In-depth experiments performed in vitro and in vivo using viable motheaten mice deficient for PTP SHP-1 have

firmly established that SHP-1 plays a pivotal role in Leishmania-infected macrophage dysfunction and the survival of this parasite within its host cell (Forget et al., 2001; Forget et al., 2005b, 2006). The development of cutaneous leishmaniasis was significantly reduced during the early stage of infection in the absence of SHP-1 and was coincident with the up regulation of inflammatory and NO-dependent protective mechanisms regulated in part by STAT-1a-dependent signaling events. Similarly, the PTP PTP1B deficient mice were found to show significant reduction of cutaneous leishmaniasis progression in correlation with augmented innate immune and microbicidal functions (Gomez et al., 2009). This latest observation suggests that both PTPs are necessary for full macrophage inactivation process upon initial interaction with the parasite and its host cells, as the absence of either PTP shows the parasite having a reduced capacity to fully shut-down the initial innate immune response storm. Phosphorylation of proteins at their tyrosyl residues can result from increases in PTK activity, decreases in PTP activity,

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or a combination of the two. As described previously (Olivier et al., 1998), the use of PTP inhibitors coupled with direct measurements of PTK or PTP activities in cell preparations is a valuable tool for investigating whether the regulation of tyrosine phosphorylation following specific stimulation is PTK or PTP regulated. In macrophages, SHP-1 can be rapidly phosphorylated on its tyrosyl residues following stimulation with colony-stimulating factor 1 (CSF-1), which suggests that this PTP may be involved in early events of growth factor signal transduction (Yeung et al., 1992). SHP-1 contains two SH2 domains in its N-terminal region that may direct this unique PTP to tyrosine-phosphorylated proteins, thereby modulating PTK-related signal transduction (Shen et al., 1991). SHP-1 is expressed predominantly in hematopoietic cells and is thought to play a major role in functions regulated by tyrosine phosphorylation, with dephosphorylation acting as a signaling terminator (Yi et al., 1992). As previously discussed, the demonstration (Olivier et al., 1998) that macrophages pretreated with the pV compound bpV(phen) were more responsive to IFN-g stimulation is consistent with previous observations concerning the use of PTP inhibitors to modulate tyrosyl phosphorylation-dependent mechanisms (Posner et al., 1994). pV treatments clearly render macrophages more responsive to extracellular stimuli; experiments performed with these compounds in vivo have revealed that modulation of PTP activities completely blocks the progression of murine visceral and cutaneous leishmaniasis (Olivier et al., 1998). NO was one of the key molecules modulated by pV treatment leading to the control of Leishmania infection (Matte et al., 2000). Collectively, these experiments have provided the first demonstration that inhibitors directed toward a signaling molecule can modulate the progression of an infection. Activation of macrophage PTPs must involve signaling events that are rapidly induced following parasite attachment to the host cell membrane. For example, activation of the macrophage SHP-1 in response to CSF-1 is accompanied by tyrosine phosphorylation of the enzyme (Yeung et al., 1992). Leishmania can also induce tyrosine phosphorylation of macrophage SHP-1 (Blanchette et al., 1999). lt is interesting that both macrophage Ca21 influx and PTP activities are rapidly inducible by Leishmania infection and that the [Ca21]i of L. donovani-infected human monocytes was abnormally elevated (Olivier et al., 1992) due to increased macrophage plasma membrane permeability for Ca21. This increase in macrophage Ca21 influx was rapidly induced following the initial Leishmania–macrophage membrane interaction. In regard to PTP activation, it has been recently observed that Leishmania metalloprotease GP63 can rapidly cleave and activate PTPs within the macrophage, passing via the lipid-raft microdomains (Gomez et al., 2009).

Downstream Regulatory Events

Internalization of Leishmania, coated or not with host opsonins such as Ab or C3b (Mosser & Edelson, 1984), is known to occur via specific macrophage receptors (i.e., FcgR, CR3, CR1, and mannose receptor) (Russell & Talamas-Rohana, 1989; Guy & Belosevic, 1993). Several reports have shown that specific ligation of macrophage receptors such as FcgR and CR3 leads to the inhibition of certain macrophage functions (Marth et al., 1997; Sutterwala et al., 1997). It has also been shown that CR3 ligation inhibits IFN-g-induced phosphotyrosine phosphorylation of STAT-1. In light of these aggregate observations, it is predictable that one or more of these ligand-receptor interactions are involved in Leishmania-induced host cell PTP activation. As previously discussed, there is supportive evidence that SHP-1 is responsible for host cell tyrosyl residue dephosphorylation, JAK2 signaling alterations, and subsequent macrophage

dysfunctions (Blanchette et al., 1999) (see Fig. 5 and 6). The central involvement of macrophage SHP-1 in these deactivating processes has been confirmed in experiments with immortalized bone marrow-derived macrophage (BMDM) cell lines from SHP1-deficient motheaten (C3HeB/FeJme/me) and viable motheaten (C57BL/6J me(v)/me(v) ) mice and from their respective littermates (C3HeB/FeJ me/1 and C57BL/6J me(v)/ 1) which were not deficient in SHP-1 (Kozlowski et al., 1993; Forget et al., 2001, 2005b). Whereas SHP-l-mediated JAK2 alteration may result in the inability to tyrosyl phosphorylate STAT-1a, and consequently abolishes its nuclear translocation, it is possible that L. donovani infection has triggered other cellular mechanisms that affect STAT-1a proteolysis. Contrary to what was initially believed, (i.e., that STAT-1a alteration was caused solely by SHP-1), IFN-g-inducible STAT-1a translocation in SHP1-deficient BMDM infected by Leishmania was not seen even if infected cells were capable of mounting a normal response to IFN-g by secreting NO (Forget et al., 2006). Observations of macrophage STAT-1a protein degraded in Leishmania-infected cells in comparison to uninfected macrophages was found to be mediated by proteasome activation following Leishmania infection (Forget et al., 2005a). Proteasomes are known to modulate several transcriptional regulators, including IkB/NF-kB activation, as well as STAT proteins. STAT-1a inactivation following Leishmania infection could involve the participation of proteasome (26S [20S1PA700,11S], responsible for ATP-dependent proteolysis) and/or other proteolytic molecules. Using the proteasome antagonists lactacystin and MG-132, it has been possible to block Leishmania-induced macrophage STAT-1a proteolysis in a dose-dependent manner (Forget et al., 2005a). A role for proteasome-mediated proteolysis involving ubiquitination in the regulation of the transcription factor STAT has been previously reported, but the participation of PTP, such as SHP-1, in this control is still controversial (Haspel et al., 1996). The fact that SHP-1 is not responsible for Leishmania-induced STAT-1a inactivation (Forget et al., 2005a, 2006) strongly suggests that SHP-1 is not involved in the regulation of this transcription factor. A molecule acting negatively on STAT-1 regulation has been recently described and named PIAS1 (protein inhibitor of activated STAT-1) (Liu et al., 1998). PIAS1 specifically associates with STAT-1 dimers, leading to their inactivation by an unknown mechanism. That this STAT-1-inhibitory molecule is activated by Leishmania infection remains to be determined.

Summary

In summary, the protozoan parasite Leishmania has developed several powerful strategies to subvert the macrophage signaling system, and, consequently, this affects the development of protective immune responses to favor parasite survival. Other unicellular and multicellular parasites have evolved different means of desensitizing immune cells to avoid detection and destruction by the immune system. This involves altering signal transduction mechanisms, driving the activation of T or B cells that are indispensable for host protection. For instance, the rodent filarial nematode Acanthocheilonema viteae secretes a phosphorylcholine-containing glycoprotein, termed ES-62, which desensitizes B and T lymphocytes to subsequent activation of several signaling pathways involving the PKC, Ras mitogen-activating protein kinase, and phosphoinositide3-kinase (PI3K) (Harnett et al., 1999). A homolog of ES-62 exists in Brugia malayi, a human filarial nematode, at concentrations equivalent to those found in the bloodstream of infected humans. It is possible that such phosphorylcholinecontaining molecules affect the signaling integrity of human lymphocytes. Theileria annulata-infected bovine leukocytes are transformed into proliferating metastatic tumors resembling a leukemia-like disease. This infection is a good example where


the subversive mechanism by which the parasite favors its dissemination and pathogenesis involves the activation of specific signaling events. For instance, data (Chaussepied et al., 1998) have shown that uncontrolled leukocyte proliferation triggered by T. annulata infection was the consequence of constitutive AP-1 transcriptional activation involving up regulation of all members of Jun/Fos protein family, associated with permanent Theileriainduced JNK activation. Recent observations revealed that GPl-anchored surface proteins and related glycoconjugates of Plasmodium, Trypanosoma, and Leishmania differentially modulate the host immune response and may affect specific signaling pathways (see previous discussion on African trypanosomes) (Tachado et al., 1999). Whereas GPI purified from Plasmodium and Trypanosoma was shown to induce PTKdependent and PKC-dependent signaling events leading to NF-kB/rel-dependent lL-1, TNF-a and iNOS gene expression, GPI substituents isolated from Leishmania mexicana were capable of antagonizing PKC activity, leading to the inhibition of TNF-a expression (Tachado et al., 1997). Similarly, the Leishmania surface molecule LPG has been extensively studied and reported to be a powerful inhibitor of PKC, leading to the inactivation of several macrophage functions regulated by this signaling pathway (Turco, 1999). More recently, several reports point toward metalloprotease GP63 as an important virulence factors that could influence several signaling events during the initial Leishmania-macrophage interaction (Gregory et al., 2008; Gomez et al., 2009). Down modulation of macrophage functions as a result of abnormal signaling also has been reported for macrophages that have engulfed malarial pigment hemozoin (Schwarzer & Arese 1996; Schwarzer et al. 1998). Toxoplasma gondii infections cause host immune response down regulation and, in particular, CD41 T-cell anergy. Expression of the immunosuppressive cytokine IL-10 may be partly responsible for this inactivation, but recent observations suggest that alterations in Ca21 mobilization in T cells isolated from Toxoplasma-infected animals directly affect the translocation of the Ca21-dependent transcription factor NF-ATc that is required for T-cell proliferation (Haque et al., 1998).

CONCLUSIONS

Parasitic protozoa regulate almost every aspect of host innate and adaptive immunity, and have evolved multiple mechanisms that permit passive and active evasion of host resistance. These mechanisms include not only the expression of variant antigens and specialized surface coats (African trypanosomes), but also the modulation of antigen expression and signaling pathways of the cells that they infect or interact with (trypanosomes and Leishmania spp.), effectively subverting or suppressing cellular mechanisms that can affect parasite survival. A variety of very distinct molecular mechanisms underlie parasite-induced antigen recognition and signaling abnormalities, and the challenge for immunologists is to more precisely determine how these events are triggered and what can be done to inhibit or regulate them.

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GENETICS OF THE ANTIMICROBIAL HOST RESPONSE

VII



37 Genetics of Antibacterial Host Defenses STEVEN M. HOLLAND

INTRODUCTION

CUTANEOUS DEFENSES

The struggle between humans and the organisms that inhabit and challenge them is constant and played out on many different fronts. Some of the interactions are distinctly hostile, such as between virulent microbes and susceptible hosts. However, the majority of encounters between us and the microbial world are benign and scripted interactions that stretch way back into evolutionary time. In fact, some cohabitations are even necessary for our very survival, such as the provision of vitamin K for the integrity of our coagulation system by enteric inhabitants. Therefore, the host response to bacteria has to be extraordinarily nuanced, with our epithelium and immune systems more finely attuned to differences between microbes than most microbiology laboratories. That is, in the normal state, we are innately aware of the differences between pathogens and commensals and respond accordingly. Failures of host–bacterial interaction typically occur in the immune compromised, either due to iatrogenic, disease, or genetic causes. Because genetic mutations offer the most specific, focal defects, allowing for mechanistic and pathophysiologic understanding, they have deservedly garnered a growing level of attention. However, it is critical to keep in mind that the overwhelming majority of bacterial infections occur in people thought to be “normal” or in the setting of iatrogenic (e.g., chemotherapy) or acquired (e.g., malnutrition) susceptibilities. In this chapter, we will review the well-characterized genetic syndromes of bacterial susceptibility with an eye toward specificity of the susceptibility, applicability of the defect to infections in the general population, and, where applicable, treatment. We will consider defects in barriers involving the skin and lung and defects of leukocyte number, trafficking, and function. Although our focus here is on bacterial defense, some of these defects also predispose to fungal and viral infections as well.

Skin constitutes our major organ and is in contact with the outside world at all times, as both a colonized and a confronted surface. The major protection afforded by the skin is due to the stratum corneum, which provides a multifunctional barrier retarding water loss and providing an antimicrobial barrier. Hydrophobic lipids (ceramides, cholesterol, free fatty acids) are deposited by the epidermal lamellar body, which delivers lipids, enzymes, proteases, and antimicrobial peptides into the stratum corneum, creating the first line of host defense. Serine protease activity is critical in stratum corneum integrity as evinced by Netherton syndrome, an autosomal recessive disease due to mutations in SPINK5, which encodes the serine protease inhibitor, lymphoepithelial Kazal-type trypsin inhibitor (LEKTI). Therefore, Netherton syndrome is due to excessive serine protease activity, leading to severe atopic dermatitis and characteristic bamboo hair. Patients with Netherton syndrome have thin and excessively permeable stratum corneum. SPINK5 single nucleotide polymorphisms (SNPs) such as Glu420Lys are over-represented in atopic dermatitis in several population-based studies, suggesting that the function of LEKTI is critical for development of atopic dermatitis (Renner et al., 2009; Elias & Schmuth, 2009). Filament aggregating protein (filaggrin, FLG) is a protein that aggregates keratins in the cytosol and attaches them to the cell walls of corneocytes. Synthesized as a large phosphorylated cationic precursor, pro-filaggrin, it is dephosphorylated and cleaved into monomers. Heterozygous, compound heterozygous, and homozygous mutations in filaggrin account for up to 50% of European cases of atopic dermatitis due to transcutaneous water loss and dehydration of the stratum corneum. Decreased filaggrin expression is common in ichthyosis vulgaris as well as in atopic dermatitis. Heterozygous mutations cause diminished filaggrin expression and milder dermatitis, whereas homozygous and compound heterozygous filaggrin mutations cause severe scaling and atopic dermatitis. Cutaneous viral infections such as molluscum contagiosum, herpes simplex (eczema herpeticum), and vaccinia (eczema vaccinatum) can complicate atopic dermatitis, as can fungal infections such as tinea corporis and infections from Malassezia furfur. Most importantly, atopic dermatitis skin is heavily colonized and often infected with Staphylococcus aureus. These susceptibilities are likely due to the

BARRIER DEFECTS

Barriers keep our epithelial surfaces free of invasive bacteria, relying on physical and biochemical factors to minimize exposure and maximize clearance of microbes. Impairment of these barriers leads to overaccumulation of bacteria and may also result in abnormal responses to the bacteria that are retained. Steven M. Holland, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1684.

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474


T helper 2 (Th2) lymphocyte profile in the skin of patients with atopic dermatitis. The Th2 cytokines IL-4 (interleukin 4) and IL-13 drive down the cutaneous antimicrobial peptides LL-37 and the b-defensins (Elias & Schmuth, 2009). IL-17 is also important in the regulation of cutaneous health, through the regulation of IL-22 and other cytokines. Many of these cytokines use signal transducer and activator of transcription 3 (STAT3) as a critical signaling molecule, explaining why atopic dermatitis is pronounced in the setting of STAT3 deficiency, otherwise known as hyper IgE recurrent infection (Job’s) syndrome. In this disease, dominant negative mutations in STAT3 lead to about 25% normal signaling through STAT3-dependent pathways, causing complex immune and somatic defects, among which are atopic dermatitis and recurrent cutaneous staphylococcal infections. One of the cardinal findings in Job’s syndrome is the lack of IL-17-committed T lymphocytes (Th17) (Paulson et al., 2008).

PULMONARY DEFENSES

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the gene that encodes the major epithelial chloride channel (Rowe et al., 2005; O’Sullivan & Freedman, 2009). Although CF has effects on the gastrointestinal tract and sweat glands, its cardinal, morbid, and mortal effects are respiratory and due overwhelmingly to infection. Located on chromosome 7 and spanning 180,000 bp, the CFTR product is a member of the ATPbinding cassette (ABC) family of genes. These proteins are membrane-spanning transport molecules, and include such members as P-glycoprotein, the mediator of chloroquine resistance in Plasmodium falciparum. The disease is best characterized in Caucasians of Northern European descent, but can occur in essentially all ethnic groups. The frequency of mutated alleles in the general Caucasian population is at least 4%, and the frequency of infection is around 1 out of 3,200 Caucasian births. CFTR mutations have been divided into six types, reflecting the physiologic consequences of the underlying mutation: type I, failure to make protein; type II, impaired maturation with early degradation; type III, impaired regulation; type IV, impaired chloride conductance; type V, diminished transcripts; type VI, accelerated degradation from the cell surface. Classes I, II, and III are considered severe. The most common mutation is deletion of phenylalanine 508 (delF508), leading to impaired folding and degradation (type II). The consequences of CFTR mutation are many and complex and correlate to large extent with severity of the mutations. Severe forms of CF are complicated by pancreatic destruction and insufficiency, cirrhosis, and severe lung disease characterized by recurrent infections, bronchiectasis, upper lobe cystic changes, and progressive respiratory compromise leading to pulmonary failure. The underlying mechanism is thought to be impaired inward chloride flux, leading to excessive chloride and sodium in the airway lumen, causing dessication of respiratory mucus, impaired ciliary function, and retained tenacious secretions. This viscous hypertonic mucus impairs antibacterial peptide activity (Goldman et al., 1997) and is receptive for bacterial, mycobacterial, and fungal growth. Aggressive control of infections in CF has led to major changes in survival and outcome. Infections in CF lung vary with age and exposure (Foweraker, 2009). Children with CF develop infections with S. aureus and H. influenzae, but these are typically treatable and do not persist into later years. In contrast, another frequent childhood pathogen, Streptococcus pneumoniae, is

uncommon in CF. Later in life, infections with Pseudomonas aeruginosa, Burkholderia cepacia complex, and Burkholderia gladioli become recurrent, antibiotic resistant, and difficult to manage. With age, and typically in the setting of milder mutations, nontuberculous mycobacterial infections, especially Mycobacterium avium complex and Mycobacterium abscessus complex occur, with attendant symptoms and progressive lung destruction (Olivier et al., 2004). Despite the fact that CF is classically considered an autosomal recessive disease, heterozygosity for CFTR mutations is not entirely silent. Heterozygous mutations are encountered at higher rates in bronchiectasis, nontuberculous mycobacterial infections, congenital absence of the vas deferens, pancreatitis, rhinosinusitis, and nasal polyps. Presumably, these reflect some form of mild haploinsufficiency. Careful studies of background genetics have identified several gene polymorphisms as potential modifiers in cystic fibrosis (Collaco & Cutting, 2008). Primary ciliary dyskinesia (PCD) represents a distinct form of pulmonary infection susceptibility (Knowles & Boucher, 2002). Cilia are highly conserved throughout evolution and play central roles ranging from the locomotion of unicellular organisms, the locomotion of sperm, the rotation of organs during embryogenesis, and the movement of mucus. Like CF, patients develop chronic bronchitis leading to bronchiectasis and infections with Haemophilus influenzae, S. aureus, and S. pneumoniae. However, mucoid Pseudomonas aeruginosa and nontuberculous mycobacterial infections may occur as patients age. They also develop chronic rhinosinusitis and recurrent otitis media. Whereas CF is caused by defects in chloride channel function, PCD is due to mutations in the genes that control cilia formation and function, leading to impaired ciliary motion. Human consequences include impaired mucus movement and clearance in the lung leading to bronchiectasis, male infertility, anomalies of organ laterality (e.g., situs inversus, heterotaxy) and congenital heart disease (Leigh et al., 2009). Mutations in eight genes have been identified causing ciliary defects so far: DNAI1, DNAH5, DNAH11, DNAI2, KTU, RSPH9, RSPH4A, and TXNDC3, but this list will clearly expand. Surprisingly, no clear genotype2phenotype distinctions have yet emerged to separate these different genes. Clinically, these patients typically present with some form of respiratory distress in the neonatal period, presumed due to failure to clear fluid after birth. However, the majority of cases get beyond this and only receive a diagnosis at around age 4, recognized because of recurrent respiratory infections or cough. Another relatively common finding in PCD is laterality defects, which manifest as either classic Kartagener’s syndrome (chronic sinusitis, chronic bronchitis, and situs inversus) or less complete versions of laterality defects, such as heterotaxy (hetero-, other; taxy, arrangement), in which the heart, liver, spleen, and abdominal viscera are randomly left2right oriented. The role for heterozygous mutations in these genes and how they may affect other genetic predispositions, like heterozygosity for CFTR, have not been determined.

IMMUNE DEFECTS

Although multiple immune genetic defects can lead to bacterial susceptibility, such as those associated with severe lymphocyte dysfunction in severe combined immune deficiency (SCID) or acquired immunodeficiency syndrome (AIDS), these syndromes have such extensive derangements of immune homeostasis that dissection of the genetic contribution to specific bacterial infections is complex. In contrast, mutations in the genes regulating the innate immune system have been very informative regarding the

37. Genetics of Antibacterial Host Defenses

specific associations of genes and infections. Therefore, we will focus on examples of innate immune defects, especially those involving phagocytic cells.

Neutropenias

Inherited neutropenias can occur in both autosomal recessive and dominant forms (Table 1). Homozygous mutations in the antiapoptotic molecule HAX1 cause autosomal recessive severe chronic neutropenia, the disease originally described by Kostmann in rural Sweden (Klein et al., 2007). HAX1 is critical for maintaining the inner mitochondrial membrane potential and protects myeloid cells from apoptosis (Zeidler et al., 2009). Some patients with severe chronic neutropenia subsequently acquire mutations in the G-CSF receptor (GCSFR) associated with the development of acute myeloid leukemia. These mutations are likely consequences of severe congenital neutropenia but are not causes of it. Dominant or spontaneous severe congenital neutropenia is due to heterozygous mutations in neutrophil elastase (ELA2, 19p13.3). Mutations in ELA2 also cause cyclic neutropenia. However, the mutations that cause cyclic neutropenia tend to be clustered around the catalytically active site of ELA2, whereas those associated with severe congenital neutropenia are located on a different side of the three-dimensional protein (Zeidler et al., 2009). ELA2 mutations are thought to cause neutropenia based on the “unfolded protein response” (Xia & Link, 2008). ELA2 mutations cause more than half of severe congenital neutropenia cases in Caucasians. Heterozygous mutations in growth factor independent 1 (GFI1) cause congenital neutropenia and monocytosis. GFI1 mutations cause dominant-negative dysregulation of C/EBP epsilon, ELA2, and the monocytopoietic cytokine CSF1 (Horwitz et al., 2007). X-linked severe congenital neutropenia (XLN) has been reported due to specific mutations in the Wiskott-Aldrich syndrome protein (WASP). In contrast to the WASP mutations that produce classical Wiskott-Aldrich syndrome or X-linked thrombocytopenia, most of which are caused by mutations resulting in reduced WASP transcription or translation, the mutation causing XLN (L270P) disrupts a WASP autoinhibitory domain, thereby creating a constitutively active mutant protein (Devreindt et al., 2001).

TABLE 1

Reticular dysgenesis is an autosomal recessive severe combined immunodeficiency, which presents as G-CSF-refractory early myeloid arrest, neutropenia, lymphopenia, and sensorineural loss. Adenylate kinase 2 (AK2) is the responsible gene; the protein localizes to the mitochondrial intermembrane space (similar to HAX1). AK2 may be important in mitochondrial energy metabolism and control of apoptosis (Pannicke et al., 2009; Lagresle-Peyrou et al., 2009). The syndrome of warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) is due to heterozygous mutations of the intracellular portion of the chemokine receptor CXCR4. These mutations lead to enhanced responses to CXCL12, CXCR4’s cognate ligand, expressed on bone marrow stromal cells. Engaged CXCR4 delays the exit of mature neutrophils from bone marrow, resulting in retained mature apoptotic neutrophils in the bone marrow (myelokathexis) and circulating neutropenia. Patients develop recurrent sinopulmonary infections, and neutrophil counts typically increase during infections. Neutrophils are also mobilized in response to steroids, epinephrine, endotoxin, G-CSF, and GM-CSF. Sustained therapy with G-CSF or GM-CSF increases the number of neutrophils in the peripheral blood and decreases the number of infections. The symptoms of warts and hypogammaglobulinemia indicate that this defect includes T- and B-cell functions as well (Kawai & Malech, 2009). The metabolism of glucose is central to most aspects of mammalian life, and to neutrophils. Mutations in glucose 6 phosphatase catalytic subunit 3 (G6PC3) cause infection susceptibility in mice and humans. The human disease is striking for the severe neutropenia in infancy and associated cardiac and urogenital abnormalities, along with prominent truncal venous patterns. These infants present with neonatal sepsis, predominantly atrial anomalies, cryptorchidism, and distinct vascular patterns. Their marrows show relatively few mature neutrophils, in comparison to those in WHIM. They do have good responses to G-CSF (Boztug et al., 2009).

IRAK4 and MyD88 Deficiencies

Signal transduction from Toll-like receptors (TLRs) and the IL-1 receptor, among others, are funneled to NF-kB activation through a variety of cytoplasmic molecules, including the

Inherited neutropenias

Disease name

475

Gene

Pattern

Manifestations

Kostmann syndrome, severe chronic neutropenia

HAX1 1q21.3

Autosomal recessive

Recurrent bacterial infections, mucosal ulcerations, onset in infancy

Severe chronic neutropenia, cyclic neutropenia

ELA2 19p13.3

Autosomal dominant

Persistent neutropenia or cyclic neutropenia every 21–28 days

Autosomal dominant neutropenia

GFI1 1p22

Autosomal dominant

Recurrent bacterial infections, mucosal ulcerations, onset in infancy

Severe congenital neutropenia

G6PC3 17q21

Autosomal recessive

Neonatal infections, structural heart defects, urogenital anomalies, truncal venous pattern

X-linked neutropenia

WAS Xp11.23

X-linked

Recurrent severe infections, monocytopenia, activating mutations

Reticular dysgenesis

AK2 1p34

Autosomal recessive

Profound neonatal susceptibility to infection, very few total leukocytes, hearing loss

WHIM syndrome

CXCR4 2q21

Autosomal dominant

Mild neutropenia, warts, low IgG, myelokathexis

476


IL-1 receptor associated kinase (IRAK) complex and myeloid differentiation primary response gene 88 (MyD88). Mutations in a critical component of the IRAK complex, IRAK4, and MyD88 lead to similar syndromes of early onset pyogenic bacterial infections with streptococci, staphylococci, and meningococci. These children often have meningitis or tissue abscesses, and case fatality rates in infancy and childhood are high. Interestingly, after patients achieve late childhood or adolescence the rates of severe infection and mortality drop precipitously (Ku et al., 2007; von Bernuth et al., 2008). An important hallmark of these syndromes is the absence of fever despite severe infection, since IL-1, a critical driver of the fever response, signals through this pathway as well.

Defects of Neutrophil Granule Formation and Content

Chédiak-Higashi syndrome (CHS) is characterized by oculocutaneous albinism, frequent pyogenic infections, neurologic abnormalities, and a relatively late-onset lymphoma-like “accelerated phase.” It is due to mutations in the lysosomal trafficking regulator gene, LYST or CHS1 (1q42.1–q42.2). Giant azurophil granules form from the fusion of multiple primary granules in neutrophils, eosinophils, basophils, and all granule-containing cells. Neutropenia occurs due to neutrophil destruction within the bone marrow. Mild mental retardation and progressive neuropathies are manifestations of the granule biogenesis defect in nonhematopoietic tissues (Huizing et al., 2008). The life-threatening accelerated phase is like other hemophagocytic syndromes with fever, hepatosplenomegaly, lymphadenopathy, cytopenias, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, and tissue lymphohistiocytic infiltration. Standard hemophagocytic syndrome therapies are effective, but, without bone marrow transplantation, it usually recurs (Eapen et al., 2007). Other diseases of neutrophil granule content include neutrophil secondary granule deficiency, Griscelli syndrome, and Hermansky-Pudlak syndrome (Huizing et al., 2008).

Defects of Neutrophil Oxidative Metabolism: Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is caused by defects in the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Table 2). The NADPH oxidase (phagocyte oxidase, phox) is a latent enzyme whose component parts are separated in the resting cell into those coembedded in the walls of secondary granules (gp91phox and p22phox) and those loosely connected in the cytoplasm

TABLE 2

Chronic granulomatous disease

Gene

Pattern and location

gp91phox CYBB

X-linked Xp21.1

p22phox CYBA

Autosomal recessive 16q24

p47phox NCF1

Autosomal recessive 7q11.23

p67phox NCF2

Autosomal recessive 1q25

p40phox NCF3 Rac2

(p47phox, p67phox, p40phox, rac). Once the complete NADPH oxidase is formed after cellular activation, an electron is taken from NADPH and donated to molecular oxygen in the phagosome, leading to the formation of superoxide. Superoxide dismutase converts this to hydrogen peroxide, which, in the presence of myeloperoxidase and chlorine, is converted to bleach (Segal et al., 2000). Phagocyte production of reactive oxygen species is apparently most critical for microbial killing due to its activation of the primary granule proteins neutrophil elastase and cathepsin G inside the phagosome. Therefore, reactive oxidants are critical intracellular signaling molecules, leading to activation of enzymatic and antimicrobial peptide killing pathways (Segal, 2008). Mutations in any of the five genes of the NADPH oxidase cause CGD. gp91phox (Xp23.1) mutations account for about two-thirds of cases. The majority of autosomal recessive cases are due to mutations in p47phox (about 25%), with mutations in p22phox and p67phox making up the remainder. Compound heterozygous mutations in p40phox were recently recognized in a single child (Matute et al., 2009). In general, mutations in the X-linked gene gp91phox are clinically more severe than those in autosomal genes. The frequency of CGD worldwide is probably around 1 out of 200,000 live births. The majority of patients are diagnosed as toddlers and young children, but cases are often recognized in older children and adults, especially in those with autosomal mutations. Infections of the lung, skin, lymph nodes, and liver are the most frequent. In North America, the infections are caused by S. aureus, Burkholderia cepacia, Serratia marcescens, Nocardia, and Aspergillus. In other countries with relevant exposures, important organisms to include in the list are Salmonella species, bacillus Calmette-Guerin (BCG), Mycobacterium tuberculosis, and Leishmania (van den Berg et al., 2009). The gastrointestinal and genitourinary tracts are frequently affected by inflammatory complications in CGD. The retinas and lungs also develop inflammatory lesions. Symptomatic, proven gastrointestinal involvement occurs in at least 43% of X-linked and 11% of autosomal recessive cases. Abdominal pain is common, as are diarrhea, nausea, and vomiting. Colonic granulomata mimic Crohn’s inflammatory bowel disease (IBD), however, extraintestinal manifestations of Crohn’s, such as pyoderma and arthritis, are typically absent (Marciano et al., 2004). Genitourinary strictures and granulomata occur in up to 18% of CGD patients, mostly those with mutations in gp91phox or p22phox.

Frequency

Comments

65–70%

Typically more severe, inflammatory bowel disease

,5%

Coexpressed with gp91phox

25%

Recurrent mutation due to nearby pseudogene

,5%

More severe than p47phox deficiency


1 case

Inflammatory bowel disease


2 cases

Defects in oxidative burst and leukocyte migration


The X-linked female carriers of gp91phox are lyonized, with two populations of phagocytes, one that produces superoxide and one that does not. Therefore, carriers have a characteristic mosaic pattern of peripheral blood neutrophils on oxidative burst testing. Infections are uncommon in these female carriers unless the normal neutrophils are below 10%, in which case these carriers are at risk for CGDtype infections. Prophylactic trimethoprim/sulfamethoxazole (5 mg/kg/day based on trimethoprim) reduces the frequency of major infections from about once every year to once every 3.5 years. It reduces staphylococcal and skin infections without increasing the frequency of serious fungal infections. Itraconazole prophylaxis is highly effective (Freeman & Holland, 2009). IFN-g (interferon gamma) also reduces the number and severity of infections in CGD by 70% compared to placebo, regardless of the inheritance pattern, sex, or use of prophylactic antibiotics. Interestingly, no significant difference could be detected in terms of in vitro superoxide generation, bactericidal activity, or cytochrome B levels. Mortality in CGD correlates with noncirrhotic portal hypertension and progressive damage of the hepatic microvasculature. Local or systemic infections, in addition to drug-induced liver injury, may be underlying conditions. A history of liver abscess, alkaline phosphatase elevations, and platelet count decrease over time and are individually associated with mortality in CGD patients (Feld et al., 2008). Successful hematopoietic stem cell transplantation (HSCT) provides a cure for CGD. Since as few as 10% of normal cells are sufficient to prevent and control infections—as shown in lyonized females—stable mixed hematopoietic chimerism is sufficient to prevent infection (Seger et al., 2002). Similarly, gene replacement or correction are attractive in CGD and are likely to be viable in the near future.

Myeloperoxidase Deficiency

Myeloperoxidase (MPO) deficiency (17q23) is a common autosomal recessive disorder with such highly variable expressivity that the vast majority of those missing the gene are clinically inapparent. It is the most common primary phagocyte disorder: 1 out of 4,000 individuals have complete MPO deficiency, and 1 out of 2,000 individuals have a partial defect. Myeloperoxidase is synthesized in neutrophils and monocytes, packaged in azurophilic granules, and used to catalyze the conversion of hydrogen peroxide to hypohalous acid (in neutrophils, the halide is Cl2 and the acid is bleach). In vitro, it is clear that MPO has a major role in defense against candida. In agreement with that, patients who have clinical findings usually have mucocutaneous, meningeal, and bone candidiasis. However, diabetes mellitus is a critical cofactor for candida species infections in the context

TABLE 3

of MPO deficiency. Definitive diagnosis is established by neutrophil/monocyte peroxidase histochemical staining or specific protein detection. There is no specific treatment for MPO deficiency; diabetes should be controlled and infections should be treated (Hansson et al., 2006).

DEFECTS OF ADHESION

Leukocyte movement from the bloodstream toward inflamed sites is necessary for tissue remodeling and prevention of severe infection.

Leukocyte Adhesion Deficiency, Type 1 (LAD1)

LAD1 is caused by mutations in the common b2 chain (CD18) of the b2 integrins (see Table 3). Each of the b2 integrins is a heterodimer composed of an a chain (CD11a, CD11b, CD11c, CD11d), noncovalently linked to the common b2 subunit, CD18. Therefore, mutations causing loss of CD18 lead to either very low or no expression of CD11a, CD11b, CD11c, and/or CD11d, causing LAD1. Severe LAD1 occurs with less than 1% of normal expression of CD18 on neutrophils; moderate LAD1 has 1% to 30% of normal CD18 expression. However, normal expression of nonfunctional b2 integrin also occurs in the case of proteinpreserving mutations. Therefore, surface expression alone is inadequate to fully characterize the severity of the lesion in LAD1; functional studies are needed. Severe LAD1 is characterized by delayed umbilical stump separation, omphalitis, persistent leukocytosis (over 15,000/ ml), and severe gingivitis and periodontitis. Recurrent infections of the skin, upper and lower airways, bowel, and perirectal area, are common and usually caused by S. aureus or gram-negative bacilli, but not by fungi. Infections tend to be necrotizing and may ulcerate. Neutrophil invasion is absent on biopsy histopathology. Impaired healing of infectious, traumatic, or surgical wounds is also characteristic of LAD1 patients. Scars have a dystrophic “cigarette-paper” appearance. Moderate phenotype LAD1 patients tend to present later in life, have normal umbilical separation, have fewer life-threatening infections, and better chances for survival. However, leukocytosis, periodontal disease, and delayed wound healing are still common. LAD1 neutrophils show diminished chemotaxis in vivo and in vitro and complement-mediated phagocytosis is severely impaired because of the absence of the complement receptor CD18/CD11b (CR3 or Mac-1) (Etzioni, 2009). Somatic reversion of selected mutations has been reported in LAD1 cytotoxic T lymphocytes (Uzel et al., 2008). Bone marrow transplantation remains the only definitive treatment, but is complicated by poor engraftment and high rates of graft versus host disease (Elhasid & Rowe, 2009).

Leukocyte adhesion defects

Disease name

Gene and location

477

Pattern

Manifestations

Leukocyte adhesion deficiency type 1

CD18 ITGB2 21q22.3

Autosomal recessive

Leukocytosis, delayed umbilical stump separation, gram positives, and enterics

Leukocyte adhesion deficiency type 2, congenital defects of glycosylation type IIc

FUCT1 SLC35C1 11p11.2

Autosomal recessive

Leukocytosis, mental retardation. characteristic facies, Bombay (Hh) blood type

Leukocyte adhesion deficiency type 3

Kindlin3 FERMT3 11q12

Autosomal recessive

Leukocytosis, bleeding diathesis due to platelet dysfunction

478


Leukocyte Adhesion Deficiency, Type 2 (LAD2); Congenital Defect of Glycosylation IIC (CDGIIC)

LAD2 or CDG-IIc is a very rare autosomal recessive defect in fucose metabolism due to mutations in the GDP-fucose transporter gene, FUCT1 (11p11-q11) (Table 3). Absent fucosylation leads to a lack of fucosylated proteins such as sialyl-LewisX, impairing the initial interaction of leukocytes with the endothelium characterized by gentle rolling adhesion. The clinical phenotype includes infections of the skin, lung, and gums, leukocytosis, poor pus formation, as well as mental retardation, short stature, distinctive facies, and the Bombay (Hh) blood phenotype. Surprisingly, the frequency and severity of infections tend to decline with time. Fucose supplementation has had variable results in LAD2 patients (Etzioni, 2009).

Leukocyte Adhesion Deficiency, Type 3 (LAD3)

LAD3 (previously known as LAD1 variant) is associated with a syndrome like Glanzmann’s thrombasthenia, a b3 integrin-related bleeding disorder (Table 3). LAD3 is due to mutations in KINDLIN3 (FERMT3), a molecule responsible for b1, b2, and b3 integrin activation in leukocytes and platelets leading to recurrent infections and bleeding (Meves et al., 2009).

RAC2 Deficiency

In one study, a single male patient with an autosomal dominant mutation in the Rho GTPase gene RAC2 (22q12.13– q13.2) had delayed umbilical cord separation, perirectal abscesses, failure to heal surgical wounds, and absent pus in infected areas despite neutrophilia. Chemotaxis and superoxide production were impaired. His neutrophils showed defective azurophilic granule release and impaired phagocytosis. Rac2 comprises more than 96% of Rac in neutrophils and is critical to both the dynamic organization of the actin cytoskeleton for movement, hence the consideration in defects of chemotaxis, and superoxide production,

hence the similarity to CGD. Bone marrow transplantation led to complete correction (Gu & Williams, 2002).

Mycobacterial Susceptibility

The mononuclear phagocyte is required to present antigen, stimulate lymphocytes, produce certain cytokines, and control infection. Mycobacteria invade and multiply within macrophages, usually leading to the production of IL-12 p70 (IL-12 p40 and IL-12 p35) and IL-23 (IL-12p40 and p19). IL-12 and IL-23 act on T and NK (natural killer) cells to phosphorylate STAT4 and produce IFN-g. IFN-g acts through its receptor to phosphorylate STAT1 and turn on interferon responsive genes. NF-kB essential modulator (NEMO) transduces signal from a wide array of cell surface receptors including tumor necrosis factor, certain TLRs, and certain cytokines, such as IL-1 and IL-18. Hypomorphic mutations in NEMO are highly associated with bacterial, viral, and nontuberculous mycobacterial infections in males. The NEMO-deficient phenotype may be complex, including hypohidrotic ectodermal dystrophy, immune deficiency, and more rarely, lymphedema and osteopetrosis. Almost 40% of males with hypomorphic mutations in NEMO develop mycobacterial infections, mostly with environmental species (Hanson et al., 2008). In heterozygous females, NEMO mutations are associated with incontinentia pigmenti (Ehrenreich et al., 2007). Patients with defects in IFNGR1 (6q23-q24), INFGR2 (21q22.1-q22.2), IL-12 receptor b1 (19p13.1), IL-12 p40 (5q31.1-33.1), STAT1 (2q32.2-q32.3), or NEMO (Xq28) are susceptible to at least Mycobacteria and Salmonella infections, but there are some very striking gene and mutation specific susceptibilities (Rosenzweig & Holland, 2005) (Table 4). Patients with autosomal recessive mutations leading to abolition of IFNGR1, IFNGR2, or STAT1 have the most severe phenotypes and present early in life, especially if they receive BCG vaccination. Those with complete defects in IFNGR1 or IFNGR2 have complete loss of signaling

TABLE 4 Mycobacterial susceptibility Disease name

Gene and location

Pattern

Manifestations

Recessive IFN-g receptor 1 deficiency

IFNGR1 6q23–q24

Autosomal recessive

Disseminated BCG, NTM infections, salmonellosis

Dominant IFN-g receptor 1 deficiency

IFNGR1 6q23–q24

Autosomal dominant

Localized BCG, NTM, especially osteomyelitis

IFN-g receptor 2 deficiency

IFNGR2 21q22.1

Autosomal recessive


Recessive STAT1 deficiency

STAT1 2q32.2–q32.3

Autosomal recessive

Disseminated BCG, NTM infections, bacterial infections, viral infections

Dominant STAT1 deficiency

STAT1 2q32.2–q32.3

Autosomal dominant

Disseminated BCG, NTM infections

IL-12p40 deficiency

IL-12p40 IL12B 5q31.1–q33.1

Autosomal recessive


IL-12 receptor b 1 deficiency

IL-12Rb1 IL-12Rb 19p13.1

Autosomal recessive


NF-kB essential modulator deficiency

NEMO IKKg IKBKG Xq28

X-linked recessive

Disseminated BCG, NTM infections, enterics, pseudomonas, CMV, PCP


through the IFN-g receptor, but retained signaling through the IFN-a receptor, and have severe susceptibility to Mycobacteria and mild susceptibility to herpes viruses and Salmonella. Those with complete defects in STAT1 have complete loss of both IFN-g and IFN-a signaling, leading to profound susceptibility to Mycobacteria, bacteria, and viruses, and early death. In contrast to those with complete defects, patients with autosomal dominant mutations in IFNGR1, typically caused by a hotspot mutation in the intracellular domain, have partial retained IFN-g signaling leading to very limited infections with nontuberculous Mycobacteria, especially osteomyelitis. The mutation causes a truncation of IFN-gR1 that preserves the extracellular ligand-binding portion but removes the intracellular signaling and receptor recycling domain. The mutant IFN-gR1 product remains stuck on the cell surface, where it binds IFN-g and interferes with the normal function of the wild-type allele, leading to a dominant negative mutant. Patients with the dominant negative mutation usually present in childhood with pulmonary nontuberculous mycobacterial infection but then may go on to develop recurrent multifocal nontuberculous osteomyelitis (Dorman et al., 2004). Very few dominant mutations in IFN-gR2 have been reported, and these seem rather severe (Rosenzweig et al., 2004). Dominant mutations in STAT1 are relatively mild and limited in infections to disseminated BCG or nontuberculous mycobacterial infections (Chapgier et al., 2006). Patients with complete IL-12 receptor b1 deficiency usually have a phenotype less severe than other complete cytokine receptor or signal molecule defects. The infection risk for nontuberculous Mycobacteria is high in childhood but wanes after age 12 for IL-12 receptor b1 deficiency (Fieschi et al., 2003). NEMO deficiency affects the signaling of TLRs, IL-1, IL-18, CD40, and TNF-a (tumor necrosis factor alpha). NEMO-impaired children also have defects in hair, tooth, and sweat gland formation (Hanson et al., 2008). Flow cytometry can detect certain IFN- gR1 mutations. Recessive mutations tend to cause loss of protein, leading to profound reductions of receptor levels on the cell surface. In contrast, in dominant forms of IFN- gR1 deficiency mutant protein is overabundant on the cell surface and easy to detect (Dorman et al., 2004). Detection of IFN- gR2 and IL-12Rb1 defects require cell culture and proliferation or sequencing. Intracellular STAT1 is phosphorylated rapidly after IFN-g stimulation, while intracellular STAT4 is phosphorylated rapidly after IL-12 stimulation, and these can be detected in flow cytometry as well. Direct detection of IL12p40 or IL-12p70 can be used for the diagnosis of patients who are deficient in IL-12p40.

479

SYNDROMES WITH IgE ELEVATION

The discovery of IgE in the 1970s led to recognition of critical components of allergy and hypersensitivity. With the identification that some children with recurrent severe staphylococcal infections and complex phenotypes had IgE elevation, the term “hyper IgE syndrome” was coined. Subsequently and not surprisingly, IgE elevation has been found to accompany many syndromes, just as staphylococcal infections have, leading to confusion about what is meant by the term hyper IgE syndrome. Recently, three distinct syndromes with overlap in IgE elevation have been identified, associated with somewhat different clinical presentations and genetic etiologies (Table 5).

STAT3 Deficiency (Job’s Syndrome; Hyper IgE Recurrent Infection Syndrome)

The hyper-IgE syndrome (HIES or Job’s) is characterized by elevated serum IgE, eczema, recurrent skin and lung infections, and somatic features including characteristic facies, scoliosis, and fractures, and is caused by dominant negative STAT3 mutations. Mutations are all protein positive and cluster predominantly in the SH2 (mediating protein–protein interactions) and the DNA-binding domains (mediating the interaction of protein with DNA) (Paulson et al., 2008). Cutaneous “cold” abscesses are common and due to S. aureus infections. While mucocutaneous candidiasis is common, systemic candidiasis is rare. Recurrent pneumonias caused by S. aureus, S. pneumoniae, and Haemophilus influenzae typically start in childhood, and are complicated by pneumatoceles and bronchiectasis. These in turn predispose to Pseudomonas and Aspergillus infections. The pneumatoceles may become secondarily infected and bleed. Scoliosis, osteopenia, minimal trauma fractures, hyperextensibility, degenerative joint disease, craniosynostosis, coronary artery aneurysms, and Chiari 1 malformations also occur frequently. The general mechanism underlying bone abnormalities is unknown, and the role of bisphosphonates in treating the osteoporosis and minimal trauma fractures in HIES is undefined. Almost all Job’s syndrome patients develop facial asymmetry, broad nose, and deep-set eyes with a prominent forehead and retain some, if not all, primary teeth. HIES patients have coronary artery aneurysms, dilatations, and tortuousities; carotid artery berry aneurysms; and early onset MRI T2-weighted hyperintensities (unidentified bright objects or UBOs). As reported in other primary immunodeficiency diseases affecting lymphocytes, both Hodgkin’s and non-Hodgkin’s lymphomas are significantly increased in Job’s syndrome.

TABLE 5 IgE elevation syndromes Disease name

Gene and location

Pattern

Manifestations

Hyper IgE, recurrent infection, (Job’s) syndrome

STAT3 17q21

Autosomal dominant

Skin and lung infections, complex somatic features

DOCK8 deficiency

DOCK8 9p24

Autosomal recessive

Severe eczema, cutaneous viral infections, cancers

Tyk2 deficiency

Tyk2 19p13.2

Autosomal recessive

Eczema, BCG, Salmonella, molluscum

Netherton syndrome

SPINK5 LEKTI 5q32

Autosomal recessive

Severe eczema, bamboo hair

480


DOCK8 Deficiency

An autosomal recessive IgE elevation syndrome due to DOCK8 mutations has severe eczema and recurrent skin and lung infections as well as cutaneous molluscum contagiosum, herpes simplex, varicella zoster, and human papilloma viruses (Table 5). Severe eczematoid rashes start early in life, although not necessarily in the newborn period. In contrast to Job’s syndrome, pneumonias due to S. aureus, H influenzae, Proteus mirabilis, Pseudomonas aeruginosa, and Cryptococcus typically heal without pneumatocele formation. This recessive disease also lacks the connective tissue, skeletal, tooth exfoliation, fracture, and characteristic facial features of Job’s syndrome (Zhang et al., 2009). Another distinct feature is that DOCK8 patients are susceptible to squamous cell and T-cell cancers, whereas those with STAT3 deficient HIES are more susceptible to B-cell lymphomas.

Tyk2 Deficiency

One case of autosomal recessive Tyk2 deficiency has been reported with IgE elevation and infection with BCG, herpes simplex, and molluscum. Tyk2 is a major signal-transducing molecule interacting with the IFN-a, IL-6, IL-10, IL-12, and IL-23 receptors (Minegishi et al., 2006).

OTHER IgE ELEVATION SYNDROMES

Netherton syndrome, due to SPINK5 mutations, also has elevated IgE, as does the syndromes of immune dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) (Torgerson, 2008).

CONCLUSIONS

There are a growing number of genetic syndromes that predispose an individual to bacterial infections. They dissect out for us the biologic pathways that are required, desirable, and dispensible for protection. The complexity of bacterial susceptibility is compounded by the extraordinary variability in bacteria themselves, as well as the ways in which we encounter them. This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH.

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38 Immunogenetics of Host Response to Parasites in Humans JENEFER M. BLACKWELL

INTRODUCTION

(reviewed in Blackwell et al., 2009a, 2009b; Burgner et al., 2006). This makes it difficult to evaluate chance statistical events from real genetic associations. In our recent review (Burgner et al., 2006), we provided comprehensive supplementary tables that listed all the studies up to that time undertaken in humans looking for susceptibility genes for leishmaniasis and malaria, while Quinnell (Quinnell, 2003) has provided a similar analysis for helminth infections. Here we discuss complexity and heritability of parasitic disease susceptibility, and how study design is evolving to provide greater statistical power to identify susceptibility to complex diseases like parasitic infections. Examples are provided (summarized in Tables 1 and 2) to demonstrate how different methods have been used to identify genes involved in innate and acquired immune responses, as well as examples of genes that determine other aspects of the host parasite relationship.

Parasitic diseases are a leading cause of human mortality and morbidity, with much of the burden falling on children (Black et al., 2003). Infectious diseases are a major selective pressure (Sabeti et al., 2002; Walsh et al., 2006) and genes involved in the immune response are the most numerous and the most diverse in the human genome (Apanius et al., 1997; Murphy, 1993), reflecting the evolutionary advantages of a diverse immunological response to a wide range of infectious pathogens. With expanding knowledge of the human genome and the development of high throughput technologies to measure human genetic variation, it has become possible to identify the numerous genes that make even modest contributions to human parasitic disease susceptibility, and to understand the complex interaction of environmental and host genetic factors. Understanding the environmental and genetic risk factors that determine why two people with the same exposure to a particular parasitic disease differ in susceptibility could provide important leads for improved therapies. Indeed, the intersect of human genetic and gene expression studies have the power to influence one of the major bottlenecks in drug development, that of choosing the best targets that represent key points of therapeutic intervention and of targeting the best possible drug to individuals who may differ in their response to therapy (Vallance & Levick, 2007). One of the major aims of genetic studies is to identify genes, mechanisms, and pathways that contribute to the pathogenesis of disease (e.g., by influencing trafficking to, or survival of, a parasite in host cell or niche or by determining the type of immune response that is made). Genetics can also provide concrete evidence for the role of modifiable environmental variables (e.g., iron) in determining disease outcome (Ebrahim & Davey Smith, 2008). Knowledge gained through both avenues can translate into improved interventions. One of the problems in reviewing the field of immunogenetics and host response to parasites just now is that most genetic studies undertaken to date have been underpowered

COMPLEXITY AND HERITABILITY

The interplay between host and parasite is complex, with multifactorial disease risk associated with multiple genetic and environmental factors. One of the first things geneticists often do is to estimate the heritable component of disease risk or of associated quantitative traits like immune response phenotypes. This provides more confidence in undertaking studies designed to find the genes involved. Twin studies are useful as the comparison of concordance of disease in monozygous versus dizygous twins provides an obvious measure of the degree of heritability of the trait. This has been used successfully to look at heritability for leprosy, tuberculosis (TB), and associated immune response traits (Jepson et al., 2001), but as far as we are aware there are no published twin studies for parasitic infections. Another way to evaluate heritability is to look for familial correlations, to determine whether disease in siblings is more highly correlated than between offspring and more distant relatives. This can be done, for example, within the FCOR program of the SAGE (Statistical Analysis for Genetic Epidemiology) software package (version 4.8, 1987). Heritability (h2) is estimated from the sibling correlations r using the equation h2 5 2r. We recently used this method to determine heritability of quantitative delayed type hypersensitivity (DTH) responses to

Jenefer M. Blackwell, Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Subiaco, Western Australia, Australia.

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leishmanial antigen in families from Natal, Brazil (Jeronimo et al., 2007). In these families, there were 440 sibling pairs with a correlation of 0.42, 212 grandparent–grandchild pairs with a correlation of 0.265, and 90 cousin pairs with a correlation of 0.13. This is consistent with genetic control of the DTH response, where first-degree relatives have a stronger correlation than second- or third-degree relatives. Estimated heritability of the DTH immune response was 84%, suggesting a substantial genetic component to variation in induration size, as determined by the DTH skin test. Others have used similar methods to look at heritability of infection intensity in helminth infections (see Bethony et al., 2001; Breitling et al., 2008; Grant et al., 2008; King et al., 2004; Williams-Blangero et al., 1997), with estimates varying from 15% to 58% depending on the study area and measure of infection intensity being examined. Other studies have looked at heritability of nonspecific and specific immune responses associated with parasitic infections. For example, Grant and colleagues (Grant et al., 2008) demonstrated 60% heritability for total IgE in an area where Schistosoma mansoni prevalence was high and heritability for S. mansoni egg counts was 31%. Stirnadel and colleagues (Stirnadel et al., 2000) were interested in heritability of antibody subclass responses to malaria antigens like RESA and MSP2, which are major vaccine candidates. Familial aggregation was observed for IgG1, IgG2, and IgG3 responses against RESA, IgG1, and IgG3 responses against the 3D7 form of MSP2, and IgG1 and IgG2 responses against the FC27 form of MSP2. Allowance for sharing of houses explained some of the nongenetic variance but not the familial aggregation. The variance of IgG3 responses against RESA and IgG1, IgG2 against MSP2 (FC27) was partly explained by sharing of HLA class II genotypes. Such host genetic variation in responses to specific parasite antigens has important implications for vaccine development. However, the paucity of antiparasite vaccines in use in humans has meant that there has not been the opportunity to look at genetic variation in response to vaccination. Variation in immune responses to viral and bacterial vaccines is highly heritable, particularly in infants (reviewed in Kimman et al., 2007).

WAYS OF MEASURING GENETIC EFFECTS

As outlined in our recent review (Blackwell et al., 2009a), traditional approaches to genetic analysis of complex diseases have included allelic association analysis of candidate genes and linkage analysis using multicase families. The linkage test, usually reported as a LOD score (logarithm of the odds for linkage), is based on genetic recombination events in families and maps disease susceptibility genes into intervals of 10 to 20 centiMorgans (cM) (10 to 20 Mb). This approach has generally been used to undertake genome-wide linkage scans (GWLS) (i.e., to search for new regions of the genome carrying susceptibility loci, the first such study being to look for genes controlling the complex disease type 1 diabetes) (Davies et al., 1994). Such studies typically genotype all members of multicase families for 400 to 500 highly polymorphic microsatellite markers spaced at 10 to 20 cM intervals across the genome. The use of so many markers means that statistical evaluation has to be robust to multiple testing. GWLS (see Table 2) have been used to identify regions of the genome that contribute to susceptibility to leishmaniasis, to malaria infection intensity, to schistosomiasis and to other helminth infections, but it can be quite difficult to fine map the etiological genes and variants under the linkage peak (e.g., Bucheton et al., 2007; Kouriba et al., 2005). Allelic association tests determine direct association between alleles at particular loci (e.g., a candidate gene), or

haplotypes of closely linked markers (i.e., markers in linkage disequilibrium [LD] with each other), and a disease phenotype. Until recently, this approach was used largely to analyze candidate genes (summarized in Table 1). However, with the advent of technologies that allow upwards of 500,000 single nucleotide polymorphisms (SNPs) to be assayed simultaneously, the so-called “SNP-chip” technology, GWAS (genome-wide association study) have become possible, again requiring the establishment of stringent thresholds to take account of multiple testing (Wellcome Trust Case Control Consortium, 2007). The first statistically powered GWAS recently published for malaria (Jallow et al., 2009) demonstrates the promise that this approach has for the future, although, interestingly, the major hit was for HbS, and only a handful of genes identified by the scan were replicated in other African populations. Allelic association is measured over smaller intervals, usually less than 1 Mb depending on the extent of LD in the population under investigation. For example, LD generally extends over larger intervals in Caucasian populations as compared to African populations (Reich et al., 2001), making it potentially more efficient to fine map etiological variants in African populations. However, the malaria GWAS (Jallow et al., 2009) found that extreme genetic heterogeneity meant that even the etiological variant at HbS was found on different haplotypes in different African populations. This means that HapMap (Gibbs et al., 2003; Thorisson et al., 2005) data derived from the Yoruba in Ibadan (YRI) population will not necessarily allow imputation of genotypes for neighboring markers, and could affect the selection and efficiency of haplotype-tagging SNPs (tag-SNPs) (Service et al., 2007; Xu et al., 2007) for association studies in African populations compared to Caucasian and Asian populations where SNP selection for association studies is quite efficient using tag-SNPs based on CEU, CHB, and JPT HapMap data (Nannya et al., 2007; Service et al., 2007; Xing et al., 2008). Allelic association studies can be undertaken using either population-based sampling (e.g., case control), or family-based collections of case-parent trios. Standard 1 and 2 degrees of freedom tests (e.g., logistic regression analysis), supported by Bayesian approaches (Wellcome Trust Case Control Consortium, 2007), are used to analyze case-control GWAS data, allowing for adjustment for covariates such as environmental variables. Family-based allelic association tests (reviewed in Ewens et al., 2008) based on the transmission disequilibrium test (TDT) (Spielman et al., 1993), which looks for a bias in transmission of alleles from heterozygous parents to affected offspring, are used to analyze case-parent trios/families. Robust tests can be applied to data from multicase families for association testing, taking pedigree or family clustering or known linkage to a region into account. Case-control sampling can be a problem in ethnically admixed populations, where mismatching of cases and controls can lead to type I errors. The TDT approach, which uses family-based controls, is therefore preferable in ethnically admixed populations although methods for checking ethnic matching in casecontrol studies are improving (reviewed in Teo, 2008). A case/pseudo-control strategy (Cordell & Clayton, 2002) and conditional logistic regression analysis can also be used for trios, where the case is the actual genotype transmitted from parents to the affected offspring, and the pseudo-controls are the one to three genotypes (depending on phase) that could have been transmitted. This allows for easy adjustment for data on environmental variables or analysis as covariates, and extension of the analysis to determine whether multiple loci/SNPs within a gene show independent main effects, or whether one SNP carries all of the information

for that gene (i.e., is a tag-SNP for markers in LD across the region of the gene associated with disease). There are many open access statistical tools available for analysis of genetic studies of complex disease, including PLINK (Purcell et al., 2007) which has been developed around the analysis of GWAS data.

SAMPLE SIZE AND POWER

One major problem with all candidate gene studies for infectious diseases reported to date is that they were underpowered (reviewed in Burgner et al., 2006). Until recently, this was a general problem in genetic analysis of complex disease, along with issues relating to study design and population history (reviewed in Cordell & Clayton, 2005; Palmer & Cardon, 2005). Small sample sizes also preclude definitive conclusions being drawn from many studies of parasitic diseases reporting no association for candidate genes. The complex nature of parasitic infections means that genetic effects are likely to be on the order of magnitude that give odds ratios (ORs) of about 1.5. Figure 1 compares power to detect association at OR 5 1.5 or OR 5 2, given different risk allele frequencies, P values, and sample sizes. This shows that 500 trios or case-control pairs have little power to detect association for small effect sizes (OR 5 1.5). Even with 1,000 trios or case-control pairs, power is limited for risk alleles with frequency less than 0.2 for an effect size (OR) 1.5, although low frequency (e.g., 0.10) risk alleles with larger effect sizes (OR greater than 2) may be detected. All published studies (reviewed in Blackwell et al., 2009a; Burgner et al., 2006) of genetics of parasitic infections typically have sample sizes of less than 300 cases, severely limiting power even for hypothesis-driven candidate genes with larger effect sizes (OR greater than 1.5).

INNATE IMMUNITY

Recent interest has focused on the role of innate immunity in driving the adaptive immune response, particularly in relation to intramacrophage pathogens. A number of studies looking at genes involved in innate immunity have been reported for parasitic diseases (Table 1), largely arising on the basis of genes and/or mechanisms shown to be important in murine studies. Notable among these are studies beginning to look at whether polymorphisms in Toll-like receptors (TLRs) that determine innate pattern recognition of molecules on pathogens are associated with susceptibility to malaria (TLR4) (Mockenhaupt et al., 2006; supported by the GWAS data [Jallow et al., 2009]) or toxoplasmosis (TLR9) (Peixoto-Rangel et al., 2009). In mice, the archetypal innate resistance gene was first identified as a gene controlling visceral leishmaniasis (VL) caused by Leishmania donovani sensu strictu (reviewed in Blackwell et al., 2001). This gene, originally designated Lsh, Ity, or Bcg, was also shown to influence innate resistance to Salmonella enterica serovar Typhimurium, Mycobacterium bovis BCG, Mycobacterium lepraemurium, and Mycobacterium intracellulare. Following its identification by positional cloning (Vidal et al., 1993), it was renamed the natural resistance associated macrophage protein (Nramp1). This is now superseded by the functional designation solute carrier family 11a (proton-coupled divalent metal ion transporters) member 1 or Slc11a1, which is consistent with the formal demonstration that the proteins encoded by murine Slc11a1 and human SLC11A1 function as proton/divalent cation (Fe21, Zn21, and Mn21) antiporters (Goswami et al., 2001; Techau et al., 2006). The protein localizes to the late endosomal/lysosomal compartment of

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FIGURE 1 Percent power (Y-axis) of N 5 500 (A) or N 5 1000 (B, C) case-control pairs to detect allelic association for a risk allele with effect size (OR) 1.5 (A, C) or 2 (B), given different risk allele frequencies (X-axis) and P-values (1022 to 1027 as indicated on key for different lines on each graph). Greater robustness to type 1 error (i.e. lower thresholds for P-values, is required for GWAS) (cf. in text).

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macrophages (Searle et al., 1998) and has many pleiotropic effects on macrophage function (reviewed in Blackwell et al., 2001). In humans, SLC11A1 has been linked to genetic susceptibility to leprosy in Vietnam and to tuberculosis in Brazil and Aboriginal Canadians (reviewed in Blackwell et al., 2003). SLC11A1 is globally associated with TB, with both 5 and 3 polymorphisms contributing independently to disease risk (Bellamy et al., 1998). SLC11A1 is associated with HIV (human immunodeficiency virus) (Marquet et al., 1999) and a wide range of autoimmune diseases in humans (reviewed in Blackwell et al., 2003). Polymorphism at SLC11A1 has been linked (Bucheton et al., 2003b; Mohamed et al., 2004) and associated (Mohamed et al., 2004) with VL in Sudan. Preliminary data from Brazil also shows association of VL with the 274C/T (x2 5 5; p 5 0.03) and 469114G/C (x2 5 4.28; p 5 0.04) polymorphisms (S. E.

Jamieson, J. M. Blackwell, M. E. Wilson, & S. M. Jeronimo, unpublished data). These data require robust analysis in a larger sample. In the Sudan, we demonstrated allelic association with 5 (GTn, 274C/T, 469114G/C) but not 3 (D543N, 3UTR TGTG, 3UTR CAAA) markers within SLC11A1. To date, only the promoter GTn is known to be functional in regulating expression of SLC11A1 (Searle & Blackwell, 1999), modulated by SNPs at 2237 bp (Zaahl et al., 2004) and 286 bp (H. S. Mohamed & J. M. Blackwell, unpublished data ) in the promoter region. The GTn functions in humans by binding hypoxia-inducible factor 1 alpha (HIF1) to a sequence element within the repeat (Bayele et al., 2007), whereas studies in mice indicate that interferon (IFN) response factor 8 regulates Slc11a1 expression through a c-myc signaling pathway (Alter-Koltunoff et al., 2008).

TABLE 1 Examples of genes related to innate, acquired immunity, or nonimmune related genes that are associated with parasitic disease susceptibility or outcomea Genes related to Innate immunity

Candidate gene SLC11A1 TLRs MBL P2RX7 CYBB NOS2A TNF IL-6 IL-10c IL-12p40 IFNGc IFNGR1c

Acquired immunity

Nonimmune related

Function Divalent cation homeostasis in macrophages, PMN and DC Innate pattern recognition receptors Mannose binding lectin Purinergic receptor P2X7 GP91phox component of respiratory burst complex Inducible nitric oxide (NO) synthase Tumour necrosis factor (TNF) IL-6 IL-10 P40 chain of dimeric IL-12 receptor Cytokine product of NK (natural killer) cells Type 1 receptor for IFN-g on macrophages

HLA Class I HLA Class II IL-2RB IL-4/13

Presentation of antigen to CD8 T cells Presentation of antigen to CD4 T cells IL-2 receptor B Cytokine product of T helper 2 cells

IL-10c

IL-10

IFNGc

Cytokine product of T helper 1 cells

IFNGR1c

Type 1 receptor for IFN-g on macrophages

COL2A1 ABCA4 CTFG

Collagen 2A1 ABC transporter of all trans retinol Connective tissue growth factor

Parasitic disease associationsb Leishmaniasis, including recently cutaneous disease (Castellucci et al., 2010) Malaria, toxoplasmosis (Peixoto-Rangel et al., 2009) Malaria Toxoplasmosis (Jamieson et al., 2010) Malaria Malaria Malaria, leishmaniasis Leishmaniasis (Castellucci et al., 2006) Leishmaniasis (Farouk et al., 2009; Salhi et al., 2008) Malaria Malaria, schistosomiasis (Chevillard et al., 2003) Malaria, post-kala-azar dermal leishmaniasis (Salih et al., 2007) Malaria, leishmaniasis Malaria, leishmaniasis Leishmaniasis (Bucheton et al., 2007) Malaria (Cabantous et al., 2009), leishmaniasis, schistosomiasis (Kouriba et al., 2005) Leishmaniasis (Farouk et al., 2009; Salhi et al., 2008) Malaria, schistosomiasis (Chevillard et al., 2003) Malaria, post-kala-azar dermal leishmaniasis (Salih et al., 2007) Toxoplasmosis (Jamieson et al., 2008) Toxoplasmosis (Jamieson et al., 2008) Hepatic fibrosis/schistosomiasis (Dessein et al., 2009)

a More exhaustive tables of candidate genes associated with malaria and leishmaniasis are presented in an online table (Burgner et al., 2006). It should be noted that most of the studies undertaken have been underpowered (reviewed in Burgner et al., 2006), and very few of these associations have been replicated. Supplementary table 3 from the malaria GWAS paper (Jallow et al., 2009) provides information on the most significantly associated SNP within 50 kb of the malaria candidate genes. b Only those references which post-date our detailed online tables of associations that accompanied our previous review (Burgner et al., 2006) are provided here. c Included under both innate and acquired immune gene lists since cells from both compartments make these cytokines.

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TABLE 2 Summary of genome-wide linkage studies (GWLS) used to identify regions of the human genome that contain susceptibility loci for parasitic infectionsa Organism (disease)

Phenotype/ Trait

Peak LOD score

Nominal/empirical P-valuesb

2.27 3.50 5.65 3.74 1.08 1.14 1.60 1.60 1.09 2.50 1.93

0.001 3 3 1025 1.68 3 1025; 1025 1.68 3 1025; 1024 0.013 0.011 0.003; 0.003 0.003; 0.003 0.013; 0.013 0.0003; 0.008 0.001; 0.029

Reference

VL VL DTH

17q21.2 9p21.1 2p14 13q13.3 15q26.2 19q13.33

Plasmodium (malaria)

Infection intensity

10p15

rs1964428

3.03

9.4 3 1025; 2.1 3 1024

(Timmann et al., 2007)

Schistosoma mansoni (schistosomiasis)

Egg count

5q33.1

D5S636

4.74

1.51 3 1026

(Marquet et al., 1996)

Trichuris trichiura (trichuriasis)

Egg count

9p24

D9S288

3.35

4.3 3 1025; 0.0142

(Williams-Blangero et al., 2008b)

18p11

D18S452

3.29

5 3 1025; 0.0162

8q23 11p14 13q33

– – –

3.03 3.19 3.37

9.4 3 1025; 0.032 6.4 3 1025; 0.022 4.1 3 1025; 0.0132

VL VL

Ascaris lumbricoides (ascariasis)

Egg count

2q34 22q12 1p22 6q27

Marker D2S142 D22S280 D1S2868 D6S281 D6S281 D17S502 D9S1118 D2S441 D13S894 D15S657 D19S246

Leishmania (leishmaniasis)

VL

Chromosome region

(Bucheton et al., 2003a) (Miller et al., 2007) (Miller et al., 2007; Jamieson et al., 2007) (Jamieson et al., 2007) (Jeronimo et al., 2007) (Jeronimo et al., 2007)

(Williams-Blangero et al., 2008a)

a A classification was proposed (Lander & Kruglyak, 1995) for reporting the results of GWLS data based on the number of times a result would be expected at random in a dense, complete genome scan. Thresholds proposed are: “suggestive linkage,” where statistical evidence would be expected to occur one time at random in a genome scan; “significant linkage,” 0.05 times; “highly significant linkage,” 0.001 times; and “confirmed linkage,” where significant linkage from an initial scan has been confirmed with a nominal P value of 0.01 in a second independent study. The first three categories correspond to point-wise significance levels of 7 3 1024, 2 3 1025 and 3 3 1027 (LOD scores 2.2, 3.6, and 5.4 respectively). Although considered to be overconservative (Sawcer et al., 1996), these thresholds serve as a guide to evaluate the significance associated with the nominal point-wise P values reported by most authors. VL, clinical visceral leishmaniasis; DTH, delayed type hypersensitivity skin test response to leishmanial antigen. b Genome-wide P-values as determined by Williams-Blangero and colleagues (Williams-Blangero et al., 2008a, 2008b).

ACQUIRED IMMUNITY

The outcome of infection with pathogenic parasites is complex, so we expect multiple genes to influence susceptibility to disease. In particular, genes that regulate induction of an adaptive T-cell response will be important. The major histocompatibility complex (H-2 in mice, HLA in humans) has provided a major focus for candidate gene studies (reviewed in Blackwell et al., 2009b), forming part of a broader analysis (Table 2) of genes that control T helper 1 (Th1) versus T helper 2 (Th2) immune responses. Like HLA, immune response genes tend to cluster in the genome, which is thought to have evolved as part of vertebrate defense strategies against infection (Trowsdale & Parham, 2004). Particular interest has focused on chromosome 5q23-q33, a region that carries the genes encoding type 2 interleukins (IL) 4, 5, 9, and 13, along with other immune-related genes. Genes in this region have been linked or associated with various outcomes of leishmaniasis, malaria, and schistosomiasis (Table 2).

NONIMMUNE GENES

Not all genes that influence the outcome of parasitic infection are likely to be immune-related genes. For example, in a study of congenital toxoplasmosis (Jamieson et al., 2008), we recently hypothesized that propensity for Toxoplasma gondii

to cause eye disease may be associated with genes previously implicated in congenital or juvenile onset ocular disease. Using mother-child pairs from Europe, and child-parent trios from North America, we demonstrated that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4 previously associated with juvenile onset retinal dystrophies, including Stargardt’s disease. Polymorphisms at COL2A1 encoding type II collagen, previously associated with Stickler syndrome, were associated only with ocular disease in congenital toxoplasmosis. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting, which provided an explanation for the pattern of inheritance and parent-of-origin effects observed. This analysis of genetic and epigenetic risk factors has provided unique insight into molecular pathways involved in the pathogenesis of disease in utero. In a similar way, Dessein and coworkers (Dessein et al., 2009) noted that abnormal fibrosis occurs during chronic hepatic inflammation and is the principal cause of death in schistosome infections. Slow or rapid development of hepatic fibrosis is regulated by genes at chromosome 6q23. A candidate gene in this region, CTFG encoding connective tissue growth factor, is strongly fibrogenic. Dessein and colleagues (Dessein et al., 2009) showed that SNP rs9402373 lying close

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to CTGF is associated with severe hepatic fibrosis (OR 2.01; confidence interval 1.51–2.7; P 5 2 3 1026) in two Chinese samples, in Sudanese, and in Brazilians infected with either S. japonicum or S. mansoni. A second SNP rs12526196, also located close to CTGF, was independently associated with severe fibrosis (odds ratio 1.94; confidence interval 1.32–2.82; P 5 6 3 1024) in the Chinese and Sudanese subjects. Variants at the two SNPs affect nuclear factor binding and hence may alter gene transcription or transcript stability. They could provide valuable markers to predict disease progression, while CTGF could provide a novel target for chemotherapeutic prevention of hepatic fibrosis.

CONCLUSIONS

The application of SNP-chip-based GWAS has been highly successful in rapidly increasing the number of loci that have been positively associated with complex diseases. The NHGRI Catalogue of Published Genome-Wide Association Studies now contains (as of January 13, 2010) 472 publications showing 2,204 SNPs associated with complex diseases that fulfill stringent criteria for genome-wide significance and at least one replication (Hindorff et al., 2009). To date, only one of these is a study of a parasitic disease (Jallow et al., 2009), but as part of phase 2 of the WTCCC, we will shortly publish GWAS data for large samples sizes of VL from India and Brazil, and for analysis of quantitative trait data for DTH responses to leishmanial antigen from Brazil. We expect that many similar studies will follow for parasitic diseases, providing a wealth of new data that will seed many novel functional studies on mechanisms of disease that can be translated into better interventions for the future. I acknowledge the many colleagues, collaborators, and members of my own laboratory who have contributed to research reviewed here. Research on leishmaniasis in my laboratory is funded by The Wellcome Trust and the NIH, and the work on toxoplasmosis has been funded by the British Guide Dogs for the Blind Association.

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39 Immunogenetics of Virus Pathogenesis SEAN WILTSHIRE, DAVID I. WATKINS, EMIL SKAMENE, AND SILVIA M. VIDAL

INTRODUCTION

variety of viruses has been obtained from twin and familial studies. For example, higher rates of concordance for hepatitis B virus (HBV) persistence (Lin et al., 1989), severity of infection with respiratory syncytial virus (Thomsen et al., 2008), and poliomyelitis (Herndon & Jennings, 1951) were found in identical rather than nonidentical twins. Among HIV (human immunodeficiency virus) positive individuals, disease progression, immune responses, and viral evolution were highly concordant between identical twins as compared to a sibling (Draenert et al., 2006). Furthermore, familial clustering has been reported for a phenotype of resistance to HIV infection in cohorts of sex workers repeatedly exposed to HIV (Plummer et al., 1999), and in rare cases of inherited susceptibility to certain types of viral infections infection (Zhang et al., 2008). Data from families or from independent individuals have been used to identify the genetic component of resistance or susceptibility to infection. Linkage analysis can track the transmission of susceptibility alleles within families and has been the preferred approach in the mapping of rare monogenetic disorders (Zhang et al., 2008). However, family approaches are not feasible in certain infectious settings, for example HIV or HCV, because of the limited familial nature of exposure. In these situations, investigators use case-control studies that rely on population-based cohorts. The ultimate goal of linkage or association studies is to find an association between polymorphic markers (such as SNPs [single nucleotide polymorphisms]) within a gene or genomic region and the phenotype of interest, which is then followed by genetic, functional, and biological testing to establish causality. The number of genes for which there is convincing evidence of association of a specific allele with susceptibility or resistance to virus infection is increasing steadily. Most of these have arisen from candidate gene studies based on a priori knowledge of the role of a gene in virus pathogenesis emanating from a body of experimental studies with cells and/or animals. The majority of host susceptibility genes, however, remain to be identified. The field has started moving towards whole-genome association strategies (Davila & Hibberd, 2009), which have been enabled by recent advances in large-scale genotyping technologies and statistical genetics. The association of thousands of SNP markers with a disease phenotype allows the identification of susceptibility loci across the entire human genome. Such studies take advantage of the numerous meiotic recombination events

Virus infections are characterized by the variability of clinical manifestations among infected patients. Environmental factors, viral dose, and virulence of the pathogen contribute at varying degrees to this phenomenon. However, a growing body of evidence indicates that the genetic makeup of the host plays a key role in the onset, progression, and ultimate outcome of infection. In this view, the role of host genetic variation comes to the forefront because highly specific allelic polymorphisms or mutations predispose certain individuals to diseases caused by viruses that do not cause overt disease in others. The mechanisms identified converge to well-defined biochemical pathways and cell types that are essential barriers against the progression of virus multiplication. These are: (i) the availability of a virus receptor; (ii) type I IFN (interferon) responses; (iii) NK (natural killer) cells, and (iv) T-cell responses, which will be reviewed in this chapter. These findings would help to determine which aspects of the antiviral response are the most promising targets for intervention. Identification of the genetic component of infectious disease susceptibility is an area of intense investigation, which, as presented in this chapter, can provide clues to fundamental questions about virus pathogenesis and the diversity, redundancy, and specialization of host mechanisms against infection. Furthermore, from a clinical standpoint, it can improve the definition of the risk profile of an individual for specific infectious disease, identify new drug targets, and, as we gain further insight into the interplay between the pathogen and the host, lead to the development of customized therapies. But first, we will briefly address some methodological approaches of the identification of susceptibility genes to virus infection.

Extracting Information from the Human Genome

A significant heritable component of a phenotype constitutes the basis for genome analysis. In humans, evidence of the heritable nature of susceptibility to infection with a Sean Wiltshire, Emil Skamene, and Silvia M. Vidal, Department of Human Genetics, The McGill Life Sciences Complex, Bellini Pavilion, Room 356, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1, Canada. David I. Watkins, Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, 6152 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706.

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that have occurred in the human population, which enable high-precision mapping. Furthermore, whole-genome association studies are completely unbiased in terms of gene function and therefore can potentially identify totally novel susceptibility genes. However, genome-wide association studies will have to address several challenges before their value in the genetic analysis of host susceptibility to infection can be established. In particular, although the associations between variants and phenotype are robust, novel experimental paradigms lag behind to determine which variants are causative and how they exert their effects.

Mouse Genetics

The mouse model represents a treasure trove for translational discovery of the molecular basis of human disease (Paigen, 2002). Indeed, excellent mouse models of experimental infection with many mouse and human viruses have been described, which recapitulate accurately several aspects of virus-induced pathogenesis (Lee et al., 2003). These responses can be studied in an environment where virus strain, virulence, dose, and route of infection can be carefully controlled. In addition, there is a plethora of immunological and biochemical reagents that can be used to characterize innate and adaptive immune responses in such infection models. Furthermore, there exists a number of inbred strains and naturally occurring mutants that can be used to search for polymorphic alleles and major gene effects that affect onset, progression, and ultimate outcome of infection. A large variety of specialized strains including recombinant inbred, recombinant congenic and large multistrain intercross are available for mapping susceptibility or resistance loci (Peters et al., 2007). Genetic studies are facilitated by publicly available genomic resources in the form of informative markers (SNPs) and haplotype maps. These resources allow forward genetic approaches that involve the use of large informative crosses and a whole-genome scanning approach, whereby host resistance loci are mapped to a specific chromosomal position. Thus, over the past 20 years, forward genetics in mice has proven useful to dissect the genetic architecture of host defenses against many infectious diseases, revealing new physiological pathways (Vidal et al., 2008). The means by which viruses are sensed, create intracellular replication blockage, and infected cells are recognized are three examples among many processes illuminated by mouse forward genetics. Nevertheless, two disadvantages of using inbred mice are that the genetic effect may be complex, with individual monogenic contributions that are difficult to delineate; and that there is limited natural variation in the pool of laboratory mice. Mouse germ-line mutagenesis by ethyl-nitrous-urea (ENU) that causes random single-point mutations has emerged as a powerful strategy to overcome these problems (Nolan et al., 2000). This approach mirrors genetic analysis of natural variation in populations by requiring no assumptions about the identity of immunological genes. Unlike natural variation, however, ENU-mutations segregate as monogenic traits facilitating both mapping and the assignment of causality. Random mutagenesis of the mouse genome suggests that there are about 300 nonredundant genes that determine resistance against murine CMV (cytomegalovirus) (Beutler et al., 2005). Therefore, many new genes are likely to be discovered in screens for aberrant immune responses or susceptibility against virus infections. Though major breakthroughs achieved through forward genetics in mice will continue to provide an ideal foundation for human studies, intrinsic differences between mice and humans as well as differences between natural and experimental conditions indicate that mouse studies will not suffice to uncover the full spectrum of medically relevant host susceptibility genes.

Over that past few decades, infectious disease immunogenetics has developed into a diverse field exploiting epidemiological, familial, animal, and evolutionary studies to identify a number of diverse host genes influencing resistance or susceptibility to infection. These findings have provided key insight into the molecular mechanisms used by cell populations that are essential for the early detection of and response to viruses. In the following sections we will review some of the most informative examples of such discoveries, with an emphasis on their implication in our evolving understanding of innate or acquired immune defenses. The mechanisms identified converge to well-defined biochemical pathways and cell types that are essential barriers against the progression of virus multiplication. These are: (i) the availability of a virus receptor; (ii) type I IFN responses; (iii) NK cells, and (iv) T-cell responses.

GENETIC CONTROL OF VIRUS ENTRY INTO THE HOST CELL

The interaction of a virus with its cellular receptors is a critical early event of the virus life cycle. Viral receptors are carbohydrates or proteins with unrelated functions in normal cells that have been usurped by the virus to gain access to the cell. There is evidence that expression of receptors on target cells is a fundamental mechanism of host susceptibility to infection with a given virus. Thus, individuals carrying autosomal recessive mutations in virus entry receptors that prevent virus entry into the cell and subsequent infectious replication, are naturally resistant to infection. In contrast, people carrying the receptor wild-type alleles, which allow a portal of entry for the virus, are intrinsically immunodeficient with regard to particular pathogens.

Resistance to Mouse Coronavirus

Infection with mouse coronavirus (mouse hepatitis virus, MHV) has served as a model to understand the viral and immunological determinants of coronavirus disease, including the recently described severe acute respiratory syndrome (SARS) (De Albuquerque et al., 2006). Inbred strains are susceptible to experimental infection with the prototype MHV (A59) strain; however, it was noted early on that the SJ/L strain is exquisitely resistant. In these mice, there is a lack of virus replication in the liver and in explanted macrophages as determined by a recessive locus on chromosome 7 locus, named Hv2, and now designated carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1) (Dveksler et al., 1993). CEACAM1 is a glycoprotein with an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) that functions as an MHV-receptor. SJ/L mice express the CEACAM1b isoform, which differs from the homologous isoform (CEACAM1a) in other mouse strains at the N-terminal virus-binding domain. The relevance of the protein in MHV pathogenesis was demonstrated in vitro by expression of Ceacam1a in SJ/L macrophages, which rendered the cells permissive to infection. Furthermore, susceptible mice with an engineered null allele at the Ceacam1a (Ceacam1a2/2) locus were rendered fully resistant to MHV (Hemmila et al., 2004). The role of CEACAM1 may go beyond its function as a virus receptor. CEACAM1 has wide tissue distribution and, owing to the ITIM domain, is involved in T-cell regulation, suggesting that CEACAM1mediated modulation of the immune response may also occur in mice infected with MHV. CEACAM1-mediated protection is highly pathogen specific. Ceacam1a2/2 mice are infected and may succumb to infection with certain MHV strains (e.g., JHM), suggesting the existence of additional strain-specific MHV receptors.

39. Immunogenetics of Virus Pathogenesis

Human coronaviruses do not bind the orthologous protein CEACAM1a. However, the N-terminal domain of the glycoprotein is the main target of several bacterial pathogens, including gram-negative and gram-positive bacteria that colonize the human mucosa (Hauck et al., 2006). These findings imply that there must be some major advantage for both virus and bacteria to specifically target the CEACAM1 protein.

Resistance to HIV

The AIDS pandemic has affected several different risk groups (e.g., male homosexuals, intravenous drug users, hemophiliacs, sex workers, and transfusion recipients). Studies of high-risk populations consistently show that a subset fails to become infected, suggesting that there are some individuals with a lower risk of infection. Many HIV restriction genes have been identified, which provide different levels of protection. Among them, mutation of the chemokine receptor 5, CCR5, gene provides the strongest single gene effect. As is typical for other successful intracellular pathogens, HIV uses highly conserved host elements for cellular entry, in this case, CD4 and either CXCR4 or CCR5 as coreceptors (reviewed in Alkhatib, 2009). After the virus envelop protein gp120 and binds to CD4, a conformational change is induced that permits binding of gp120 to one of two coreceptors. The first coreceptor, CCR5, serves for the entry of macrophage (M)-tropic HIV-1 strains into cells, whereas the second, CXCR4, gives access of T-cell (T)-tropic HIV-1 strains into CD41 T cells. For reasons that are not completely clear, most primary HIV-1 isolates are predominantly CCR5 tropic and gradually tend to become CXCR4 tropic during late infection. In the wake of the discovery of CCR5 as a coreceptor, several groups identified a 32-bp deletion that introduces a premature stop codon into the CCR5 chemokine-receptor locus. D32-CCR5 deletion introduces a stop codon in the second extracellular loop of CCR5. This mutant encodes a receptor with only four transmembrane segments that remains in the cytoplasm, resulting in a null allele. Therefore, in homozygotes, there is no coreceptor-binding site for HIV-1 and the virus does not enter macrophages. In heterozygotes, there is less than half the level of mature CCR5s on the cell surface than in wildtype homozygotes (Lederman et al., 2006). Individuals carrying a homozygous D32-CCR5 mutation are highly protected against HIV infection across populations. Importantly, their blood cells are resistant to infection with viruses that used CCR5 for entry. HIV infection in people homozygous for D32-CCR5 is extremely rare, and when it does occur, it is caused by viral strains that can use CXCR4 for viral entry. Thus, congenital absence of CCR5 protects against acquisition of HIV infection. Heterozygous individuals carrying a copy of D32-CCR5 and a wild-type allele are marginally protected from HIV infection, but they have an attenuated course of infection and slower progression to clinical stages of AIDS in most cohorts. This partial resistance has been attributed to a gene dosage effect. Other rare mutations in CCR5 that result in a dysfunctional protein have been described. Although homozygous individuals have not been identified for these rare alleles, they can play a protective role when associated with D32-CCR5 in some high-risk seronegative persons (Lederman et al., 2006). The D32-CCR5 mutation has a frequency of 5% to 25% in Caucasians, but is virtually absent in Africans and Asians, suggesting a single origin of the mutation after these populations diverged. Most recent findings suggest that the mutation appeared 2,900 years ago, and that its frequency in Western Europe has remained virtually unchanged at about

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10% between the Bronze Age and Middle Ages. The high frequency and geographical constraints of the mutation suggests that a selective advantage in favor of D32-CCR5 might have existed, although its nature has remained speculative (Sabeti et al., 2005). The HIV pandemic is certainly too recent to have played a role in D32-CCR5 selection, but other viruses such as smallpox may have been implicated. Of note, CCR5 is also used as an entry receptor by simian immunodeficiency virus (SIV) and CCR5 variants have been identified in several monkey species (Palacios et al., 1998). A deletion allele encoding a receptor that is absent from the cell surface has been found in sooty mangabeys monkeys, the natural hosts of SIV. Other frequent CCR5 variants exhibiting altered coreceptor function have been found in monkey species developing nonpathogenic SIV infections. In this case, SIV infection constitutes a likely candidate for selective pressures acting against functional CCR5. These results suggest that there is not a strong disadvantage associated with a null CCR5 allele, in neither primates nor humans, perhaps owing to the redundancy of chemokine and chemokine receptor systems. New experimental findings, however, indicate a complex role of CCR5 in innate immunity against a number of diverse pathogens, including West Nile virus (WNV) (Hedrick & Verrelli, 2006). In this model, the accumulation of leukocytes in the brain, lacking in Ccr5-deficient mice, correlates with the protective antiviral response. These data are consistent with the increased frequency of CCR5D32 homozygotes found among WNV-infected individuals, suggesting caution in the use of therapeutic interventions through inhibition of the CCR5 molecule. Nevertheless, several CCR5-targeteted viral inhibitors have been shown to have antiviral activity in clinical trials (Kuritzkes, 2009) and to provide important benefits in cases of ARV (AIDS-associated retrovirus)-drug resistance. Moreover, a recent case of an HIV-positive subject who received a histocompatible bone-marrow transplant, which was also homozygous for the CCR5-D32 allele, had cleared the virus for 20 months after the intervention (Hutter et al., 2009). In a remarkable example of personalized medicine, this result suggests that it might be possible to eliminate the latent cellular reservoir for HIV.

Resistance to Parvovirus B19

Viruses and other pathogens frequently exploit carbohydrates as cellular receptors, including some histoblood group antigens. By definition, their products reside on the erythrocyte surface and are highly polymorphic in human populations. Thus, variation in the enzyme genes for carbohydrate biosynthesis can exert strong effects on susceptibility or resistance to specific pathogens. For example, parvovirus B19, a significant human pathogen that causes fetal loss and severe disease in immunocompromised patients (Corcoran & Doyle, 2004), is classified as an erythrovirus due to its ability to infect red blood cell precursors of the bone marrow. The virus binds to the globoside P antigen to enter and replicate in erythroid progenitor cells, inhibiting erythropoiesis. P and its precursor Pk are antigens of the P blood group system, which have, in addition to erythrocytes, a high-level expression on fibroblasts, uroepithelial cells, heart cells, and placenta. Variation in the P system may result in total absence of P antigens, the null p phenotype, or expression of the Pk1 precursor only, the Pk1 phenotype. These phenotypes are very rare (1 in 200,000) resulting in severe transfusion reactions and recurrent spontaneous abortions. However, two cross-sectional epidemiological studies in Swartzentruber Amish patients, who have a higher prevalence of the p phenotype than the general population, indicated that individuals who lack erythrocyte P antigen are resistant to parvovirus B19. These studies showed that no patient with the blood-group

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p or Pk1 phenotypes had evidence of previous infection with parvovirus B19, despite normal seroprevalence levels of B19 in the rest of the community. Furthermore, ex vivo infection with parvovirus B19 inhibited erythroid colony formation in cultures of bone marrow from donors expressing the P antigen but had no effect in cultures from donor erythroid progenitor cells that did not express P antigen, indicating that the latter were not susceptible to the cytotoxic effect of parvovirus B19 and had no viral DNA. In 2000, the genes for both Pk and P synthase were identified. Individuals deficient in P antigen carry mutations in the B3GALNT1 gene, causing a lack of functional P synthase and, consequently, express increased levels of the precursor Pk. Persons without any P system antigen have mutations in the A4GALT gene, causing a lack of Pk synthesis, and the rare p blood group phenotype. Further studies on patients with the rare p and Pk blood group phenotypes indicated that the majority of mutations identified correspond to unique alleles, emphasizing the genetic heterogeneity of the glycosyltransferase loci underlying the P blood group system. The medical relevance of P blood group antigens goes beyond parvovirus infections. In epithelial cells, these macromolecules act as cellular receptors for uropathogenic Escherichia coli, with Pk binding the verotoxins implicated in hemolytic uremic syndrome. Moreover, recent exciting data has linked expression of Pk on monocytic cells, where it is designated CD77, with resistance to infection with M-tropic and T-tropic HIV-1 viruses, indicating that Pk may provide protection against HIV-1 infection (Hillyer, 2009).

Resistance to Norwalk Virus

Noroviruses or Norwalk-like viruses are the leading cause of viral acute gastroenteritis in humans. They constitute a heterogenous group of highly infectious viruses noted for causing repeated reinfection and outbreaks in communities, schools, and cruises. However, some individuals remain uninfected even after challenged with high doses, and this resistance may cluster in families, suggesting that inherited factors may protect from infection. Studies of volunteer and authentic infections have shown that resistance to norovirus infections is associated with null alleles at the FUT2 gene at 19q13 (Le Pendu et al., 2006). FUT2, also known as the Secretor gene, codes for the a(1,2)-fucosyltransferase needed to assemble aspects of ABO blood group antigens. This is accomplished, in concert with genes of the FUT3 and ABO loci. In individuals of the secretor phenotype (Se1), FUT2 determines the secretor status, which is the presence of ABO antigens on the surface of epithelial cells and bodily fluids. Nonsecretor individuals are homozygous for nonfunctional FUT2 alleles. Norwalk-like viruses readily bind to histoblood group antigens present on the surface of gut epithelial cells from secretor individuals but cannot attach to cells from nonsecretors. Various FUT2 null alleles have been described that widely differ in human populations. In populations of European and African descent, the most frequent null allele is a G482A nonsense mutation. About 20% of Caucasians are homozygous for the A allele. The nonsense mutation C571G is mainly found in the Pacific Islands, whereas weak secretor individuals are common among Chinese, Japanese,

FIGURE 1 Human resistance or susceptibility to noroviruses. Phylogenetic analysis has shown that the most variable domain of norovirus nucleocapsid corresponds to the carbohydrate ligandbinding domain, named the P2 site. Variation in norovirus strains is depicted by different shading of the P2 site in the nucleocapsid. Noroviruses bind to carbohydrates (represented by geometric shapes) regulated by the FUT2, FUT3, and ABO loci, which are polymorphic in the population. The cell receptor for Norwalk virus is a 1,2 linked-fucose (grey circle) controlled by the FUT2 gene. Therefore, individuals homozygous for nonfunctional FUT2 alleles (FUT22/2) are fully protected against Norwalk virus infection. However, these individuals are susceptible to norovirus strains that bind fucose residues (white circles) controlled by the FUT3 gene, or N-acetylgalactosamine or galactose residues (white triangles) depending upon ABO gene polymorphisms (adapted from Le Pendu et al., 2006).


Australian aborigines, and African-Americans. The skewed prevalence, together with signs of balancing selection in the coding FUT2 sequence, suggests selection in response to specific infections or environment (Le Pendu et al., 2006). The FUT2 G482A allele was shown to be fully penetrant against infection with rare Norwalk strains and in outbreaks associated with other common genotype II norovirus strains as nonsecretor individuals developed an infection. However, other enzymes may serve as susceptibility factors as well, since secretor-negative individuals have antibodies against human noroviruses and develop clinical signs when challenged with certain noroviruses. Moreover, among FUT2-positive individuals, those of the B blood group type were less likely to be infected by Norwalk virus than A or O individuals, suggesting that the ABO locus modulates sensitivity within the secretor-positive group. However, the influence of the ABO phenotype was more or less extended with other norovirus strains. Finally, a portion of the susceptible secretor-positive population remains resistant to infection, suggesting that additional factors afford protection from Norwalk virus challenge. Furthermore, phylogenetic analysis of GII.4 norovirus strains indicates that both antigenic drift and recombination have contributed to the evolution of new epidemic strains in the last 20 years. Notably, the most variable region corresponds to the surface-exposed carbohydrate ligand-binding domain of the norovirus nucleocapsid. Binding assays indicated that GII.4 noroviruses can bind to carbohydrates regulated by FUT2, FUT3, and ABO loci (Lindesmith et al., 2008). This suggested that variation in the capsid ligand binding is tolerated because of the wide range of related carbohydrates that can serve as ligands. On the host side, the set of alleles at the FUT2, FUT3, and ABO loci result in the generation of various carbohydrate ligands that stratify the population. As a result, a given virus strain is expected to target only a fraction of the population. Thus, the very high diversity of noroviruses and histoblood antigens are strongly suggestive of host–pathogen coevolution (Fig. 1). Furthermore, the secretor status is also associated with susceptibility towards other infectious agents including gram-negative and gram-positive bacteria, influenza A and B viruses, respiratory syncytial virus, echovirus and HIV. Thus, although the origins of the blood-group glycosyltransferases remain uncertain, it is evident that they significantly diversify the mucosal glycotopes exposed to microbes; and therein may be found a potential explanation for their existence.

GENETIC CONTROL OF IFN-MEDIATED IMMUNITY IN VIRAL INFECTION

Antiviral type I IFN was discovered in 1957 as a factor interfering with influenza virus infection. Chorio-allantoic membrane cells were rendered resistant after exposure to heat inactivated virus. This interference occurred maximally if the cells had some time to react, and activity was transferrable into the media. Since then, considerable research has been devoted to understanding the molecular mechanisms through which a cell can sense infection, conduct this information, and generate effector molecules in response. Viral presence is first sensed through two major classes of receptor. The first class consists of membrane bound Toll-like receptors (TLRs) and the second class consists of two cytosolic RNA helicases: retinoic acid-induced protein I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). Signals from these sensors are relayed through adaptors such as MyD88 and TRIF. Signaling cascades arrive at transcription factors such as IRF3 or ISGF3, which initiate an antiviral response. During viral infection, this response consists

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of alarm signals, such as IFN itself, which in turn directly induce antiviral proteins such as the MX, PKR, or OAS1. Understanding key components in this pathway has been advanced by the identification of mutations in mouse models and in human patients (Fig. 2).

Sensing

Direct pathogen sensing remained elusive until the discovery that the Toll gene in Drosophila was found to be essential for recognition of fungi and led to the activation of NF-kB. The existence of at least one mammalian homolog raised the possibility that TLRs might play a similar role in mammals. Bolstering this hypothesis, insensitivity to bacterial lipopolysaccharide in C3H/HeJ and C57BL/10ScCr mice, attributed to the Lps locus, was found to be due to a defect in TLR4. Therefore, germ-line encoded receptors could sense specific pathogens’ associated molecular patterns (PAMPs) and lead to relevant and immediate immune responses. Of the 10 human TLRs (TLR1–10) and 11 mouse TLRs (TLR1–7, 9, 11–13) identified to date, TLR9 receptor was the first identified as one that recognized viruses. The ligand to TLR9 was originally found to be DNA containing unmethylated CpG motifs, a common feature of bacterial, but not mammalian, DNA. The genome of herpes simplex virus (HSV) also contains unmethylated CpG DNA, making it a reasonable candidate for TLR9 recognition. Experiments in vitro found that HSV-2 DNA was sufficient to stimulate IFN production and this effect was abrogated in the absence of TLR9. Recognition was independent from viral replication, suggesting that the unmethylated CpG DNA alone was directly recognized. Even so, when TLR9 deficient mice were infected with HSV-2, they were not significantly more susceptible from wild-type mice. In contrast to HSV infection, a TLR9 mutant generated through N-ethyl-N-nitrosourea (ENU) mutagenesis (cpg1) was highly susceptible to another herpes virus, mouse cytomegalovirus (MCMV), as measured by viral replication in the spleen, cytokine production, and survival. Other TLR recognizing viral nucleic acids include TLR7 (in mice) and TLR8 (in humans), which recognize single-stranded viral RNA and TLR3, which recognizes double-stranded RNA (Beutler et al., 2006). The TLRs reside within the endoplasmic reticulum and within endosomes, requiring endosomal acidification for proper function. Shared features within the TLR family include an ectodermal leucine-rich repeat domain (LRR) as well as an intracellular domain shared between TLR and the interleukin 1 (IL-1) receptor (TIR domain) that mediates signal transduction with the TLR adaptor molecules (more below). The link between TLR function and human disease begins in an ENU mutant mouse model. The triple-D mouse (3d) was found to be unresponsive to ligands of TLR3, 7, and 9. The 3d mutation was found to render mice hypersusceptible to MCMV and two bacterial infections. Positional cloning revealed 3d to be Unc93b1, which encodes a membrane spanning protein of previously unsuspected function. More recently, UNC93B1 has been demonstrated to deliver TLR7 and 9 to the endolysosome, where they can then interact with downstream signaling molecules. The role of this protein in human health was demonstrated in two unrelated patients identified with a loss of function mutations in UNC93B1 who developed HSV-1 encephalitis (HSE) (Casrouge et al., 2006). HSE is a relatively rare (1 in 250,000 persons) complication of HSV-1 infection, which occurs in otherwise healthy patients. A defect in type I IFN signaling in response to ligands of TLR7 and TLR9 (as in 3d) led to the identification of two distinct recessive mutations in UNC93B1 in the patients, both leading to truncated protein (1034del4

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and 781G.A). Low production of IFN in peripheral blood mononuclear cells (PBMC) in response to multiple TLR7 and 9 agonists was accompanied by a diminished response to multiple viral pathogens in vitro. Additionally, patient fibroblasts were found to be much more susceptible to both HSV-1 and another neurotropic virus, vesicular stomatitis virus (VSV). This effect was complemented with the addition of IFN- a, consistent with the hypothesis that deficiency was in recognition and not generation of a response. UNC93B1 deficiency is responsible for susceptibility to at least HSE in humans, and in vitro deficiency occurs with multiple viral infections. That individual TLRs are nonredundant in viral pathogenesis became clear with the identification of a naturally occurring variant of TLR3 in patients who also developed HSE. Two unrelated patients were identified with HSE, after excluding other immunodeficiencies (including UNC93B1), it was discovered that both share a substitution in TLR3 at residue 554 (P554S). This mutation occurred in the extracellular LRR domain in a region thought to be critical for RNA binding and multimerization. Fibroblasts derived from the P544S patients had reduced or absent production of response to polyinosine polycytidylic acid (polyI:C), a TLR3 agonist. Further, fibroblasts were more susceptible to viral replication by both HSV-1 and vesicular stomatitis virus (VSV); this effect was complemented by the addition of purified type I IFN. These patients were otherwise immunologically normal and had not developed chronic infections by other viruses, which can be explained in part by two observations. The first is that peripheral blood mononuclear cells (PBMC) from these same patients responded normally to an array of viruses, indicating a degree of redundancy in viral sensing in the blood. The second is that HSV viral particles are not transported through the blood where they would encounter PBMC during the course of HSE. Therefore, a specific defect in TLR3 may provide just enough of a crack in the human immunological defense for HSV to develop into HSE, but functional redundancy seems to be able to compensate during other infections.

Signaling

Once a pathogen-associated molecular pattern has been detected through one of the TLRs, this signal is relayed through adaptors in a signaling cascade, ultimately reaching transcription factors, which will initiate molecular counter measures. Signal conduction of viral recognition by TLRs is so far known to occur through two distinct pathways, both of which depend on the intracellular TIR domain (O’Neill & Bowie, 2007). TLR7, 8, and 9 begin signaling by interacting with the myeloid differentiation primary response 88 (MyD88) adaptor molecule, MyD88 then interacts with IL-1 receptor associated kinase 1 (IRAK1), IRAK4, TRAF6, IKK-a, leading to the activation of transcription factors IRF7 and NF-kB (Beutler

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et al., 2007, 2006; O’Neill & Bowie, 2007). The existence of a MYD88-independent pathway was suggested, as both TLR3 and TLR4 were able to stimulate an IFN response in the absence of MyD88. Investigations based on homology of the TIR domain led to the identification of TRIF. TRIF was shown to potently activate IFN-b in vitro, and interact with TLR3 and a TRIF molecule with deleted functional domains to dominantly suppress several TLRs. Confirmation of the relevance of MyD88-independent, TRIF-dependant immunity in response to viral pathogens came with the identification of the Lps2 mouse, again generated by ENU mutagenesis. The Lps2 mouse was unresponsive to ligands of not only TLR3 but also TLR4, as measured by IFN-b, and nitric oxide production, as well as activation of the STAT-1 transcription factor. Positional cloning determined that the Lps2 phenotype was due to a single base pair deletion in the C-terminus of Trif, a frame shift that resulted in the loss of 24 amino acids. Mice carrying a homozygous Lps2 mutation were more resistant to E. coli LPS toxicity and significantly more susceptible to infection by MCMV in vivo and by Vaccinia in vitro. TRIF interacts directly with TLR3, and indirectly through TRAM with TLR4. From TRIF, the signal leads to NF-kB activation through two pathways, one involving TRAF6 (as in MyD88-dependent) and another through RIP1. TRIF then signals in a pathway dependent on TRAF family-member-associated NF-kB activator binding kinase 1 (TBK1), and leads to the activation of the transcription factor IRF3 and the production of type I IFN. Converging on the TBK1 activation of IRF3 and IRF7 are the cytosolic RNA helicases, which activate TBK1 through TRAF3 and the mitochondrial protein IPS1. Once type I IFN is produced, it is secreted and signaled through the type I IFN receptor (IFNAR1 and IFNAR2) in an autocrine and paracrine manner. Upon ligand binding, the IFN receptor initiates a signaling cascade through the activation Janus kinases (JAK), including JAK1 and TYK2. The relevance of JAK signaling in combating viral infection was revealed by the identification of a natural variant of TYK2 in an individual with hyper IgE syndrome (HIES). The patient was susceptible to a constellation of pathogens, including bacteria, fungi, and viruses, including affliction by recurrent otitis media, infection with molluscum contagiosum (a pox virus), as well as HSV infection of skin and mucosa. The patient’s T cells were unable to produce IFN-g in response to IL-12 and did not respond to IFN-a, suggesting a defect at a common point in the two pathways. One such molecule was TYK2, which was sequenced, revealing a 4-base pair deletion resulting in a frame shift and loss of most of the protein. JAK1 and TYK2 phosphorylate STAT1 and STAT2, which, together with IRF9, form a trimer called IFN-stimulated gene factor 3 (ISGF3), which translocates to the nucleus. In the nucleus, ISGF3 will bind to IFN stimulus response elements (ISRE), a promoter which activates hundreds of genes to respond to infection (Sadler & Williams,

FIGURE 2 Genetically nonredundant molecular pathways in the type I IFN response to viral infections. (a) Upon viral infection, viral nucleic acid is initially detected by two classes of receptors (in black). The TLR class of receptors are located in the endosomal compartment and require UNC93B for maturation. TLRs 3, 7, and 9 bind pathogen-associated molecular patterns (PAMPs) such as double-stranded RNA, viral ssRNA, and viral dsDNA. The cytosolic helicases RIG-I and MDA5 perform similar functions in the cytoplasm. (b) Upon binding a PAMP, a signal is relayed through an adaptor molecule (light grey, black border) such as MYD88, TRIF, or IPS-1 to a series of kinases (dark grey, light border), and ultimately to transcription factors (dotted border) such as IRF3, IRF7, or NFkB, which induce the expression of type I IFN (white). (c) IFN signals to self and neighboring cells through the IFN receptor, which initiates a signaling cascade involving JAK and TYK2, these induce formation of the ISGF3 transcription factor from STAT1, STAT2, and IRF9. The ISGF3 transcription factor leads to the production hundreds of genes including antiviral effector molecules such as Mx, PKR, and OAS1b. (d) Genetically determined nonredundant genes in whole pathways are shown in bold and underlined.

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2008). STAT1 deficiency has been recovered as essential in resistance to MCMV in mouse model (domino) (Crozat et al., 2006). The domino mouse responds to infection by VSV or MCMV infection with a normal production of IFN; however, IFN-stimulated genes (ISG) were not detected, consistent with the focal role of STAT1 in IFN signaling and ISG production. Mice of domino/1 genotype were phenotypcally normal, whereas no complementation occurred between homozygous domino mice bred with Stat12/2 mice. STAT1 deficiencies, resulting in increased susceptibility to viral infection, have been documented in five separate instances. Two of these patients were unrelated, suffered from mycobacterial disease, and died during infancy of viral infection (one HSV-1 and one unknown). Cell lines derived from the two were similarly unresponsive to IFN and did not produce ISGs in response to in vitro infection. The third patient again suffered from disseminated mycobacterial infection, as well as fulminant Epstein-Barr herpes virus (EBV), which required antiviral treatment, though this may have been due to treatment with an immunosuppressant for an organ transplantation. The patient was able to clear low virulence viruses without aid including oral polio vaccine, parainfluenza, and a rhinovirus, indicating a level of redundancy in certain low virulence viruses. The patient died of multiorgan failure by 11 months of age and was never infected with HSV, though cells from the patient were highly susceptible to HSV and VSV after treatment with IFN-a. Of the other two patients documented with STAT1 deficiency, one developed several severe herpes infections, including CMV and VSV, both requiring hospitalizations, as well as HSV gingivostomatitis. Both developed severe Salmonella infections, together with severe viral infections, suggesting a possible STAT1 deficiency. Directed sequencing uncovered a single polymorphism (P696S) that induced differential splicing into a shorter and untranslated form with no residual activity, as well as a low abundance, full-length transcript. The presence of low abundance fully functional transcript was thought to account for the low, but not abrogated, response to IFN in these two individuals.

Effectors

Hundreds of genes are stimulated by IFN, though the functions of many are unknown. Among those with known functions, only a small handful have characterized nonredundant roles in antiviral immunity. Well before the description of IFN in 1957, genetic evidence in the form of mice susceptible or resistant to infection strongly suggested that host genetics was responsible for variable susceptibility to viral infection. One strain of mice (A2G) was created accidentally through an illegitimate mating, which occurred at some point between 1942 and 1950. Years later, in 1963, it was found that A2G mice were significantly more resistant to influenza infection. The A2G resistance was ascribed to a single dominant gene (Mx) and it was found that this resistance could be abrogated through the administration of a potent polyclonal antiserum against partially purified IFN (AIF). That IFN mediated the Mx phenotype was surprising, since A2G mice were specifically resistant only to influenza virus, whereas IFN was produced generally in response to all viruses. A unique 75kD protein that was highly expressed by Mx carrying mice was identified and expressed maximally by 4 hours post-stimulation in vitro, coinciding quite neatly with the original IFN effect observed (Isaacs & Lindenmann, 1957). Lacking a complete genome sequence, the coda sequence of Mx was obtained by fractionating whole RNA through electrophoresis, translation in a cell free system, and identification of a single correct protein using a specific antibody. When Mx cDNA was expressed

in cells otherwise susceptible to IFN, it was found to be sufficient to confer resistance to influenza. The localization of Mx to chromosome 16 came last and was found not through positional cloning, but using cDNA as a probe in hybridized cell lines containing one or more mouse chromosomes. Since the identification and localization of the Mx gene, it has been found to be a GTPase involved in either sequestering viral mRNA in the nucleus or in sequestering of viral capsid proteins (MacMicking, 2004). A total of four families of IFN-inducible GTPases have been found, including the p47, p65, Mx, and VLIG families—with the exception of Mx—their roles in vivo remain largely unclear. Other effectors include the OAS1 and RNaseL system (Sadler & Williams, 2008). Natural resistance to flaviviruses had been known to exist in certain inbred strains of mice since the 1920s. The Flv (also called Wnv) locus was an autosomal dominant, IFN-dependant gene present in a minority of inbred laboratory strains that conferred resistance to WNV and yellow fever virus (YFV). The resistant allele was fixed onto a congenic background of a susceptible strain, and, through linkage analysis, was found to reside on chromosome 5. Further refinement by linkage analysis revealed a perfect concordance between susceptibility and a premature stop codon in the L1 isoform 29-59 oligoadenylate synthetase (Oas1b). OAS was known to produce the oligormerization of ATP by catalyzing 29-59 phosphodiester bonds formation. The presence of 29-59 A, in turn, affects the dimerization and activation of latent RNaseL, which degrades single-stranded RNA. In addition to degradation of viral genome or antigenomes, RNaseL may also produce small dsRNA, which reactivate sensor molecule such as RIG-I and MDA-5, thus enhancing IRF3 signaling. RNaseL deficiency leads to increased susceptibility to several RNA viruses in mice. Other IFN-stimulated genes (ISG) (Sadler & Williams, 2008) include PKR, a kinase that inhibits translation upon binding of viral RNA by phophorylation of EIF2a, which blocks GDP recycling. PKR has been found to be involved in response to multiple RNA viruses. The highly induced ISG15 protein is an ubiquitin homolog that is bound to a number of substrates and may be a regulator of IFN signaling. ISG15 deficiency in mice results in increased susceptibility to influenza, a and g herpes virus, as well as sindbis virus. HIV is incapable of infecting certain cell lines in the absence of the Vif gene. Through cDNA subtraction, the Apo3g gene encoding a member of the APOBEC family was identified as a resistance factor in the absence of Vif. APOBEC3G prevents efficient reverse transcription by first being packaged into progeny virions, and then deaminates cytidine residues to uracil, causing an excess of C-T substitutions (KewalRamani & Coffin, 2003). Vif seems to counteract this by targeting APOBEC3G for degradation. However, in the macrophage, APOBEC3G can be potently induced by IFN-a, leading to resistance of the macrophage to infection. This effect seems to be specific as it is reversible with APOBEC3G siRNA knockdown.

Viral Escape

Viral evasion strategies of the type I IFN system have been documented at every level, underscoring how critical this system is in defense (reviewed in Bowie & Unterholzner, 2008; Takaoka & Yanai, 2006). In order to decrease IFN production, multiple viruses may be hidden from TLRs and cytosolic helicases by sequestering proteins that camouflage nucleic acids. Adaptor molecules, kinases, and transcription factors are degraded, cleaved, or otherwise prevented from signaling in order to block signal transmission. IFN signaling is also hampered by competition for IFN receptors


and inhibition of JAK or Tyk2 signaling. ISGF3 formation may be blocked by interfering with IRF9 binding, whereas other viruses interfere with or degrade STAT1/STAT2 to the same effect. Effector molecules are no exception: examples of interference with PKR, OAS/RNaseL, MxA, and APOBEC1 have all been documented with more sure to follow.

NK RECEPTOR REPERTOIRE AND MHC CLASS I ALLELES CAN INFLUENCE NK CELL RESPONSES AND OUTCOME AFTER VIRAL INFECTION

An important consequence of type I IFN secretion is the activation of natural killer (NK) cells, a central component of the innate response against virus infection (Lee et al., 2007). NK cells monitor the body for aberrant cells, which they can eliminate through cytotoxic activity and secretion of proinflammatory cytokines/chemokines. Target cell recognition and NK cell activity depend on a complex array of inhibitory and activating germ-line encoded receptors. Among them, inhibitory receptors of MHC class I molecules, named killer immunoglobulin receptors (KIR) in humans and lectin-related receptors (Ly49) in mice, play a preponderant role (Fig. 3). Down regulation of class I, as frequently observed in tumor cells or virally infected cells, confers vulnerability to NK-cell-mediated recognition and killing. This mode of recognition was first referred to as the “missing self” hypothesis (Karre, 2008). However, lack of selfrecognition is not sufficient to stimulate NK-cell-mediated

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killing, which also requires specific activating signals (Lanier, 2005). Unraveling the genetic basis of differential susceptibility to infection with mouse cytomegalovirus (MCMV) in inbred mouse strains has proven extremely informative to understand the interface of NK cell–pathogen interaction (Pyzik et al., 2008). Of the several MCMV resistance loci mapped using informative mouse crosses, Cmv1 and Cmv3 have been characterized at the gene level as the stimulatory Ly49 family of activating NK cell receptors of MHC class I, involved in specific recognition of virus-infected cells.

Cmv1 Requires Activation of the Ly49H-m157 Axis

The Cmv1 locus on distal chromosome 6, identified by Scalzo and Yokoyama (Scalzo & Yokoyama, 2008), constitutes one of the best-studied examples of a single mouse gene determining host resistance against virus infection. Cmv1 controls an early stage of MCMV replication and presents two alleles: a dominant resistance allele, Cmv1r, and a recessive susceptibility allele, Cmv1s. Strains of the C57BL background carry Cmv1r, which restricts virus load in the spleen, lung, and liver by more than 1,000-fold as compared to mouse strains carrying Cmv1s. Expression of Cmv1-determined resistance is mediated by NK cells, which limit viral replication via secretion of IFN-g and exocytosis of perforin and granzyme-containing granules. Identification of Cmv1 required high-resolution mapping in close to 4,000 informative progeny mice, cloning of the critical interval in yeast and bacterial artificial chromosomes, and development of monoclonal antibodies for detailed characterization of Cmv1r mice. Ultimately, Cmv1 was found to encode

FIGURE 3 The human and mouse major histocompatibility complex (MHC) and natural killer complex (NKC). (a) The MHC is a genomic region central to the immune response found in most vertebrates, including on the short arm of human chromosome 6 and proximal mouse chromosome 17. The MHC encodes proteins that help to distinguish between self and nonself protein components. MHC class I molecules (human HLA-A, HLA-B, and HLA-C; mouse H2-D, H2-K, H2-L) present endogenous antigens to CD81 T cells and class II molecules (human HLA-DP, HLA-DQ, HLA-DR; mouse H2-P, H2-A, H2-E) present exogenous antigens to CD41 T cells. A number of other proteins that support these two pathways also map to the MHC, such as the class III complement (C) proteins and inflammatory cytokine genes (TNF), and the antigen processing proteins (TAPBP, TAP/LMP). MHC class I proteins also serve as ligands to MHC class I NK cell receptors, as a set of molecules that modulate NK cell activity. (b) Two distinct structural families of NK receptors that bind to MHC class I ligands have been identified: the human killer immunoglobulin-like receptors (KIRs) encoded on chromosome 19, and the mouse killer cell lectin-like receptors (KLRs or Ly49s) on chromosome 6. Both families contain activating and inhibitory members that perform similar functions. The presence of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic tail of the receptor inhibits NK lysis. In contrast, activation requires the recruitment of an adaptor molecule (DAP12) through a charged amino acid in the transmembrane domain. DAP12 contains an immunoreceptor tyrosine-based activation motif (ITAM), which triggers NK cell activating pathways. Inhibitory receptor genes (human KIR3DL3, KIR3DP1, KIR2DL4, KIR3DL2; mouse Ly49q, Ly49e, Ly49i, Ly49g, Ly49c, Ly49a) are generally conserved. In contrast the number of activating receptor genes (in brackets) is highly variable among individuals.

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Ly49H, an activating DAP12-associated NK cell receptor. Ly49H directly binds to a viral glycoprotein, m157, which is expressed on the surface of MCMV-infected cells soon after infection. The essential role of the Ly49H/m157 recognition mechanism in controlling MCMV infection was confirmed by the transfer of MCMV-resistance to genetically susceptible mice by Ly49h transgenesis, while MCMV-resistance was abolished in C57BL/6 mice with mutant Dap12 or lacking Ly49h expression. In addition, C57BL/6 mice displayed a susceptible phenotype during infection with an MCMV deletion mutant lacking m157 (Dm157) . All NK cells become activated after MCMV infection and release cytokines and chemokines, including IFN-g, MIP-1a, MIP-1b, RANTES, and ATAC. However, unlike the NK cells from MCMV-sensitive mouse strains or the Ly49H2 NK cells from C57BL/6 mice, Ly49H1 NK cells have more persistent activation and undergo massive expansion with almost 80% of NK cells expressing Ly49H by day 8 postinfection. Proliferation of Ly49H1 NK cells is both DAP12 dependent and m157 dependent. Ly49H1 NK cells are required for efficient control of MCMV replication, and their presence is crucial to limit systemic production of cytokines, to protect attrition of the CD81 T cell compartment, and to maintain normal cell spleen microarchitecture. Moreover, recent data indicate that experienced Ly49H1 NK cells during MCMV infection are long lived and convey significantly enhanced protection than naïve Ly49H1 NK cells using a model of neonatal cytomegalovirus infection (Sun et al., 2009).

Viral Escape

Passage of MCMV through resistant C57BL/6 mice results in mutation of the gene encoding a mutation of the gene encoding m157. Sequencing of MCMV isolated from wild mice revealed frequent loss-of-function mutations in m157, indicating that there is a selective pressure against the expression of m157 in MCMV-infected cells. These findings begged the question of why MCMV would maintain a gene that confers selective disadvantage to its survival. The virus protein m157 also binds the inhibitory receptor Ly49I from the MCMV-susceptible 129/J mice, suggesting that m157 may have evolved as a mechanism to escape NK cell killing by targeting inhibitory receptors in certain susceptible mice. In support of this hypothesis, the crystal structure of m157 has revealed that this viral protein has significant homology to MHC class I molecules. Moreover, it was found that the affinity of m157 for the inhibitory Ly49I receptor is higher than the affinity of its MHC class I ligand, suggesting that the virus has co-opted a mouse MHC class I gene as a means to escape NK-cell-mediated responses by engaging inhibitory Ly49 receptors. It has been proposed that the activating Ly49 receptors evolved more recently than the inhibitory Ly49 receptors. It is conceivable that during the co-evolution of MCMV with its host, which predates the radiation of mammals, the pathogen imposed selective pressures, driving the diversification of the Ly49 receptor family, with the creation of activating receptors.

Cmv3: Activation of the Ly49P-H2-Dk-m04 Axis

The impact of MCMV on the mouse genome has been further supported by the characterization of Cmv3, a locus identified in MA/My mice (Pyzik et al., 2008). This mouse strain possess an NK-cell-dependent resistance to MCMV infection. However, MA/My mice lack Ly49h and diverge genetically from the resistant strain C57BL/6 in the region of Cmv1. Genetic analysis indicated that resistance is imparted by a major Ly49-linked locus, named Cmv3, that accounts for a 100-fold decrease of splenic viral titers. However, expression of Cmv3 is strictly H2-dependent because MCMV-resistance is only observed in mice with a specific

combination of Ma/My alleles at the Ly49 locus and MHC (H2k). Functional candidate gene testing using a cell reporter assay to detect stimulation of Ly49 receptors isolated from MA/My mice showed that another DAP12-associated receptor, Ly49P, responds to MCMV-infected cells. Ly49P activation was observed only against MCMV-infected targets carrying the H2k haplotype, and recognition of these cells was blocked by an H2-Dk-specific monoclonal antibody. Additional experiments using target cells expressing H2 chimeras between H2-Db and -Dk, or infected with a panel of MCMV deletion mutants (including the virus mutant Dm04 MCMV, which lacks the m04 gene) showed that Ly49P recognition of the infected cell required both the presence of the H2-Dk peptide-binding platform and the surface expressed viral component m04. Although surface expression of m04 complements Ly49P stimulation by target cells infected with Dm04 MCMV, m04 expression alone is not sufficient, indicating that additional factor(s) to m04 and H2-Dk are required for Ly49P activation. Nevertheless, NK-cell-dependent MCMV-resistance of MA/My mice was abrogated during infection with Dm04 MCMV, implicating NK cells in host protection through a Ly49P-H2-Dk-m04 axis of recognition (Kielczewska et al., 2009). Significantly, the study of host resistance to MCMV indicates that activating members of the Ly49 family of MHC class I NK cell receptors can distinguish specific pathogen determinants. Although they have analogous functions, Ly49 receptors in mice and KIRs in humans are structurally distinct. Nevertheless, they share many molecular characteristics in terms of gene diversity, gene regulation, protein expression, and signaling function. Thus, research on Ly49 receptors has provided an excellent framework to study the KIR function in response to virus infections (Fig. 4).

KIR and their MHC Class I Ligands in Host Response to Virus Infections

In humans, combinations of activating KIR and MHC class I ligands that stimulate NK cell cytokine secretion and target cell cytolysis have been found to be beneficial during virus infection. Moreover, binding studies and tertiary structure analysis have shown that inhibitory KIR bind their cognate HLA ligand with different strengths, which determines a hierarchy of inhibition with varied impact on the host response (Parham, 2005). The importance of KIR inhibitory signals in the control of viral infection has been postulated in the case of a 7-year-old patient (Gazit et al., 2004), who presented symptoms identical to those observed in patients with NK-cell deficiency (Biron et al., 1989). The patient was hospitalized because of recurrent herpes virus infections (CMV, varicella zoster, EBV). All laboratory tests were normal, including B cell, T cell, and NK cell counts. However, expression of KIR2DL1 was unusually detected on the entire population of NK cells. Furthermore, all NK clones from the patient expressed KIR2DL1 and were unable to kill targets cell transfected with the HLA-C2 molecules corresponding to the patient genotype. This suggested that overexpression of an inhibitory NK cell receptor in the presence of its high affinity ligand defines a novel immune deficiency associated with vulnerability to herpes virus infections. Inhibitory KIR-ligand combinations that send relatively poor inhibitory signals might also be important in regulating NK cell activation. Such a mechanism has been proposed to explain KIR-HLA mediated protection against infection with hepatitis C virus (HCV). Among intravenous drug users infected with HCV, homozygosity for both KIR2DL3 and group HLA-C1 allotypes was found to be associated with increased resolution of infection. This combination is predicted to give the weakest KIR-mediated inhibition of NK cells and may


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FIGURE 4 Models of NK cell receptor-mediated response in mice (top). In mice, a diverse array of MHC class I NK cell receptors of the Ly49 family are critical for the host response against MCMV. (A) The mouse strain 129/J is susceptible to MCMV infection. This strain possesses the ITIM-containing inhibitory receptor Ly49I, which binds the virus MHC class I-like protein, m157. In this case, Ly49I-mediated inhibitory signals prevent NK cell activation. (B) MCMV-resistance in C57BL/6 mice is mediated by the DAP12-associated Ly49H receptor, which also binds to m157. Upon m157 recognition, Ly49H triggers NK cell-mediated cytotoxicity via perforin and granzyme (black circles), cytokine secretion mainly through IFN-g (white circles), and cell proliferation leading to clearance of MCMV-infection. (C) MCMV-resistance in MA/My strains is mediated by a different activating receptor, the DAP12-associated Ly49P receptor. Ly49P recognition of the infected cell requires the presence of both a host MHC class I molecule, H2Dk, and an MCMV-encoded protein, m04. Arrows in the NK cell diagram indicate activating signals emanating from stimulatory receptors. Human NK cell receptors in virus infection (bottom). In humans, a diverse array of MHC class I NK cell receptors of the KIR family, in concert with their MHC class I ligands, determine the host response against viruses. The outcome of infection depends on the strengh of signals elicited by receptor/ligand pairs. (D) A patient with a genotype for the high-affinity interaction between inhibitory KIR2DL1 and HLA-C2 presents severe and recurrent herpes infections (HCMV, VZV, EBV) associated with NK cell impaired cell-mediated cytotoxicity and cytokine production against cognate target cells. (E) HCV-infected drug users with a genotype for the low-affinity interaction between inhibitory KIR3DL3 and HLA-C1 are relatively protected against chronic HCV infection. The weak inhibitory signals are thought to lower the threshold for NK cell activation. (F) HIV patients encoding the compound genotype for the activating KIR3DS1 receptor and its cognate ligand, HLA-Bw4, have delayed progression to AIDS. Upon challenge with HIV-infected cognate cells, KIR3DS11 NK cells from these patients proliferate and elicit secretion of cytokines (white circles), and cytotoxic granules (black circles). Arrows in the NK cell diagram indicate activating signals emanating from stimulatory receptors.

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afford protection by lowering the threshold of NK cell activation to clear HCV infected cells. In a more recent study, the protective effect of inhibitory KIR was shown to be further enhanced by the presence of KIR2DS4 in combination with its putative ligand, HLA-C2 (Bashirova et al., 2006). In the context of HIV infection, numerous studies have addressed the role of the KIR genes in combination with their HLA ligand genotypes in the control of viral load, rate of CD41 T cell decline, and disease progression (Carrington et al., 2008). Although different conclusions have emerged, the best powered studies by Martin and colleagues (Martin et al., 2002) have found that a combination of HLA-B Bw4–80I with either activating KIR3DS1 or inhibitory KIR3DL1 were associated with slow progression to AIDS and lower viral means in a cohort of 1,000 HIV seroconverters. The protective effect of KIR3DS1/ Bw4–80I was substantiated with functional data measuring inhibition of HIV replication in two-cell assays where KIR3DS1 positive NK cells inhibited HIV replication in Bw4–80I positive T cells to a significantly greater extent than any other effector/target cell combination. Moreover, NK cells from recently HIV-infected persons who carried at least one KIR3DS1 allele showed higher IFN-g production and degranulation against MHC class I-negative target cells compared to those without KIR3DS1. This functional and genetic data supported a model in which virus control depends on the functional interaction between KIR3DS1 on NK cells with Bw4–80I on HIV infected target cells. The result of a protective effect of the inhibitory receptor KIRDL1/B4-80I is counterintuitive. However, recent data from mouse and human studies indicate that inhibitoryreceptor pairing during NK cell development are indeed critical to acquiring their activation potential, a mechanism termed NK cell licensing. Thus, responsiveness of NK cells against HIV-infected target cells would correlate with the presence of functional KIRDL1-HLA B4-80I interactions. This hypothesis has been recently supported by functional data demonstrating that NK cells expressing the activating receptor KIR3DS1 and, to a lesser extent, the inhibitory re-

ceptor KIR3DL1 undergo specific expansion in acute HIV-1 infection in the presence of HLA-B Bw4-80I, their putative HLA class I ligand. A possible interpretation of these results is that, similar to Ly49H during MCMV infection, both KIR3DS1 and KIR3DL1 NK cells play a critical role in the control of natural HIV-1 infection, and they rapidly expand during HIV infection depending on the interaction with their ligand on infected cells (Alter & Altfeld, 2008).

MHC CLASS I ALLELES AND TCR REPERTOIRE CAN INFLUENCE CD81 T RESPONSES AND OUTCOMES AFTER VIRAL INFECTION

MHC class I alleles dictate how an individual responds to a pathogen. These molecules are central to the function of the immune system and determine which pathogen-derived peptides are recognized by the infected host. Given the enormous diversity encoded by the MHC class I region, almost every infected individual’s response to any particular pathogen is different. The products of the MHC class I loci are, therefore, one of the major factors influencing a successful outcome after a pathogen infection. The TCR that binds to these MHC class Ipeptide complexes can also vary from individual to individual. It is, therefore, not surprising that certain individuals respond to pathogens better than others. There are HIV-positive individuals, for example, who control viral replication to undetectable levels (elite controllers, ECs). Similarly, Indian rhesus macaques can also control SIV replication after infection, recapitulating this EC phenomenon in a tractable animal model of HIV infection (Goulder & Watkins, 2008). The resolution of acute phase viremia is temporally associated with the appearance of virus-specific CD81 T lymphocytes in both humans and macaques (VandeWoude & Apetrei, 2006). Several studies have shown a relationship between strong CD81 T lymphocyte responses and EC status in HIV-infected individuals. More recently, investigations into the phenotypes of HIV-specific CD81 T cells have suggested that controllers retain multiple effector functions, which are lost in progressors (Fig. 5). MHC-I alleles such as

FIGURE 5 Factors affecting development of elite control (EC) or rapid progression (RP) in HIV and SIV infection (top). Effective function of cytotoxic (CD8) T cells has been shown to depend on allele of MHC, characteristic peptides presented to T cells by MHC, a broadly specific CD8 response to pathogen and a more rapidly expanding response. Rapid progression of HIV to AIDS has been correlated with allele of MHC, as well as an immunodominant response and delayed expansion of CD8 T cells (bottom).


HLA-B*57 and HLA-B*27 have been correlated with EC status, whereas HLA homozygosity and HLA-B*35 have been associated with rapid progression (Fig. 5). This suggests that HLA-B*57 and HLA-B*27 may restrict especially potent antiviral CD81 T lymphocyte responses. Interestingly, viral escape from an immunodominant CD81 T lymphocyte response restricted by HLA-B*27 was correlated with loss of control of viral replication and progression to disease. Perhaps, most strikingly, studies in SIV-infected macaques have shown that transient depletion of CD81 cells in infected macaques using anti-CD8 antibodies has a marked effect on virus replication. During chronic infection, CD81 cell depletion results in a rapid increase in viral loads of one to three orders of magnitude. In addition, CD81 cell depletion in the acute phase resulted in increased peak virus titers and higher set-point viremia (Benito et al., 2004). Recently, in vivo CD81 cell depletion in macaque ECs transiently broke control of viral replication, resulting in virus replication in all six depleted macaques. However, since the neutralizing antibody used in these studies also depletes CD81 NK cells, it is still not clear as to which of these two CD81 cell populations is largely responsible for control of viral replication. Furthermore, genetic data from the analysis of large cohorts of HIV-infected patients have implicated CD81 NK cells in control of HIV replication. As discussed above, interactions between KIR3DS1 and certain HLA-B molecules have been shown to delay disease progression and combinations of KIR3DL1 alleles, and certain HLA-B locus alleles can influence both AIDS progression and viral load (O’Brien & Nelson, 2004). Functional data also support the influence of KIR allotype and HLA type on expansion of NK cells and killing of HIV-1-infected target cells. Taken together, these results implicate CD81 cells in the control of HIV replication and in the development of EC status.

The Role of HLA-B*57 and -B*27 in Control of HIV-1 Replication

A variety of studies have shown associations between two HLA class I alleles and the slowing of HIV disease progression. However, it has been very difficult to determine the exact mechanisms by which HLA-B*57 and HLA-B*27 influence control of HIV replication. Interestingly, a fraction of individuals expressing these HLA molecules become ECs after infection. Unfortunately, it has been impossible to predict which of the HLA-B*57 or HLA-B*27 positive individuals will become ECs or which will progress to disease with an average time course. Studying the genetic, immunological, and virological basis for such natural control could provide insight into the mechanisms by which replication of these viruses can be contained, thereby informing future vaccination strategies.

A Nonhuman Primate Model for Understanding ECs

There are many difficulties inherent to studying HIV-infected humans. Viral control appears to be mediated during resolution of acute phase viremia, with the appearance of CD81 T-cell responses in HIV-infected humans. It is therefore generally accepted that immune responses generated during this period are critical to viral containment. However, HIV is rarely diagnosed during acute infection, making the study of immune responses involved in the initial control of HIV replication extremely difficult. Acute phase studies are further complicated by the diversity of HIV isolates with which individuals might be infected. AIDS research with nonhuman primates provides an animal model to complement human studies. Importantly, there are examples of successful containment of pathogenic immunodeficiency virus replication in the macaque. Researchers working with SIV-infected macaques have

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direct control over key variables such as virus strain, host genotype, and route of infection. Perhaps, most importantly, the events of acute infection can easily be studied in detail. Macaques can also be challenged with mutant viruses, experiments that are impossible to perform in humans. There are four different outcomes after SIVmac239 infection. Macaques can progress to disease quickly with high plasma virus concentrations (rapid progressors, RPs) or control replication of SIV to less than 1,000 vRNA copies/ml (ECs). Most macaques have chronic phase viral loads of approximately 500,000 vRNA copies/ml (progressors, Ps), while a small number become controllers (controllers, Cs). We have previously described the role of Mamu-B*17 in the development of EC status. Approximately 30% of Mamu-B*17 Indian rhesus macaques control SIVmac239 replication and become ECs. This rhesus MHC class I molecule binds peptides with a tryptophan at its carboxy terminus, similar to the peptide binding motif of HLA-B*57 (Fig. 5). We have recently defined an additional MHC class I (MHC-I) allele associated with control of SIV replication in our cohort of SIVmac239-infected macaques. Mamu-B*08 (6 of 16) was overrepresented in our EC cohort, implicating this MHC-I molecule in the control of viral replication (Fig. 5). Intriguingly, the peptide binding motif of Mamu-B*08 is almost identical to that of HLA-B*27 (Table 1). Mamu-B*08 shares the HLA-B*27 binding profile, a notion supported by the fact that all eight Mamu-B*08restricted CD81 T-cell epitopes currently described fit the peptide binding requirements defined for HLA-B*27. Furthermore, peptides that bind to HLA-B*27 bind to Mamu-B*08 with high affinity.

The Unusual Nature of HLA-B*27 and HLA-B*08-Bound Peptides

Almost all of the peptides bound by HLA-B*27 and HLAB*08 have arginine at position 2 (Fig. 5). Interestingly, many of the immunodominant epitopes also have an additional arginine or another basic amino acid, lysine, at position 1. This has led Herberts and colleagues (Herberts et al., 2006) to test the hypothesis that these peptides with two N-terminal basic amino acids are more resistant to degradation by cytosolic peptidases. Indeed, peptides with dibasic amino acids were long lived and efficiently presented by HLA-B*27. Interestingly, several of the SIV-derived Mamu-B*08-bound peptides have either arginine or lysine at P1. Thus, the nature of the MHC-bound peptide may dictate the success of the Tcell response and outcome after viral infection.

Immunodominance and TCR Usage in the Control of Viral Replication

Only a fraction of HIV-infected HLA-B*57 and HLA-B*27 positive patients become ECs. Similarly, approximately 20% of Mamu-B*17 and 50% of Mamu-B*081 macaques control replication of SIV. Thus, these alleles have important roles in the development of EC status, but other factors clearly must play a part in control of viral replication. While most MHC-I molecules are capable of binding 5 to 15 different epitopes in HIV or SIV, typically each individual only mounts 3 to 10 CD81 T-cell responses per MHC-I molecule. Some responses are high frequency (immunodominant) whereas others are barely detectable (subdominant). Unfortunately, the rules governing whether a CD81 T-cell response is immunodominant or subdominant are poorly understood (Yewdell, 2006). Recently, a Mamu-B*081 macaque that failed to control viral replication mounted a strong, immunodominant, response largely against only a single epitope. In the same study, two macaques that became ECs mounted broad CD81 T-cell responses against several epitopes. While these data are preliminary and involve only

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TablE 1

Susceptibility loci with function

Pathogen

Species

Locus

Gene

Human immunodeficiency virus (HIV)

Human

APOBEC3G

Parvovirus B19

Human

P

B3GALNT1, A4GALT


Human

D32-CCR5

CCR5

Norwalk virus

Human

ABO

FUT2


Human

HLA-B*35


Human

HLA-B*57 and HLA-B*27

Human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV) Hepatitis C virus (HCV)

Human

KIR2DL1

Human

KIR2DL3 (and) HLA-C1

Hepatitis C virus (HCV)

Human

KIR2DS4 (and) HLA-C2


Human

Simian immunofeficiancy virus (SIV)

Human

KIR3DS1 (or) KIR3DL1 (and) HLA-B bw4-80I Mamu-B*17 (or) Mamu-B*08

Herpes simplex virus (HSV)

Human

Murine hepatitis virus

Mouse

Ceacam1hv2

Ceacam1

Mouse cytomegalovirus (CMV)

Mouse

Ly49hCmv1

Ly49h

Mouse cytomegalovirus (MCMV)

Mouse

LY49pCmv3

Ly49p (and) H2-dk

Influenza virus

Mouse

Mx1Mx

Mx1

West Nile virus (WNV), yellow fever virus (YFV)

Mouse

FlvOas1b, WnvOas1b

Oas1b

Mouse cytomegalovirus (MCMV)

Mouse

Tlr9CpG1

Tlr9

Mouse cytomegalovirus (MCMV), vaccinia

Mouse

TrifLps2

Trif

TLR3

Functions APOBEC1 is a cytidine deaminase that causes C to T substitutions in HIV but is counteracted by HIV protein Vif. P antigen is a blood-grouping marker that is also used as a viral receptor by PVB19. Individuals with mutations in the p system do not synthesize full p antigen and are resistant to PVB19. CCR5 is a coreceptor for HIV in the macrophage, individuals with a mutation (D32-CCR5) are resistant because D32-CCR5 does not reach the cell surface. a(1,2)-fucosyltransferase is involved in assembly of ABO blood group antigens, which are required for Norwalk virus attachment HLA-B*35 is a MHC class I molecule that presents peptide to cytotoxic T cells. It is associated with rapid progression to AIDS. HLA-B*27 and HLA-B*27 are MHC class I molecules that present peptides to cytotoxic T cells. They are both associated with enhanced HIV clearance (elite controllers). Strong overexpression of the inhibitory NK cell receptor KIR2DL1 and impaired NK ability to counter several viral infections. Combination of weakly inhibiting NK receptor KIR2DL3 and MHC class one HLA-C1 associated with improved resolution of HCV infection. Combination of activating NK receptor KIR2DS4 and MHC class one HLA-C2 associated with improved resolution of HCV infection. Combination of either activating KIR3DS1 or inhibitory KIR3DL1 and HLA-B bw4-80I are associated with slower progression to AIDS. Mamu-B*17 is a macaque MHC molecule similar to HLA-B*57 (and Mamu-B*08 with HLA-B*27), 30% of carriers go on to become elite controllers. TLR3 is a pathogen associated pattern sensor for double stranded RNA, deficiency is associated with Herpes Simplex Encephalitis (HSV-1) Ceacam1 is a glycoprotein receptor of MHV. SJ/L mice carry a version (Ceacam1b) which does not allow viral infection. Ly49H is a NK cell receptor that recognizes MCMV viral protein m157 and induces proliferation and killing of infected cells. Ly49P is a NK cell receptor that recognizes MCMV in the presence of viral protein m04 and host MHC H2-Dk. Mx1 is an IFN-induced GTPase that interferes with influenzae replication. Oligoadenylate synthase catalyzes synthesis of 29-59 A. This activates latent antiviral RNaseL. Deficient mice are susceptible to flavivirus. Cpg1 mice are deficient in TLR9, a membrane bound pathogen-associated molecular pattern sensor specific for unmethylated CpG DNA as found in viruses and bacteria. Adaptor relays TLR3 signal to downstream signaling molecules. Trif deficient mice are susceptible to MCMV and vaccinia.


39. Immunogenetics of Virus Pathogenesis TablE 1

505

(Continued)

Pathogen

Species

Locus

Gene

Mouse cytomegalovirus (MCMV), herpes simplex virus (HSV)

Mouse & Human

Unc93b13d

Unc93b1, UNC93B

Mouse cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), herpes simplex virus (HSV), vesicular stomatitis virus (VSV)

Mouse & Human

Stat1domino

Stat1, STAT1

a few animals, there is at least a suggestion that the development of broad CD81 T-cell responses might play a role in development of EC status. In the absence of correlates of immune control, recent studies have attempted to understand efficacious antigenspecific CD81 T-cell responses. The function of CD81 T cells is impacted by viral replication in a bidirectional interaction. Not only can CD81 T cells affect viral replication, but high levels of viral replication can also stimulate CD81 T cells, leading to exhaustion. Furthermore, HIV and SIV preferentially infect activated antigen-specific CD41 T cells, leading to their destruction and resulting in reduced CD41 T-cell help for CD81 T cells. Any epitope-specific T-cell population is defined by its composite clonotypes. Given that the interface between the virus and adaptive T-cell immunity occurs through the T-cell receptor (TCR), it is probable that the efficacy of a virus-specific CD81 T-cell population is dependent on how TCRs engage the viral antigen. The nature of the TCR expressed by antigen-specific CD81 T cells might therefore play an important role in protective immunity (Fig. 5). In a recent study led by Danny Douek and David Price (Price et al., 2009), TCR usage of the immunodominant GagCM9-specific T-cell response was followed in a cohort of vaccinated Mamu-A*011 macaques that were subsequently challenged with SIVmac239. Interestingly, the expression of public TCRs correlated with the ability to control viral replication after challenge. These public TCRs (TCRs expressed by CD81 T cells from several different animals) might therefore be more effective in the control of viral replication. Indeed, evidence suggests they are more “cross-reactive” and thus might be able to recognize variant viruses.

The Rate of T-Cell Expansion During the Acute Phase may Determine Disease Outcome

The timing and magnitude of the CD81 T-cell response may also be a critical determinant of protection (Fig. 5). Studies on infection kinetics led to the “too late concept;” CD81 T-lymphocyte responses develop too late to prevent dissemination of virus throughout the body (Reynolds et al., 2005). In natural history studies of intravaginal transmission using tetramers to stain antigen-specific CD81 T cells, it was found that the SIV-specific CD81 T-lymphocyte responses against Mamu-A*01-restricted immunodominant epitopes in Gag (GagCM9) and Tat (TatSL8) together account for 70% of the CD81 T-cell response in acute SIV infection (in Mamu-A*011 macaques) and were only detectable several days (in cervicovaginal tissues and throughout the lymphatic tissues) after the peak of viral replication. However, tetramer-positive cells subsequently increased rapidly after 14 days postinfection, and the increases were correlated with a decline in both plasma viremia and the frequency of SIV-

Functions UNC93b1 is required for at least TLR7 and TLR9 function. Humans with UNC93B1 deficiency were susceptible to herpes simplex encephalitis (HSV-1) and vesicular stomatitis virus (in vitro). STAT1 is a transcription factor responding to type I IFN. Humans deficient in STAT1 are susceptible to several pathogens and are unresponsive to IFN-a treatment.

RNA1 cells. Additionally, the percentage of positive lymphocytes for the TatSL8 tetramer was inversely correlated with the quantity of virus in the cervix. By combining in situ tetramer (IST) staining with in situ hybridization (ISH) for virus particles, effector to target ratios (E:T) could readily be estimated in vivo, in order to demonstrate that tetramer-positive cells colocalized with productively infected SIV-RNA1 cells. Host defenses were mounted too late to prevent the establishment of a systemic infection in these natural history studies in Mamu-A*011 macaques that progress to disease normally. CD81 T-cell responses following peak replication could substantially reduce the number of infected cells. The hypothesis that follows from this analysis is that control or loss of control in infected Mamu-B081 animals that become ECs could be a function of the E:T ratios, with high ratios associated with control of viral replication and colocalization of CD81 T cells and infected cells.

Viral Escape

The role of CD81 T lymphocyte escape—that is, viral sequence variation resulting in diminution of recognition by CD81 T lymphocytes—in HIV and SIV pathogenesis is now well established (Saez-Cirion et al., 2007). However, the full extent of escape is still perhaps underappreciated. Recent studies show substantial escape from CD81 T-cell responses in immunodeficiency virus infection. Data from the SIVinfected macaque model have conclusively shown that CD81 lymphocyte-driven escape occurs frequently. Escape mutations are selected during both the acute phase and throughout the chronic phase of SIV infection. Viral variation is detected in many epitopes targeted by CD81 T cells. This variation may be selected for with varying kinetics following infection, though, for a given epitope, the same escape mutation is often found in multiple animals (Goulder & Watkins, 2004). While there is considerable debate surrounding the role of viral escape from CD8 1 T lymphocytes in the development of EC status, this area remains largely unexplored, especially in rhesus ECs. Escape in an HLA-B*27-restricted epitope has been shown to be associated with progression of disease, but there does not appear to be any relationship between amino acid replacements in HLA-B*57-positive patients and EC status. This latter study was carried out in chronically infected patients and it was difficult to follow the ontogeny of the escape mutations in these patients. The rhesus macaque model offers an ideal opportunity to follow the development of escape mutants after infection with a cloned virus.

CONCLUSIONS

The majority of HIV-infected individuals who control HIV replication express either HLA-B*57 or HLA-B*27. Similarly, more than 90% of Indian macaques that control SIV

506


replication express either Mamu-B*17 or Mamu-B*08. However, not all individuals who express these protective alleles control viral replication (Fig. 5). The first and foremost challenge is to understand the mechanisms of MHC class I mediated control. Is this due to the nature of the bound peptide, the individual’s TCR repertoire, the speed with which CD81 T cells respond to the pathogen, or NK cells? The second issue will be to address why certain individuals with “protective” MHC class I alleles do not control viral replication. While these two phenomena may be connected, it is possible that a completely different set of loci may be important contributors to MHC class I-associated control. Over the past few decades, infectious disease immunogenetics has developed from MHC disease-association studies to a diverse field exploiting epidemiological, familial, animal, and evolutionary studies to identify a number of diverse host genes influencing resistance or susceptibility to infection. The available data indicate a highly polygenic basis for host susceptibility to many virus infections. Recent studies, however, demonstrate that human genetics of common infectious diseases also involves single gene determinism. Furthermore, the study of differential susceptibility to virus infection in animal models has fueled the field by providing key insight into the molecular mechanisms and the cell populations that are essential for the detection of and response to viruses. The discoveries have converged to mechanisms involving receptor availability, type I IFN response, and NK and CD81 T cytotoxic response, which together define the contours of the antiviral response. In addition to understanding immunobiology, these advances are being translated into possible new therapies. Chemotherapies designed to mimic viral receptors may slow or block progression of disease; for example, several CCR5 antagonists are presently in development (Barbaro et al., 2005). Knowledge of innate pattern recognition systems has led to new adjuvants being used to stimulate immunity during vaccine development (Guy, 2007). Direct TLR agonists are in use to treat several conditions, including cutaneous viral infection, and recombinant IFN is in use as a treatment for hepatitis B and C infection. As specific molecules recognized by germ-line encoded receptors on NK cells and by adaptive elite controlling CD8 T-cell receptors are discovered, they will suggest strong candidates for vaccination. Though each system is involved in response to any particular infection, the remarkable specificity of which mechanisms are essential for resolution suggests that therapies will need to be tailored to pathogens and individuals. A consequent theme in immunobiology is the character of essential genes of being “public” (i.e., responding to several pathogens or involved in several pathways) or “private” (i.e., only required in response to a single pathogen) (Quintana-Murci et al., 2007). The presence of this public/private dynamic coupled with the many viral diseases with unknown host determinants suggest that the silhouette of antiviral immunity presented above leaves many details to be filled in. To this end, in recent years, a broadened approach has been undertaken by many to include recent evolutionary selection, interspecies differences in response, and genome-wide association with common diseases. Pathogens are considered one of the most powerful forces shaping the evolution of the human genome; furthermore, numerous studies have shown that genes or loci involved in host defense against infection present signs of positive or negative selection. These are polymorphisms that change the amino acid sequence of a protein, alleles that show geographically restricted distribution, and loci that present longrange linkage disequilibrium (Dean et al., 2002). The first study of positive selection in humans was on genes encoding

the major histocompatibility complex. Since then several candidate genes, including blood group antigens, killer cell immunoglobulin-like receptors (KIR), and, more recently, TLR genes have demonstrated signs of selective pressures most likely imposed by pathogens. The availability of largescale catalogs of genetic variation has stimulated genomewide scans for selection in several species with the premise that selection signatures are likely to be valuable signposts for gene variants that might influence medically relevant traits. In the context of infectious diseases, location of selected genes could aid in the effort to isolate the genetic variants that underlie susceptibility or resistance to infection, and parse essential from redundant immune genes. Numerous genome-wide studies in human populations have indeed shown that genes involved in host–pathogen interactions are among the most common targets of adaptive evolution. Moreover, the results have shown that individual candidate genes may participate in common pathways. This is the case of selected genes in some African populations, LARGE and DMD, which encode proteins critical for the function of a-dystroglycan, the cellular receptor of Lassa fever virus. Comparative genomics of the human and chimpanzee genomes has also revealed that many of the genes showing the strongest evidence for selection are involved in immune defenses such as the ABOBEC3F gene. The ABOBEC3G gene, whose product targets HIV and SIV, has previously shown to be under positive selection, demonstrating the ancient evolutionary pressures imposed by retroviruses. Another example of a primate-selected gene is the HIV restriction gene TRIM5a. In this case, positive selection identified a segment of the TRIM5a protein likely to represent the antiviral interface, an assumption that was confirmed by further experimental analysis. Thus, mapping the patterns of adaptive evolution has the potential to generate a strong hypothesis not only with respect to the mechanisms and pathways critical for host defense but also regarding gene function. The first applications of genome-wide association approaches to identify the variants involved in the response to HIV and HCV have spurred much optimism because previously unrecognized host determinants of pathogenesis have been identified (Ge et al., 2009). The continuous accumulation of genetic information will present an unprecedented opportunity for the advancement of human health; however, how this information can be translated into treatments remains persistently challenging (Iadonato & Katze, 2009).

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40 Viruses, Autoimmunity, and Cancer† MEGHANN TEAGUE GETTS, LIES BOGAERT, W. MARTIN KAST, AND STEPHEN D. MILLER

VIRUSES AND AUTOIMMUNITY

evidence that such an etiology is feasible (see Table 2). In humans, disease concordance rates for mono- and dizygotic twins indicate that although genetics plays an important role in disease susceptibility, environmental factors, including microbial infections, may also play a role. Certain regions of the world confer a heightened risk for developing MS to their inhabitants that does not necessarily appear to be related to ethnic considerations, suggesting that infection with a microbe common to those particular areas may be a key event that precipitates the onset of MS. Specifically, inhabitants of North and Central Europe, the northern United States and Canada, and Southeastern Australia and New Zealand are at increased risk of developing MS compared to people living in other regions of the world. This putative microbial infection seems to occur just before or during adolescence, as people who migrate between regions of differing risk retain the level of susceptibility conferred during preadolescence and adolescence (Kurtzke, 1993). There are also several instances where MS appears to have been introduced to a previously isolated part of the world by a newly arriving population of people. A cogent example is the case of the Faroe Islands, an archipelago located between Iceland and Norway where, prior to 1943, there were no documented cases of MS. This was peculiar due to the islands’ similarity, both in climate and in ethnic origin of most of the inhabitants, to the regions that suffer the highest risk for developing MS. In 1940, British troops occupied these islands, and the first cases of MS appeared a few years thereafter, suggesting that MS was brought about by the emergence of an unknown transmissible infection that was introduced to the inhabitants by the British troops. After 1943, the inhabitants of the Faroe Islands experienced several epidemics that continued for decades after the troops departed in 1945 (Kurtzke, 1993). Several other cases of a sudden increase in the number of MS cases in physically isolated locations, including Iceland, the Shetland-Orkney Islands, and Key West, Florida, have been reported and support the hypothesis that an infectious agent may play a critical role in the initiation of MS (Kurtzke, 1993). Due to the evidence that MS is caused or at least precipitated by a transmissible infection, the search for such an infectious agent has been rigorous. To date, no one microbial infection has been shown to have a direct cause-andeffect relationship with MS, but multiple pathogens have

Epidemiological Evidence Supporting Infectious Etiologies for Autoimmune Diseases

Autoimmune diseases occur when the immune system recognizes a self-tissue as foreign and elicits a self-destructive inflammatory response in an attempt to destroy it. For many autoimmune diseases, the cells and immunological mechanisms that drive disease are at least partially understood, but the actual initial trigger that causes autoreactive cells to become activated is not known. The initiating event has long been thought to be of infectious origin, and various autoimmune diseases are associated to various degrees with possible infectious triggers. Rarely can a direct cause-and-effect relationship between a single infectious agent and a particular disease be established, but a convincing amount of circumstantial evidence often associates a disease with one or several pathogens. Even when an autoimmune disorder can be attributed to a particular infection with confidence, as is the case for group A streptococcus and rheumatic fever, the exact mechanism through which the pathogen initiated the autoimmune disease is not always clear. The autoimmune disorder multiple sclerosis (MS) is an instructive example in which several pieces of evidence suggest that a microbial infection is the trigger for disease, with most evidence pointing to viral infections (Oldstone et al., 1996) (see Table 1). Infectious triggers for MS-like diseases have been studied in several animal models, providing theoretical Meghann Teague Getts and Stephen D. Miller, Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611. Lies Bogaert, Departments of Molecular Microbiology & Immunology and Obstetrics & Gynecology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033. Department of Surgery and Anesthesiology of Domestic Animals, Faculty of Veterinary Medicine, University of Ghent, Merelbeke, B-9820, Belgium. W. Martin Kast, Departments of Molecular Microbiology & Immunology and Obstetrics & Gynecology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033. Cancer Research Center of Hawaii, University of Hawaii, Honolulu, HI 96822. † M.T.G. and L.B. are co-first authors who made equal contributions, and W.M.K. and S.D.M. are co-senior authors.

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AUTOIMMUNITY AND CANCER TABLE 1 Selected murine models of infection-induced autoimmune diseases

Multiple sclerosis

Mechanism(s) of disease initiation or exacerbationb,c

Infectious agenta

Disease modeled TMEV

TMEV expressing PLP139 TMEV expressing PLP139 mimics LCMV (in mice expressing LCMV protein in CNS) Semliki Forest virus EAE 1 bacterial superantigen staphylococcal enterotoxin B (SEB)d Type 1 diabetes

Myocarditis

Bystander activation and epitope spreading Molecular identity Molecular mimicry Molecular identity Molecular mimicry Superantigen

Coxsackie B4 LCMV (in mice expressing LCMV protein in pancreas) Pichinde virus (in mice expressing LCMV protein in pancreas)d

Bystander activation Molecular identity

Mouse cytomegalovirus

Bystander activation or molecular mimicry Molecular mimicry Molecular mimicry

Coxsackie B3 virus Chlamydia

Molecular mimicry

Stromal keratitis

HSV

Molecular mimicry and/or bystander activation

Rheumatoid arthritis

Collagen-induced arthritis (CIA) 1 murine arthritogenic mycoplasma superantigen (MAM)d

Superantigen

a TMEV, Theiler’s murine encephalomyelitis virus; LCMV, lymphocytic choriomeningitis virus; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; HSV, herpes simplex virus. b Unless otherwise noted, the infectious agent has been shown to trigger the indicated disease. c References to these murine models of infection-induced autoimmune diseases can be found in Munz et al., 2009. d The indicated infectious agent does not cause disease, but results in exacerbation of disease established by other means.

been reported to be associated with the disease (Table 1). A large body of published data points to a link between MS and a herpesvirus, the Epstein-Barr virus (EBV) (Munz et al., 2009; Salvetti et al., 2009). Studies have shown an increased susceptibility to MS in patients that have had EBV-induced infectious mononucleosis. Furthermore, a relationship exists between symptomatic episodes of MS and EBV reactivation. However, none of these items of evidence, singly or collectively, constitutes a definitive demonstration of a causeand-effect relationship, perhaps because more requirements exist for the elicitation of disease than merely the presence of a single pathogenic agent. One commonly held theory is that infection with one or more of several possible viral or bacterial agents is required for the trigger of other cellular events that may eventually lead to MS in a genetically susceptible individual. As with MS, the etiology of autoimmune type I diabetes (T1D) is also associated with microbial (particularly viral) infection, and also is based on circumstantial and anecdotal evidence, along with a lack of a definitive cause-and-effect relationship. Like MS, twin studies indicate that genetics along with some environmental factors are important players in the development of disease. Anecdotal evidence includes the association of an outbreak in 1993 of T1D in patients under the age of 14 years in Philadelphia that occurred 2 years after an outbreak of measles in the same population of children (Zipris, 2009). T1D has also been associated with

other viruses, including coxsackie B virus infections, both in animal models (Table 1) and in humans (Table 2). MS and T1D are only two examples of autoimmune disease where multiple lines of evidence implicate an infectious etiology. Theories abound as to how the autoimmune disease was initiated, but there is, as of yet, no definitive evidence. Multiple other examples of such associations between a human autoimmune disease and a particular microbial infection exist (Table 2).

Potential Mechanisms of Infectious Triggering of Autoimmune Disease

Various animal models of both MS and T1D tell us that, in principle, infections can trigger autoimmune responses. The generation of an autoimmune response, however, must be distinguished from the elicitation of overt autoimmune disease as a direct result of microbial infection. The latter is not only more difficult to identify, but is also likely to be more rare. Triggering an autoreactive response is the first step toward overt autoimmune disease, however, and it requires one essential element: that immune cells exist within the human body that have the potential to recognize selftissues as foreign. It is generally accepted that most autoreactive T cells are deleted in the thymus during development. However, autoreactive T cells can avoid deletion and make their way to the periphery in humans and animals via several potential

40. Viruses, Autoimmunity, and Cancer TABLE 2 Selected autoimmune diseases and their proposed viral associations and potential mechanismsa Disease Multiple sclerosis

Proposed viral association Measles EBV HHV-6

Type 1 diabetes

Rheumatoid arthritis

Coxsackie virus

Proposed mechanismb,c None suggested Molecular mimicry/ bystander activation Molecular mimicry

Rubella

Bystander activation, molecular mimicry Molecular mimicry

EBV

None suggested

Parvovirus

None suggested

SLE

EBV

Molecular mimicry

HTLV-1associated myelopathy

HTLV-1

Molecular mimicry

Stromal keratitis

HSV-1

Molecular mimicry

a

EBV, Epstein-Barr virus; HHV, human herpesvirus; SLE, systemic lupus erythematosus; HTLV, human T-cell leukemia virus; HSV, herpes simplex virus. b For those diseases in which no proposed mechanism has been suggested, evidence for involvement of the indicated virus in disease pathogenesis includes association of increased antiviral responses or increased detectable virus at site of diseased tissue and/or epidemiological evidence. c References to these virus-associated human autoimmune diseases can be found in Munz et al., 2009.

mechanisms. The first possibility is that the cognate selfantigen of a particular T cell was not expressed within the thymus, and therefore this self-reactive cell was not negatively selected for deletion. Alternatively, some T cells that make their way to the periphery might be highly specific for a microbial antigen but, due to TCR degeneracy, also have an affinity for a self-antigen that was not high enough to result in thymic deletion. While establishing firm links between an autoimmune disease and a causative microbial infection has been difficult, investigations into the actual mechanisms by which microbial infections could initiate autoimmune responses, using animal models, have been very instructive. The initial trigger of such an autoreactive response could occur through several mechanisms, including molecular mimicry, bystander activation, epitope spreading, or super-antigen activation (Fig. 1). Evidence, mostly from animal models, exists for each, but the most widely studied mechanism of infection-induced autoimmunity that is supported by the largest body of evidence is molecular mimicry.

Molecular Mimicry

Although T-cell receptors (TCRs) are considered to be “specific” for a particular antigen, they do exhibit a great degree of degeneracy, allowing recognition of and response to different peptides of a sufficient degree of similarity in shape and charge distribution in the context of a major histocompatibility complex (MHC) molecule (Wucherpfennig & Strominger, 1995). While such degeneracy is likely to be an important feature underlying immunological processes, such as thymic selection and the ability to recognize a vastly diverse array of pathogen-derived antigens, it also implies that a T cell specific for a foreign antigen could be cross-reactive with one or more self-antigens. This

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cross-reactivity, particularly within the environment of an inflammatory response to a pathogen, could result in the induction of autoimmunity. A similar phenomenon occurs with regard to the B-cell receptor, as monoclonal antibodies that recognize both microbial and self-antigens have been identified (Fujinami et al., 1983). The idea that microbial and self-antigens can “mimic” each other in such a way to induce cross-reactive responses was first proposed by Fujinami and Oldstone (Fujinami & Oldstone, 1985) and is now accepted to be a general phenomenon (Wucherpfennig & Strominger, 1995). There are many animal models in which molecular mimicry has been shown to serve as the trigger for autoimmune disease. Examples include Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD), in which intracerebral infection with TMEV leads to an autoimmune demyelinating disorder that mimics human MS (Miller et al., 1997); herpes simplex virus (HSV)-associated stromal keratitis, in which HSV infection leads to T-cell-mediated blindness in both humans and mice (Zhao et al., 1998); diabetes models (Christen et al., 2004); autoimmune demyelinating disease associated with Semliki Forest virus (SFV) (Mokhtarian et al., 1999); and autoimmune myocarditis associated with infection with coxsackie virus (Gauntt et al., 1995), murine cytomegalovirus (Lawson, 2000), or Trypanosoma cruzi (Tibbetts et al., 1994). One useful method for investigating molecular mimicry is the use of molecular identity models. Molecular identity is achieved when a microbial protein or epitope is transgenically expressed in a particular tissue. Generally, animals are not susceptible to the development of spontaneous autoimmune disease under these conditions; however, autoimmunity in the tissue expressing the transgenic protein can be induced by infecting the animal with the microorganism from which the protein is derived (Ohashi et al., 1991; Oldstone et al., 1991). These models do not necessarily aim to closely replicate a particular disease state, but do serve to reveal potential mechanisms by which immune responses to infections could lead to autoimmunity through molecular mimicry. Though artificial, these models have served the important function of demonstrating that T cells specific for a “self” antigen can become activated in the presence of infection with a microbe containing an identical antigen. In the presence of an infection, which provides the appropriate innate immune signals, overt autoimmunity can occur. In a molecular identity model of diabetes, infection can ultimately result in autoimmune disease even when the microbial antigen (LCMV glycoprotein) is also expressed in the thymus. Thymic expression of an antigen allows the normal mechanisms of negative selection to significantly decrease the number of high-affinity T cells that are specific for the microbial antigen. Therefore, a more physiological scenario, in which T cells with a low affinity for self antigen that have escaped negative selection and exist in the periphery, could potentially lead to autoimmune disease through molecular mimicry. TMEV-IDD occurs due to the activation of T cells specific for proteolipid protein 139–151 (PLP139–151), a self-myelin epitope. Models that more directly address the mechanism of molecular mimicry have utilized several bacterial and viral peptides that mimic PLP139–151. Peptides derived from microbes such as Haemophilus influenzae or murine hepatitis virus that mimic PLP139–151 are able to induce a rapid onset, severe demyelinating disease that is similar to that induced by infection with TMEV expressing the native PLP139–151 peptide itself (Croxford et al., 2005; Olson et al., 2001). Bacterial peptide mimics of the myelin basic protein (MBP) epitope 85–99, which are derived from several pathogens including

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FIGURE 1 Mechanisms of infection-induced autoimmunity. Circulating T cells are generally specific for some pathogen-derived or otherwise foreign antigen (virus antigen recognized by a virusspecific T cell, in the example here); however, some circulating T cells also possess a capacity for recognizing self-tissues (autoreactive T cell). A virus infection (for example) could elicit autoimmunity through several mechanisms. Molecular mimicry (A) occurs when T cells are cross-reactive with self and viral antigens, so that a viral infection can activate a T cell that is capable of recognizing a self-antigen as foreign. Bystander activation (B) occurs when tissue damage results in the release of self-antigens that are recognized by autoreactive cells. Such tissue damage could occur due to inflammatory mediators, including cytokines that are released by infected cells and other cells detecting the presence of a pathogen through PAMPs. By extension, epitope spreading can occur when the autoreactive response spreads to other self-antigens, exacerbating the autoimmune process. Superantigen-induced activation of autoreactive cells (C) could occur if autoreactive cells are present within the population of T cells that are nonspecifically activated by a superantigen.

Mycobacterium tuberculosis, Bacillus subtilis, and Staphylococcus aureus, induce demyelinating disease in mice that transgenically express a human MBP85–99-specific TCR as well as the relevant HLA class II molecule (Greene et al., 2008). Molecular mimicry was also shown in a model of diabetes in which lymphocytic choriomeningitis virus (LCMV) nucleoprotein was expressed under the control of the rat insulin promoter. Infection with Pichinde virus, which contains an epitope that mimics a subdominant epitope in the LCMV nucleoprotein, accelerated autoimmune disease that had already been established by previous infection with LCMV (Christen et al., 2004). An example of a model of molecular mimicry-induced autoimmunity in which autoimmune disease occurs in the absence of genetic manipulation is HSV-induced stromal keratitis, wherein corneal-antigen-specific T-cell responses are induced following corneal HSV infection (Zhao et al., 1998). In human disease, evidence suggests that as in animal models, pathogens can prime T cells that subsequently cross-react with self-antigens to cause autoimmunity. At present, most of the evidence for molecular mimicry in humans is circumstantial. During symptomatic EBV infection, high viral loads are associated with a heightened risk

for development of MS (Munz et al., 2009; Salvetti et al., 2009) and MS patients possess clonally expanded T cells specific for EBV-encoded nuclear antigen 1 (EBNA1). In addition, EBNA1-specific T cells recognize myelin antigens more readily than other autoantigens that are not associated with multiple sclerosis (Lunemann et al., 2008). Human herpesvirus 6 (HHV-6) has also been linked to the development and pathogenesis of MS, as HHV-6 possesses an amino acid sequence identical to myelin basic protein (MBP) residues 86–102 (Tejada-Simon et al., 2003), although whether this has any relevance for disease remains unclear. Other examples of the potential for molecular mimicry in the form of microbial epitopes that mimic self-epitopes that may be associated with disease states in animals and humans are noted in Tables 1 and 2. Although a great deal of evidence from animal models clearly demonstrates that molecular mimicry can theoretically induce autoimmune disease, no single autoimmune disease in humans has been demonstrated to be a direct result of molecular mimicry between microbial and selfpeptides. If infection(s) do trigger an autoimmune response through cross-reactive epitopes, they may be cleared long

40. Viruses, Autoimmunity, and Cancer

before evidence of autoimmune responses arises, making the identification of such infections extremely challenging.

Bystander Activation and Epitope Spreading

Bystander activation occurs when autoreactive T or B cells are stimulated by APCs that have become activated within the inflammatory milieu of a pathogenic infection. Thus, selfantigen, which is obtained from tissues undergoing immunemediated destruction or subsequent to APC uptake of local dying cells, is presented to epitope spreading, which is a form of bystander activation, and occurs when an immune response that is initiated by a stimulus that can include microbial infection, trauma, autologous transplanted tissue, or autoimmunity, “spreads” to include responses directed against a different portion of the same protein (intramolecular spreading) or a different protein (intermolecular spreading) (Vanderlugt & Miller, 2002). Through this mechanism a broader set of T cells are activated. Such spreading can be helpful during a response to a pathogen or tumor, but epitope spreading can potentially cause or contribute to disease when spreading to and within self-proteins occurs. In animal models, epitope spreading proceeds in a predictable manner in that, for certain disease states, the order of responses to individual epitopes can be consistently predicted, and that more immunodominant epitopes are the first to elicit responses, followed by less dominant epitopes. Such spreading occurs in experimental autoimmune encephalomyelitis (EAE), a noninfectious model of multiple sclerosis, as well as in TMEV-IDD and in the nonobese diabetic (NOD) mouse model of type 1 diabetes (Munz et al., 2009; Vanderlugt & Miller, 2002). These examples of epitope spreading include those scenarios in which spread occurs from autoantigen to autoantigen, but the inflammatory environment of a microbial infection may also elicit autoimmunity by increasing presentation of self-antigens alongside inflammatory signals, and resulting in spread from a microbespecific response to an autoreactive response. Superantigen-induced activation of autoreactive T cells is perhaps the broadest form of bystander activation. Superantigens are microbially derived, and cross-link MHC class II molecules on APCs with a particular Vb domains on TCRs. Thus, an entire population of T cells of varied specificities is activated, and a subset of T cells that are specific for a selfantigen could be activated within this population (Wucherpfennig, 2001). There are multiple examples in which superantigens are involved in diseases such as EAE, arthritis, and inflammatory bowel disease, making superantigens another mechanism by which bystander activation can at least exacerbate autoimmunity in mouse models (Munz et al., 2009). In these studies, staphylococcal, mycoplasma, and enteric microbiota-derived superantigens were shown to amplify, but not initiate, autoimmune T-cell responses (Table 1). The retrovirus HERV-K18, which is transactivated by EBV and encodes superantigens, has been associated with MS (Tai et al., 2008). This potential link is somewhat weakened by the fact that the T-cell populations that are known to be affected by HERV-K18 superantigens are not particularly expanded in MS patients. However, the superantigen-mediated method of autoreactive cell activation is certainly a possible mechanism, and merits more study in human diseases.

Pathogens as Adjuvants

The ability of pathogens to activate the immune system can certainly contribute to any of the mechanisms discussed above, either during the initial encounter of an autoreactive cell with its autoantigen, or once autoreactive cells have already become activated through molecular mimicry, a form of bystander activation, or some other mechanism. In this way, pathogens could further contribute to the driving of an

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autoimmune response through pathogen-induced general activation of the immune system and provision of antigens that specifically stimulate immune responses, and which may crossreact with self-antigens and therefore cause autoreactive immunopathologies. Pattern-recognition receptors (PRRs) are a group of molecules that recognize various molecular patterns on bacteria, viruses, and fungi. These receptors include Tolllike receptors (TLRs), nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), RIG-I-like helicases and a subset of C-type lectin receptors, which together confer the ability of the host to defend against invading pathogens (Ishii et al., 2008). Engagement of PRRs initiates signaling pathways that result in cellular activation, allowing an increase in antigen-presenting capacity and the expression of costimulatory molecules on antigen-presenting cells (APCs). The production of type I interferons (IFNs), proinflammatory cytokines, and chemokines is also triggered by PRR ligation, and these molecules initiate and direct the immune system to the appropriate type of response to the invading pathogen. Clonal expansion of T and B cells that are specific for pathogen antigens is driven by such molecules and by the microbial antigens themselves. Thus, pathogens act as adjuvants by triggering early innate immune responses, increasing APC function, and providing a source of antigen for T- and B\-cell activation and function. Microbial infection therefore elicits a potent inflammatory environment. If autoreactive cells are present within this inflammatory environment, the initiation and/or escalation of an aberrant, destructive autoreactive immune response can easily be triggered.

Future Perspectives

Infections affect the immune response in many ways, and mechanisms such as molecular mimicry and bystander activation are certainly not the only ways in which pathogens trigger or accelerate autoimmune disease. There may be numerous other mechanisms through which immune responses to infections result in or accelerate autoimmune diseases. Since evidence of autoimmunity is likely to become clinically apparent only after a considerable period of subclinical autoimmune responses, an autoimmunity-triggering pathogen may have been cleared from the system for some time, and may therefore be difficult to identify. On the other hand, some diseases are so closely associated with the presence of an infectious agent that whether or not autoimmune responses are occurring, as opposed to simple pathogen-induced tissue destruction, is difficult to establish. As an additional complicating factor, it is important to remember that these mechanisms can be interrelated, nonmutually exclusive, and dynamic, so the idea of microbial infection eliciting autoimmunity must be viewed not as a defined event that occurs via a particular mechanism, but as a process that can occur through many pathways simultaneously and/or sequentially. As a result, it can be difficult to distinguish among all of the postulated mechanisms, even in seemingly simple animal models. In the much less simple scenario of human autoimmune disease, identification of a specific pathogen trigger, or even a general mechanism through which autoimmunity ensues, can seem nearly impossible. However, it is important to continue to attempt to identify the potential triggering pathogens so that we can not only achieve a better understanding of autoimmune disease processes, but so that we can also make important steps toward preventing or treating these serious diseases.

VIRUSES AND CANCER Historic Context

For centuries, cancer has been believed to have an infectious origin, as evidenced by records of “cancer houses” in which many dwellers developed a certain cancer. Observations that

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married couples sometimes could be affected by similar cancer types and that cancer appeared to be transmitted from mother to child lent further support to an infectious etiology of tumors. However, during the 19th century, extensive investigation failed to demonstrate a carcinogenic role for bacteria, fungi or parasites, leading to the belief that cancer is not caused by an infectious agent (McLaughlin-Drubin & Munger, 2008). The first successful transmission using cellfree extracts was done for oral dog warts in 1898, followed by human warts in 1907. One year later, it was demonstrated that leukemia in birds could be transmitted using extracts of leukemic cells or serum from diseased birds. In 1911, Rous produced solid tumors in chickens using cell-free extracts from a transplantable sarcoma. In the 1950s, murine leukemias were found to be induced by viruses. Over the next 2 decades, numerous additional animal oncogenic retroviruses inducing lymphoproliferative disorders in cattle, cats, and rodents were isolated (Javier & Butel, 2008). After the successes in the animal tumor virus field, scientists were drawn to the field of human tumor viruses. In 1964, Epstein-Barr virus (EBV) was demonstrated to be caused by virus infection by electron microscopy in cells cultured from Burkitt’s lymphoma, but it was not until the early 1980s that the causal relationship between other viruses and cancer was acknowledged. At that time, human T-lymphotropic retrovirus type 1 (HTLV-1) was linked to adult T-cell leukemia; a compelling link between persistent hepatitis B virus (HBV) infection and liver cancer was established and human papillomavirus (HPV) types 16 and 18 were isolated from human cervical cancer specimens (reviewed in Kalland et al., 2009). Since then, the link between other viruses and cancer has been established, and many more viruses have been associated with cancer, although a causal relationship has not been demonstrated yet for all of these viruses.

Viral Carcinogenesis and the Role of the Immune System

Carcinogenesis represents a complex, multistep process, and the field of tumor virology has provided groundbreaking insights into the causes of human cancer. Approximately 15% to 20% of all human cancers are associated with viral infections. Oncogenic viruses can contribute to different steps of the carcinogenic process, and the association of a virus with a given cancer can be anywhere from 15% to 100% (Parkin, 2006). However, the presence of viral DNA within a tumor or seroepidemiological studies revealing elevated antibody titers against the respective infection represents a hint but clearly is not proof for an etiological relationship. It is clear that Koch’s postulates to prove that an infection is the cause of a specific disease, based on the isolation of the infectious agent, its in vitro propagation, the reinoculation into a susceptible animal host and the induction of symptoms analogous to those observed in the diseased patient cannot be applied to tumor viruses (zur Hausen, 1999). As a result, different guidelines have been proposed to aid in establishing a causal relationship between viruses and human cancers (Table 3). Although some of these guidelines are difficult to meet and may exclude indirect contributions to cancerogenicity (e.g., by continuing immunosuppression; HIV infection), the guidelines are nonetheless useful when evaluating a putative association between a virus and cancer. Although viruses often contribute to cancer, it appears that they are not sufficient for carcinogenesis. Many years or even decades of viral latency may pass between the primary infection and tumor appearance. No known virus infection in humans exists where the exposure to human cells results in immediate malignant transformation. In addition, the

TABLE 3 Criteria for defining a causal role for an infection in cancera • Epidemiological plausibility and evidence that a virus infection represents a risk factor for the development of a specific tumor • Regular presence and persistence of the nucleic acid of the respective agent in cells of the specific tumor • Stimulation of cell proliferation upon transfection of the respective genome or parts thereof in corresponding tissue culture cells • Demonstration that the induction of proliferation and the malignant phenotype of specific tumor cells depends on functions exerted by the persisting nucleic acid of the respective agent a

Data from zur Hausen, 1999.

majority of the infected individuals either clear the respective infection by immunological intervention or may harbor viral DNA for a lifetime within specific cells without any clinical symptoms (zur Hausen, 1999). Thus far, no human tumor virus has been identified that does not require modifications of host cell DNA prior to tumor formation. Cofactors such as host immunity and chronic inflammation as well as additional host cellular mutations must therefore also play an important role in the transformation process (Kalland et al., 2009). One key feature of oncogenic viruses is their ability to infect, but not kill, their host cell. In contrast to many other viruses that cause disease, oncogenic viruses have the tendency to establish long-term, persistent infections. The long-term interaction between virus and host is a key feature of the oncogenic viruses, as they set the stage for a variety of molecular events that may contribute to eventual virus-mediated tumorigenesis. Commonly, the induced mutations affect cellular pathways engaged in the control of these persisting virus infections. Additionally, oncogenic viruses have evolved strategies for evading the host immune response, which would otherwise clear the virus (McLaughlin-Drubin & Munger, 2008). One virus with an indirect but prominent effect on cancer development is the human immunodeficiency virus (HIV) (Engels, 2009). In particular, the risk of Kaposi sarcoma (KS) and non-Hodgkin’s lymphoma (NHL) is vastly higher among HIV-infected persons than in healthy persons. Cervical cancer risk is also elevated among HIV-infected women, though to a lesser degree. All three malignancies are caused by oncogenic viruses, namely human herpesvirus type 8 (HHV-8) for KS, EBV for the two most common AIDS-associated NHL subtypes, and HPV for cervical cancer. The effect of HIV is probably through immunosuppression, allowing other viruses to escape control and thereby increase their viral load (Parkin, 2006). Elevated risk is also observed among solid organ transplant recipients, another immunosuppressed population (Grulich et al., 2007). Another mechanism that viruses can use to induce cancer is “hit-and-run,” wherein the virus will contribute to one specific step in the carcinogenic process, after which it is no longer needed for further tumor development and disappears from the lesion. Additionally, tumor cells lacking viral sequences will experience a positive selection due to a decreased immunogenicity, resulting in a growth advantage. In those cases of transient infection, a causal link between the virus and the cancer is much more difficult to establish. Simian virus 40 (SV40), a monkey polyomavirus introduced in the human population by contaminated polio vaccines, has been proposed to induce cancer using a hit-and-run mechanism. The large T antigen (Tag) induces chromosomal aberrations,


leading to an accumulation of genetic alterations, rendering the presence of viral transforming functions unnecessary. SV40-uninfected cells may subsequently have a proliferative advantage and become the prevalent population in the tumor. However, the current evidence is inadequate to accept or reject a causal relationship between SV40-containing polio vaccines and cancer (Barbanti-Brodano et al., 2004). The intact immune system is capable of recognizing and eliminating primary tumors, with an important role for lymphocytes and the cytokines they produce. Tumors are imprinted by the immunological environment in which they form, resulting in the generation of tumors that are better able to withstand the tumor-suppressing action of the immune system by eliminating tumor cells of intrinsically high immunogenicity but leaving behind tumor variants of reduced immunogenicity that have a better chance of surviving in the immunocompetent host. The alterations that must occur during the immunologic sculpting of a developing tumor are probably facilitated by the inherent genetic instability of tumors. Some of the likely targets of the immunologic process that sculpts tumors are genes encoding tumor antigens, components of the major histocompatibility complex (MHC) pathways that process and present antigens, or components of the IFN-g receptor signaling pathway. It is likely that immunologic sculpting of tumors occurs continuously, but the major effects of this process are most prominent early when the tumor is perhaps histologically, but not clinically, detectable (Dunn et al., 2002).

Human Papillomavirus Life Cycle and Pathogenesis

Human papillomaviruses (HPV) will be used a model to illustrate how a virus can induce cancer. Specific high-risk subtypes of this virus, predominantly types 16 and 18, have been associated with cervical cancer, as well as other anogenital and a proportion of head-and-neck cancers. HPV is responsible for over 200,000 cancer deaths each year worldwide, and the high-risk types represent one of the most important causes of human cancer after tobacco smoking (Kalland et al., 2009). HPVs are characterized by a small, nonenveloped, icosahedral capsid. Their genome is a circular double-stranded DNA molecule of 7,500 to 8,000 base pairs, with 6 early genes involved in replication and regulation of transcription (E1, E2) or with transforming capabilities (E5, E6, E7), and 2 late genes encoding the capsid proteins (L1, L2). Their genomes are replicated in the nuclei of host cells using host cellular machinery (zur Hausen, 2002). The virus replication cycle itself is an immune evasion mechanism that avoids the host defense system. During acute virus infection, replication of the virus genome is strictly linked to the state of differentiation of the infected cell. The virus initially infects the basal keratinocytes. The early genes are then expressed in the undifferentiated basal and suprabasal layers. Viral DNA is replicated in the differentiating spinous and granular layers and expression of the late structural proteins is limited to the terminally differentiated cells of the squamous layer, where the new virus particles are encapsidated and released into the environment as the cells shed (Stanley, 2008). During the initial phase of infection, HPV is present as an episome, but in the majority of anogenital cancers, HPV has integrated into the host genome. This results in the inactivation of the E2 open reading frame and a loss of its repressor function for E6 and E7 transcription, which allows for an accumulation of genetic changes. The molecular pathogenesis of cancer caused by the high-risk HPV types is not fully understood and, although they are self-sufficient to induce carcinogenesis, the infection itself is not able to induce the malignant transformation of infected cells (Kanodia et al., 2007). The

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development of cervical cancer and the invasive phenotype is a multistep process, including persistent HPV infection of the metaplastic cervical epithelium, clonal progression of the infected epithelium to cervical precancer, or further invasion (Sheu et al., 2007).

The Immune Response After HPV-Infection

As outlined in the section above, the HPV replicative cycle is strictly tied to the keratinocyte differentiation program. Several factors minimize or prevent exposure of the virus to the immune system. First, HPV replicates its DNA in undifferentiated cells and forms new progeny only in external differentiated epithelial cells, making it hard for the immune system to capture the highly immunogenic viral particles and mount an effective immune response. Early proteins are produced in insufficient quantities and/or are not accessible for immune recognition (Stanley, 2008). Second, the life cycle of HPV is nonlytic and therefore does not elicit any proinflammatory signals that activate Langerhans cells (LCs) to present virionderived antigens to the immune system and induce migration of effector cells into the local environment (Tindle, 2002). Third, there is no blood-borne phase of the HPV life cycle, so the immune system outside the epithelium has little opportunity to detect the virus (Kanodia et al., 2007). Fourth, papillomavirus late genes contain codons that mammalian cells rarely use, resulting in the inhibition of abundant papillomavirus capsid proteins in mammalian basal-epithelial cells by the restricted availability of the appropriate tRNAs (Tindle, 2002). Thus, HPV controls the expression levels of its gene products and levels of expression of capsid proteins are minimized, and/or expression of these proteins to differentiated epithelium is delayed (Kanodia et al., 2007). Further mechanisms of immune escape include molecular mimicry of HPV16 E7, which has a high and widespread similarity to several human proteins, which might be one cause of limited immunogenicity of E7 (Tindle, 2002). T-cell-mediated immune responses against specific E6 and E7 peptides of oncogenic HPVs are believed to play a central role in cervical carcinogenesis, although the nature of the tumor associated and/or specific antigens recognized by T cells on autologous cervical carcinoma cells are still unknown. Differential Th cell responses to HPV16 E7 correlate significantly with either viral clearance and regression of disease or with persistence in patients with cervical neoplasia. Failure to mount an effective immune response is related to the inefficient activation and priming of the innate and adaptive immune response of the host, which facilitates viral persistence. MHC class I expression and antigen presentation is down regulated in HPV-associated tumors, which may result in the induction of HPV-specific T-cell anergy (Kanodia et al., 2007). Furthermore, cervical cancer patients often have an impaired CD41 T-cell response against the early antigens E2 and E6 (de Jong et al., 2004). Additionally, these patients have an increased number of regulatory T cells, which might assist in the failure of the immune system to control the development of HPV-induced cancer (Visser et al., 2007). Since HLA molecules are responsible for the presentation of foreign antigens to the immune system, they play a central role in the immune recognition and subsequent clearing of virally infected cells. Therefore, there is a strong a priori biological plausibility supporting a role of HLA antigens in the development of HPV-related cervical cancer. Some HLA class II genes, important in the regulation of the immune response to viral and other infections, have consistently been found to be protective against cervical cancer (Hildesheim & Wang, 2002). However, no consistent association has been found between HLA class II alleles and an increased risk of disease.

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The host milieu in infected cervical epithelium provides a tolerogenic state for HPV antigens and permits progression of high-grade disease to frank malignancy (Patel & Chiplunkar, 2009). Cervical cancer typically originates in the transformation zone (TZ) at the squamous/columnar junction. The development of squamous intraepithelial lesions (SIL) and, subsequently, cervical cancer is preferentially associated with a local type II (interleukin 4/6 [IL-4/ IL-6]) and/or immunosuppressive (IL-10) cytokine pattern, not a cell-mediated type I immune response, which is more appropriate for tumor immunity. The density of LCs in the TZ is reduced and TZ-derived LCs show lower levels of proliferation and IL-2 production and higher levels of the immunosuppressive cytokine IL-10 in the presence of allogeneic PMBCs in comparison to the exocervix. The immunosurveillance within the epithelium of the TZ may be intrinsically perturbed due to the altered expression of chemokines and cytokines and the concomitant reduced density of LCs. Since tumors undergoing apoptosis, as opposed to necrosis, are capable of rendering dendritic cells (DCs) tolerogenic, the progressive increase in apoptosis as SIL progresses to cancer may contribute to the tolerization of the immune response (Giannini et al., 2002). An important role in immune evasion by HPV can be attributed to an impaired function of LCs. LCs are able to bind and internalize HPV VLPs in a manner similar to DCs; however, in contrast to DCs, they do not become activated. LCs are unable to initiate an E7-specific T-cell response and LCs are proposed to be unable to initiate a CD81 response to not only HPV VLPs, but also to intact HPV. The development of SIL is associated with a local Th2 cytokine profile, whereas there is an absence of a cell-mediated immune response, or Th1 response, which is preferred for tumor immunity (Fausch et al., 2002). A prominent effect of the immune system on HPVinduced disease is observed in people with the rare condition, epidermodysplasia verruciformis (EV). EV is an autosomal recessive genodermatosis and is characterized by persistent, refractory, disseminated skin lesions resembling flat warts, or presenting as maculae of various colors. EV begins in infancy or childhood and is associated with a high risk of skin cancer, predominantly associated with HPV-5 and HPV-8 and other related beta-papillomavirus types (Orth, 2006). Patients have a mild but demonstrable defect of cell-mediated immune responses. It initially seemed as if the HPV types found in EV were EV-specific types that contributed to the development of skin cancer. However, as techniques progressed, these and related HPV types were also detected in patients with profound immunosuppression following organ transplantation and even in the majority of immunocompetent individuals, not only in SCCs but also in normal skin and plucked hairs (Sterling, 2005). However, the virus is transcriptionally active and the viral DNA is more readily detectable in EV patients compared to organ transplant patients and immunocompetent individuals (Berkhout et al., 2000). EV can thus be considered a primary defect of innate immunity resulting in an abnormal susceptibility to infection by specific HPV types (Orth, 2006). These findings clearly show that a well-functioning immune system is an important component in reducing the effects of HPV infection on cell biology.

Other Viral Infections Associated with Human Malignancies Epstein Barr Virus (EBV)

EBV is a highly prevalent virus worldwide, with 95% seroconversion by young adulthood in most countries. Most childhood infections are clinically silent, but postadolescent EBV

infection frequently results in acute mononucleosis. During primary infection, a polyclonal expansion of B cells occurs, resulting in EBV-specific cellular and humoral immune responses. Latency is established in a subset of B cells, which can be interrupted under certain conditions to produce reactivation of infection (Persing & Prendergast, 1999). In immunocompromised, genetically predisposed, or environmentally loaded individuals, infected cells increase in number and B-cell growth control pathways are activated, inducing transformation and leading to malignancies such as Burkitt lymphomas, nasopharyngeal carcinomas, posttransplant lymphomas, and gastric carcinomas (de Martel & Franceschi, 2009). EBV-associated malignancies follow distinct geographical distributions and occur at particularly high frequency in certain racial groups, indicating that host genetic factors may influence disease risk. In the United States, the most common EBV-associated tumor is non-Hodgkin lymphoma, associated with immunosuppression, typically in the context of HIV-infection or in transplant recipients. Immune evasion strategies of EBV include nonlytic infection, restricted protein expression, absence or nearabsence of cell-adhesion molecules, and down regulation of MHC class I expression (Tindle, 2002).

Hepatitis B Virus (HBV)

It is estimated that over 400 million people worldwide are chronic carriers of HBV. The vast majority of infections are asymptomatic, and the noncytopathic HBV does not cause a significant immune response after the initial infection, presumably at least in part because HBV entry and expansion do not induce the expression of any cellular genes. Nonetheless, HBV is a major etiological factor in the development of hepatocellular carcinoma (HCC), as 5% to 10% of infected adults and most infants born to infected carrier mothers will develop chronic active hepatitis which can, in turn, lead to cirrhosis, liver failure, or HCC (Persing & Prendergast, 1999). This development is accelerated by the exposure to environmental carcinogens including aflatoxin B, cigarette smoke, and alcohol (Parkin, 2006). HBV-associated HCC is a combination of HBV encoded oncogenic activities along with the synergistic effects of chronic inflammation. Liver injury mediated by persistent weak cytotoxic T lymphocyte responses against HBV, and the resulting regenerative responses, create a mitogenic and mutagenic environment for DNA damage that leads to HCC (Tindle, 2002).

Hepatitis C Virus (HCV)

HCV infects approximately 2% of the population worldwide, although the prevalence of HCV varies by geographical location. Persistent HCV infection is associated with hepatitis, hepatic steatosis, cirrhosis, and hepatocellular carcinoma (HCC). In the vast majority of infected individuals, HCV establishes a persistent and life-long infection via highly effective viral immune evasion strategies. Some of the HCV proteins counteract the cellular antiviral response through a variety of mechanisms, and HCV is also very effective in subverting T-cell mediated adaptive immunity. Infection with HCV causes active inflammation and fibrosis, which can progress to cirrhosis and ultimately lead to tumor development. Duration of infection and the degree of persistent liver injury appear to be major determinants of hepatocellular cancer development. Host immunogenics or other factors may play a role in susceptibility to viral infection, as well as in determining the severity of infection (Persing & Prendergast, 1999). Numerous cofactors of the development of HCV-associated HCC exist, including coinfection with HBV, excessive alcohol consumption, and host factors. HCV causes genome instability, suggesting that certain HCV proteins may have a mutator function (de Martel & Franceschi, 2009).


Kaposi’s Sarcoma Herpesvirus (KSHV)/Human Herpesvirus 8 (HHV-8)

Kaposi’s sarcoma (KS) is an angioproliferative disease and is one of the three AIDS-defining malignancies, together with non-Hodgkin lymphoma and cervical cancer. Before the onset of the HIV-epidemic, KS was only rarely observed, and most cases can be attributed to the immunosuppressive effects of HIV. Human herpesvirus type 8, more commonly known as Kaposi’s sarcoma herpesvirus (KSHV), is recognized as a necessary but not sufficient factor for the development of KS (Javier & Butel, 2008). It establishes life-long latency in B cells, and its neoplastic potential, especially in immunocompromised individuals, is well-established. Several immune evasion genes are expressed, resulting in MHC class I down regulation, inhibition of the host IFN response and inhibition of complement-mediated lysis of infected cells. Together, these immune evasion strategies ensure lifelong viral persistence in the host, and subsequently contribute to KSHV-associated pathogenesis (McLaughlin-Drubin & Munger, 2008).

Human T-Lymphotropic Virus (HTLV)

HTLV type 1 is a retrovirus infecting millions of people worldwide and is associated with various neoplastic manifestations, such as adult T-cell leukemia (ATL). Malignant transformation is most commonly seen in Japan, the Caribbean, and South America (Javier & Butel, 2008). The long clinical latency, together with the relatively low cumulative lifetime risk of a carrier developing ATL, indicates that HTLV-1 infection is not sufficient to elicit T-cell transformation. While the exact cellular events remain unclear, a variety of steps, including virus, host cell, and immune factors, are implicated in the leukemogenesis of ATL. The multifunctional viral accessory protein Tax is supposed to be the major transforming protein of HTLV-1 because of its deregulatory effects in multiple cellular transcriptional signaling pathways (McLaughlinDrubin & Munger, 2008).

Polyomaviruses

For a very long time, polyomaviruses (PyV) have been proposed to have a causal link with cancer, because under experimental conditions, cells which are nonpermissive for viral replication can be transformed by PyVs. However, their ubiquitous nature, a long exposure time, and a very low rate of cancer development after natural infection have always made it difficult to establish a causal link between these viruses and human cancer. The oncogenic potential of PyVs is mediated by the large and small T antigens, which bind to pRB, p53, and other tumor suppressor proteins (Lee & Langhoff, 2006). PyVs have been associated with human disease, yet a consistent and causal link with cancer for three of them (BKV, JCV, and SV40) has not been established (zur Hausen, 2008). Recently, a new PyV has been shown to be monoclonally integrated in 80% of Merkel cell (MC) carcinomas, a rare but aggressive human skin cancer that typically affects elderly and immunosuppressed individuals, a feature suggestive of an infectious origin (Feng et al., 2008). Although the presence of MCPyV DNA in Merkel tumors does not clearly prove a causal involvement, these tumors seem to represent the first human malignancy with a relatively consistent presence of integrated sequences of a specific type of polyomavirus (zur Hausen, 2008).

Future Perspectives

Over the past decades, more and more viruses have been causally linked to different forms of human and animal cancer. Today, one cancer case in five is caused by an infectious agent. With techniques developing rapidly, more associations between viruses and malignancies can be expected in the coming years. The research of virus-induced tumors has led

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to the discovery of many oncoproteins and has elucidated various aspects of cancer biology. Virus-induced tumors have the advantage that they can potentially be prevented by the use of prophylactic vaccines, which are already into use for HBV and more recently for HPV. Additionally, the presence of viral material in a tumor allows specific targeting of therapeutic agents against the transformed cells.

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41 The Role of Bacterial and Parasitic Infections in Chronic Inflammatory Disorders and Autoimmunity STEFAN EHLERS AnD GRAHAM A. W. ROOK

INTRODUCTION

good evidence that diminished exposure to microorganisms that drive immunoregulatory pathways is one important factor (Rook, 2009). Many types of organisms appear to be involved, and their nature and the mechanisms they activate to prevent or mitigate CIDs are discussed in this chapter.

The incidence of chronic inflammatory disorders (CIDs) is rising in wealthy, developed countries. CIDs encompass classical autoimmune diseases, in which the immune system recognizes and attacks host tissue (e.g., multiple sclerosis, ankylosing spondylitis), atopic and allergic diseases (e.g., eczema, asthma), as well as other chronic inflammatory syndromes (e.g., psoriasis, sarcoidosis, Crohn’s disease). In addition to genetic factors, environmental triggers play a major role in the development of CIDs. It is therefore perhaps no surprise that many CIDs predominantly become manifest in barrier organs such as the skin, lung, and gut. Here, infectious agents may initiate, exacerbate, or regulate allergic and autoimmune responses. While this tenet has been substantiated for a number of viral infections (see chapter 40), evidence is relatively sparse that bacterial and parasitic infections actually cause, trigger, or enhance CIDs. Ascribing infectious causation to CIDs may be particularly difficult when there is little if any acute pathology near the onset of infection, when infection and actual disease manifestation are separated in time, or when more than one infectious agent is involved in driving CIDs (Cochran et al., 2000). In addition, it has become increasingly complicated to obtain suitable, long-lived animal models that will mimic long-delayed chronic disease after infection. Only rarely, then, has the pathophysiology of a particular CID been attributed to a specific molecular target structure of an infectious agent (see this chapter); more often, nonspecific, bystander activation is thought to cause, or exacerbate preexisting, disease, the microbial trigger being linked only in a circumstantial way. On the other hand, failure to terminate redundant immune responses can certainly contribute to CID. There is

INFECTIONS AS PROMOTERS OF AUTOIMMUNE DISORDERS Mechanisms of Microbe-Induced Inflammation

Microbe-associated molecular patterns (MAMPs) on the surface of microorganisms are recognized by pattern recognition receptors expressed by immune cells. For example, Toll-like receptors (TLRs), located on the surface of host cells (TLR1, -2, -4, -5, -6) or in intracellular compartments (TLR3, -7, -8, -9), recognize lipopeptides and lipoproteins (TLR1, TLR2, TLR6), double-stranded viral RnA (TLR3), lipopolysaccharides (TLR4), flagellin (TLR5), single-stranded viral RnA (TLR7, TLR8), and CpG-motifs in DnA (TLR9). The intracellular nOD-like receptors recognize peptidoglycan fragments, and retinoic-acid-inducible-gene (RIG)-like helicases recognize viral RnA in the cytoplasm. Ligationinduced signaling events trigger inflammatory responses, such as cytokine and chemokine secretion, adhesion molecule expression, and the regulation of costimulatory receptors on the surface of immune cells. Inflammatory cells are recruited, microbicidal effector molecules are produced, and antigen presentation to T and B cells occurs. The presence of an infectious agent thus signals danger, which is translated into an inflammatory milieu highly conducive to pathogen elimination and effector and memory cell differentiation (Fig. 1) (Kumar et al., 2009). This microenvironment, however, may also facilitate aberrant immune responses to selfantigen. Aberrant helper T-cell activation has been implicated in the pathogenesis of many autoimmune diseases. Generally, T helper 1 (Th1) responses are required for macrophage activation to eliminate intracellular pathogens, while T helper 2 (Th2) responses drive B-cell antibody production for neutralizing and eliminating extracellular microbes. The balance of effector T-cell responses is key for maintaining health and ensures the absence of excessive inflammation, which drives

Stefan Ehlers, Microbial Inflammation Research, Research Center Borstel, Parkallee 1, D-23845 Borstel, Germany, and Molecular Inflammation Medicine, Institute of Experimental Medicine, ChristianAlbrechts-University, Arnold-Heller-Str. 3, D-24105 Kiel, Germany. Graham A. W. Rook, Centre for International and Medical Microbiology (CIMM), Windeyer Institute for Medical Sciences, University College London (UCL), 46 Cleveland Street, GB - London W1T 4JF, United Kingdom.

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FIGURE 1 This simplified scheme shows possible immune responses to microbial challenge. They either contribute to differentiation of antibacterial effector mechanisms (top) or may trigger or exacerbate chronic inflammatory disorders by molecular mimicry or bystander activation (CID) (bottom).

autoimmunity (Fig. 1). In experimental models of multiple sclerosis (allergic encephalomyelitis) or autoimmune arthritis, Th17 responses, characterized by T-cell production of IL(interleukin)-17, IL-21, and IL-22, are critical elements driving prolonged cellular infiltration and tissue erosion (Hofstetter et al., 2009; Peck & Mellins, 2009). For a while, Th17 responses appeared to be exclusively detrimental to the host, indicating a clearly defined target for immunotherapy. However, similar to other arms of adaptive immunity, Th17 are just another two-edged sword, their deleterious capacity being offset by the antimicrobial protection they afford in some, but not all, infections (Cooper, 2009). There are several mechanisms by which microbial encounter may lead to CID (Fig. 1). First, the pathogen itself may persist and chronically trigger an inflammatory response. In this case, CID would amount to a futile effort at eradicating the infectious agent, but the etiology and pathophysiology would, in essence, not differ from other chronic infectious diseases (e.g., latent and reactivating tuberculosis). Second, the infectious agent may exhibit structural elements that are similar enough in three-dimensional conformation to selfantigen that the pathogen acts as a self-mimic. T or B cells that are activated in response to microbial encounters would then be cross-reactive to self and lead to direct tissue damage (so-called molecular mimicry). Third, a persisting pathogen or the immune response against it may cause cell or tissue damage; the antigens released from damaged tissue (“hidden epitopes”) would then serve to initiate a self-specific immune

response (“epitope spreading”). Finally, naturally occurring autoimmune T or B cells may get activated in a nonspecific way by the inflammatory milieu created by microbial structures, such as TLR ligands (“bystander activation”). In some cases, therefore, infections may merely prepare a fertile field for antigen-driven autoimmune processes (von Herrath et al., 2003). The above-mentioned scenarios may sometimes occur in a sequential way and are not mutually exclusive. The following section discusses representative examples of CIDs promoted by selected bacterial or parasitic infections to illustrate some of the relevant molecular and immunological pathways involved.

Persistent Infection

Until only 30 years ago, stomach ulcers were thought to be a chronic inflammatory ailment due to psychosocial stress, dietary mistakes, and possibly autoimmune responses. With the discovery that Helicobacter pylori infection is a prerequisite for lesion formation, antibiotic therapy to eradicate H. pylori has been highly successful in reducing disease prevalence (Cover & Blaser, 2009). It should therefore always be taken into consideration that any chronic inflammatory disorder may still have a microbial agent at its origin (Cochran et al., 2000).

Crohn’s Disease and Mycobacterium paratuberculosis

Crohn’s disease (CD) is a chronic inflammatory disorder affecting the walls of the ileum and colon. Histopathological

41. The Role of Bacterial and Parasitic Infections in Chronic Inflammatory Disorders and Autoimmunity

hallmarks are epithelial ulceration, granuloma formation, submucosal fibrosis, and development of necrotizing fistulas. The incidence of CD is rising steadily in industrialized countries (currently 6 to 7 per 100,000 people), and an environmental contribution to disease manifestation is likely, since the concordance rate in monozygotic twin studies does not exceed 50%. CD bears a strong resemblance to a granulomatous ileitis in cows with diarrhea and wasting (termed Johne’s disease), the etiological agent of which is Mycobacterium avium subspecies paratuberculosis. A number of publications have documented the presence of M. avium subspecies paratuberculosis in tissues of patients with CD, by culture and by molecular methods (reviewed in Mendoza et al., 2009; Packey & Sartor, 2009). In some instances, recovery of M. avium subspecies paratuberculosis has been as high as 46% in CD patients, 45% in those with ulcerative colitis, and in 20% without IBD (inflammatory bowel disease) (naser et al., 2004). Although this may just reflect how abundant M. avium subsp. paratuberculosis is in the environment, it is intriguing that variations in genes encoding intracellular antibacterial defenses (such as nOD2, an intracellular peptidoglycan receptor, and ATG15L1, a component of the autophagy machinery) are associated with susceptibility to CD and thus are supportive of an infectious etiology at least in a subset of patients. It bears mentioning that several reports have not been able to verify a strict association of the detectability of M. avium subsp. paratuberculosis with manifest CD (reviewed in Packey & Sartor, 2009). In addition, two recent studies found no association between the presence of M. avium subsp. paratuberculosis and nOD2 mutations (Bernstein et al., 2007; Parrish et al., 2009). M. avium subsp. paratuberculosis infection does occasionally occur in humans (Greenstein, 2003; Hermon-Taylor et al., 1998), but whether this truly progresses to Crohn’s disease is unproven. There has been no direct evidence for horizontal or vertical transmission. CD is less common in rural areas, although maximal exposure would be expected in farming environments. The beneficial clinical responses to anti-TnF (tumor necrosis factor) treatment in Crohn’s disease are at variance with CD being a chronic mycobacterial infection, because the latter often exacerbate under anti-inflammatory therapies. In CD patients, there are highly variable, often low-level serologic or cellular responses to M. avium subsp. paratuberculosis (nakase et al., 2006), although one recent report did detect Th1/Th17 phenotype cells with specificity against M. avium subsp. paratuberculosis antigens in CD patients (Olsen et al., 2009). In a study comprising 235 patients with CD, ulcerative colitis, irritable bowel syndrome (IBS), and no disease, the presence of M. avium subsp. paratuberculosis insertion sequence 900 DnA was correlated with cytokine secretion in intestinal biopsy samples. TnF levels were higher in CD patients with M. avium subsp. paratuberculosis, but there was no correlation between the presence of M. avium subsp. paratuberculosis and IL-2, IL-12, IL-10, and interferon gamma (IFn-g) secretion (Clancy et al., 2007). One unresolved question is whether CD is a paucibacillary M. avium subsp. paratuberculosis infection, making the fulfillment of Koch’s postulates very difficult, or whether M. avium subsp. paratuberculosis organisms might be present in larger numbers in blood vessels, lymph vessels, and lymph nodes, adipocytes in the mesentery, or the circumference of fistulas outside the bowel walls (Pierce, 2009). In biopsies taken from CD patients, a variety of bacterial DnA (other than M. avium subsp. paratuberculosis DnA) can be found (Ryan et al., 2004), reflecting disturbed host–flora interactions and general dysbiosis, which might, by itself, maintain the inflammatory response and cause sporadic

523

exacerbation. In addition, selective persistence of adherent/ invasive Escherichia coli strains has been documented in CD that may contribute to barrier disruption (Barnich & Darfeuille-Michaud, 2007). Macrolides, in combination with rifabutin, ethambutol, or clofazimine, are the most effective drugs for treatment of M. avium subsp. paratuberculosis infection. One meta-analysis suggested that antimycobacterial treatment might be effective in maintaining remission achieved by corticosteroid therapy (Borgaonkar et al., 2000). A large (213 patients), randomized, placebo-controlled trial demonstrated a short-term benefit at 16 weeks of treatment, but did not provide evidence for a sustained advantage of antibiotic combination therapy (Selby et al., 2007). The short-term improvement was interpreted to result from nonspecific, antibacterial effects on the bowel flora mitigating inflammatory sequelae. Thus, despite intense effort, the hypothesis that M. avium subsp. paratuberculosis is a cofactor in CD pathogenesis has neither been validated nor been entirely refuted.

Atherosclerosis and Chlamydia pneumoniae

The obligate intracellular bacterium Chlamydia pneumoniae is a respiratory pathogen that causes acute pneumonia, bronchitis, sinusitis, or pharyngitis. Reinfections during an individual’s lifetime are common, giving rise to a seroprevalence of up to 80% in adults. C. pneumoniae has been detected in atherosclerotic plaques, but not in healthy vascular tissue. Specifically, PCR detection, electron microscope techniques, and immunohistochemical staining of aortic lesions repeatedly demonstrated the presence of C. pneumoniae within atheromatous lesions (Kuo et al., 1993). Cultivation from atheromatous plaques provided definitive evidence that the lesions harbor viable bacteria (Maass et al., 1998). It is thought that following ingestion by alveolar macrophages, C. pneumoniae is transported to pulmonary lymphoid tissue from where it enters the blood stream and is transported within mononuclear cells to activated endothelia. There, infection is transmitted to endothelial cells and smooth muscle cells of the vascular wall, causing or exacerbating localized inflammation (Kern et al., 2009). Atherosclerosis is now generally classified as a chronic inflammatory disease. Macrophages, having engulfed oxidized low-density lipoprotein (foam cells), are at the core, causing endothelial dysfunction, proliferation of vascular cells, and vascular wall inflammation. When C. pneumoniae is transported into a nascent plaque, infected mononuclear cells will contribute to perpetuate the local inflammation by stimulating proatherosclerotic proteins (PAI-1, tissue factor, IL-6) in vascular cells (Dechend et al., 1999), which, among other effects, promotes intravasal coagulation. This contributes to the attraction, attachment, and transendothelial migration of monocytes and lymphocytes. Once C. pneumoniae has infected the smooth muscle cells of the vessel, IL-6, fibroblast growth factor, and chemokines are induced, further aggravating the vicious inflammatory cycle (Coombes & Mahony, 1999). Macrophages within the atheromatous plaque, infected with C. pneumoniae, enhance their expression of metalloproteinases, which may ultimately lead to destabilization and subsequent rupture of the plaque. In hyperlipidemic apolipoprotein E-deficient mice, repeated infection with C. pneumoniae significantly accelerated progression of preexisting atherosclerotic lesions (Blessing et al., 2000). Data from rabbit models also indicate that C. pneumoniae preferentially infects predamaged areas and mostly fuels atherosclerotic plaque progression rather than initiating it (Fong et al., 1997).

524


Although a case for C. pneumoniae infection as an accelerator can be inferred from these data, it should be kept in mind that bacterial DnA from many other species has been identified within atheromatous plaque. This reflected the microbiota of the individual’s bowel or buccal flora (including fungi) and may be a consequence of a physiological bacteremia (Ott et al., 2006); homing to atherosclerotic plaques would thus be a reflection of the fact that activated macrophages, having engulfed bacteria, migrate to sites of inflammation by virtue of the adhesion molecules and chemokine receptors they express. C. pneumoniae would then be found most often in the plaques because it represents a fairly common and recurring infectious agent. Interestingly, Helicobacter pylori also ranks highly as a trigger factor for atherosclerotic plaque formation. Here, a persistent T cell and antibody response against a particular region in the H. pylori heat shock protein 60 (HSP60) sequence is considered to drive progression of atherosclerosis because stressed cells of the vascular endothelium, expressing endogenous HSP60, come under attack due to the misdirected T-cell response (Okada et al., 2007). Experimentally, H. pylori infection can also induce atherosclerosis in a hyperlipidemic mouse model (Ayada et al., 2009). Trials in patients with coronary artery disease with antibiotics effective against C. pneumoniae to prevent cardiovascular events have not uniformly demonstrated a beneficial effect (reviewed in Mussa et al., 2006). One study in patients with unstable angina showed that roxithromycin treatment significantly reduced the incidence of recurrent angina, myocardial infarction, and early death. no treatment benefit for secondary prevention of coronary heart disease was seen in two smaller trials with azithromycin. In two large randomized, multicenter prospective trials, patients with either stable coronary artery disease or acute coronary syndrome received azithromycin or fluoroquinolone, respectively, for at least 1 year. no significant risk reduction with regard to death due to coronary artery disease, myocardial infarction, unstable angina requiring rehospitalization, coronary revascularization, or stroke was found. It has been pointed out, however, that IFn-g stimulates C. pneumoniae to enter the state of nonreplicating persistence (at least in monocytes), thereby rendering it refractory to treatment with currently available antibiotics. Future trials will therefore need to use drugs that specifically target pathways involved in Chlamydia persistence. Meanwhile, an alternative view holds that atherosclerosis, rather than being a consequence of any specific infectious insult, might rather represent an immunoregulatory disorder whereby a constant low-grade inflammatory response against invading microorganisms derived from the skin, mouth, or gut is not sufficiently controlled (reviewed in Rook & Stanford, 1998) (see below).

Asthma and Chlamydia pneumoniae

As in the case of atherosclerosis, there is a debate as to whether the association between C. pneumoniae and asthma is causal or coincidental. There is a significant relationship between the markers of asthma severity, including lung function and symptom scores, and elevated antibody levels to C. pneumoniae, but not to other common respiratory agents. C. pneumoniae was found to cause stasis of ciliary movement in bronchial epithelial cells, which may specifically contribute to disease severity. Due to its capacity to induce cytokine synthesis, C. pneumoniae may amplify inflammation and exacerbate asthma symptoms (von Hertzen, 2002).

Sarcoidosis and Mycobacterial Catalase-Peroxidase

Sarcoidosis is a systemic disease characterized by noncaseating epithelioid granulomatous inflammation with pulmonary

involvement in over 90% of patients. The disease may present as an acute inflammatory syndrome with a high remission rate, or as an unremitting multiorgan granulomatous inflammation associated with progressive fibrosis and organ failure. Persistent mycobacterial infections have repeatedly been implicated in the pathogenesis of disease because some studies identified mycobacterial 16SrRnA in disease lesions. However, these reports have not been confirmed by other, more recent studies with a more sophisticated methodology. In a limited proteomics approach, tissue antigens were identified that reacted with immunoglobulin fragments from the sera of sarcoidosis patients in 9 out of 12 sarcoidosis tissues, but only 3 out of 22 control tissues (Song et al., 2005). One of these tissue antigens was confirmed by MALDI-TOF (matrix-assisted laser desorption ionization–time of flight [mass spectrometry]) as Mycobacterium tuberculosis catalase-peroxidase (mKatG). Material reactive with mKatG-specific antibodies was specifically detected in sarcoid lesions, but not in tissues from Wegener’s granulomatosis. T-cell responses to mKatG were prominent in two clinically distinct sarcoidosis patient cohorts from Sweden and the United States, with higher frequencies of mKatGreactive, IFn-g-expressing T cells in the blood of sarcoidosis patients compared with nontuberculosis-sensitized healthy controls (Chen et al., 2008). mKatG-reactive CD4 Th1 cells preferentially accumulated in the lung in sarcoidosis patients. It remains unclear which mycobacterial species is the initial infecting agent and why remnant mycobacterial antigens, such as mKatG, persist in the lung and drive inflammatory responses.

Persistent Response to Infection: Molecular Mimicry by Microbial Structures (Table 1) Acute Rheumatic Fever and Streptococcus pyogenes

Infections with group A streptococci (GAS) (Streptococcus pyogenes) cause exudative tonsillitis and a sore throat, and in approximately 1.2% of children, it may be followed by acute rheumatic fever (ARF) with inflammation of the heart. Myosin has been identified as the dominant autoantigen in the heart, and myosin-reactive antibodies derived from patients with ARF are cross-reactive to both M protein (a major virulence factor of GAS) and the carbohydrate epitope N-acetyl-beta-d-glucosamine (Glcnac). Patients may also develop cross-reactive antibodies to other heart proteins such as tropomyosin, laminin, and vimentin (reviewed in Cunningham, 2008). In addition, T cells recovered from heart lesions of ARF patients were shown to recognize streptococcal M protein and heart tissue-derived proteins. Binding of cross-reactive antibodies increases the expression of vascular cell adhesion molecule 1 that reacts with very late antigen 4 on infiltrating T cells. Together with complement activation, this further increases tissue migration of mononuclear cells and enhances local inflammation, causing heart valve destruction. Mice, rats, and rabbits immunized with M protein or bacterial components all developed cardiac lesions (Cromartie & Craddock, 1966; Murphy & Swift, 1950), contributing to the evidence that mimicry is a major mechanism of pathology in human rheumatic heart disease. Infections with GAS have also been associated with the development of movement and behavioral disorders such as Sydenham’s chorea, Tourette’s syndrome, and obsessivecompulsive disorder (Kurlan, 1998). Molecular mimicry between basal ganglia and GAS-derived proteins is the major postulated mechanism of disease induction. Indeed, patients with these disorders often have antibodies to the basal ganglia of the brain, and rabbits immunized with

41. The Role of Bacterial and Parasitic Infections in Chronic Inflammatory Disorders and Autoimmunity TABLE 1

Examples of molecular mimicry driving autoimmune inflammation

Microorganisma

Microbial Antigen

Host antigen

Disease

References

OspA

LFA-1

Lyme arthritis

Flagellin

Myelin basic protein

neuroborreliosis

Campylobacter jejuni

Lipooligosaccharide

Gangliosides

Guillain-Barré syndrome

Mycoplasma pneumonia

Glycolipids

Galacto-cerebroside

Escherichia coli; other fimbriated bacteria

FimH

LAMP2

Pauci-immune focal necrotizing glomerulonephritis

Kain et al., 2008

Group A streptococci

N-acetylglucosamine

Myosin, tropomyosin, laminin, vimentin

Acute rheumatic fever

Reviewed in Cunningham, 2008

Haemophilus influenzae; Helicobacter pylori; Neisseria gonorrheae

TLRVYK-related structures

Beta-2glycoprotein-I

Antiphospholipid syndrome

Reviewed in Blank & Shoenfeld, 2004

Saccharomyces cerevisiae

Phosphopeptidomannan of cell wall

Borrelia burgdorferi

Reviewed in Bolz & Weis, 2004

Aspinall et al., 1994 Kusunoki et al., 2001

Helicobacter pylori

CagA protein

Platelet-associated IgG

Idiopathic thrombocytopenic purpura

Takahashi et al., 2004

Klebsiella pneumoniae

Capsular polysaccharide

Type I, II, IV collagen

Ankylosing spondylitis

Reviewed in Rashid & Ebringer, 2007

Novosphingobium aromaticivorans; Lactobacillus delbrueckii

Various proteins

PDC-E2 on bile ducts

Primary biliary cirrhosis

Selmi & Gershwin, 2009

Many urinary tract pathogens Trypanosoma cruzi

a

525

Bogdanos et al., 2009 B13 protein, Cha peptide FL160

Myosin

Chagas’ cardiomyopathy

Cunha-neto et al., 2006

48kDa nerve protein

Chagas’ megacolon

Cunha-neto et al. 1996 Van Voorhis and Eisen, 1989

This is not a comprehensive list. For further references, see also Blank et al., 2007 and Ercolini & Miller, 2009.

streptococcal M protein developed antibodies that were cross-reactive with several human brain proteins (Bronze & Dale, 1993). In CSF from Sydenham’s chorea patients, dual-specific antibodies were detected that react with both the immunodominant carbohydrate epitope on GAS cell walls (GlcnAc) and with lysoganglioside GM1 on the surface of neurons (Kirvan et al., 2003). In addition, signaling via GlcnAc-reactive antibodies from the sera of patients with pediatric autoimmune neuropsychiatric disorders associated with streptococci (PAnDAS) was inhibited by lysoganglioside GM1 (Kirvan et al., 2006). A subset of SJL mice primed with GAS homogenate developed movement and behavioral disorders, formed antibody deposits in their brains and had serum antibody reactive to several regions of the brain (Hoffman et al., 2004).

Chronic Chagas’ Cardiomyopathy and Trypanosoma cruzi

Chagas’ disease is caused by infection with the protozoan Trypanosoma cruzi. In its chronic phase, the disease manifests as irreversible cardiomyopathy. Since T. cruzi antigens and DnA can also be detected in infected people who remain

asymptomatic, the tissue destruction characteristic of chronic Chagas’ cardiomyopathy (CCC) is considered to be largely autoimmune in character (Cunha-neto et al., 2006). CCC is histopathologically characterized by mononuclear cell infiltrates, with more CD81 T cells than CD41 T cells present (Higuchi et al., 1987). Reports of local production of IFn-g, TnF-a, IL-4, and IL-6 suggest bystander inflammatory tissue destruction as a contributing event in CCC. PBMCs (peripheral blood mononuclear cells) from CCC patients showed cytotoxicity against noninfected cardiac myocytes and cytokine production against cardiac tissue homogenate (Mosca et al., 1985). Antibodies to the cardiac protein galectin-1 were found in both the sera and cardiac tissue of CCC patients; levels correlated with severity of cardiac damage. The T. cruzi protein B13 elicits cross-reactive responses to cardiac myosin in CD41 cells and B cells (Cunha-neto et al., 1996). Cross-reactive antibodies were present in 100% of CCC patients but only 14% of asymptomatic infected individuals. Mouse models support the contention that CCC is an autoimmune syndrome triggered by T. cruzi. Chronically infected susceptible mice develop CD41 T cells that

526


proliferate in response to cardiac myosin (Rizzo et al., 1989). A murine CD41 T-cell line, which was cross-reactive with both cardiac and T. cruzi-derived proteins, caused severe heart inflammation when transferred into infected or antigen-challenged nude mice (Ribeiro-Dos-Santos et al., 2001). T cells from T. cruzi-infected mice were reactive to both the SAPA antigen on T. cruzi and the homologous, newly identified Cha autoantigen (Girones et al., 2001). Transfer of these T cells into naïve mice produced anti-Cha autoantibodies and heart lesions. A subset of Chagas’ disease patients develop motor dysfunction of the gastrointestinal tract, possibly via destruction of neurons of the enteric nervous system. Antibodies raised in rabbits against a flagellum-associated surface protein on T. cruzi (FL-160) were cross-reactive with a 48-kDa protein found exclusively in nervous system tissue (Van Voorhis & Eisen, 1989).

Guillain-Barré Syndrome and Campylobacter jejuni

Guillain-Barré-Syndrome (GBS) is a paralytic illness that affects both myelin and the axons of the peripheral nervous system. Several microorganisms have been associated with GBS development. However, based on structure-biological comparisons, the outer membrane LPS (lipopolysaccharide) from Campylobacter jejuni most closely mimics host gangliosides (Aspinall et al., 1994). Priming of mice, rats, and rabbits with C. jejuni LPS produced corresponding antiganglioside antibodies (Goodyear et al., 1999). One report in rabbits immunized with C. jejuni LPS demonstrated flaccid limb weakness that was associated with antibodies to the ganglioside GM1 and peripheral nerve pathology identical to that seen in GBS (Yuki et al., 2004). Patients infected with Mycoplasma pneumoniae often develop antibodies to glycolipids which may cross-react with galactocerebroside (Kusunoki et al., 2001). Associated antibodies to GM1 have also been reported for H. influenzae infection (Mori et al., 2000).

Autoimmune Vasculitis Syndromes, Antineutrophil Cytoplasmic Antibodies, and Fimbriated Bacteria

Several severe inflammatory vasculitis syndromes are associated with autoantibodies to neutrophil cytoplasmic antigens (AnCA), among them Wegener’s granulomatosis, microscopic polyangiitis, or pauci-immune crescentic focal necrotizing glomerulonephritis (FnGn). In the latter, for example, injury is caused by neutrophils and macrophages that localize in glomerular capillaries without detectable immune deposits (Jennette & Falk, 1997). Experimentally, antibodies to myeloperoxidase and proteinase-3 activate primed neutrophils and cause neutrophil-dependent endothelial injury in vitro, and administration of antibodies to myeloperoxidase provokes FnGn in rodents. Recently, autoantibodies to lysosomal membrane protein-2 (LAMP-2) were identified as a new AnCA subtype that cause pauci-immune FnGn in rats (Kain et al., 2008). In addition, a monoclonal antibody to human LAMP-2 was found to induce apoptosis of human microvascular endothelium in vitro. The autoantibiodies in FnGn patients were highly specific for a LAMP-2 epitope that bears 100% homology to the bacterial adhesin FimH. It is noteworthy that infections with fimbriated pathogens, such as E. coli, were shown to occur commonly before the onset of FnGn. Rats immunized with FimH developed pauci-immune FnGn due to antibodies against LAMP-2, formally demonstrating that FimH may operate as a molecular mimic for LAMP-2.

Primary Biliary Cirrhosis and Diverse Bacterial Infections

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized by progressive destruction of intrahepatic bile ducts, resulting in chronic accumulation of bile, leading ultimately to fibrosis, cirrhosis, and liver failure (Selmi & Gershwin, 2009). PBC is characterized by the presence of serum antimitochondrial antibodies (AMA). An increased prevalence of urinary tract infections in patients with PBC, the presence of bacterial products in mononuclear cells surrounding damaged bile ducts, and the serum crossreactivity to a number of causative agents in mostly urinary tract infections have implicated E. coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Staphylococcus minnesota, Mycobacterium gordonae, Neisseria meningitidis, and H. pylori in the pathogenesis of PBC. In addition, a xenobiotic-metabolizing, ubiquitous aerobic gram-negative bacterium, Novosphingobium aromaticivorans, as well as Lactobacillus delbrueckii have been implied (Bogdanos et al., 2009; Selmi & Gershwin, 2009). All of these microorganisms elicit a specific antibody reaction to pyruvate dehydrogenase complex E2-like molecules (PDC-E2) present on bile duct cells (Padgett et al., 2005). Experimental immunization with N. aromaticivorans can induce autoreactive AMA and chronic PBC-like T-cell-mediated autoimmunity against small bile ducts in a murine model of PBC (Mattner et al., 2008). Since the liver is the main detoxifying organ, it is possible that modified PDC-E2-like structures locally elicit antibodies of the IgA type. The latter are transported to the vascular side of the bile duct cell where they react with PDC-E2, located on the luminal surface of the cell membrane. The tissue injury observed in PBC is thought to be the consequence of both the direct cytopathic effects of autoreactive T cells and the initiating of an apoptotic signaling cascade due to antibody complexes on the surface of the bile duct cell.

BACTERIA AND HELMINTHS AS INHIBITORS OF INFLAMMATION “Old Friends” (Table 2)

Diminished exposure to microorganisms that drive immunoregulatory pathways is one factor that contributes to the rising incidence of CIDs in wealthy, developed countries (Fig. 2) (Rook, 2009). Many types of organisms appear to be involved, but they can be considered under three overlapping headings. First, since all immune responses are terminated by a switch to immunoregulation, the overall burden of infection, even if transient and sporadic, might contribute to the overall development of regulatory circuits. Mechanisms are discussed later. Second, a number of clinical and epidemiological studies implicate diminished exposure to specific groups of microorganisms in the current increase in immunoregulatory disorders (Correale & Farez, 2007; Matricardi et al., 2000; Pelosi et al., 2005; Seiskari et al., 2007; Umetsu et al., 2005). The organisms highlighted are all thought to have been present since at least the paleolithic era, and probably present throughout mammalian evolution, and have been designated “old friends” (Rook, 2009). They tend to be relatively harmless, and to have reliable modes of transmission, such as the orofecal route. Several of them can persist in carrier states, or are so abundant in the environment, particularly in mud and untreated water, as to become “pseudocommensals,” that will have been consumed regularly and inevitably in milligram

41. The Role of Bacterial and Parasitic Infections in Chronic Inflammatory Disorders and Autoimmunity TABLE 2

527

Some of the bacteria and helminths shown to inhibit chronic inflammatory disorders in animals and humans

Organism

Host

Type of evidence

Disease

Reference

Bacteriaa Human

Clinical correlations

Allergic disorders

Mouse

Experimental

Asthma model

Mycobacterium vaccae

Mouse Dog

Experimental Clinical trial

Asthma model Eczema

Zuany-Amorim et al., 2002 Ricklin-Gutzwiller et al., 2007

Acinetobacter lwoffii; Lactococcus lactis

Mouse

Experimental

Asthma model

Debarry et al., 2007

Salmonella

Human

Epidemiology

Allergic disorders

Pelosi et al., 2005

Helicobacter pylori

Human

Epidemiology

Allergic disorders

Matricardi et al., 2000

Bacteroides fragilis

Mouse

Experimental

Models of inflammatory bowel disease

Mazmanian et al., 2008

Firmicutes and Bacteroidetes

Human

Clinical correlations


Frank et al., 2007

Mouse

Experimental model

Type 1 diabetes

Wen et al., 2008

Human

Reduced counts correlate with ileal CD Therapeutic in mouse colitis model

Crohn’s disease

Sokol et al., 2008

Human

Clinical studies


Packey & Sartor, 2009

Mouse

Many papers & experimental models

Allergies Allergies, models of inflammatory bowel disease, autoimmunity, etc.

Reviewed in Rook & Witt, 2008 Forsythe et al., 2007; O’Mahoney et al., 2008

Trichuris suis

Human

Clinical trials


Summers et al., 2005

Various helminths

Human

Clinical correlation

Multiple sclerosis

Correale & Farez, 2007

Heligmosomoides polygyrus

Mouse

Experimental

Asthma model

Wilson et al., 2005

Schistosoma mansoni

Mouse

Experimental

Anaphylaxis model

Mangan et al., 2004

Trichinella spiralis; Heligmosomoides polygyrus

Mouse

Experimental

Type 1 diabetes, nOD mouse

Saunders et al., 2007

Flarial nematode; Acanthocheilonema viteae

Mouse

Experimental

Asthma model & dextran sodium sulphateinduced colitis

Schnoeller et al., 2008

Mycobacterium tuberculosis (or BCG)

Faecalibacterium prausnitzii (a member of the phylum Firmicutes)

Mouse Various probiotics (especially Lactobacillus and Bifidobacter)

Reviewed in Rook & Witt, 2008

Sokol et al., 2008

Helminthsa

a

This is not a comprehensive list. They are a subset of the examples described in the main text.

quantities throughout our evolutionary history. The orofecally transmitted organisms highlighted in recent studies are particularly illuminating (Matricardi et al., 2000; Pelosi et al., 2005; Seiskari et al., 2007; Umetsu et al., 2005). They include H. pylori, Salmonella, hepatitis A virus (HAV), and Toxoplasma gondii. These organisms, and also the helminths that were universal throughout mammalian development, might have developed roles in driving immunoregulatory circuits as a consequence of their long evolutionary association with the mammalian immune system, and of the latter’s

need to tolerate organisms that are inevitably present and that cannot be eliminated (Rook, 2009). Third, the gut microbiota include a crucial (and partly overlapping) group of immunoregulatory organisms. The idea that modulating the intestinal microbiota can modulate chronic inflammatory disorders has a long history. In 1985, Kohashi and colleagues observed that the susceptibility of rats to adjuvant arthritis depended on the nature of the gut flora (Kohashi et al., 1985). It was also established 10 years ago that the intestinal microbiota are required for successful induction,

528


FIGURE 2 Epidemiological studies and laboratory screening for immunoregulatory properties have highlighted organisms that were present throughout the evolution of the mammalian immune system, but are relatively depleted from the modern environment. These include organisms that undergo orofecal transmission, ubiquitous environmental organisms (i.e., “pseudocommensals” in mud, untreated water, and fermented foods), helminths, and gut microbiota. In wealthy developed countries where exposure to these is diminished, CID may develop in individuals with genetic predisposition to dysregulation of Th1/Th17 or Th2 effector mechanisms. This is a classical gene– environment interaction.

by the oral route, of tolerance to ovalbumin (Sudo et al., 1997). Germ-free mice have poorly developed gut-associated lymphoid tissue (GALT), little IgA in the intestine, and striking defects in immunoregulation, leaving them susceptible to several types of gastrointestinal inflammation (Round & Mazmanian, 2009). The recent work of Kasper and colleagues has shown that immunoregulatory function can be restored to germ-free mice by administering a single polysaccharide from Bacteroides fragilis (Mazmanian et al., 2008). This is not a unique property of Bacteroides because similar findings have been reported using E. coli (Bouskra et al., 2008) or segmented filamentous bacteria (Talham et al., 1999). The dominant immunoregulatory role of the microbiota was confirmed in a study in nonobese diabetic (nOD) mice. These animals develop spontaneous autoimmune destruction of the b cells in the pancreas. When the gene encoding MyD88, a critical signal transducer of TLRs, was knocked out, this did not occur. Further experiments demonstrated that this was not due to direct impairment of the response to b cells, but rather an indirect effect via changes in the gut microbiota (Wen et al., 2008). Similarly, in addition to its known role in Th1-cell differentiation, the transcription factor Tbet controls the response of the mucosal immune system to commensal bacteria by regulating production of TnF by colonic dendritic cells (DCs). When Tbet was deficient, a colitis occurred that could be transferred to wild-type mice (Garrett et al., 2007). Indeed, there is increasing evidence that the bowel flora is involved in the pathogenesis of IBD. A recent metagenomic study compared the microbiota of patients with IBD with that of non-IBD controls and

revealed a statistically significant depletion of Firmicutes and Bacteroidetes, suggesting distortion of the microbiota (dysbiosis) (Frank et al., 2007). Similarly, antibiotics or fecal diversion (which limits access of fecal flora to the mucosa) help to relieve symptoms. Direct evidence of an immune response to the gut contents is provided by the observation that patients have raised levels of antibodies to gut commensals (Macpherson et al., 1996). An experimental model has shown that colitis can be induced by the presence of a single CD41 T-cell clone specific for a bacterial epitope (Kullberg et al., 2003). These points suggest that the microbiota provide the targets of an inappropriate immune response. Perhaps dysbiosis may lead to defective regulation of the response to commensals, particularly those “pathobionts” that have latent pathogenic potential (i.e., Helicobacter, Clostridium, and Enterococcus species). Interestingly, a species that is depleted in CD (Faecalibacterium prausnitzii) was therapeutically active when given orally in a mouse model (Sokol et al., 2008).

Changes in Exposure to Microorganisms that are Relevant to Immunoregulation

Many of the relevant changes are a consequence of the rapid shift from the hunter-gatherer environment, to which humans are adapted, to the modern concrete inner city. Loss of exposure to soil and animals seem particularly important (Rook, 2009). Changes in exposure can involve partial or complete elimination of the microorganism, or a change in the dose of the organism or its components, or a delayed infection. Elimination of most helminths, and of HAV,


is well established. A change in dose of microbial components can switch the lymphocyte types that are driven by their adjuvant effects. For instance, doses of LPS, dsRnA, or C. pneumoniae determine whether Th2 responses are increased or decreased (Schroder, 2009). Delayed infection can be crucial because it can occur after levels of antibody acquired from the mother have waned. For instance, HAV is harmless in small babies, but often fatal in those over the age of 50 (Harrison et al., 2009). The timing of virus infections is relevant to whether they enhance or inhibit type 1 diabetes (Filippi et al., 2009; Harrison et al., 2008). It is not known whether this is also true for bacterial infections. The gut microbiota can be modified by dietary changes, reduced exposure to the gut flora of others (hygiene), severe gastrointestinal infections, antibiotic use, or changes in the immune system, such as the findings with MyD88 and Tbet discussed above (Garrett et al., 2007; Wen et al., 2008). Obesity is increasing in wealthy countries due to inappropriate diet and lifestyle changes. Obesity is associated with a distinct pattern of changes in the gut microbiota (Turnbaugh et al., 2009), and in view of the regulatory effects of these organisms, this might contribute to the increased incidence of inflammatory disorders in obese individuals. Antibiotics have profound effects on the nature and diversity of the intestinal microbiota (Dethlefsen et al., 2008). In a model of food allergy, it was shown that TLR4-mediated recognition of the commensal flora inhibited allergic sensitization to orally administered allergen. However, the animals became susceptible to induction of food allergy if the composition of the bacterial flora was altered by antibiotic treatment beginning at 2 weeks of age, or if the TLR4 gene was disrupted (Bashir et al., 2004). As far as humans are concerned, there is epidemiological evidence that antibiotic use in young children can predispose to allergies (Farooqi & Hopkin, 1998), and allergic children have diminished numbers of colonizing lactobacilli in their intestinal microbiota (Bjorksten et al., 1999). A long-lasting imbalance in the microbiota can also follow a severe gastrointestinal infection, especially if this was treated with antibiotics. This induced dysbiosis can persist for many years and is associated with an increased incidence of irritable bowel syndrome (IBS) and perhaps Crohn’s disease (Marshall, 2009). Thus, changes and, in particular, reductions in microbial exposures are taking place in the developed countries as a result of changing diet and lifestyle, and increased use of antibiotics (Dethlefsen et al., 2008; Guarner et al., 2006).

Mechanisms Regulatory T Cells

Immune responses are terminated by a switch from effectors to regulatory cells. These include multiple cell types (innate Tregs, induced Tregs, TR1, Th3) that will not be discussed in detail here. Thus, in theory, any infection will expand Treg populations. Human and murine CD251 Tregs express TLR2, 4, 5, 7, and 8 (Caramalho et al., 2003; van Maren et al., 2008). Pretreatment of Tregs with endogenous hsp60 (TLR2), lipopolysaccharide (LPS) (TLR4), or flagellin (TLR5) has been reported to enhance their suppressive functions. PAM3Cys (TLR1/2) and single-stranded RnA (ssRnA) (TLR8) temporarily abrogate the suppressive capacity while the Treg population expands (Sutmuller et al., 2006; van Maren et al., 2008). Thus, when the TLR2 agonist disappears (indicating eradication of the pathogen) an expanded population is present that regains suppressive function, and contributes to termination of the response. These findings suggest that some regulatory cells can be at least partly activated by nonspecific pathways, such as TLR

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agonists and IL-2. Many other experiments suggest that initial activation requires contact with the epitopes for which their T-cell receptors are specific (Groux et al., 1997; Zuany-Amorim et al., 2002). However, once activated, in the continuing presence of the epitope, nonspecific bystander suppression can occur due to the release of nonspecific mediators such as IL-10, TGF-b, IL-35, perforin, granzyme B, and adenosine monophosphates (van Maren et al., 2008).

Nonspecific Effects of Increased Levels of Treg

Any increase in Treg numbers and activation might contribute to background anti-inflammatory activity. There is evidence that Tregs play a role in driving repair following nonspecific inflammatory stimuli such as transient ischaemia (Gandolfo et al., 2009) or endotoxin. This might be due to suppression of autoimmune responses to self-epitopes exposed by the inflammation, but it might be a nonspecific effect of increased background levels of anti-inflammatory mediators. This type of mechanism might explain why two different viruses (coxsackievirus B3 (CVB3) and lymphocytic choriomeningitis virus (LCMV) that trigger immunoregulatory mechanisms but no b cell damage, were able to protect prediabetic nOD mice from T1D. The viruses caused increased numbers of “invigorated” CD41CD251 Tregs that produced TGF-b (Filippi et al., 2009). In this experimental system a second mechanism was also identified. The viruses caused transient up regulation of PD-L1 on lymphoid cells, which prevented the expansion of diabetogenic CD81 T cells expressing PD-1. Full protection from T1D resulted from synergy between PD-L1 and CD41CD251 Tregs (Filippi et al., 2009). It would be surprising if other organisms, including bacteria, did not exert similar effects, though the importance of “invigorated” Tregs in nonviral contexts remains unknown.

Treg Adjuvants

As outlined earlier, the current reworking of the hygiene hypothesis suggests an additional role for the groups of microorganisms that have a very long evolutionary association with the mammalian immune system, and thus had to be tolerated. Many of these, including gut commensals (Bifidobacterium infantis) and saprophytic mycobacteria (M. vaccae) can be shown to induce Tregs in vivo (O’Mahony et al., 2008; Saunders et al., 2007; Wilson et al., 2005; Zuany-Amorim et al., 2002), or to cause DCs to mature into cells that preferentially drive Treg formation in vitro (Smits et al., 2005; van der Kleij et al., 2002). This mechanism is likely to explain the observation that patients with multiple sclerosis (MS) who picked up helminth infections (that were not treated) were found to develop circulating populations of T cells that released IL-10 or TGF-b in response to myelin peptides in vitro (Correale & Farez, 2007). This is not a bystander effect due to stimulation of Tregs that recognize the organism, but rather, an adjuvant effect, causing induction of Tregs specific for epitopes that are not present in the organism.

Balance of Th17 to Tregs in the Gut

Th17 cells are often implicated in autoimmune pathology. The expression of IL-17 in the gut depends on the nature of the gut microbiota. Genetically identical inbred mice from different suppliers had different ratios of expression of Th17 cells and Tregs. This turned out to be attributable to the presence of different microbiota, particularly the relative abundance of Bacteroidetes. The latter seem to drive Th17 cells, and the striking immunological differences between the mice waned when they were housed together or when microbiota were transferred (Ivanov et al., 2008).

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There are numerous mechanisms that monitor the microbiota. Dendritic cells (DCs) in the subepithelial dome (SED) of Peyer’s patches capture antigen by interacting with microfold (M) cells or by extending transepithelial projections into the lumen. Murine lamina propria CD11chi DCs can be separated into two subpopulations based on the expression of CD11b: CD11chiCD11b1 DC cells induce proinflammatory Th17 cells, while CD11chiCD11b2 DCs induce anti-inflammatory cells (Denning et al., 2007). The latter DCs also express the integrin alpha chain, CD103, which is involved in de novo conversion of Foxp3-CD41 cells to Foxp31 Treg cells (Coombes et al., 2007). Conversion of DCs to this tolerogenic phenotype is driven locally by TGF-b and retinoic acid (RA), some of which is released by epithelial cells (Iliev et al., 2009). Indeed, epithelialcell-conditioned DCs were capable of inducing Tregs that were protective when adoptively transferred to mice with experimental colitis (Iliev et al., 2009). Like the epithelial cells, these CD1031 DCs express aldh1a2, the gene encoding RALDH2. This enzyme is involved in conversion of dietary retinal to RA, which induces expression of gut-homing receptors (CCR9 and a4b7) and enhances development of FoxP31 T cells and IgA B cells. Importantly, CD1031 DCs with similar phenotype and functional properties were present in human MLn, though they have not yet been studied in detail (Jaensson et al., 2008). TLR9 is another relevant sensor of microbiota. TLR92/2 mice have more Foxp31 cells, and less Th17 and Th1 cells in gut-associated lymphoid tissue. TLR9 agonists such as CpG, or DnA extracted from gut bacteria inhibit Treg development. If microbiota are depleted by antibiotic treatment, development of Th1 and Th17 cells is reduced, but can be restored by administering DnA (Hall et al., 2008). Thus, the DnA of microbiota acts as an “adjuvant” for local homoeostatic inflammatory responses by limiting induction of Tregs. Interestingly, earlier work had suggested that TLR9 was essential for immunoregulatory effects of some probiotics. The inflammatory response in the lungs of allergic mice was diminished by gavage with living Lactobacillus reuteri before allergen-challenge, but this protective effect was not seen in TLR92/2 animals (Forsythe et al., 2007). TLR9 was also implicated in the protective effect of DnA extracted from probiotics in a model of dextran sodium sulphate (DSS)-induced colitis (Rachmilewitz et al., 2004). Recent work provides an explanation for this apparent paradox. Activation of DCs via TLR9 suppresses induction of Foxp3 expression in T cells, but enhances expression of IL-10 (Hall et al., 2008; Maynard et al., 2009). Therefore, in appropriate experimental models, TLR9 agonists might exert anti-inflammatory effects, but via IL-10 rather than via Foxp31 Tregs. Another critical factor might be secretion of ATP. This causes DCs to secrete IL-23, which maintains Th17 cells. Enteric nervous system function is also driven via ATP sensors (Atarashi et al., 2008). Some members of the microbiota may induce IL-25 in intestinal epithelial cells, and this down regulates IL-23, so secondarily, reducing maintenance of Th17 cells (Zaph et al., 2008).

Regulatory Macrophages

Regulatory microorganisms can also operate via macrophages. During helminth infections there is expansion of the population of alternatively activated macrophages, activated by Th2 rather than Th1 cytokines (nair et al., 2005). Such macrophages are able to inhibit lymphocyte proliferation in a contact-dependent manner (Loke et al., 2000), and

may be responsible for preventing inflammation in mucosal surfaces such as the lung. Recently, a new colon-infiltrating macrophage population induced by Schistosoma infection was shown to prevent colitis in mice (Smith et al., 2007). An increase of CD11b1CD11c2 macrophages in the lamina propria of infected mice was associated with limited intestinal inflammation in DSS-induced colitis and transfer of these mononuclear cells also inhibited inflammation in noninfected recipients. This effect was independent of T cells and Tregs. Similarly, prevention of allergic sensitization and airway hyperresponsiveness by filarial cystatin, a secreted protease inhibitor from filarial nematodes, was dependent on macrophages and also on IL-10 (Schnoeller et al., 2008).

Regulatory B Cells

Helminths also induce regulatory B cells. In the mouse, there is a B-cell subset in the GALT that expresses CD1b (Mizoguchi et al., 2002). These B cells exert anti-inflammatory effects via release of IL-10. These IL-10-producing CD1dhiCD51 regulatory B cells, which can also suppress EAE in the mouse model (Matsushita et al., 2008), have recently been designated B10 cells (Yanaba et al., 2008). S. mansoni infection prevented anaphylaxis in a mouse model, and this suppression of the effector phase of the allergic response was mediated by IL-10-secreting B cells (Mangan et al., 2004). Evidence for the existence of regulatory B cells in humans is less strong. However, depletion of human B cells using rituximab can occasionally exacerbate ulcerative colitis and trigger psoriasis, both of which are Th1-mediated conditions, suggesting that rituximab removed a regulatory factor (Yanaba et al., 2008). Moreover, there are IL-101 B cells in humans, and B cells from MS patients are relatively deficient in their capacity to produce IL-10 (Duddy et al., 2007). Again, a link with immunomodulatory microorganisms can be shown, because this defect was corrected in MS patients developing intestinal helminth infections, but not in patients infected by T. cruzi (Correale & Farez, 2007).

Th1/Th2 Balance

It was thought that lack of infections driving Th1 was leading to overproduction of Th2 cells. This was never a strong theory. First, Th1 cytokines such as IFn-g are present in large quantities both in asthma (Krug et al., 1996) and in established atopic dermatitis (Klunker et al., 2003). Secondly, profound defects in the IL-12 or IFn-g (Th1) pathways do not lead to an increased incidence or severity of allergic disorders, implying that, in humans, type 1 cytokines are not crucial regulators of Th2 responses (Lammas et al., 2000). Thirdly, superimposing polarized Th1 cells onto a Th2mediated inflammatory site can lead to synergistic inflammation rather than to down regulation of immunopathology (Hansen et al., 1999). Other examples of synergy between Th1 and IL-4 responses have been reported (Lawrence et al., 1998; Van Kampen et al., 2005). It may be that when IL-4 is associated with immunosuppression it is coming from a subset of Tregs that simultaneously release other regulatory mediators (Tiemessen et al., 2007). In reality, the Th1/Th2 balance hypothesis has been untenable since as early as 1998 (Rook & Stanford, 1998) by which time it had been well-documented that there was a simultaneous increase in Th1-mediated (or perhaps Th17-mediated) chronic inflammatory diseases (e.g., type 1 diabetes, MS, IBD) (Bach, 2002), occurring in the same countries as the increases in allergic disorders (Stene & nafstad, 2001). Interestingly, at the individual level, developing a Th1mediated disorder may be associated with a reduced likelihood of developing a Th2-mediated disorder and vice versa


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(Tirosh et al., 2006; Tremlett et al., 2002). Thus, at the level of the individual, the Th1/Th2 balance might be important. Th1-mediated and Th2-mediated chronic inflammatory diseases are increasing in parallel in developed countries but whether an individual living in such a country develops a Th1- or Th2-mediated disorder (or no disorder) will depend on his or her genetic constitution and the Th1/Th2 balance evoked by his or her previous immunological history. This is represented by the “double see-saw” in Fig. 2. Similarly, in animal models, the antiallergic effect is sometimes attributed to induction of a Th1 bias (Debarry et al., 2007). Moreover, individuals infected by helminths, which enhance Th2 responses, are paradoxically less likely to have allergic sensitization or allergic disorders, and treating the infection leads to increased allergic sensitization (Yazdanbakhsh et al., 2002). It has been argued that the helminths merely cause secretion of IL-10 and so suppress other responses. However, we now know that the situation is more complex and interesting. Helminths can act as Treg adjuvants, as discussed above.

recruited 54 patients with an ulcerative colitis disease activity index (DAI) of at least 4. Patients were randomly assigned to receive placebo or 2,500 T. suis ova orally at 2-week intervals for 12 weeks. The primary endpoint of the trial was a decreased DAI. After 12 weeks of therapy, improvement was documented in 13 of 30 patients treated with the ova compared with 4 of 24 patients given placebo (p50.04) (Summers et al., 2005b). A similar study was performed in 29 patients with active Crohn’s disease (Summers et al., 2005a). Formal trials with T. suis are now planned in other chronic inflammatory disorders such as allergies and MS. Moreover, small companies marketing the ova have appeared, and many individuals are now testing the material informally on a patient-by-patient basis. Pritchard and his colleagues in the United Kingdom have determined the maximum load of hookworm (Necator americanus) that can be tolerated without adverse effects (Mortimer et al., 2006). A phase 1 trial in allergic rhinoconjunctivitis has been completed (Blount et al., 2009), and further studies are in progress for allergies, MS, and IBD.

Interactions with Other Changes in Modern Lifestyles

REFERENCES

Other aspects of modern life must contribute, and are likely to interact with, and amplify the immunoregulatory deficit resulting from the changes to our microbial environment. Diet and obesity are associated with modified gut flora (Turnbaugh et al., 2009). Psychological stress also modulates gut flora and gut permeability, while both obesity and stress result in greater release of proinflammatory cytokines (referenced and reviewed in Rook, 2008). Similarly, vitamin D is involved in driving regulatory cells (Xystrakis et al., 2006). Deficiency of vitamin D is extremely common and increasingly implicated in the increases in chronic inflammatory disorders. Finally, pollution, particularly dioxins, which drive Th17 cells via the aryl hydrocarbon receptor (Veldhoen et al., 2009) will also encourage inflammatory responses.

Implications for Therapy and Prevention Manipulating the Intestinal Microbiota

The previous sections make it clear that there is considerable potential for modulating inflammatory disorders by changing the intestinal microbiota. There is good evidence for efficacy of this approach in animal models, and examples have been given in previous sections. Evidence for efficacy in humans is more tenuous. The most convincing data come from studies of gastrointestinal disorders (Preidis & Versalovic, 2009), while studies in autoimmune and allergic disorders are less convincing (Rook, 2009). nevertheless, the microbiota’s powerful influence over the immune system is such that this is clearly set to develop into one of the major therapeutic tools in the future. We need to define and select appropriate probiotic strains and to combine their use with appropriate antibiotics and prebiotics that favor growth or colonization by selected organisms. Moreover, as we learn more about how the composition of the microbiota is regulated, it will become possible to manipulate it indirectly by means of diet and drugs that modulate gut physiology. It will also be logical to use combinations of these strategies. Interestingly, the U.S. national Institute of Health website lists no fewer than 170 studies of the probiotic approach as of July 2009 (http://clinicaltrials.gov/ct2/results?term=probiotic).

Helminths as Therapeutic Agents

Trials have been performed in inflammatory bowel disease, using ova of the pig whipworm, Trichuris suis. One study

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42 Theileria-Induced Leukocyte Transformation: an Example of Oncogene Addiction? MARIE CHAUSSEPIED AND GORDON LANGSLEY

INTRODUCTION

in bovine T and B lymphocytes (Dobbelaere & Heussler, 1999). T. annulata infects monocytes/macrophages and B cells of bovine, ovine, and caprine origin (Dobbelaere & Heussler, 1999; Schnittger, 2000), but only bovine leukocytes are transformed. The macroschizont stage is largely responsible for the pathology of Theileria infections (Hooshmand-Rad, 1976). The intracellular multinucleated parasite induces the proliferation of its host cell, leading to the clonal expansion of the parasitized cell population (Hulliger et al., 1964; Nichani et al., 1999). The synchrony between host cell and parasite cell cycles, together with the tight association of the macroschizont with the host cell spindle microtubules, ensures that each daughter cell inherits a parasite at the completion of mitosis (Hulliger et al., 1964). Environmental signals trigger the differentiation of the intracellular parasite to the microschizont stage; merogony is coupled to the disruption between host cell and parasite growth as the host cell ceases to divide (Hulliger et al., 1966; Schmuckli-Maurer et al., 2008; Shiels, et al., 1992). The released merozoites infect erythrocytes, where they differentiate into piroplasms that will eventually be ingested by a tick during a blood meal. Like Plasmodia, in their mammalian hosts, Theileria parasites are haploid. Diploid parasites and genetic recombination only take place in the insect vector that are ticks—Hyalomma for T. annulata and Rhipicephalus for T. parva. It is the distribution of the two tick species that determines the distribution of the two diseases.

Tropical theileriosis and East Coast Fever are tick-borne diseases of cattle caused by the protozoan parasites Theileria annulata and Theileria parva, respectively. The high mortality and morbidity, the deficit in livestock production, and the cost imposed by measures necessary to control these parasitic infections account for the heavy economic burden they impose on endemic countries in Asia, Africa, and southern Europe. Both theilerioses display similar clinical features manifested initially by the enlargement of the lymph node draining the tick bite followed by a generalized lymphadenopathy, which is accompanied by general symptoms that include fever, anorexia, and respiratory distress (Forsyth, 1999). Whereas imported animals (e.g., Holstein-Friesian cattle of the species Bos taurus) are extremely susceptible to these parasitic infections, with death occurring in up to 90% of the cases, indigenous cattle, including domestic buffalo, such as Sahiwals (species Bos indicus) as well as crossbred animals, appear significantly resistant and develop mild forms of the disease (Glass & Jensen, 2007). Theileria sporozoites are injected with the tick saliva during a blood meal. In the draining lymph node, they bind to and infect mononuclear cells wherein their rapid development involves a defined sequence of events, including, remarkably, the dissolution of the enclosing parasitophorous vacuole membrane (Shaw, 2003). At the end of this developmental process, the intracellular trophozoite freely resides in the cytoplasm of the host cell, closely associated with an array of host cell microtubules (Shaw, 2003). In the case of T. parva, these initial invasion and developmental steps can be successfully completed in caprine (goat) lymphocytes in vitro after proteolytic cleavage of surface proteins (Syfrig et al., 1998). However, intracellular development to the next multinucleated stage of the parasite, the so-called macroschizont, will only take place

INTERPLAY BETWEN HOST AND PARASITE DETERMINANTS

The restricted range of mammalian host cells that permit the full intracellular development of Theileria underscores the complexity of the interactions that need to be established between the infected cell and the parasite. The ability to experimentally infect in vitro caprine peripheral blood cells by T. parva that does not result in their transformation shows that blast transformation of parasitized bovine lymphocytes that occurs at the macroschizont stage

Marie Chaussepied and Gordon Langsley, Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. Inserm, 1016, Paris, France. Laboratory of Comparative Cell Biology of Apicomplexa, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France.

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is a process that can be separated from initial invasion and parasite establishment in the host cell cytosol (Syfrig et al., 1998). These data suggest that permissive host cell factors determine the outcome (transformation) of the invasion of the target cell. This notion is further illustrated by the observation that although T. parva sporozoites can enter and develop within bovine dendritic cells (DCs) and macrophages up to the macroschizont stage, they do not undergo blast transformation, unlike T and B cells (Dobbelaere & Heussler, 1999; Shaw, 1993). Moreover, only a subset of peripheral blood mononuclear cells (PBMC) infected in vitro with T. parva sporozoites and harboring a schizont will undergo clonal expansion (Rocchi et al., 2006). These data point to another restriction point beyond macroschizont development that will affect the result of target cell infection by Theileria. Extensive cell loss occurs in the lymph node medulla following T. annulata sporozoite injection (Campbell et al., 1995). Whether death of schizont infected-cells contributes to this cell loss has not been examined; however, it is tempting to speculate that early host determinants affect the full development of the intracellular parasite and the survival and amplification of the successfully parasitized cell population (Campbell et al., 1995). The importance of host determinants in the pathology of theileriosis is exemplified by the severity of the disease that affects B. taurus animals, as compared to the mildness of the symptoms in B. indicus (Glass et al., 2005). Although B. taurus animals were shown to produce higher levels of acute phase proteins (Glass et al., 2005), T. annulatainfected cell lines established from resistant and susceptible cattle exhibited no difference in proinflammatory cytokine mRNA levels (McGuire et al., 2004). Interestingly though, large scale transcriptomic analyses of monocytes isolated from B. taurus and B. indicus animals revealed differences in the transcriptional profile at both the basal, resting state, and after T. annulata infection (Jensen et al., 2008). How these differentially expressed genes affect the clinical manifestations of a T. annulata infection remains to be determined. Immunization against T. annulata is based on live vaccine lines that are obtained by serial in vitro cultivation of virulent field isolates (Morrison & McKeever, 2006). Animals injected with serially passaged T. annulata-infected cells develop a subclinical infection and become immune to subsequent challenge (Morrison & McKeever, 2006). Darghouth and colleagues remarked that transformed cell lines can only be established in culture with infected leukocytes isolated from lymph node biopsies when calves were injected with attenuated cells, whereas peripheral blood taken from virulent isolate-injected calves can also give rise to transformed lines of T. annulata-infected cells (Darghouth et al., 1996). This raises the possibility that the migratory and invasive properties of T. annulata-infected cells account for the pathology of theileriosis. Accordingly, we have recently shown that TGF-b (transforming growth factor beta) signaling promotes invasion of T. annulata- and T. parva-infected cells in in vitro assays. Moreover, TGF-b1 and TGF-b2 transcript levels in B. taurus and B. indicus derived T. annulata-infected cell lines correlated with sensitivity or resistance to parasite infection (Chaussepied et al., in press). Restriction fragment length polymorphism (RFLP) and glucose phosphate isomerase (GPI) isoenzyme-banding pattern analyses of parasite populations revealed that in vitro attenuation of T. annulata-infected isolates is accompanied by a reduction in the polymorphism of the parasite population present in the isolate (Darghouth et al., 1996; Sutherland et al., 1996). In light of these observations, one

can imagine that certain parasite genotypes are associated with the initial host cell (metastatic) phenotype and that these virulent genotypes are lost upon attenuation (i.e., there are Theileria virulence genes) (Rocchi et al., 2006). Differential mRNA display experiments performed on a T. annulata-infected cloned cell line and a derivative defective in merozoite formation identified a limited number of differentially expressed parasite genes (Oura et al., 2001), but whether some of these contain virulence genes is undetermined. The availability of an annotated T. annulata genome sequence (Pain et al., 2005) now offers the opportunity to approach on a whole genome scale basis the question of Theileria-dependent virulence using DNA microarrays to screen for genes differentially expressed between attenuated and virulent T. annulata-parasitized cells (see concluding remarks).

A PARASITE-INDUCED HEMATOLOGICAL CANCER?

Hanahan and Weinberg have proposed that for a cancer to develop it must overcome the natural, physiological barriers to malignant progression through the acquisition of a limited number of capabilities (Hanahan & Weinberg, 2000). These include self-sufficiency in growth signals, insensitivity to growth inhibitory signals, evasion of programmed cell death, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis (Hanahan & Weinberg, 2000). Looking at Theileria infection through this prism, can theileriosis be dubbed a cancer-like disease? In 1964, Hulliger and colleagues reported the longterm (several months) ex vivo maintenance of T. annulata and T. parva explants from infected animals in tissue cultures (Hulliger et al., 1964). Parasitized cells obtained following in vitro infection can similarly be propagated in culture for very long periods of time. Theileria-infected cells, regardless of cell type or parasite species, proliferate in vitro without the need for exogenously applied mitogenic or antigenic stimulation (Dobbelaere & Heussler, 1999). Indeed, a number of autocrine loops involving cytokines such as IL-2 (interleukin 2), GM-CSF (granulocyte-macrophage colony-stimulating factor), or TNF-a (tumor necrosis factor alpha), and cytokine receptors have been described that contribute to the proliferation of Theileria-infected leukocytes in vitro (Baumgartner et al., 2000; Dobbelaere & Heussler, 1999; Guergnon, 2003a). Both phenomena, immortalization and growth factor independence, rely strictly on the presence of live, intracellular macroschizonts and are suppressed upon elimination of the parasite with the parasiticidal drug, buparvaquone (Fig. 1) (Dhar et al., 1986). Depending on the cell type, parasite death can result in phenotypic reversal, as is the case for T. annulata-infected macrophages that remain viable while having ceased proliferation. Upon parasite elimination, they reacquire some of their lineage-specific characteristics and functional properties (Jensen et al., 2009; Sager et al., 1997). In contrast, T. parva-infected T lymphocytes undergo massive apoptosis upon parasite elimination without any obvious sign of proliferative arrest, whereas cured T. parva- and T. annulata-infected B lymphocytes initially arrest in the G1 phase of the cell cycle before dying of apoptosis (Guergnon, 2003b; M. A. Chaussepied & G. Langsley, unpublished data) (Fig. 1). These data have been interpreted as evidence for the absence of heritable alterations of the host cell genome occurring as a result of Theileria-induced immortalization. Strikingly though, bovine lymphosarcoma cell lines

(BL3 and BL20) infected with T. annulata and treated with buparvaquone do not revert to their original BL3 and BL20 lymphosarcoma status, but behave like parasitized primary B cells (i.e., they arrest in G1 before dying of apoptosis) (Guergnon, 2003b; Chaussepied & Langsley, unpublished data) (Fig. 1). This behavior of Theileriainfected cells upon drug-induced parasite elimination is reminiscent of what happens in mice cancer models that harbor a conditional oncogene. Oncogene deinduction caused the regression of the tumor that took the form, depending on the tissue type, of differentiation, apoptosis, or dormancy (Jonkers & Berns, 2004). This phenomenon has been coined “oncogene addiction” to underlie the absolute requirement for the permanent activity of a given oncogene for tumor maintenance (Weinstein, 2002; Weinstein & Joe, 2008). One could similarly argue that although bovine lymphosarcoma cells are likely to harbor genetic alterations, their infection with Theileria has rendered them “Theileria-addicted”. How Theileria parasites perturb and rewire the normal signaling circuitry in parasitized cells is the subject of active research. The invasive nature of Theileria-infected leukocytes is evidenced by post-mortem findings in animals having undergone acute, lethal sporozoite-induced infection: T. annulata macroschizont-harboring cells are found in both lymphoid (lymph nodes, spleen, and thymus) and nonlymphoid (liver, kidney, lung, abomasum, adrenal glands, pituitary gland, brain, heart) organs (Forsyth et al., 1999). When T. parva and T. annulata-infected leukocytes are injected subcutaneously into immunocompromised mice, they form invasive tumor masses that infiltrate the underlying muscles (Fell et al., 1990; Irvin et al., 1975; Lizundia, 2006). Moreover, foci of parasitized leukocytes that have disseminated are found in various organs (Fell et al., 1990; Irvin et al., 1975; Lizundia et al., 2006). Contrast echography reveals that the subcutaneous tumors formed in mice by T. parva-transformed B lymphocytes are highly vascularized (Renault & Langsley, unpublished data) (Fig. 2). Indeed, Theileria tumor masses can reach a very significant volume, yet histology does not show necrotic areas (Lizundia et al., 2006). The angiogenesis triggered by Theileria-infected leukocytes is likely to foster local tumor growth and facilitate dissemination. Matrix metalloproteases (MMPs) have been shown to mediate the metastatic behavior of Theileria-infected leukocytes in scid mice (Somerville et al., 1998). T. annulatainfected cells secrete various proteases, including MMPs, in very large amounts whose activity can be detected in the supernatant of cells in culture (Baylis et al., 1992). Importantly, long-term culture of T. annulata-infected field isolates that leads to attenuation is associated with a significant reduction of these MMP activities (Baylis et al., 1992; Hall et al., 1999). MMPs participate, via proteolytic degradation of various substrates, in multiple aspects of tumor cell biology, including local tumor growth and proliferation, angiogenesis, as well as invasion and metastasis (McCawley & Matrisian, 2000). For example, the release of diffusible VEGF-A upon proteolytic processing of its ECM-sequestered form probably accounts for the ability of MMP9 to prompt an angiogenic switch (Bergers et al., 2000; Lee et al., 2005). Also, another proangiogenic factor, TGF-b, is activated by MMPs-mediated proteolytic cleavage of its latent form (Elliott & Blobe, 2005; Pepper, 1997). Therefore, both MMPs and TGF-b seem to be essential factors in the pathogenicity of Theileria infection by regulating a similar aspect of Theileria-transformed leukocytes biology—their invasive behavior (Darghouth et al., 1996; Chaussepied et al., in press).

FIGURE 1 Time course analysis of the cell cycle distribution of buparvaquone-treated Theileria-infected cell lines. Theileriainfected cell lines were kept untreated or treated with buparvaquone. After the indicated time, the cells were fixed and DNA was labeled with propidium iodide. Cell cycle distribution was analyzed by flow cytometry.

42. Theileria-Induced Leukocyte Transformation: an Example of Oncogene Addiction?

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FIGURE 2 Contrast echography following intravenous injection of microbubbles that allow blood vessels to be detected. The image was made with the help of Gilles Renault of the small animal imagery platform at the Cochin Institute. T. parva-transformed B cells were injected into thighs of Rag2/ gammaC mice (Lizundia, 2006) and tumor development followed every week for 8 weeks by contrast echography. The highly vascularized tumor shown is at 6 weeks postinjection.

Although TGF-b signaling can be growth-suppressive in hemopoietic cells, notably via the transcriptional induction of cyclin-dependent kinase (CDK) inhibitors, or the blockade of c-myc transcription (Li et al., 2006), pharmacological inhibition of TGF-b signaling in T. parva-infected B lymphocytes does not enhance their proliferation in vitro, suggesting that Theileria-induced transformation has rendered the infected leukocytes refractory growth inhibition by TGF-b (Chaussepied et al., in press). Similarly, although T. parva-transformed T cells coexpress the death receptor Fas (CD95) and its ligand FasL, they are resistant to Fas-mediated apoptosis, whereas their buparvaquone-treated counterparts exhibit a time-dependent increase in FasL sensitivity (Kuenzi et al., 2003). The resistance of T. parva-infected T cells to Fas-induced cell death has been attributed to the parasite-dependent expression of the antiapoptotic proteins c-FLIP and XIAP (Kuenzi et al., 2003).

IN SEARCH FOR PARASITE ONCOGENE: PROBING PARASITE-TARGETED ONCOGENIC PATHWAYS

A recurrent theme in Theileria research has been the search for putative parasite oncogene(s). T. parva and T. annulata genomes are predicted to contain about 4,000 proteincoding genes, out of which only 38% can be assigned with a putative function based on homology searches (Gardner et al., 2005; Pain et al., 2005). The straightforward, naïve approach aimed at identifying parasite orthologs of viral oncoproteins using bioinformatics analysis of T. parva and T. annulata predicted proteomes did not yield any pertinent candidate (Shiels et al., 2006). In the pregenome era, a number of laboratories (including our own) undertook the characterization of the signaling pathways active in Theileria-transformed leukocytes. One rationale was that both parasites would use a similar mechanism to transform B lymphocytes, T lymphocytes, and monocytes/macrophages, and therefore the activated intracellular signaling pathways would be similar regardless

of the parasite species or the cell type considered. One obvious drawback to this strategy was an underestimation of the different contributions that cell type-specific autocrine stimulations make to the activation status of the different types of infected leukocyte, which has significantly hindered the identification of common signaling pathways directly impacted by Theileria parasites. A number of receptor-associated and cytosolic kinases, as well as transcription factors, were found to be permanently active in various Theileria-infected cell lines (Table 1; Fig. 3). Yet, for most of them, evidence for the involvement of parasite-originated signals in their activation is lacking. Theileria-dependent activation of NF-kB is of particular interest (Ivanov et al., 1989; Palmer et al., 1997). This ubiquitously expressed family of transcription factors is induced by a large array of stimuli among which are inflammatory cytokines (Pahl, 1999) (www.nf-kb.org). Theileria infection is associated with elevated expression of several proinflammatory cytokines, including IL-1a and TNF-a; TNF-a receptor signaling was shown to contribute to NF-kB transcriptional activity in T. parvainfected B lymphocytes (Brown et al., 1995; Collins et al., 1996; Guergnon, 2003a; Yamada et al., 2009). NF-kB activation entails the nuclear translocation of cytoplasmic NF-kB dimers (Hoffmann & Baltimore, 2006). In the so-called “canonical” pathway of NF-kB activation, the proteosomal degradation of the inhibitor protein I-kB is primed by its phosphorylation that is mediated by a multiprotein I-k B kinase (IKK) complex comprised of two kinases, IKKa and IKKb, and a scaffold protein, NEMO (also called IKKg) (Hoffmann & Baltimore, 2006). In Theileria-infected cells, interfering with IKK function using dominant negative mutants of its components partially inhibited NF-kB transcriptional activity (Heussler et al., 2002). Remarkably, IKKa, IKKb and NEMO were found to colocalize with phosphorylated I-kBa in discrete foci at the surface of the intracellular parasite in T. parva- and T. annulata-transformed cells (Heussler et al., 2002; Schmuckli-Maurer et al., 2009). How recruitment of the IKK complex to the surface of the macroschizont might stimulate its kinase activity is at present unknown. Also, the kinetic of IKK signalosome association with the parasite surface has not been investigated. This question is particularly relevant given the well-established role of NF-kB in protecting cells from apoptosis (Van Antwerp et al., 1996). Indeed, inhibiting NF-kB activity in T. parva-transformed T lymphocytes causes apoptotic cell death (Heussler et al., 1999). Although the subcellular distribution of the IKK complex in Theileria-infected cells was found to be specific for the transforming stage of the parasite and could no longer be observed following differentiation to the microschizont/merozoite stage, it has not been examined at early time periods following Theileria entry as the trophozoite establishes itself into the infected cell and develops to the macroschizont transforming stage (Heussler et al., 2002). The case of Theileria-dependent AP-1 activation represents another example where there exists some suggestion for a straightforward influence of the parasite. T. parvaand T. annulata-infected leukocytes possess high levels of Jun and Fos proteins that form AP-1 dimers, AP-1 DNA binding activity, and AP-1 transcriptional activity (Botteron & Dobbelaere, 1998; Chaussepied et al., 1998). The AP-1 family of transcription factors positively regulates numerous genes implicated in various aspects of the tumorigenic process, including cellular proliferation, cell survival, invasion, and angiogenesis, thus accounting for its oncogenic activity (Eferl & Wagner, 2003). Expression

42. Theileria-Induced Leukocyte Transformation: an Example of Oncogene Addiction? TABLE 1.

541

Kinases and transcription factors activated in Theileria-infected leukocytes.

Type of factor

Experimental Evidence

Cell type

Function

Reference(s)

Receptor-associated kinases Hck

T. parva-infected B lymphocytes

Kinase activity

JAK


Inhibitor study

Proliferation

Baumgartner, 2003

PI3-K


Kinase activity

Proliferation

Baumgartner, 2000

AKT

T. parva-infected T lymphocytes, T. annulata-infected macrophages

p-AKT(S473) & p-AKT(T803) WB; Kinase activity

Proliferation

Heussler, 2001

CK2

T. parva-infected T lymphocytes, T. parva-infected B lymphocytes, T. annulata-infected cells

Kinase activity

Survival, proliferation

Dessauge, 2005b; ole-MoiYoi, 1993; Shayan & Ahmed, 1997

JNK

T. parva-infected B and T lymphocytes, T. annulatainfected macrophages

Kinase activity

Survival

Chaussepied, 1998; Galley, 1997; Lizundia, 2005

PKA

T. annulata-infected B lymphocytes

Kinase activity

AP-1 (Jun, Fos, ATF)

T. parva-infected B lymphocytes, T. annulata infected macrophages

Transcriptional activity (reporter gene assay); DNA binding activity

Tumorigenicity, invasiveness

Adamson, 2000; Botteron & Dobbelaere, 1998; Chaussepied, 1998; Lizundia, 2006

E2F

T. parva-infected B lymphocytes, T. annulata-infected B lymphoctes

Transcriptional activity (reporter gene assay)

Survival

Chaussepied et al., in preparation

c-Myc



Survival

Dessauge, 2005a

NF-kB

T. parva-infected B and T lymphocytes,

-Transcriptional activity (reporter gene assay) -DNA binding activity

Survival

Guergnon, 2003a; Heussler, 1999; Ivanov, 1989; Machado, 2000

NOTCH-CSL/ RBP-Jk



ND

Chaussepied, 2006

STAT3


p-STAT3 WB

ND

Dessauge, 2005a

Dessauge, 2005a

Cytosolic kinases

Guergnon, 2006

Transcription factors

of a dominant negative c-Jun mutant and functional inhibition of AP-1 in T. parva-transformed B lymphocytes had no effect on their proliferation in vitro, but these AP-1-low cells were impaired in tumor formation and dissemination when injected into immunocompromised mice (Lizundia et al., 2006). Whether the inability of T. parva-infected B cells with reduced AP-1 activity to form invasive tumors is due to defective in situ proliferation, survival, or vascularization has not been examined. Remarkably, the in vitro attenuated T. annulata-infected field isolates display qualitatively altered AP-1 DNA binding activity as well as significantly lower AP-1 transcriptional activity compared to their virulent, preattenuation counterparts (Adamson et al., 2000). As in vitro attenuation was shown to lead to selection and simplification of parasite genotypes, this suggests that Theileria

macroschizont-derived signals could prompt AP-1 activation (Darghouth et al., 1996; Sutherland et al., 1996).

TRAFFICKING INFORMATION BETWEEN THEILERIA AND ITS HOST CELL

The situation of Theileria parasites inside their leukocyte host is unique. Unlike other Apicomplexa like Plasmodia or Toxoplasma, Theileria macroschizonts are not enclosed in a parasitophorous vacuole, but rather are exposed in the cytosol of the infected cell (Shaw, 2003). This implies that the plasma membrane of the parasite lies in direct contact with the cytoplasm of the host cell and that secretion products from the parasite end up in the host cell cytoplasm. Although no parasite “oncogene” has been identified to date, a few parasite-encoded proteins have been characterized that localize to the host cell

542


FIGURE 3 Activated signaling pathways in Theileria-infected cells. Receptor-associated kinases including JAK, PI3-K, and Src are classically activated upon growth factor receptor engagement. In Theileria-infected cells, cytokine autocrine loops participate in Theileria-dependent proliferation and may contribute to the activation of receptor-associated, as well as cytoplasmic kinases among which are JNK, CK2, and PKA. Transcription factors like STAT, c-Myc, and AP-1 are positively regulated by phsophorylation mediated by JAK, CK2, and JNK, respectively. Phsophorylation controls their nuclear translocation, their stability, and/or their transactivating potential. Whether the intracellular macroschizont directly modulates the activation status of receptor-associated and cytosolic kinases or their target transcription factors is hypothetical. In contrast, the localization of the IKK signalosome formed by IKKa, IKKb, and NEMO at the surface of the macroschizont suggests a direct influence of the parasite over the activity of this kinase complex that is responsible for IkBa phosphorylation and degradation and the ensuing nuclear translocation of the NF-kB p50-p65 dimer.

cytoplasm or nucleus (Fig. 4) and may participate in processes essential for the biology of the intracellular schizont. One critical aspect of Theileria parasites is that multiplication at the leukocytic stage of their life cycle relies on the ability of the macroschizont to be distributed in each daughter cell as the host cell completes mitosis. During the anaphase and telophase of the host cell mitosis, the intracellular schizont is being pulled between the two spindle poles. Interestingly, Schneider and colleagues reported the isolation of a parasite cDNA encoding a T. annulata secretory protein (TaSE) expressed at the piroplasm and schizont stages (Schneider et al., 2007). Immunolocalization studies showed that TaSE is found within the intracellular macroschizont, associated with the parasite plasma membrane, as well as in discrete areas in the infected cell cytoplasm where it colocalizes with aTubulin staining (Schneider et al., 2007). Importantly, TaSE localizes to host cellular structures that are essential for the organization of the mitotic spindle—the centrosome in interphase cells and the centriole at the spindle poles in anaphase (Schneider et al., 2007). In telophase cells, TaSE is also seen at the midbody (Schneider et al., 2007). In a heterologous system, ectopically expressed TaSE displays the same distribution (Schneider et al., 2007). The function of TaSE has not been determined, but given its localization, it could play a role in the organization of the microtubule network associated with

the Theileria macroschizont and the partitioning of the parasite between the daughter cells at the end of mitosis. Another important aspect of Theileria biology inside the infected leukocyte relates to its ability to alter host cell gene expression. In this respect, the identification of Theileria DNA binding proteins localized in the nucleus of the parasitized cell is of particular significance. The TashAT gene cluster that include genes for TashAT1-3 and TashHN1-2 encodes polypeptides with AT-hook DNA binding domains; some of them were found to localize in the nucleus of the infected host cell (Swan et al., 1999, 2003, 2001). Interestingly, their expression was down regulated as macroschizont to microschizont and merozoite differentiation was induced, while levels remained high in Theileria-infected cell clones defective for merozoite production (Swan et al., 1999). As parasite differentiation to merozoites is accompanied by a cessation of host cell proliferation, the concomitant down regulation of TashATs expression raises the possibility that this protein family may be involved in the maintenance of the proliferative state of the infected cell. However, how ectopically expressed TashAT family members within differentiation-competent Theileria-infected cell lines affect their response to the differentiation inducers has not been examined. AT-hook DNA binding domains are found in a number of proteins that organize the chromatin and

42. Theileria-Induced Leukocyte Transformation: an Example of Oncogene Addiction?

543

FIGURE 4 Theileria-encoded polypeptides exported to the host cell. TaSE protein was shown to localize to the parasite surface but was also found associated with microtubules in discrete areas. TashAT are found in the nucleus of the infected cell. There has been some suggestion that the catalytic subunit of T. parva CK2 might be exported into the host cell.

nucleosome architecture (Reeves, 2009); these include the HMGA family of high mobility group (HMG) proteins that are involved in regulating gene transcription, notably via the orchestrated assembly of the so-called enhanceosome multi-protein DNA complex. In addition, overexpressed HMGA proteins were shown to foster tumor development and metastasis (Reeves, 2001). AT-hook motifs are present also in a number of proteins that form the ATP-dependent chromatin remodeling complex (Reeves, 2009). At present, little is known about TashAT and TashHN polypeptides. Although the host cell nuclear pool of TashHN was shown to be modified by phosphorylation, neither the mechanism nor the role of this modification have been examined. Peptide sequence analysis predicts both cyclin-docking motifs and CDK phosphorylation sites, suggesting a role for parasite or host cyclin-CDK complexes in TashHN modification. The functional significance of their nuclear localization has not been determined either. The absence of consensus AT-hook motifs in the T. parva orthologs of TashHN and TashHNs, questions the importance of these proteins in Theileriadependent changes in the host cell transcriptome. Another parasite-encoded AT-hook nuclear protein is the SuAT1 polypeptide, whose open reading frame forms part of the large cluster of SuAT1 and TashAT-like genes in the T. annulata genome (Pain et al., 2005; Shiels et al., 2004). Unlike the TashAT family members that have been studied, SuAT1 expression is up regulated as merozoite differentiation is induced (Shiels et al., 2004). The endogenous SuAT1 protein localizes to the nucleus of the parasitized leukocytes at the time following merogony induction when host cell proliferation has ceased. Ectopically expressed SuAT1 was also found in the nucleus of a transfected bovine macrophage cell line (BoMAC cells) (Shiels et al., 2004). Interestingly, the phenotype of SuAT1 expressing BoMAC cells is reminiscent of the one adopted by cells undergoing a permanent growth arrest termed “replicative” or “oncogene-induced senescence,” where cells appear enlarged and flattened with a large nucleus (Hayflick, 1965). Moreover, HMG proteins were recently shown to contribute to senescence notably via

the stable repression of genes whose products are required for proliferation (Narita et al., 2006). Remarkably, Shiels and colleagues reported that a single cell line was established upon stable transfection of BoMAC cells with SuAT1 expression vector, and furthermore, these cells exhibited a proliferative defect compared to empty vector transfected cells (Shiels et al., 2004). This raises the possibility that the SuAT1 expressing clone acquired some mutations that allowed the cells to partially bypass the detrimental effect of SuAT1 overexpression. The development of Theileria inside its host cell involves highly interdependent interactions between the intracellular parasite and the parasitized cell. For example, in proliferating, macroschizont harboring cells, the parasite replicates its DNA after the host cell’s S phase has been completed but before mitosis starts, while the host cell is in the G2 phase of the cell cycle. On the other hand, during merogony, parasite division and host cell division are uncoupled and host cell DNA replication ceases (Shiels et al., 1992). Whereas a lot of effort has been put into understanding how the intracellular parasite influences the behavior of the infected cell, virtually nothing is known about whether and how the parasitized host cell may signal to the intracellular parasite. Strikingly, intraerythrocytic Plasmodium berghei, which is isolated from the red blood cell’s cytoplasm by a parasitophorous vacuole, imports an enzyme, d-aminolevulinate dehydrase (ALAD), required for the parasite’s heme biosynthetic pathway (Bonday et al., 1997). Similarly, Toxoplasma gondii parasites have recently been described to co-opt host cell calpains to facilitate their escape from infected cells (Chandramohanadas et al., 2009). It is tempting to speculate, therefore, that Theileria macroand microschizonts might similarly incorporate host cell components required for the biology of the parasite inside the leukocyte. As proteomic analysis has now been performed on a number of Apicomplexa, and a protocol enabling the purification of the intracellular macroschizont exists (Baumgartner et al., 1999), a proteomic dissection of purified Theileria macroschizonts could give new insights into this complex host–parasite interaction (Wastling et al., 2009).

544


CONCLUSIONS

With the determination of the genome sequences of T. parva and T. annulata in 2005 (Gardner et al., 2005; Pain et al., 2005), combined with the availability of the B. taurus genome (http://www.ncbi.nlm.nih.gov/projects/genome/ guide/cow/), research on Theileria-infected bovine leukocytes has entered the postgenomic era. These resources make it possible to screen both bovine and parasite microarrays and this is indeed being done in the context of a Wellcome Trust funded network in a study entitled “An Integrated Approach for Sustainable Methods to Control Tropical Theileriosis” (http://www.theileria.org/). Screening of bovine arrays will allow the identification of host cell genes that are either up or down regulated during infection and leukocyte transformation. Screening of T. annulata microarrays with mRNA isolated from virulent and attenuated macrophage vaccine lines should allow the identification of parasite genes whose expression is down regulated during host cell attenuation (i.e., loss of metastatic potential). Such parasite genes represent potential Theileria virulence genes, whose “oncogenic” potential can be tested by reintroducing them into infected attenuated macrophages and screening for gain of function (i.e., restored virulence). However, it is clear that to fully exploit the benefits of the postgenomic era, efficient transfection of Theileria parasites is necessary to knockout or knockdown such virulence genes. Following the initial report of transient transfection of T. annulata (Adamson et al., 2001) Theileria-specific transfection technology has not evolved and is clearly a brake on the study of this very interesting host–parasite combination that is the only example of one eukaryotic cell being able to transform another eukaryotic cell. Some of the work discussed in this review received support from the Wellcome Trust Animal Health Initiative (GR075820MA) on “An Integrated Approach for the Development of Sustainable Methods to Control Tropical Theileriosis.”

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43 Systems Vaccinology: Using Functional Signatures To Design Successful Vaccines TROY D. QUEREC AnD BALI PULENDRAN

INTRODUCTION

studies used histopathological correlates of protection, however, new components and interactions within the immune system continue to be identified, and technology has given vaccinologists an increasing diversity of tools with which to measure immune responses. This has led to an interest in using functional signatures not just as correlates of protection, but also to understand the signaling networks involved in different types of immunological protection. Vaccines operate by stimulating the innate immune system to develop acquired immune responses. Professional antigen presenting cells such as macrophages and dendritic cells (DCs) are key orchestrators of this process (Takeda et al., 2003; Pulendran, 2005; Colonna et al., 2006; Steinman & Banchereau, 2007). When the system is in surveillance equilibrium, DCs continuously sample their local environment as sites of pathogen entry into the body such as the skin, mucosal tissues, and blood. Inoculation methods introduce the vaccine into these sites, where it is taken up and processed by the DCs, which become activated and migrate into the draining secondary lymphoid tissue. The DCs process the antigens into peptides for loading onto major-histocompatibility (MHC) molecules. The displayed MHC-peptide complexes then signal antigen-specific T cells via the T-cell receptor (TCR). The MHC-TCR interaction determines which T cells are to be targeted, and the adjuvant delivered in the vaccine determines the accompanying costimulatory and cytokine signals that the DC will present to a targeted T cell. These costimulatory signals determine the nature of the T-cell response (Steinman & Banchereau, 2007; Cook & Holmes, 2005). When a vaccine adjuvant activates the appropriate costimulatory pathways, CD81 T cells can differentiate into cytotoxic T lymphocytes (CTLs), whose main role is to destroy infected or cancerous cells, and CD41 T cells can differentiate into various subsets that either activate CD81 T-cell responses, antibody production, and macrophage activation, or negatively regulate other immune responses.

Vaccination seeks to imitate or improve upon the acquired immune responses induced by a natural infection to stimulate long-term protective immunological memory, so that upon exposure to the infectious organism morbidity and mortality will be reduced or prevented. The pathway to modern vaccine design began with the ancient observation that individuals that had previously survived a particular infection were often unaffected when there was a new outbreak of that infection. Those observations led to the practice of variolation, in which dried puss or scabs from smallpox victims was introduced into the nose or skin of an infected individual (Plotkin, 2005). Many of these variolated individuals were protected from full-blown smallpox, thus reinforcing the refinement of the technique, but up to 3% died of smallpox induced by the variolation (Artenstein & Grabenstein, 2008). The first modern vaccination occurred when Edward Jenner induced protection to smallpox by inoculating individuals with the related bovine pathogen, cowpox or vaccinia. These “vaccinated” individuals did not develop smallpox (Hochstein-Mintzel, 1977; Baxby, 1999). Functional signatures of immune responses are experimentally defined cellular and molecular measurements, which indicate that the immune system has received sufficient stimulus to move from a surveillance equilibrium to an activated state that will provide future protection against the target disease. Analyzing the functional signatures of vaccines aids vaccine development in two ways: (i) by monitoring the efficacy of vaccines as they are refined and (ii) by revealing the mechanisms of vaccine action, such that they can be applied to the development of future vaccines. The original hallmark of vaccine efficacy was its impact on symptomatic infections, severity of disease, and mortality rates. However, it is not always possible to monitor the impact of a vaccine upon exposure to an infectious organism. The concept of correlates of protection was developed as an objective criterion that a certain threshold of a functional immunological response induced would be necessary and sufficient to protect the vaccinee against infection. Early

FUNCTIONAL SIGNATURES AS CORRELATES OF PROTECTION

As the field of vaccinology developed and new disease targets for vaccine research were selected, neutralizing antibodies were thought to be the primary correlate of

Troy D. Querec and Bali Pulendran, Emory Vaccine Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329.

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protection against the morbidity and mortality caused by most pathogens. Many of the early successful vaccines protected against diseases that could be prevented with antibodies (Plotkin, 1999). These included viruses, such as hepatitis, that circulate through the bloodstream before reaching their target organ (Jack et al., 1999; Van Damme & Van Herck, 2007) and yellow fever (Wheelock & Sibley, 1965; Reinhardt et al., 1998; Lang et al., 1999); diseases like diphtheria (Ipsen, 1946) and tetanus (Looney et al., 1956), which are caused by bacterial toxins that can be neutralized by antibodies; pathogens like influenza (Dowdle et al., 1973; Mostow et al., 1973) and rotaviruses (Jiang et al., 2008), which colonize mucosal surfaces; and rabies virus (nagarajan et al., 2008), which can be neutralized by antibodies before it reaches neuronal axons. The original method used to determine the antibody activity induced by vaccination was to measure the passive transfer of immunity by injecting serum from a vaccinated animal to an unvaccinated animal prior to a challenge with the pathogen. In the development of the yellow fever vaccine 17D, such animal experiments played a dual role, both ensuring the vaccine strain was safely attenuated and did not cause disease in the recipients of the vaccine, and confirming that the vaccine was able to induce protective immunity as measured in the passively protected serum recipients (Theiler & Smith, 1937). As antibody-generating vaccines became more prevalent, simple standardized methods were developed to measure the antibody responses. These methods include antibody titers measured through ELISAs (enzyme-linked immunosorbent assay) and hemagglutination inhibition and functional measures of activity such as neutralization and opsonophagocytosis (Table 1). In addition to being relatively easy to perform and replicate, antibody assays have a basic, linear interpretation. The tests result in a single value that has to be considered. Antibody measurements above a certain threshold are considered to be protective against a disease. Thus, one goes directly from a single antibody measurement to determining if a protective immune response was induced or not.

Despite the common use of antibody assays to measure the efficacy of current vaccines, protective vaccination may not always be correlated to the humoral immune response. Varicella virus vaccination efficacy is usually determined by measuring antibody titers using serum neutralization or ELISA. However, persistent varicella-specific CD41 T cells have been shown to be indicators of protection from varicella virus infection and have been suggested as possible additional or alternative correlates of protection in children and the elderly (LaRussa et al., 1996; Levin et al., 2008). Influenza virus protection may also be better correlated to CD41 T-cell responses as well. Elderly individuals that have strong influenza-specific CD41 T-cell responses are less likely to develop flu regardless of postvaccination antibody titers (McElhaney et al., 2006). While antibody titers did not distinguish between elderly subjects that did or did not develop flu, those subjects with high IFn-g (interferon gamma):IL-10 (interleukin 10) ratios following ex vivo influenza stimulation of PBMCs (peripheral blood mononuclear cells) were more likely to be protected from influenza illness. Those patients with high numbers of CMV (cytomegalovirus)-specific T cells are less likely to have reactivation of CMV when on immunosuppressive drugs to prevent rejection of donated organs (Sester et al., 2001; Bunde et al., 2005). In fact, many diseases that are a top priority for vaccine development—such as HIV (human immunodeficiency virus), TB (tuberculosis), and malaria— are believed to require strong T-cell responses for protection (Pantaleo & Koup, 2004; Hoft, 2008; Reyes-Sandoval et al., 2009). These realizations have led to interest in measuring T cells as correlates of protection. Measuring the functional signature of the T-cell response as a correlate of protection is not as straightforward as the antibody titers that have been used historically. The complications with using T cells as correlates of protection largely stems from diversity within the T-cell population. There are the two main lineages of T cells: CD81 and CD41. Activation of antigen-specific T cells induces cell proliferation and differentiation into effector cells, effector

TABLE 1 Methods to measure antibody correlates of protectiona Vaccine

Test

Correlate of protection

Diphtheria Hepatitis A Hepatitis B Hib polysaccharide Hib conjugate Influenza Lyme Measles Pneumococcus

Toxin neutralization ELISA ELISA ELISA ELISA HAI ELISA Microneutralization ELISA; opsonophagocytosis

Polio Rabies Rubella Tetanus Varicella

Sn Sn Immunoprecipitation Toxin neutralization Sn; gpELISA

0.01–0.1 IU/ml 10 mIU/ml 10 mIU/ml 1 mg/ml 0.15 mg/ml 1/40 dilution 1,100 EIA U/ml 120 mIU/ml 0.2–0.35 mg/ml (for children); 1/8 dilution 1/4–1/8 dilution 0.5 IU/ml 10–15 mIU/ml 0.1 IU/ml  1/64 dilution; 5 IU/ml

a Traditional correlates of protection measuring antibody responses use a simple threshold which, when achieved or exceeded, is assumed to be a signature of protective immunization. Either the concentration of the antibody was measured by ELISA or immunoprecipitation, or some measure of antibody activity was measured such as neutralization of the pathogen or its toxin. These tests have become well standardized and relatively straightforward to perform. Adapted from Plotkin, 2008.

43. Systems Vaccinology: Using Functional Signatures To Design Successful Vaccines

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FIGURE 1 Future correlates of protection may integrate multiple measurements. Using multiple cocorrelates of protection (e.g., A, B, C), immunological protection would increase (dark shading) as any of the individual correlates increased in strength, but the greatest level of protection is achieved when cocorrelates combine. These cocorrelates may be, for example, neutralizing antibody titer, T-cell proliferation, T-cell IFn-g to IL-10 ratio, or frequency of trifunctional T cells.

memory cells, and central memory cells, all of which are important for the immediate and/or long-term immune responses (Sallusto et al., 1999; Harari et al., 2004). Recently activated T cells can be phenotypically monitored by measuring up regulation of CD38 and HLA-DR or the expansion of peptide-MHC tetramer staining cells (Callan et al., 1998; Appay et al., 2002; Miller et al., 2008). Differentiation into effector and memory phenotypes can be detected by expression of CD45RA, CD62L, CD127, and CCR7, in addition to other markers. However, the frequency of differentiated T-cell phenotypes may not be specific enough to be used as a correlate of protection; the functional activity of the cells may also be important. Measures of T-cell function include IFn-g, IL-2, TnF-a (tumor necrosis factor alpha), and perforin production, as well as other measures of cell proliferation and cell-mediated cytotoxicity. Thus, there are numerous T-cell functional signatures that can be measured as correlates of protection in lieu of the traditional antibody response. Furthermore, it may not provide sufficient specificity to measure a single functional response such as IFn-g production; however, using a functional signature combining two or more activities should provide more specific and reliable correlates of protection (Harari et al., 2006). Finally, it may be necessary to depart from the simple linear functional signature model developed for antibody titers, where a predetermined threshold is used for assessment. Instead of using a set threshold of a single variable to determine vaccine efficacy, cocorrelates of protection may be more appropriate, where it is the balance among multiple variables that indicates efficacy (Qin et al., 2007). For instance, an appropriate level of protection may be estimated to be achieved when two conditions are met: (i) the frequency of Th1 CD41 effector memory cells meets a given threshold, and (ii) CD81 T cells are of the trifunctional IFn-g1/IL-21/TnF-a1 type. In other cases, it may be the interaction between various cocorrelates and not independent levels of each that provides a functional signature of vaccine efficacy. For instance, for control of

viruses or intracellular pathogens, the lower the neutralizing antibody titer induced by a vaccine, the higher the cytotoxic T-cell response needs to be to enhance the likelihood of protection (Fig. 1). The possibility of measuring the innate immune response as a correlate of protection has only recently been considered. The innate immune response programs the quality and strength of the acquired immune response, and particular innate immune functional signatures may serve as indicators that the vaccine induced the appropriate quality and sufficient strength of activation to induce protective acquired immunity. It has been shown with yellow fever vaccine 17D that molecular signatures in the blood 3 to 7 days postvaccination, corresponding with vaccine viremia and activation of the innate immune pathways, may be used to predict the peak frequency of activated virus-specific T cells and long-term neutralizing antibody titers (Querec et al., 2009). A benefit of using functional signatures of innate immunity as correlates of protection is that they occur quite early after vaccination compared to the development of memory T cells and antibody responses, which can take weeks, months, or years. Being able to determine vaccine efficacy in a short time is useful for many reasons. One potential problem with vaccine trials is the need to conduct these clinical studies over long time frames. Having a shorter study period increases the probability of retaining all the subjects for the duration of the study, increasing the proportion of subjects that are tracked from vaccination through the final time point. In addition, measuring the functional signature of vaccine-induced innate immunity makes high throughput screening of vaccine candidates more feasible. The short duration of time required to measure innate immune activation relative to the endpoints of acquired immunity means: (i) shorter duration to analyze each batch of vaccine candidates, (ii) potentially less resources and costs devoted to the early stage analysis of each vaccine candidate, and (iii) quicker refinement of vaccine formulations and delivery methods.

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Apart from a lack of inducing sufficient protection, another common reason that vaccines fail are the severe side effects. These side effects are usually associated with overactivation of certain components of the innate immune system (Gupta et al., 1993; Pulendran et al, 2008). Thus, functional signatures of innate immunity may be used to screen adjuvants or as cocorrelates of protection along with parameters of acquired immunity for complete vaccines (antigen and adjuvant). Functional signatures may not only help in the design of protective vaccines but may also help to limit the deleterious side effects.

VACCINE SIGNALING NETWORKS GIVE INSIGHTS FOR DESIGN

The greater utility of measuring the innate immune response to successful vaccination may not be as a correlate of protection but in understanding how to program the immune system to give the desired acquired immune response. Vaccines are used to relay two pieces of information to the acquired immune system about an antigen: (i) that a response should be made to the antigen and (ii) the nature of that response as influenced by the adjuvant. Until recently, most of the focus of vaccine development has been on the antigen. Instructing the nature of the immune response either came by chance as whole pathogen preparations (either live-attenuated or inactivated) contain natural adjuvants that are retained from the parental pathogen or through the default choice of the synthetic adjuvant alum. Alum is the classic synthetic adjuvant. Until recently it was the only adjuvant approved for human use, but it still remains the most commonly used human adjuvant (Lindblad, 2004). It was first used over 80 years ago to enhance the immunogenicity of diphtheria toxoid for vaccination. Through electrostatic and ligand exchange interactions, alum absorbs antigen onto aluminum oxyhydroxide or aluminum oxyphosphate crystals that form in suspension. Effective vaccination depends on the absorption of the antigen onto the adjuvant; failed or inconsistent vaccination is often blamed on undeveloped antigen-alum complexes during the preparation or dissociation of the complexes in vivo. It is suggested that alum works by serving as a depot of antigen in the body possibly for longer than 3 months, and it may facilitate the phagocytosis of the antigen by DCs through the crystalline antigen-alum complex. It has also been suggested that alum could cause necrosis in the inoculated tissue, which indirectly activates DCs through danger signals in the form of host inflammatory mediators (De Gregorio et al., 2008; Kool et al., 2008; Aimanianda et al., 2009; De Gregorio et al., 2009). The details of this mechanism are only recently being revealed. Alum operates through the nALP3 inflammasome to activate caspase-1, which in turn processes proIL-1b and pro-IL-18 into their activated forms (Eisenbarth et al., 2008; Li et al., 2008; Kool et al., 2008). The inflammatory properties of IL-1b and IL-18 help to promote antigen-specific T-cell proliferation and antibody production. Alum-based vaccines are able to induce IL-4 and IL-5 mediated Th2 responses and the corresponding IgG1 and IgE antibody responses, but they are incapable of inducing the Th1 CTL, IFn-g, and IgG2a responses believed to be required for modern vaccine targets. Because of this bias, the focus of alum use in vaccines is in cases where neutralizing antibody responses are sufficient for protection. The extent to which signaling through nALP3 inflammasome is critical for adaptive immune responses stimulated by alum remains controversial. Recent discoveries have identified alternative ways to stimulate the immune system than using alum by studying the natural adjuvants found in pathogens. The controlled manipulation of purified forms of natural adjuvants should

allow for the induction of more natural immune responses (Kwissa et al., 2007). These natural adjuvants are called pathogen-associated molecular patterns (PAMPs) or, more generally, microbe-associated molecular patterns (MAMPs) and are ligands for pathogen/pattern recognition receptors (PRRs) of host cells, particularly DCs. Many PAMPs and PRRs are evolutionarily conserved, indicating that they are key mechanisms for activating the immune system (Takeda et al., 2003; Pulendran, 2005; Steinman & Banchereau, 2007). The PAMPS are microbial components of cells walls, genomic nucleic acids, and protein motifs that are required for microbial survival and therefore cannot be discarded by pathogens despite their detection by the immune system. The eukaryotic hosts have learned to recognize these MAMPs as non-self and, thus, potentially dangerous. Recent advances in immunology suggest that the innate immune system is a critical determinant of the strength and quality of the adaptive immune response, and so, modern adjuvant research has turned towards stimulating PRRs (Pulendran, 2005; Steinman & Banchereau, 2007). Within the innate immune system, DCs, occupy a preeminent position, as they play critical roles in sensing microbial stimuli, and initiating and modulating adaptive immune responses. DCs of an immature type are scattered throughout the body, particularly at the portals of pathogen entry, where they are equipped to sense microbial signature through PRRs, such as Toll-like receptors (TLRS), c-type lectin-like receptors (CLRs), nODlike receptors (nLRs), and RIG-I-like receptors (RLRs). TLRs are a family of at least 11 receptors in mammals, which recognize distinct microbial components. For instance, on the cell surface, lipopolysaccharides (LPS) from Escherichia coli are recognized by TLR4, whereas LPS from leptospira and Porphyromonas gingivalis and the yeast cell wall component zymosan activate TLR2 (Pulendran, 2005). Furthermore, in endosomes, unmethylated DnA from bacteria and viruses are recognized by TLR9, single-stranded RnA is recognized by TLR 7/8, and double-stranded RnA is recognized by TLR3. The CLRs are a family of cell-surface calcium-dependent carbohydrate-binding proteins. The CLRs DC-SIGn, SIGn-R1, and langerin can all detect HIV and Mycobacterium in addition to other pathogens. nLRs are a family of 20 cytoplasmic proteins that recognize bacterial components, including pieces of the cell wall and flagellin. Finally, RLRs are cytoplasmic RnA sensing proteins, which detect specific types of single-stranded and double-stranded RnA viruses. Thus PRRs recognize many different kinds of PAMPs in distinct cellular compartments. Furthermore, stimulation of the PAMPs activates different kinds of innate immune response, which then influences the quality of the acquired immune response (Kwissa et al., 2007). Signaling through TLR7 and 8 or TLR9 induces robust IL-12p70 from CD8 a-DCs and IFn-a production from plasmacytoid DC, which promote the cross-presentation of exogenous antigens and the stimulation of CTLs (Pulendran, 2005; Steinman & Banchereau, 2007). In contrast, stimulation of TLR2 by several ligands has been shown to stimulate IL-10, and induce Th2 and Treg responses (Dillon et al., 2004; Manicassamy et al., 2009). CLRs have also been implicated in inducing a regulatory response or skewing the Th1/Th2 balance towards Th2 through the production of IL-10 and TGF-b (transforming growth factor beta) (Pulendran, 2005). Through the induction of IL-18 and IL-1b, nLRs promote inflammation and may promote a Th2 response. RLRs stimulate the production of IFn-a, which induces an antiviral state in cells and activated macrophages and natural killer (nK) cells. Emerging from this work on the interactions between PAMPs and PRRs are new classes of synthetic adjuvants


for potential vaccine use. Under the brand name AS04, monophosphoryl lipid A (MPL), an LPS derivative, is used in combination with alum in Cervarix, GlaxoSmithKline’s recently approved human papillomavirus (HPV) vaccine. Thus far, MPL is the only adjuvant besides alum used in an FDA licensed vaccine. It is also approved in Europe for use in the hepatitis B vaccine Fendrix and is being tested in malaria vaccines, among other uses. Many additional adjuvants have the potential for human use (Pink & Kieny, 2004). With the growing number of adjuvants at our disposal to mimic natural infections, we need a frame of reference as to how to use them for maximum efficacy. Turning to the functional signatures of innate immunity induced by some of our most successful vaccines is beginning to shed light on this area. new studies on the old vaccine yellow fever 17D (YF17D) have provided interesting insights (Gaucher et al., 2008; Querec et al., 2008). YF-17D is an experimentally attenuated live-virus vaccine, and represents one of the most successful vaccines ever developed (Pulendran, 2009). Despite the lack of understanding at the time it was created about how it induced an immune response, it is one of the most effective vaccines available with immunity lasting

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at least 10 years and as long as 35 years in some reports (Pulendran, 2009). It has been demonstrated to stimulate a mixed Th1/Th2 response, as well as a robust cytotoxic T-cell response. The vaccine activates multiple TLRs, including TLR2, 7, 8, and 9, as well as non-TLR PRRs such as RIG-I and MDA-5 (Querec et al., 2006, 2008), which results in the activation of plasmacytoid DCs and myeloid DCs (Fig. 2). Recently, two independent groups, including ours, applied systems biological approaches to identify early molecular signatures induced by YF-17D after human vaccination (Querec et al., 2008; Gaucher et al., 2008). The goals of our study were to apply systems biological approaches to: (i) obtain novel biological insights about the mechanism of action of YF-17D and; (ii) to determine whether it was possible to identify molecular signatures early after vaccination, which could predict the later immunogenicity of the vaccine (i.e., to identify biomarkers of vaccine efficacy). The latter goal addressed a critical challenge in vaccinology, namely that of identifying vaccinees that would respond suboptimally to a vaccine. These studies evaluated peripheral blood mononuclear cells (PBMCs) from humans (who had not been previously

FIGURE 2 Innate correlates of YF-17D immunogenicity identified by systems biological approaches. YF-17D stimulates polyvalent functional modules of innate immune activation. For example, YF-17D is sensed by TLRs 2, 7, 8, and 9 (which are expressed on distinct subsets of DCs), as well as by RIG-I and MDA-5, resulting in a balanced Th1/Th2 response. YF-17D also induces robust antiviral responses including PKR, OAS 1, 2, 3, L, TRIM5, and complement cascade components such as C1Qb. In addition, EIF2AK4, a key player in the integrated stress response, is present in the signatures that predict the CD81 T-cell responses to YF1-7D. Consistent with this, the expression of other genes involved in the integrated stress response (e.g., calreticulin, protein-disulfide isomerase family A, members 4 and 5) also correlate with the magnitude of the CD81 T-cell response. Consistent with the induction of a stress response, YF-17D induces phosphorylation of eiF2a and the formation of stress granules. Finally, YF-17D induces TnFRSF17 (BCMA), a receptor for BlyS/BAFF, known to regulate B-cell responses.

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vaccinated with YF-17D or infected with yellow fever, and thus immunologically naïve to the vaccine) at various time points following vaccination. Strikingly, the magnitude of the antigen-specific CD81 T-cell responses, and the neutralizing antibody titers measured at day 15 or 60 post-vaccination varied markedly between individuals (Querec et al., 2008). Transcriptomics of total PBMCs revealed a molecular signature comprised of genes involved in innate sensing of viruses and antiviral immunity in most of the vaccinees. Of note, both studies demonstrated that in addition to activating the RnA sensing endosomal TLRs, the gene expression of the cytoplasmic double-stranded RnA receptors of the 2,5-OAS family members 1, 2, 3, and L, and the RLR members RIG-I and MDA-5 were all up regulated in the blood of human vaccinees (Fig. 2). Two key transcription factors that mediate type I IFn responses, IRF7 and STAT1, are also up regulated. Members of the ISGylation pathway, which preserve essential proteins from being degraded during the IFn-induced cellular antiviral state, were increased, including ISG15, HERC5, and RIG-B. Contrasting to all the antiviral receptor and up regulation, YF-17D also upregulated LGP2, which competes with RIG-I and MDA-5 for binding of viral RnA, and thus acts as a negative regulator of antiviral responses. Another PRR group where both positive and negative regulation is induced by YF-17D is in the complement cascade. The complement signature of YF-17D included the up regulation of genes for C1q and its feedback inhibitor C1IN and the increased gene expression of the C3a receptor 1 with corresponding increase in the C3a protein in plasma (Fig. 2). Thus, YF-17D activates multiple pathogen surveillance mechanisms in several cellular compartments: extracellular, cell membrane, cytoplasmic, and vesicular (Fig. 2). We then used additional bioinformatics approaches to identify gene signatures that correlated with the magnitude of antigen-specific CD81 T-cell responses and antibody titers, and which could predict the magnitude of these responses in an independent second trial of YF-17D vaccination in humans. We observed that several signatures for CD81 T-cell responses from the first trial were predictive with up to 90% accuracy in the second trial and vice versa. Of the genes present in these predictive signatures, EIF2AK4 is known to be a critical player in the integrated stress response (Kedersha & Anderson, 2007) and regulates protein synthesis in response to changes in amino acid levels by phosphorylating the elongation initiation factor 2 (eIF2a) (Fig. 2). This results in a global shut down of translation of constitutively active proteins, by redirection of their mRnAs from polysomes to discrete cytoplasmic foci known as stress granules (SGs), where they are transiently stored (Kedersha & Anderson, 2007). Consistent with this, YF-17D induced the phosphorylation of eIF2a, and formation of stress granules (Querec et al., 2008). Moreover, several other genes involved in the stress response pathway were observed to correlate with the CD81 T-cell response (Fig. 2). These observations support the hypothesis that the induction of the integrated stress response in the innate immune system might play a key role in shaping the CD81 Tcell response to YF-17D. Experiments to test the hypothesis are currently underway. In the case of antibody responses, TNFRSF17, a receptor for the B-cell growth factor BLyS/ BAFF (known to play a key role in B-cell differentiation), was a key gene in the predictive signatures. Thus, taken together, these studies provide a global description of the innate and adaptive immune responses that are induced after YF-17D vaccination and stimulates the generation of testable hypotheses about the biological mechanisms that regulate the magnitude and nature of the immune response to YF-17D (Fig. 2).

SYSTEMS VACCINOLOGY

The systems biological approaches used with YF-17D may be broadly applicable in vaccinology in the identification of molecular signatures of vaccine efficacy. This could be especially useful in identifying vaccinees with suboptimal responses among high-risk populations, such as infants or the elderly, or in immunocompromised individuals such as HIV-infected or transplant patients. However, whether the signatures identified with YF-17D can also predict the immunogenicity of other vaccines remains to be determined. In principle, there could be an “archetypal” signature that predicts the T-cell mediated responses of all vaccines, and another archetypal signature that predicts the antibody-mediated responses of all vaccines (Color Plate 11A). However, our preliminary data with the flu vaccines suggest that this is unlikely to be the case (nakaya et al., in preparation). Alternatively, each vaccine could have a very specific signature that was unique only to itself (Color Plate 11B). However, it seems most likely that vaccines that stimulate a similar mechanism of protective immunity will induce similar molecular signatures. For example, vaccines that induce polyfunctional CD81 T cells will have a common innate signature of immunogenicity Color Plate 11C). Other vaccines may predominantly stimulate effector T cells that produced Th2 cytokines and these may have a different innate signature. Thus, there could be a cluster of signatures that predict various facets of T-cell immunogenicity. For B-cell immunogenicity, vaccines that stimulate long-lived plasma cells produce high affinity antibody, and may share a common innate signature. Others that relied on opsonophagocytic antibodies for protection may have a different innate signature; still others may require high affinity antibodies, and a different signature might correlate with these (Color Plate 11C). Thus, one would have a cluster of signatures that predict various aspects of B-cell immunogenicity. Similarly, there could be a different cluster of signatures that predict protective immunity that is not mediated by Tor B-cell dependent mechanisms, but by other mechanisms mediated perhaps by nK cells, DCs, or stress response pathways (Color Plate 11C). The identification of such predictive signatures will facilitate not only the rapid screening of vaccines, but also the stimulation of novel hypotheses about how vaccines mediate protective immunity. The realization of these challenges will ultimately lead to the development of a “vaccine chip,” (Color Plate 12), which could consist of a few hundred genes, capable of identifying predictive signatures for all the correlates of immunogenicity and protection, identified in the database mentioned above.

KEEPING BIOLOGICAL COMPLEXITY IN MIND

The traditional model of adjuvants in vaccinology is one based on a linear chain of causes and effects. An adjuvant is used to activate a PRR, thus stimulating a downstream immune response. However, biological systems are more than just the sum of the genes and proteins, and understanding the behavior of complex biological systems cannot be achieved by only focusing on individual components. This concept is summarized in the term “emergent properties,” which has been defined as “properties that are not present in the isolated component and cannot be predicted, deduced, or calculated from the properties of the parts” (Van Regenmortel, 2007). In order to use functional signatures to design vaccines, vaccinologists need to move beyond merely understanding each of the parts of the immune system with which the vaccine interacts; there needs to be an understanding how the different parts of the immune system interact with each other. Complex behavior


of biological systems cannot be understood by studying parts in isolation (Weng et al., 1999; Van Regenmortel, 2004a, 2004b). The immune system, as with all biological systems, have redundancies, feedback and feedforward regulation, and synergism, which all impact how the instruction of the vaccine are processed (Kitano, 2002). In a mouse model of Helicobacter pylori vaccination, combinations of adjuvants with different modes of action greatly increases antibody responses compared to using single adjuvants (Moschos et al., 2004). Synergism is achieved with TLR agonists when both the downstream signaling molecules TRIF and MyD88 are activated (napolitani et al., 2005). Furthermore, the functional signatures may need to be thought of as more than just a three-dimensional network of genes, proteins, and cells. A four-dimensional analysis may provide a better understanding of vaccine mechanisms—the spatial dimension of vaccine stimulation as it travels among the cells in the immune system or the signaling networks within cells, plus the fourth-dimension of time (Bork & Serrano, 2005). This dimension of time includes the rates, duration, and sequence of immunological signaling. Correlated functional signatures, while serving as a tool to screen potential vaccine candidates, are not as important as establishing direct causal links between vaccine components, innate immune signatures, correlates of protection, and ultimate immunity to disease. Knowledge of direct causal links can be exploited to rationally design vaccines, as well as to serve as screening tools of potential vaccines. The causal link may not be between just one factor and the result. Multiple factors may combine to give a particular result. That is why multidimensional modeling and multifactorial algorithms are important tools for advancing the study of vaccines.

FROM FUNCTIONAL SIGNATURES TO SUCCESSFUL VACCINES

Functional signatures of vaccines can play a role in multiple aspects of vaccine design. As a correlate of protection, functional signatures serve as an indicator of vaccine efficacy. This application has historically been limited to acquired immune responses, but there may be a role for innate immunity in the early stages of multiple vaccine candidate evaluation. Functional signatures of innate immunity to vaccines have the greatest potential utility in understanding the mechanisms of successful immune induction while limiting deleterious side effects. Lessons learned in these studies will lead to improved adjuvants and selective combinations of adjuvants to achieve particular goals. In this context, we have recently designed nanoparticle-based vaccines, similar in size and composition to a virus, containing a specific combination of TLR ligands. Our results indicate that particles containing TLR4 1 TLR7 ligands induce a synergistic enhancement in the magnitude, quality, and persistence of T responses and antibody responses, which last for nearly 2 years after vaccination (Kasturi et al., submitted). These results indicate that synthetic vaccines that resemble the live viruses may recapitulate the immunogenicity of the best empirically derived vaccines. Finally, as the field advances, new functional signatures will probably increase in complexity from the simple threshold antibody titer that has been historically used. This is a reflection of the inherent complexity of immune responses and will yield functional signatures that incorporate balanced humoral, T-cell, and innate immunity measurements. We thank the National Institutes of Health (grants U54AI057157, R37AI48638, R01DK057665,U19AI057266, HHSN266 200700006C, NO1 AI50019, NO1 AI50025) and the Bill & Melinda Gates Foundation for their generous support of our work.

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44 Meeting the Challenge of Vaccine Design To Control HIV and Other Difficult Viruses BARNEY S. GRAHAM AND CHRISTOPHER WALKER

INTRODUCTION

impossible or lead to other prevention solutions. In the case of human immunodeficiency virus type 1 (HIV-1), determining the potential risk of infection to an individual would involve universal testing and diagnosis to define which partners are infected. If this were accomplished, barrier precautions may then be a more attainable prevention strategy than passive prophylaxis with drugs or biologics. Therefore, active immunization or vaccination is a much preferred intervention for both personal and public health. What makes a viral pathogen “difficult” from the standpoint of vaccine development? In Table 1 we review properties of viruses that may predict success or failure in a viral vaccine development effort. Vaccines work by subtly altering the specificity, magnitude, kinetics, or location of the natural immune response. By reducing the inoculum size from a viral exposure with antibody or more quickly clearing virus-infected cells with CD81 T cells (CTL) the host infection can remain subclinical. When the viral pathogen is characterized by limited structural variation, susceptibility to antibody and CTL, long incubation times, and accessibility, then vaccine interventions will be easier to employ. Therefore, it is not surprising that when natural immunity is able to clear an infection in the majority of individuals with mild or no illness, any additional contribution of vaccine-induced immunity may have a profound effect on illness in the overall population. High levels of vaccine-induced efficacy may be an indication that the dynamics of the host–pathogen interaction during natural infection is balanced near the threshold of virulence. These are usually viruses that have been coevolving with humans throughout the ages. Examples include measles, mumps, polio, and rubella, where vaccination is highly efficacious.

There are many ways to address a viral pathogen from a public health perspective. The type of response depends on the potential for exposure to the virus, its lethality, and its capacity to spread. Defining the preferred countermeasures can help prioritize the development of approaches, and should take into consideration the biological characteristics of the virus and its pathogenesis. For viruses with a short incubation period between exposure and disease expression, prevention through vaccination or other forms of prophylaxis will be needed to have a significant impact on morbidity (e.g., paramyxoviruses, orthomyxoviruses, filoviruses, and flaviviruses). When the pathogenesis involves latency, treatment is unlikely to achieve a cure, and prevention through vaccination would be ideal (e.g., lentiviruses, herpesviruses). Passive prophylaxis as an alternative for active immunization may work when the risk is temporary and can be predicted. Passive prophylaxis works for respiratory syncytial virus (RSV) prophylaxis, because it is given to a select population (infants) with known risk for a limited period of time, can be controlled by the caregiver, and is not dependent on patient compliance. Passive prophylaxis has also been successful in the immediate postexposure setting for pathogens with a relatively long incubation period, particularly in combination with active vaccination (e.g., rabies, hepatitis B, hepatitis A). For diseases where the risk is less well-defined and the treatment period is open-ended, passive prophylaxis faces many challenges in addition to compliance failure. Ongoing, passive prophylaxis introduces potential iatrogenic risks to the otherwise healthy individual, a particular concern for certain subpopulations such as children or females of childbearing potential. Also, ongoing passive prophylaxis on a large scale may not be financially feasible. Defining the risk well enough to justify passive prophylaxis may be logistically

FAILURE OF NATURAL IMMUNITY

Viruses that have evolved to frequently establish persistent infection are rarely successful targets for vaccine prevention. HIV-1 and hepatitis C virus (HCV) are examples of viruses that can effectively evade immune defenses and establish persistent replication. Virtually all viruses have evolved mechanisms to alter or evade host

Barney S. Graham, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892. Christopher Walker, Nationwide Children’s Hospital and the Departments of Pediatrics, Pathology, and Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43205.

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IMMUNE INTERVENTION TABLE 1 Characteristics of difficult viruses and those with successful preventive vaccines Difficult Characteristics Infection is not self-limited, has a high frequency of severe disease, and often leads to persistence. Virus has evolved multiple mechanisms to alter and evade host immune responses The host can be reinfected.

T cells play a critical role in immunity, and there is significant genetic variation.

Site of infection is the same as major target organ for disease. Viral genome is integrated or immunoprivileged sites are infected. Animal model fails to recapitulate pathogenesis of human disease. There is a long delay between initiation of infection and onset of adaptive cellular immunity.

immune responses. Avoidance of type I interferons (IFNs) either directly or through alteration of common signaling pathways is one of the most common adaptations. Loss of this important immune modulator affects innate and adaptive immune responses, providing a window of opportunity for virus replication to proceed. Many of the most difficult viral vaccine targets have evolved multiple mechanisms of evading critical immune effector mechanisms important for viral clearance, like CTLs. HIV-1 and herpesviruses have well established mechanisms for down regulating MHC class I expression or transport of peptides into the ER (endoplasmic reticulum) to prevent CTL recognition. Again, there are exceptions. Poxviruses may have the greatest diversity of immune avoidance genes, and yet variola was the target of our most successful viral vaccine. The clearest evidence that a virus is capable of significant immune evasion is whether or not the host can be reinfected. If reinfection or superinfection is a common feature of viral pathogenesis, it suggests that there is capacity for significant immune modulation (as mentioned above), genetic variation, or both. Genetic variation allows a virus to escape epitope-specific antibody or T-cell responses. Functional and structural plasticity, particularly in surface proteins, will render antibodies useless that may have effectively neutralized a virus with the original structural motif. Flexibility in essential processes like uncoating, replication, and assembly may allow sufficient change in potential T-cell epitopes to escape recognition by T cells capable of clearing earlier versions of the virus.

GENETIC DIVERSITY AND IMMUNE EVASION

Most successful vaccine efforts have targeted viruses with a single or finite number of serotypes and have been based on establishing a protective antibody response. It is possible that even for viruses that have multiple mechanisms for evading or modifying host immunity, that if vaccine antigen can be the first to induce virus-

Characteristics Amenable to Vaccination Infections are typically benign and selflimiting, although death and clinical residua may occur. Host immune function is not significantly compromised by infection. After recovery, protection against subsequent disease (although not infection) is complete, often lifelong. Protection is mediated primarily by neutralizing antibodies, and antigenic serotypes are stable and monotypic or limited in number. Pathogenesis includes an obligatory viremia before infecting the target organ. Virus usually does not persist and is not integrated in cell genome. A useful animal model is generally available. Induction of adaptive immune responses occurs immediately after infection.

specific responses, the incoming pathogen may not have time to deploy its defenses in the setting of preexisting immunity. There has never been a viral vaccine licensed based on its ability to elicit protective T-cell responses. It is likely that T cells play a role in protection afforded by some vaccines, particularly replication-competent live virus products, but their role is secondary to antibody. Importantly, CTLs can only recognize and respond to a viral pathogen after cells have become infected, so T-cell-based vaccine protection cannot prevent infection. It is possible for effective CTL responses to rapidly clear virus-infected cells resulting in abortive infection, but infection must first occur for this effector mechanism to be employed. This is one of the many ways in which T-cell-based immunity is complementary to B-cell-based or antibody-based immunity. Teleologically, antibodies are designed to prevent infection while CTLs are designed to clear virus-infected cells. Antibody responses and T-cell responses also differ in the way they accommodate genetic variation in the response to viral infection (Fig. 1). Antibody responses tend to be very type specific. This is what has allowed serotype classification of viruses to be established. Subtle changes in structure have a large impact on accessibility and affinity of antibody binding. There are very few examples of conserved structural motifs across viral serotypes that can be targeted for cross-reactive antibodies. Recent efforts to identify these types of structures in HIV-1 and influenza are based on atomic level resolution of proteins (Kwong & Wilson, 2009). In contrast, the T-cell repertoire and antigen presentation apparatus results in a high frequency of epitope recognition across viral subtypes. Conversely, when considering the genetic variability of the host, most people have the B-cell repertoire and capacity for somatic mutation to recognize a particular structure that would be presented in a vaccine no matter how restricted in size. However, because humans have a limited number of MHC (major histocompatability complex) alleles for antigen presentation, and because T-cell receptors have much less capacity for somatic mutation and depend on

44. Meeting the Challenge of Vaccine Design to Control HIV and Other Difficult Viruses

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FIGURE 1 Immune strategies for coping with genetic diversity. The immune system is designed to accommodate genetic diversity among individuals and genetic diversity of microbes. T-cell epitopes are relatively conserved across viral subtypes compared to antibody epitopes. The implication is that vaccine-induced T-cell mechanisms may be of particular value for highly diverse viruses. Conversely, antibody-mediated protection against a specific structure may be achievable across diverse population groups.

a finite repertoire, restricting the antigenic content of a vaccine may not provide the epitopes needed by some individuals to make the responses required for protective immunity. In settings where virus-specific T cells are essential for controlling virus replication, the implication is that there is (i) extreme genetic variation in viral surface proteins (lentiviruses and the flavivirus HCV); (ii) multiple entry options or avenues of virus transmission and spread that can evade antibody (herpesviruses); or (iii) extreme virulence where small inocula can be lethal within days (filoviruses). The consequence for antiviral vaccine development is that for antibody-based vaccine protection, the design would need to accommodate serologically distinct virus subtypes, and the design of T-cell-based vaccines needs to take into consideration the distribution of human leukocyte antigen (HLA) alleles in future vaccine recipients. Most viral infections for which we have vaccines are self-limited and do not integrate, become sequestered, or infect immunoprivileged tissues to prevent access by adaptive immune responses. An exception to this is the varicella vaccine, which has been successful both in preventing infections in children and in preventing herpes zoster (shingles) (i.e., escape from latency) in adults. In the case of varicella, there was extensive historical evidence that a single infection provided lifelong immunity from a second infection and that herpes zoster was often associated with a distinct deficiency of T-cell immunity, suggesting that adaptive immunity could effectively protect against infection. The capacity for integration or infection of immunoprivileged sites affects the window of time in which vaccine-induced adaptive responses must act in order to prevent disease or persistent viral infection. If protection can be based on preexisting antibodies that can prevent infection, then it may be possible to overcome this challenge. If antibody is only able to reduce inoculum size and T cells are required for clearance, then the timing and location of the T-cell response may be critical for adequate protection.

TIMING AND LOCATION OF PATHOGENIC EVENTS

When the cells or tissue associated with virus transmission and initial infection are distinct from the tissue and cells of the primary target organ of virus-induced disease,

it is a significant advantage for successful vaccine development. The prototypic example is polio, where virus transmission and replication occur in intestinal epithelial cells, but the major target cell for disease expression is the anterior horn cell. Paralytic polio can be prevented by having relatively small amounts of neutralizing antibody in serum that interrupts the viremia required for the virus to reach the target organ. In contrast, when the initial transmission event occurs in the target for disease expression, with no separation in time or space, the threshold for establishing preexisting immunity is much higher. For example, respiratory viruses directly infect the target organ without an intervening viremia. To prevent disease, preexisting antiviral immunity has to prevent infection or control it within hours. Giving passive antibody more than 24 hours after infection does not change disease expression in most animal models of respiratory virus infection, whereas passive postexposure prophylaxis for hepatitis B, which has a longer incubation time, is very effective.

ANIMAL MODELS

Animal models are critical for the preclinical evaluation of vaccines. They are used to define a safety profile and evidence of potential efficacy, which helps to establish an ethical basis for clinical evaluation of candidate products. However, animal models must be interpreted with care and consideration given how they recapitulate the pathogenesis of human disease. The authenticity of the model depends on appropriate simulation of (i) route and inoculum size required for transmission, (ii) cellular tropism, (iii) viral burst size and replication dynamics, (iv) kinetics and patterns of spread within the host, (v) immune response patterns and mechanisms of viral immune evasion, (vi) histopathology manifestations, and (vii) patterns of clinical disease expression. Another important consideration is whether the vaccine and viral challenge is based on the candidate clinical product and human pathogen, or whether it requires a species-specific homologous vaccine–virus system developed in parallel with the human product. Using the actual vaccine and human pathogen in the animal model is a distinct advantage, but there are usually compromises in the parameters of disease pathogenesis that must be

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accommodated when the clinical vaccine product and human virus challenge can be used. When vaccinemediated protection is based on antibody, the animal model is likely to be more reliable. As mentioned above, the specificity and functional properties of antibody are more likely to be preserved across genetic variations in the host. When T-cell-mediated immunity is important for protection, particularly when the challenge virus is not a human pathogen, the animal model may be useful for defining patterns of immunity and principles of vaccination, and for rank ordering candidate human vaccines, but it may not be adequate for product selection. The prototypic example of animal modeling in establishing the basis for vaccine efficacy in humans is polio. In this case, the live-attenuated Sabin human vaccine candidate could be tested for its ability to induce an antibody response to protect against paralysis caused by human poliovirus challenge. In this review, we discuss the immunobiology of HIV-1, HCV, and RSV, which have evaded vaccine development, and discuss potential solutions for these elusive viral pathogens. HIV-1 and HCV represent different virus families with distinct properties, although many of the issues that need to be solved for vaccine development are similar, so they will be discussed together. Collectively, they have caused enormous morbidity and mortality. RSV will be discussed separately, highlighting some unique challenges it poses for vaccine development. Finding ways to vaccinate against these types of viruses would have a positive influence on the public health.

VACCINE DEVELOPMENT FOR HIV-1 AND HCV WILL BE DIFFICULT

HIV-1 has taken a greater toll than any other virus in modern history, despite the fact that it evolved and spread during a time when molecular tools were available for diagnosis and studying basic pathogenesis of disease. Although there has been dramatic success in developing antiretroviral drugs, there is not a licensable vaccine candidate on the near horizon. HIV-1 has all the features discussed under “difficult” viruses (Table 2), and there have been notable vaccine scientists who have suggested vaccine development would be impossible (Sabin, 1992). HCV is also an enormous public health problem as it has infected about 200 million people globally. The majority (about 60% to 70%) have life-long persistent infections that greatly increase the risk of serious liver disease

TABLE 2

Biological challenges for HIV vaccine development

• Genetic diversity – Antigen selection – Immune escape • Lack of natural immunity – No evidence of viral clearance and recovery from HIV infection – Evidence of superinfection • Infection of immunologic “first responders” – Infection of antigen presenting cells – Interference with antigen presentation – Rapid destruction of memory CD41 T lymphocytes • Infection of immunoprivileged sites, sequestered virus, and latency • Failure to induce neutralizing antibody

including hepatitis, cirrhosis, and hepatocellular carcinoma. Persistent infection is currently treated with type I IFN and ribavirin, but it is toxic, expensive, and often fails to achieve a sustained virological response. HCV has all of the properties associated with a difficult vaccine target (Table 1) with one notable exception. Infection is self-limited in some individuals, suggesting that immunity is sometimes effective. Features that distinguish successful immune responses from failed responses to HCV have not yet been identified, however, thus complicating vaccine development. HIV-1 and HCV display extreme genetic diversity, more than any other known viral pathogen. Prior to discovery of these highly mutable RNA viruses in the 1980s, influenza had been the prototype of a viral vaccine target with genetic variation occurring through both point mutations and reassortment of genes. Taking into account all 16 influenza hemagglutins, the genetic diversity is comparable to all the recognized subtypes and recombinant forms of HIV-1 (Kwong & Wilson, 2009). However, a dendrogram representing the genetic divergence of an annual global influenza epidemic is about the same size as one representing the HIV-1 diversity within a single infected individual (Korber et al., 2001; McCutchan 2000). HCV also displays extreme genetic diversity (Ray & Thomas, 2009). There are two major consequences of this relevant to vaccine development for HIV and HCV. First, if virus replication is able to proceed, most neutralizing antibody and T-cell epitopes are susceptible to Darwinian selection pressure, leading to escape from adaptive immune responses. Secondly, the ongoing epidemic has created a vast array of potential virions with diverse antigenic structures that may be transmissible. Therefore, it is difficult to identify a set of antigens that can protect against highly divergent HIV and HCV variants circulating in humans. Lack of natural immunity is a legitimate concern for the prospects of achieving vaccine-induced protection. There is no evidence that complete viral clearance and cure from HIV infection has ever occurred based on natural immune responses, even with the assistance of potent antiretroviral treatment. Only one case, in which an HIV-infected patient who underwent two rounds of ablative chemotherapy and bone marrow transplantation from a donor with the D32 CCR5 mutation, has achieved a proven cure from HIV infection (Hutter et al., 2009). Even more daunting is that there is indirect evidence from the frequency of recombinant strains and direct clinical evidence, that superinfection with alternative strains of HIV-1 commonly occurs in chronically infected subjects (Smith et al., 2005). As noted above, this is not the case for HCV. About 30% of infected humans and chimpanzees, the only animal with known susceptibility to infection with the virus, will spontaneously clear the infection. Moreover, there is evidence that spontaneous resolution of HCV infection can protect humans and chimpanzees from persistence if reexposed to the virus (Bassett et al., 2001; Major et al., 2002). This is perhaps best established in the chimpanzee model of infection, where animals that successfully terminate virus replication appear to have very durable protection against chronic hepatitis C. The magnitude and duration of viremia are often substantially reduced upon rechallenge of immune animals, even with a different HCV genotype. Importantly antibody-mediated depletion of CD41 or CD81 T cells from immune chimpanzees resulted in loss of protection so that infections that would normally clear in a few days were prolonged or


even persisted (Bowen & Walker, 2005a). While these observations provide direct support for rapid control of HCV infection by naturally primed memory T cells, it is important to emphasize that even this protection can sometimes fail. In one remarkable example, an animal that cleared a primary infection resisted persistence during 11 subsequent challenges with homologous and heterologous HCV genotypes (Bukh et al., 2008). Chronic hepatitis C was ultimately established in this individual by reinfection with the very first HCV strain in the challenge series (Bukh et al., 2008). Similar mechanisms of immune protection may also be operational in humans as there is evidence that those who have cleared one HCV infection are at much lower risk of persistent infection when compared with individuals who have no history or prior exposure to the virus (Osburn et al., 2009). However, cross-protection in humans is not absolute, and reasons for failure of natural immunity in these cases are poorly understood. The immune response to HIV and HCV is characterized by innate and adaptive effector mechanisms that are subverted before infections are cleared. The primary site of replication for these small RNA viruses is very different, but they have developed similar strategies to disarm innate immune responses that shape and regulate the effectiveness of adaptive effector mechanisms. Proteins encoded by HIV and HCV have the capacity to prevent innate signaling and alter the function of dendritic cells. As an example, the HCV NS3 protease can destroy signaling intermediates like TRIF and IP-10 that are critical for initiation of the innate immune response by the TLR3 and RIG-I sensing pathways, respectively (Gale & Foy, 2005). HIV proteins can have similar effects on TLR-mediated innate signaling and skewing of cytokine production patterns (Coleman & Wu, 2009). Perhaps the single most important barrier to successful vaccination against HIV and HCV is destruction and/or dysfunction of CD41 T cells (Bowen & Walker, 2005b; Douek et al., 2003). Loss of this critical T-cell subset in either infection is associated with priming of CD81 T cells that are functionally anergic and/or drive mutational escape in targeted HIV-1 and HCV epitopes (Bowen & Walker, 2005b; Petrovas et al., 2009). Moreover, effects on the B-cell compartment are apparent in both infections but are most apparent in chronic HIV infection where polyclonal B-cell activation, B-cell exhaustion, and loss of resting memory B cells is observed (Moir & Fauci, 2009). Preventing HIV-1 infection of CD41 T helper (Th) cells, particularly those that are activated, presents a challenge for vaccine strategies to prevent persistence or slow the rate of disease progression. The problem is just as difficult in HCV infection, where loss of CD41 T-cell help during acute hepatitis C is perhaps the best predictor of virus persistence. Remarkably, only those CD41 T cells that target HCV are inactivated, and they are not thought to be susceptible to direct infection by the virus. Our very incomplete understanding of how HCV causes failure of Th cells is perhaps the greatest hurdle to the development of vaccines to replicate naturally acquired immunity that prevents persistence. Vaccination will also be challenging in settings where the virus has adapted to hide from immune effectors. For HIV, the most important is the establishment of latent infection in resting memory CD41 T cells (Blankson et al., 2002). This is the reservoir that results in recurrent viremia when antiretroviral therapy is stopped in chronically infected people. There is also extracellular virus sequestered in lymph nodes, bound to follicular dendritic

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cells (DCs) through complement receptors, that is not susceptible to antibody neutralization or recognizable by T cells (Keele et al., 2008). In addition, infection of immunoprivileged organs like the brain and the eyes, which are relatively protected from immune effectors, may be a sanctuary for HIV-1. HCV has no capacity to integrate into the host genome as its replication strategy does not involve a DNA intermediate. It is likely that the virus must replicate to survive, although it is clear that persistence is possible in the human host even when replication is too low to detect using the most sensitive detection methods available.

REASONS WHY HIV-1 AND HCV VACCINE DEVELOPMENT MAY STILL BE POSSIBLE

Preventing initial infection may not a reasonable expectation for highly mutable RNA viruses like HIV and HCV, although it may be possible to rapidly clear virus-infected cells to achieve abortive infection. This is illustrated by HCV, where reinfection occurs even in those individuals who have natural immunity and are much less susceptible to persistent infection (Osburn et al., 2009). Another example comes from the recent phase III efficacy trial evaluating a recombinant canarypox vector combined with rgp120 that showed a modest reduction in acquisition. Antiviral effector functions must be amplified before the CD41 T-cell compartment is permanently inactivated or, in the case of HIV, latency is established. A key goal of vaccination against HCV persistence is to accelerate the development of protective immune responses. Adaptive cellular immune responses are delayed for very long periods of time in humans and chimpanzees. Two to three months can elapse before CD41 and CD8 1 T cells are detected in circulation and HCV replication is at least transiently contained (Rehermann, 2009). This long gap between initiation of HCV replication and the onset of cellular immunity, which has not yet been explained, may allow the virus to “outpace” the host response and successfully adapt for lifelong persistence (Rehermann, 2009). Accelerating host immunity by vaccination is predicted to tip the balance in favor of the host, resulting in more frequent resolution of infection. As noted above, the ability of HIV to establish latency presents a unique challenge. The goal for this virus may be to prime functional effector responses by vaccination so that early control of HIV-1 infection may result in reduced disease expression and fewer latently infected cells. While latency is a serious impediment to curing HIV-1 infection, there is a window of time during which latency may not be fully established. Latently infected cells are typically resting CD4 1 memory T cells (Finzi et al., 1999). In vitro, it takes days for an activated T cell to become quiescent and down regulate some of the transcription factors, and modify the chromatin structure to support the maintenance of latency. Therefore, preexisting vaccine-induced immunity may have days and perhaps a few weeks to clear virus-infected cells before latency is established. The interval between infection and the establishment of latency is an important piece of information for vaccine development that is missing. There is evidence both from natural history studies in HIV-1-infected humans and from evaluation of SIV (simian immunodeficiency virus)-infected nonhuman primates that there are natural and vaccine-induced

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immune responses that can result in control of viral replication and delayed disease progression. HIV-1 is known to cause an early loss of gut-associated CCR51 CD41 T cells (Veazey et al., 1998) and loss of integrity of the intestinal barriers to bacterial translocation (Brenchley & Douek, 2008). These events are thought to be important for fueling the T-cell activation that drives HIV-1 replication, disease progression, and infecting others during the time of peak viremia. These early events may also make the HIV-1 infected host more susceptible to superinfections that may not occur if preexisting immunity was not accompanied by activated T cell targets and diminished mucosal integrity.

OPPORTUNITIES FOR ADVANCING VACCINES FOR HIV-1 AND HCV

There is a general consensus that protection against HIV and HCV will be best provided by a vaccine that elicits cellular and humoral immunity. Vaccines that elicit a T-cell response have received the most attention in recent years, particularly for HIV.

Vaccines and Cellular Immunity

Recombinant virus vectors that express HIV proteins, including a poxvirus and an adenovirus, have advanced to human clinical trials with mixed success. A vaccine efficacy trial (the “Step” study) utilizing a recombinant adenovirus serotype 5 (rAd5) vector expressing Gag, Pol, and Nef was stopped at an interim analysis in 2007 because of a higher frequency of HIV-1 infections among vaccinees in the subgroup of men-who-have-sex-with-men (Buchbinder et al., 2008). Subjects received three injections of the rAd5 mixture and had a high frequency of HIV-1-specific CD81 T-cell responses (McElrath et al., 2008). On average, T-cell responses to about two to three epitopes per subject were elicited. The increased infection rate in vaccinees was most closely associated with being uncircumcised. While the cause of the increased rate of HIV-1 acquisition is not well understood, this study showed that the preexisting CD81 T cells elicited in a majority of subjects by this particular vaccine did not lead to reduced viral loads in subjects who became infected with HIV-1. Extensive subgroup analysis is ongoing. Despite the failure of the rAd5 vaccine in the Step study, there are several reasons to believe that induction of CD81 T cells with sufficient breadth and functionality should have a significant impact on disease progression in HIV-1 infected individuals. First, CD81 T-cell response in primary HIV-1 infection is temporally associated with a reduction in viral load (Koup et al., 1994). Secondly, the rate of disease progression is associated with HLA phenotype. For example, persons with HLA-B57 and HLAB27 alleles have slower disease progression, whereas those with HLA-B35 have more rapid progression (Carrington & O’Brien, 2003). In addition, persons with heterozygous HLA alleles generally have lower viral loads than those with homozygous alleles (Carrington et al., 1999), suggesting that the ability to present and respond to more epitopes may hold a benefit. There is pressure on CTL epitopes to mutate, and, when they do, immune control of HIV-1 viral replication is diminished. Nonhuman primate (NHP) studies have shown CTL escape is directly related to increased viral load (Barouch et al., 2002), and vaccine-induced CTL responses are associated with lower viral loads, preservation of CCR51 CD41 T cells in the gut, and delayed disease progression and survival advantage (Letvin et al., 2006).

A trial with 16,402 Thai subjects (both men and women) evaluated a combination of recombinant canarypox vector priming with monomeric gp120 boost using antigens matched to the subtype B and subtype E recombinant strains that are endemic to the region. In early phase clinical trials, this product elicited detectable CD81 T-cell responses, including cytolytic activity in about 25% of the subjects (Thongcharoen et al., 2007). The initial report indicated a 31.2% reduction in acquisition with no effect on viral load setpoint among those who became infected with HIV-1. This hint of efficacy provides an unprecedented opportunity to explore immune correlates of protection. There is one more HIV vaccine efficacy trial testing the concept that induction of preexisting CD8 1 T cells will lead to abortive infection or will reduce viral load in infected subjects. This study evaluates a combination of DNA priming followed by a single rAd5 boost in North American men. The product uses envelope antigens from subtypes A, B, and C, and Gag, Pol, and Nef antigens from subtype B. In preliminary studies, this product was shown to induce detectable CD81 T-cell responses in more than 70% of subjects, and the majority of vaccine-induced T cells are polyfunctional in the sense that they secrete multiple cytokines, or chemokines, or degranulation markers in addition to IFN-g. The results of this study will be available in 2013 (B. S. Graham., unpublished data). To date, there have been no efficacy trials of vaccines to protect against persistent HCV infection. Vaccines that stimulate T-cell immunity can dramatically reduce primary acute phase viremia in experimentally infected animals (Folgori et al., 2006; Rollier et al., 2007) but HCV can sometimes persist nonetheless (Rollier et al., 2007). A role for vaccine-induced T cells in controlling viremia was clearly documented in one notable preclinical experiment involving chimpanzees, where an rAd5 vector expressing nonstructural proteins of HCV dramatically reduced acute phase viremia when compared with unvaccinated controls (Folgori et al., 2006). All vaccinated animals cleared the infection after a prolonged period of low-level HCV replication (Folgori et al., 2006). Vaccines based on the rAd platform with nonstructural HCV proteins are now advancing to early phase human clinical trials to establish safety and immunogenicity. For T-cell-based vaccines to achieve their promise against HIV and HCV, several steps must be taken to prepare for future efficacy trials. First, a more precise understanding of the determinants of CTL-mediated protection—and the conditions under which it might fail—should be developed in animal models. Defining a functional T-cell correlate that could be evaluated and optimized in smaller clinical trials would guide vaccine design and accelerate development for HIV, and in particular HCV, where it may be difficult to assemble large cohorts for efficacy trials. Exploration and development of new assays to measure additional T-cell functions beyond cytokine production, cytolytic activity, and proliferation would also be helpful in evaluating vaccines designed to elicit protective cellular immunity. For both viruses, defining the breadth of response, the number of epitope responses needed for each incoming virus, and the extent of cross-reactive responses needed to defend against the diversity of potential infecting stains will be important for the next generation of T-cell-based vaccines. Defining additional epitopes for which a fitness cost is incurred for escape mutations (Goepfert et al.,


2008; Uebelhoer et al., 2008), and quantifying the relationship between the number of epitope-specific responses and CTL-mediated control of viral replication with more precision are critical ongoing goals for the field. While this process is well advanced for HIV, there is a paucity of data on HCV. New antigen designs employing in silico recombination to select “mosaic” antigens with optimal epitope coverage, and evaluation of new vector combinations and formulations using creative variations of schedule and delivery route may also provide new approaches to induce broader, more effective CTL response (Fischer et al., 2007). Additional challenges remain for development of HIV and HCV vaccines, some of them specific to each virus. One of the confounding characteristics of the NHP model of SIV infection is that macaques have three to four times more MHC class I alleles than humans. This allows them to make a CD81 T-cell response to several-fold more epitopes than humans. In addition, the model needs to be extended to include more authentic challenge approaches, including reproducible low-dose mucosal challenges with standardized virus stocks comprised of monoclonal, oligoclonal, or quasispecies compositions of viral subtypes. The modest efficacy result with the ALVAC/rgp120 vaccine may provide guidance on the stringency and relevance of current model systems. Secondly, there needs to be intensified focus on the early events after infection. It is likely that the dynamics of viral replication and immune response during the first 14 days after HIV-1 infection will determine the long-term outcome for the subject. Defining the exact window between the time of infection and establishment of the permanent latently infected reservoir of quiescent CD41 memory T cells could be done in the NHP model and would provide guidance for the timing and type of immune responses that will be needed to achieve abortive infection. The focus on early infection events should include intensive characterization of viruses that make it through the bottleneck described for transmission. This could significantly simplify the complexity of the antigenic content currently thought to be required for a preventive vaccine. For HCV, the chimpanzee model of infection is close to ideal for the study of vaccines and protective immunity from a biological perspective. However, these animals are rare and endangered. While the model can be used for proof of concept experiments, too few are available for systematic development of vaccines to prevent HCV persistence. A larger problem may be our poor understanding of how HCV inactivates CD41 T cells in humans and chimpanzees to establish a persistent infection. The possibility that some vaccine candidates will prime CD41 T cells that are more susceptible to virus-mediated inactivation, thus leading to enhanced persistence, cannot be excluded in the absence of this information.

Vaccines and Humoral Immunity

Given the history of successful antiviral vaccines, it is imperative to design antigens that can induce broadly cross-reactive neutralizing antibodies against HIV and HCV. Failure of HIV-1 and HCV infection to induce neutralizing antibody to a contemporary virus highlights a key scientific challenge that must be overcome. There are multiple features of the HIV gp160 envelope glycoprotein that make it a difficult target for neutralization (Burton et al., 2004), not the least of which is the paucity of trimers on the viral surface. HIV-1 gp160 is highly

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variable, heavily glycosylated, and extremely flexible until after it engages CD4. Most antigenic sites are not important functionally, and the critical structures required for entry and membrane fusion are elusive antigens that are poor inducers of antibody responses. In addition, the most important structures are not easily accessible targets for neutralizing antibodies even if they can be induced. The first vaccine efficacy trial for HIV-1 utilized a monomeric gp120 in alum. This product induced high levels of antibody that could neutralize type-specific virus, but not primary isolates. The neutralizing activity was primarily mediated through binding the V3 loop in gp120, and much of the antibody binds sites on gp120 that are not accessible on the native trimer. The complete lack of efficacy in this study indicated the need for a better understanding of envelope structure, antibody specificity, and antibody function. There have been broadly neutralizing monoclonal antibodies identified that can effectively block infection with SIV or SIV–HIV chimeric virus (SHIV) when delivered passively (Mascola et al., 2000). In addition, there are conserved structures that have been defined in the CD4 binding site in the outer domain of gp120, and the membrane-proximal domain of gp41 (Kwong & Wilson, 2009). These structures have been defined at the atomic level and the specific residues, angle of insertion, and requirement for membrane contact have been described for interacting with broadly neutralizing antibodies. In persons who have naturally produced a broadly neutralizing polyclonal antibody response, the specificity of the neutralizing activity is usually focused on the CD4 binding site (Li et al., 2007). Therefore, structures exist that could be exploited for vaccine antigens, and the challenge is whether the structures can be built and delivered in a way (Kwong & Wilson, 2009) that would elicit sufficient levels of broadly neutralizing antibody to pierce the Achilles’ heel of HIV-1 at the bottleneck of transmission. With advances in analytical capabilities and the pace of crystallizing proteins and the growing database of structures, this seems more possible than ever before (Kwong & Wilson, 2009). Structure-based design of HIV-1 envelope antigens that focus responses on the CD4 binding site and membrane-proximal domain should be advanced into clinical trials as soon as feasible. The concept of epitope scaffolding is a particularly attractive concept for HIV-1. A well-defined epitope is fitted onto another protein scaffold to hold it in the precise conformation needed for antibody recognition. Only the amino acids required for antibody contact are retained. This approach is attractive because the selected structures can be fitted on multiple protein scaffolds to allow boosting and focusing of the antibody response on the desired epitope. It is also particularly appealing for HIV-1 because it is an approach that uniquely is able to elicit an HIV-1-specific antibody response without an HIV-1-specific CD41 T-cell response. Theoretically, this would reduce the number of susceptible target cells for the virus in the early stages of infection. However, epitope-focused immunization strategies for viral vaccines have not been licensed before. As noted, all of the currently licensed vaccines are based on live-attenuated, whole-inactivated, or virus-like particle platforms that are inducing a broad array of epitopes and immune effectors on a complex particle. Until recently, the known broadly neutralizing monoclonal antibodies against HIV-1 all have unusual biophysical properties. They have long CDR3 loops, hydrophobic

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or charged residues, or posttranslational modifications in the CDR3 binding domains, VH domain swapping, or other unusual properties (Burton et al., 2004). This suggests that part of the explanation for their rarity is the unusual ways in which they were produced and selected. Although new broadly neutralizing human monoclonal antibodies against HIV-1 have been identified that have more typical physical and chemical properties, and that may guide the development of vaccine antigens (Walker et al., 2009; Wu et al., 2010), evaluation of how adjuvants, immunomodulators, or delivery platforms and routes may influence the biochemistry and structure of antibody during the induction phase has not been adequately explored. The technology for analyzing the clonotypic repertoire of the B-cell (and T-cell) response is now accessible and needs to be applied more extensively to the evaluation of vaccines. In addition to pursuing the quest for broadly neutralizing antibodies, other ways in which antibody could be used to impede HIV-1 at the point of entry need to be explored. Since transmission efficiency is so poor, and usually there is only a single virion involved in a transmission event, it is possible that nontraditional antibody functions could impact this process. In particular, the potential for antibody-induced virus aggregation or reduced mobility in mucus should be explored. This becomes particularly important since reduced acquisition in the ALVAC/rgp120 trial was noted. Development of vaccines that elicit effective humoral responses against HCV has been hindered until recently by the lack of robust assays to measure functional neutralizing antibodies. Neutralization of HCV infectivity for chimpanzees was first demonstrated using serum antibodies from humans with chronic hepatitis C (Farci et al., 1994). This animal model for measuring neutralization has obvious limitations. Lentiviral particles pseudotyped with the HCV envelope glycoproteins have provided important new insights into humoral immune responses (Stamataki et al., 2008). However, measurement of cross-neutralization requires the construction of many unique pseudotypes with envelope proteins that sometimes fail. Very few HCV isolates replicate in cell culture and so the repertoire of viruses available for neutralization studies is limited. Despite these difficulties, it is clear that the heavily glycosylated HCV E2 protein does elicit neutralizing antibodies against continuous and discontinuous epitopes in natural HCV infection. Neutralizing antibodies develop more rapidly in infections that spontaneously resolve versus those that persist, but whether they influence infection outcome or are a marker of broader protective immune response is not known (Stamataki et al., 2008). Human monoclonal antibodies can broadly neutralize infectivity of several HCV genotypes for mice carrying human hepatocytes, suggesting that broadly protective antibodies might be elicited by vaccination (Law et al., 2008). Vaccines to elicit neutralizing antibodies against HCV, most notably adjuvanted recombinant HCV E2 protein, do protect chimpanzees from infection with homologous HCV isolates (Houghton & Abrignani, 2005). They may also promote resolution of infection, although it is not clear if such a protective effect is mediated by the vaccineinduced antibodies or by the potent CD41 T-cell response primed by the adjuvanted vaccine.

Vaccines and Immunity in Specialized Tissue

Perhaps the greatest hope for controlling HIV-1 infection through preventive vaccination is its relatively low transmission efficiency. On a population level, hundreds of exposures are needed for each transmission event (Gray et al.,

2001). If HIV-1-induced disease was not clinically imperceptible until years after infection, and if urbanization, high population densities, poverty, global mobility, and other social factors did not collude to spread the virus, HIV-1 would not have been able to become so widespread. Inefficient transmission is related to a bottleneck during entry or early replication events such that most people are infected with only a single virion, and even in those with mucosal ulcers, there are only a handful of viral genotypes that can be detected during the first few weeks after infection (Haaland et al., 2009). It is not yet known whether there are structural or antigenic properties of transmitted viruses that allow viral entry, or whether the selection of transmitted virus is stochastic. If there are distinct characteristics of transmitted virus, it may reduce the antigenic complexity needed for an effective vaccine. It is possible that a minor additional reduction in transmission efficiency would have a major impact on the R0 of the epidemic, and if infection can be blocked at the point of transmission, the other difficult biological features of HIV-1 immune evasion and latency become immaterial for the individual. Blocking infection at the point of transmission is the “Battle of Thermopylae” for vaccine development. The bottleneck through which single virions pass, involves the complex environment of the mucosa where the early destruction of memory CCR51 CD41 T cells occurs in the gut mucosa, and where disruption of mucosal integrity leads to translocation of bacteria that may fuel the ongoing T-cell activation associated with HIV-1 disease progression (Brenchley et al., 2006). Therefore, it is important for us to better understand the immunobiology of mucosal tissue. While most immunogenicity evaluation of candidate vaccines is focused on cells and plasma available from blood, it has been shown that parenteral immunization with gene-based vectors can result in HIV-1-specific T cells in mucosal tissue (Musey et al., 2003). It is also possible to achieve detectable IgG antibody in mucosal secretions through transudation even with parenteral immunization. However, the localization, functional properties, mobility, and capacity for amplification of vaccine-induced humoral or cellular responses in mucosa are not well defined. The mucosal environment of the vagina is also unique, and the transmission bottleneck may be at its most vulnerable in women. Another reason to focus on the point of transmission in women is the finding that women have a VL set point about 3-fold lower HIV-1 genome copies than men (Sterling et al., 2001). This could be because the transmission bottleneck is even more restricted and viruses that successfully infect have phenotypic properties that result in lower fitness. Alternatively, the path through the mucosal environment of the female genital tract may result in different kinetics of viral replication and effector amplification and a different set of dynamics resulting in the lower VL set point. It is also possible that there are genetic differences in the balance of effector mechanisms used in women that may result in a lower VL than seen in men. Future efficacy trials must include cohorts of women to explore these issues. For HCV, the key to solving the problem of anergy induced in CD4 T cells may be understanding the immunobiology of the liver. Most vital organs have evolved mechanisms to protect against an overly aggressive immune response. The liver is unique in that a significant fraction of its cellular content is devoted to professional antigen-presenting cells, and, like the spleen, is considered a reticuloendothelial organ. It is also exposed to


large antigen loads through the portal vein circulation, and is effectively able to clear pathogens that escape the gut before they can do much harm. However, what may be good for managing bacteria and endotoxins may be counterproductive for managing viral infections. The nature of innate immune responses in the liver may also be a factor given their influence on generation of adaptive T-cell immunity.

Genetic Basis of Immunity

There is a new opportunity with the tools available in genetic analysis and transcriptional profiling to evaluate patterns of immune response and immunity on an individual and population basis. Defining polymorphisms associated with adverse events or with immune response patterns would be of value for the field of vaccinology in general, and may be necessary for achieving HIV-1 and HCV vaccine development. If there is a genetic basis for immune responses associated with delayed disease progression or vaccine efficacy, it could eventually lead to modifications in vaccine design that would allow a greater portion of the population to respond favorably. Likewise, if a genetic or transcriptional signature can be associated with qualitative aspects of immunity and be correlated with disease outcome, it may be possible to evaluate and revise vaccines in a more rapid cycle.

The Role of Empiricism

While developing a preventive vaccine against HIV-1 may be as futile as piling Pelion on Ossa, we have to try. This disease, like no other, has penetrated and exposed the most vulnerable aspects of our humanity and challenged our scientific fortitude. Its dramatic influence on public health among adolescents and young adults has compromised our most productive and promising members of society with severe social, economic, and political consequences. Even though we have learned more about HIV-1 than any other known virus, basic research is still critical. There are points of vulnerability during virus transmission and the first few days of infection. Winning the battle early in each susceptible host is essential to preventing the insurgency of chronic infection. Basic research will ultimately illuminate aspects of viral pathogenesis that can be exploited. However, even as we learn how to describe HIV-1 pathogenesis with more precision, there will always be additional outstanding questions, and deeper layers of insight to attain. Consequently, vaccine development is unlikely to progress without empirical attempts to achieve efficacy. This has been made evident by the results of the ALVAC/rgp120 trial, which started under serious controversy and criticism. Having a modest efficacy signal in a clinical trial will now allow basic and translational research to define immune correlates of protection and to understand the predictive value of animal models.

CHALLENGES FOR RSV VACCINE DEVELOPMENT

Discovered in 1956 and the most common cause of childhood hospitalization, RSV is another special case in which an important viral pathogen has been a difficult vaccine target. Like HIV and HCV, for RSV, there is not an animal model that faithfully recapitulates the pathogenesis of the human virus. Rodent models can be used to assess immunogenicity, but are relatively nonpermissive and require large inocula delivered directly into the airway for infection. Therefore, general

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patterns of immune response and basic immunological concepts can be studied in mice and cotton rats, but neither these model systems nor African green monkeys nor surrogate models of bovine RSV or pneumonia virus of mice can be used to accurately predict pathogenicity in humans. RSV has a unique set of challenges distinct from HIV and HCV that will be discussed here briefly. First, the peak age of hospitalization for RSV-induced bronchiolitis is 2.5 months of age. Therefore, immunization to prevent severe illness would have to happen near the time of birth. The immaturity of the neonatal immune system for induction of new responses, the dampening effect of maternal antibody, and the diminished capacity for somatic mutation to achieve high affinity antibody responses makes RSV immunization in this age group logistically and biologically challenging (Williams et al., 2009). Secondly, RSV has evolved multiple mechanisms to interfere with induction and effector functions of type I IFNs. Two nonstructural genes, NS1 and NS2, are devoted to inhibiting these processes, and they are first in the gene order, signifying their importance. Other RSV protein interactions that affect TLR or SOCS signaling contribute to the overall effect on suppressing IFN activity (Oshansky et al., 2009). Third, despite a relatively low level of genetic variation, RSV is able to reinfect humans throughout their lives (Hall et al., 1991). This may be related to conditions 1 and 2 above. The combined effect of the first infection happening at such an early age and the interference with cells and pathways critical for induction of immune responses does something to limit the magnitude and durability of protective immunity throughout the lifetime of the host. Understanding why natural RSV infection does not provide solid immunity against reinfection is a fundamental step needed to successfully develop an RSV vaccine. Fourth, a formalin-inactivated whole virus vaccine that was tested in the 1960s did not prevent infection and resulted in vaccine-enhanced illness in the youngest cohort (including two deaths). This phenomenon is thought to be related to poor neutralizing antibody induction, immune complex formation, and aberrant cellular responses that included Th2 CD4 T cells and eosinophilia (Delgado et al., 2009; Graham et al., 1993). Safety and liability issues, together with practical and biological challenges, have imposed a series of hurdles that both delay and prolong the RSV vaccine development process. However, RSV does have one great advantage compared to other difficult viral vaccine targets in that passively administered neutralizing monoclonal antibody can prevent infection and reduce disease severity. Therefore, if vaccines can be developed that induce effective neutralizing antibody without induction of T-cell responses that may cause safety concerns, there may be a way forward for RSV vaccine development. Atomic level structural information on neutralizing antibody epitopes may provide structure-based vaccine design approaches that can elicit RSV-specific antibody responses without induction of RSV-specific T-cell responses (McLellan et al., 2010). There are also new gene-based vector strategies that can control the specificity and phenotypic properties of RSV-specific T-cell responses and potentially preempt the immunoregulation imposed by natural infection. These approaches may not only provide mechanisms to safely induce neutralizing antibody and disease-sparing T-cell responses to protect against RSV-mediated disease in neonates, but also establish a new immunological paradigm in the host that can achieve more durable protection against reinfection.

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FIGURE 2 Evolution of viral vaccines. The 15 available antiviral vaccines have taken over 200 years to develop. Most of the rapid advances have followed the emergence of new technologies that made vaccine development possible. Vaccines developed because of the discovery of cell culture and the ability to propagate viruses in vitro are noted in green. Vaccines developed as a consequence of tools made available by molecular biology are noted in red. Rapid advances in fields ranging from genetics to computational biology may open new possibilities for developing vaccines against difficult and emerging viral pathogens. The list of future technologies is to suggest areas in which technical advances may provide an impetus for development.

CONCLUSIONS

Of the 15 genera of viruses for which there is a licensed vaccine available, 13 are based on using whole virus—of those, 8 are live-attenuated virus, 3 are whole-inactivated virus, and 2 others (influenza and polio) utilize both platforms. The only two products that are not based on whole virus are for hepatitis B and HPV, both of which comprise virus-like particles. Therefore, all licensed antiviral vaccines utilize the structures and/or antigens of whole virion particles for immunization. This implies that antiviral immunity may be multidimensional and multivalent in ways that are not currently recognized. It is likely that there are mechanisms of vaccine-induced immunity that go beyond neutralizing antibody and cytolytic T-cell recognition, and that there are aspects of specificity that go beyond epitopes and antigenic sites. Ironically, there are successful bacterial vaccines based on subunits (carbohydrate capsules) or secreted toxins that belie the complexity of the pathogen. It may be that for “difficult” viral pathogens, a specific point of vulnerability will need to be identified instead of trying to induce a generic response to the entire virion. Vaccine development for difficult viruses may require a high level of precision in defining the molecular mechanisms of virulence and the structural and functional basis for virus transmission and entry. There are still many viral diseases that have significant impact on public health for which vaccines are not available. Many of these remaining viral targets could be classified as “difficult” based on the properties outlined

in the introduction. We will inevitably be faced with emerging viral diseases for which vaccines would benefit the public health. Addressing these challenges will require new paradigms for discovery, development, manufacturing, and distribution (Graham et al., 2009). It may also require a different level of understanding for viral pathogenesis and immunity. Many prior successful vaccine development efforts have been achieved through empirical and incremental improvement in clinical efficacy using products based on whole virions, without understanding the underlying mechanisms of protection. To develop vaccines for the “difficult” viruses and to be prepared for emerging infections, we need to move from the age of empiricism to the age of rational design based on understanding fundamental immunology, structure, and function. In addition, we need to be creative in applying new technologies to vaccine science. There is a pattern that emerges when the history of successful vaccine development is examined (Fig. 2). The first vaccines for smallpox, rabies, and yellow fever were motivated by specific diseases and were generally achieved by the focused intent of a small number of individuals. However, most of the vaccines developed in the modern era have been more opportunistic. New technologies based on cell culture and molecular biology provide novel vaccine solutions for those prepared to see the potential utility. It is likely that discoveries in structural biology or novel technologies and insights from other fields of science and engineering will provide future solutions for some of our most vexing vaccine design challenges.


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45 Immune Intervention Strategies against Tuberculosis PETER ANDERSEN AND STEFAN H. E. KAUFMANN

LEARNING FROM IMMUNITY TO NATURAL INFECTION FOR VACCINE DESIGN

highly efficacious in killing invading microorganisms. DCs are the most active APCs. In contrast, macrophages or DCs are less efficacious in antigen presentation or bacterial killing, respectively. In humans, M. tuberculosis persists lifelong within macrophages and DCs, independent of whether it causes disease or not (Russell, 2007). Hence, this pathogen must have developed stratagems that allow it to undermine or evade its elimination. After engulfment, bacteria reside in phagosomes that undergo a maturation process from early to late phagosome, the latter one becoming a phagolysosome after fusion with lysosomes (Russell, 2007). Bacterial killing primarily takes place during later stages of phagosome maturation, and degradation of killed bacteria is primarily a function of lysosomal enzymes after phagolysosome fusion. M. tuberculosis arrests phagosome maturation at an early stage by maintaining a neutral pH in the phagosome. Even though M. tuberculosis impairs processing and presentation of antigens through the MCH class II pathway, required for CD4 T-cell stimulation, this impairment is incomplete. In fact, CD4 T cells are stimulated profoundly and are considered the major players in protection against TB (Cooper, 2009; Flynn & Chan, 2001). In macrophages, M. tuberculosis changes from a metabolically active stage to a dormant stage, characterized by a slow-to-absent replication rate and highly reduced metabolic activity. This change in metabolism is the result of a shift in the genetic program, which also results in expression of a different antigen repertoire. Obviously, this has direct consequences for vaccine design (Andersen, 2007; Kaufmann, 2006a). Egression of M. tuberculosis from the phagosome into the cytosol was been proposed some decades ago but only recently demonstrated experimentally (van der Wel et al., 2007). The pathogen not only evades intraphagosomal killing but also enters a milieu rich in nutrients. Residence in the cytosol could also conveniently explain activation of CD8 T cells in TB because foreign antigens in the cytosol are readily loaded onto MHC class I molecules, which present antigenic peptides to CD8 T lymphocytes. A role for CD8 T cells in TB has been convincingly demonstrated (Cooper, 2009; Flynn & Chan, 2001). The innate immune system senses invading pathogens as well as endogenous inflammation by means of so-called pattern recognition receptors. The most important ones are the Toll-like receptors (TLRs), comprising ca. 10 different receptors with specificity for distinct microbial molecular

Compared to most other infectious diseases, 10 million new cases of active tuberculosis (TB) annually is a horrifyingly high death rate. However, of the 2 billion individuals infected with Mycobacterium tuberculosis, a mere 10% of infections transform into active TB disease (World Health Organization [WHO], 2009). These comparisons also illustrate the effectiveness of naturally acquired immunity in containing M. tuberculosis and thus preventing TB disease outbreak. The immune response is quite effective in preventing disease. Unfortunately, it is insufficient in eradicating the pathogen, which persists lifelong in the host independent of whether it causes disease or not (Cooper, 2009; Flynn & Chan, 2001; Kaufmann, 2006b). As a corollary, better understanding of the efficient immune response underlying containment of M. tuberculosis and the inefficient immune response underlying outbreak of active TB disease can provide guidelines for rational vaccine design. Prevention or delay of TB disease outbreak could be achieved by vaccines that mimic natural immunity in resistant individuals. Even more desirable would be a vaccine that not only prevents disease outbreak, but even achieves sterile eradication of the pathogen. Such a vaccine, however, would depend on vaccine-induced immunity that is even better than immunity caused by natural infection.

Innate Immunity as an Early Effector and Instructor for Acquired Immunity

Principally, M. tuberculosis is transmitted via aerosols (i.e., small droplets around 2mm) containing a few M. tuberculosis organisms that reach the alveolar space deep in the lung. There, the first host cells that M. tuberculosis encounters are alveolar macrophages and interstitial dendritic cells (DC). These two cell types are either called professional phagocytes, to underline their role as effector cells in defense, or termed antigen-presenting cells (APCs) to emphasize their capacity to present antigen in a way that stimulates T-cell responses (Trombetta & Mellman, 2005). Macrophages are Peter Andersen, Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark. Stefan H. E. Kaufmann, Department of Immunology, Max Planck Institute for Infection Biology, Chapritéplatz 1, D 10117 Berlin, Germany.

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patterns. Several TLRs form heterodimers, thus broadening the spectrum of recognized molecular entities. The TLRs induce different but overlapping functions. Principally, these functions can be categorized as proinflammatory or as instructive. Proinflammatory responses induce antibacterial defense mechanisms and inflammation. Instructive signals define the quality and quantity of the ensuing acquired immune response (Akira et al., 2006). Sensing of invading microbes or of inflammation stimulates (i) production of cytokines and chemokines in macrophages and DCs and (ii) surface expression of receptors, which primarily interact with counterreceptors on the surface of lymphocytes. Cytokines, chemokines, and surface-expressed costimulatory molecules orchestrate stimulation of the appropriate acquired immune response.

Acquired Immunity

Principally, two types of lymphocytes exist: B lymphocytes, which produce antibodies and hence are responsible for humoral immunity, and T lymphocytes, which interact with other cells by cognate interactions and via cytokines and hence are responsible for cellular immunity. Both types of lymphocytes are also stimulated during infection with M. tuberculosis. However, it is generally believed that T lymphocytes are the major mediators of protection in TB (Cooper, 2009; Flynn & Chan, 2001; Maglione & Chan, 2009). Tuberculosis is a local disease. Although it can afflict any organ, in the vast majority (more than 80% of all forms of disease), TB is localized to the lung. Only in immunocompromised individuals, notably in newborns, can M. tuberculosis disseminate and spread to numerous organs to cause miliary TB. In the affected organ, M. tuberculosis is contained within granulomatous lesions composed of macrophages and DCs as well as T and B lymphocytes (Ulrichs & Kaufmann, 2006). In these organs, the immune response is orchestrated, and the close vicinity of T cells, DCs, and macrophages facilitates efficient interactions. The granuloma is enclosed by a fibrous wall, which shields it from the surrounding tissue. In the highly organized solid granuloma, M. tuberculosis resides within macrophages, which restrict its growth to a minimum. Solid granulomas can contain the tubercle bacilli over decades. To sustain mycobacterial containment over such a long period of time, macrophages fuse to form multinucleated giant cells or Langhans cells. During this containment, both bacteria and host cells will die and the center of the granuloma becomes necrotic. The hypoxic conditions in the necrotic center of a granuloma trigger dormancy in M. tuberculosis (Russell, 2007). The efficacy of the solid granuloma is demonstrated by the fact that active TB disease will not break out. Rather the individual remains latently infected harboring dormant M. tuberculosis with lowto-absent metabolic activity and low-to-absent replication. However, latency is an active process depending on an efficacious immune response, and once immunity breaks down, macrophages in the granuloma release bacteria, which grow unrestrictedly, causing damage to the cells within the granuloma. Cell detritus accumulates and solid caseous material becomes liquefied, which serves as a nutrient-rich source for extracellular growth of M. tuberculosis. The fibrous wall becomes leaky and bacteria can disseminate to other tissue sites via the bloodstream and to the environment via the alveolar system. The patient has become contagious. Recent evidence suggests that the granuloma not only coordinates effector functions but also acquires the capacities of lymphoid organs—a so-called tertiary lymphoid organ develops where naïve T cells and B cells are primed to become memory cells (Day et al., 2010). Hence, the granuloma acquires a degree of autonomy. This raises important questions regarding interpretation of data obtained with peripheral blood cells.

In other words, do immune markers and biomarkers in the blood fully reflect the situation in the granuloma given that it has a degree of autonomy? Recent studies in the model of Mycobacterium marinum infection of zebra fish have allowed deeper insights into the role of granulomas (Davis & Ramakrishnan, 2009). These studies revealed that at early stages of development, granulomas fail to contain, but rather serve, the pathogen. This is because macrophages, which are recruited to the granuloma during its formation, rapidly become infected. Subsequently, however, infected macrophages die and attract additional mononuclear phagocytes. Hence, new granulomas can be formed by infected macrophages migrating to other tissue sites. Note however that once T lymphocytes have started to take over command of the granuloma, containment prevails, and thus the granuloma primarily benefits the host. Antibodies produced during natural infection can attack free M. tuberculosis (Abebe & Bjune, 2009). Opsonization of M. tuberculosis by antibodies induces a stronger oxidative burst and this mechanism may contribute to protective immunity by increasing the killing of M. tuberculosis (Kaufmann, 2007). However, only a few M. tuberculosis organisms are freely available to antibodies because they live inside macrophages, which, in turn, are contained within granulomas. When granulomas liquefy, enormous numbers of bacteria can live freely on the liquefied caseum; however, some do reach the bloodstream and thus become potential antibody targets. It is likely that different T-cell populations contribute to protection, though at different strengths. The number one players are the CD4 T cells of T helper type 1 (Th1). These T cells produce interferon gamma (IFN-g) as a marker cytokine of protection because this cytokine activates macrophages (Serbina et al., 2008). Additional cytokines of relevance to protection against M. tuberculosis are interleukin 2 (IL-2), which stimulates T cells, and tumor necrosis factor alpha (TNF-a), which synergizes with IFN-g in macrophage activation (Serbina et al., 2008). These Th1 cells are members of a larger family of CD4 T lymphocytes, which all recognize antigenic peptides in the context of MHC II molecules (Mosmann & Sad, 1996). The different T cells can be distinguished on the basis of their marker cytokines. The Th17 cells produce IL-17, which stimulates a rapid effector response, mostly carried out by neutrophilic granulocytes (Korn et al., 2009). They may contribute to protection against TB to a minor degree, yet little is known about Th17 cells in TB. The Th2 cells produce the marker cytokines IL-4 and IL-5, which are involved in B-cell maturation and antibody secretion and also stimulate basophilic and eosinophilic granulocytes (Mosmann & Sad, 1996). These latter effectors cells are critical for infection with helminths but considered irrelevant for defense against TB. Perhaps more importantly, Th1 and Th2 cells counterregulate each other. Hence, Th2 cells are generally seen as an unwanted component of cell-mediated immunity in TB. Regulatory T (Tregs) cells produce cytokines, which inhibit Th1 cell responses, namely, tumor growth factor beta (TGF-b) and IL-10 (Belkaid & Tarbell, 2009). Principally, Treg cells are considered important regulators, which deactivate or dampen immunity to avoid collateral damage after pathogen eradication. This important function of Treg cells has been elucidated in acute infections (Belkaid & Tarbell, 2009). In chronic infections such as TB, pathogens persist but are actively controlled by T cells. Hence, weakening of cell-mediated immunity by Treg cells can lead to TB disease outbreak. Obviously, Treg cells are considered undesirable elements of a vaccine-induced immune response to TB, but may, however, serve to dampen an exaggerated inflammatory response in the later stages of chronic TB.

45. Immune Intervention Strategies Against Tuberculosis

Principally, CD8 T cells express cytolytic functions (i.e., they kill target cells) (Harty et al., 2000). In addition, CD8 T cells produce certain Th1 type cytokines, notably IFN-g and TNF-a. Lysis of target cells hidden in macrophages can facilitate the release of M. tuberculosis into the extracellular space, thus making them accessible for more efficient effector cells, such as newly immigrant blood monocytes. Perhaps more importantly, the cytolytic T lymphocytes (CTL) produce perforin and granulysin, which not only lyse target cells, but can also directly kill M. tuberculosis inside macrophages (Stenger et al., 1998). Thus, CD8 T cells appear as an important addition to Th1 cells with overlapping cytokine spectrum and the unique capacity to directly kill M. tuberculosis. Observations on the contribution of CD8 T cells in protection against TB in experimental models have raised the question as to how antigens are delivered to MHC I molecules. This enigma was partly solved with the recent demonstration that M. tuberculosis can egress into the cytosol where its antigens can be loaded onto the MHC I presentation machinery (van der Wel et al., 2007). An additional pathway, which allows for MHC I presentation, is named cross-priming (Winau et al., 2005). Macrophages infected with M. tuberculosis undergo apoptosis, which results in the formation of apoptotic vesicles by the dying cells (Schaible et al., 2003; Winau et al., 2006). These apoptotic vesicles contain antigenic cargo from M. tuberculosis (i.e., proteins and glycolipids of mycobacterial origin). DCs in the vicinity of apoptotic macrophages can take up these vesicles and release the antigens directly into the MHC I processing machinery. This pathway takes advantage of the high phagocytic activity of macrophages with the potent antigen-presentation capacity of DCs. It is likely that cross-priming is responsible for potent stimulation of both CD4 and CD8 T cells (Winau et al., 2005). Unconventional T cells recognize nonproteinaceous antigens. First, the CD1-restricted T cells recognize glycolipids, which are abundant in the mycobacterial cell wall (Brigl & Brenner, 2004). These T cells typically have a Th1 phenotype and some have been shown to express cytolytic activity. The CD1-restricted T lymphocytes express a canonical T-cell receptor comprising an a and a b chain. In contrast, the so-called gamma/delta (gd) T cells express an alternate T-cell receptor composed of a g and a d chain (Hayday, 2000). The gd T cells typically express Th1 functions and sometimes cytolytic activities. It is likely that the CD1-restricted T cells and the gd T cells contribute to protective immunity in TB, although to a minor degree. Precise definition of their role remains elusive due to a lack of valid experimental animal models. Aside from antigen recognition by the T-cell receptor, activation of T cells requires costimulatory signals delivered through receptors and counterreceptors on APCs and T lymphocytes. Many of these receptors belong to the B7 family (Greenwald et al., 2005). Some of them stimulate and others dampen T-cell stimulation. In addition to these costimulator-signaling molecules, cytokines produced by APCs instruct T-cell development (Romagnani, 2005). The cytokine IL-12, and to a lesser degree IL-18, serves as a marker cytokine for Th1 cell differentiation (Goriely et al., 2008; Romagnani, 2005). The cytokine IL-4 promotes Th2 cell development, and a combination of TGF-b and IL-6 promotes Th17 and Treg cell development with high TGF-b concentrations favoring Treg cells and low TGF-b concentrations favoring Th17 cell maturation (Belkaid & Tarbell, 2009; Korn et al., 2009). The latter example already illustrates the complexity of T-cell stimulation, which cannot be nailed down to a single costimulatory molecule or to a single cytokine but rather has to be viewed as an outcome

573

of a complex network of different combinations of cytokines and costimulatory molecules.

Memory and Suppression

The first step of T-cell development is the formation of effector T cells, which actively combat invading pathogens. Immunity induced by chronic M. tuberculosis infection as well as immunity induced by vaccination, however, has to be long lived. This is achieved by a subsequent maturation step, which leads to the formation of memory T (TM) cells. The cytokines IL-7 and IL-15 are critical for the sustenance of the TM cell response (Pulendran & Ahmed, 2006). In contrast, TGF-b dampens TM cell persistence (Belkaid & Tarbell, 2009). The TM cells are characterized by the array of cytokines as well as cytokine and chemokine receptors they express (Sallusto et al., 2004). The mosaic of chemokine receptors also reflects the biological function of TM cells, namely their capacity to either preferentially reside in draining lymph nodes (central memory T cells or TCM cells) or to directly enter peripheral tissue sites suffering from infection and/or inflammation (effector memory T cells or TEM cells). Even though we do not fully understand the functional capacities that render TM cells unique, current evidence suggests that they are typically polyfunctional (i.e., a single T cell concomitantly produces several cytokines relevant to protection). In TB, this includes IL-2, IFN-g, TNF, and granulocyte-macrophage colony stimulating factor (GM-CSF) in different combinations (Foulds et al., 2006; Lindenstrom et al., 2009; Mueller et al., 2008). According to current knowledge, vaccines should induce such polyfunctional TM cells and avoid activation of suppressive mechanisms, notably, Treg cells (Foulds et al., 2006; Kaufmann, 2006b, 2007; Pulendran & Ahmed, 2006). Vaccines will not persist for prolonged periods of time and hence bear a minor intrinsic risk of causing collateral damage. In contrast, chronic infection with M. tuberculosis requires containment by an active immune response. Hence, in TB, suppression of immune responses to avoid collateral damage and sustenance of protective T-cell effector responses to avoid outbreak of active TB disease compete with each other. The driving forces for the balance between these two mechanisms is incompletely understood, although it is generally assumed that suppression is harmful because it favors TB disease outbreak due to weakened immunity.

DTH and Protection: Similarities and Differences

In 1890, Robert Koch described specific elicitation of skin reactions in individuals infected with M. tuberculosis (Kaufmann & Winau, 2005). This so-called tuberculin reaction is still used today to identify infected individuals and forms the basis for the estimation that 2 billion people have been infected with M. tuberculosis (WHO, 2009). Principally, this so-called delayed type hypersensitivity (DTH) reaction is induced by circulating CD4 T cells of Th1 type, which attract blood monocytes to the site of antigen deposition. The time between administration of an antigen mixture, typically the purified protein derivative (PPD), and measurement of a reaction takes about 2 to 3 days for the optimum response to develop. DTH measures the encounter of the immune system with M. tuberculosis and hence cannot distinguish between latently infected individuals and patients with active TB disease. Moreover, the broad variety of antigens in PPD does not allow a distinction between infection with M. tuberculosis and vaccination with BCG (bacille Calmette-Guérin). Further, DTH is almost exclusively mediated by CD4 T cells, whereas protection against M. tuberculosis also includes CD8 T cells. Finally, partial autonomy of granulomas raises the question as to whether

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recirculating T cells sufficiently represent the highly coordinated network of immune interactions in the granuloma (Ulrichs & Kaufmann, 2006).

BCG, THE CURRENT VACCINE

Mycobacterium bovis BCG is the only vaccine currently available against TB. BCG is an attenuated strain of M. bovis obtained after more than 13 years of continuous in vitro passage (Calmette & Plotz, 1929). The genetic background for its attenuation was completely unknown until advances in molecular biology allowed direct comparisons between the genomes of M. bovis and the various strains of BCG. It became clear that the long-term in vitro propagation of BCG had resulted in the loss of a large number of gene segments clustered in different regions of difference (RD) (Behr et al., 1999). The most important of these deletions is the RD1 encoding the important T-cell antigens and virulence factors ESAT6 and CFP10, and this deletion resulted in the original attenuation of M. bovis and is present in all strains of BCG. After its discovery, BCG was evaluated extensively and found to be both safe and efficient in a number of experimental animals including guinea pigs, rabbits, and nonhuman primates. The first clinical testing of BCG demonstrated that this vaccine was highly effective in protecting against TB in children, and today, about 4 billion people have received BCG, which makes this vaccine the most widely used vaccine worldwide. Although the vaccine is well established, discussions of its benefits and drawbacks have never ceased. In particular, the fact that although BCG is generally found to have a very high efficacy in Europe, the efficacy of this vaccine has generally been disappointing in trials conducted in the developing world (Fine, 1995). Current estimates of its efficacy against adult pulmonary TB range from 0% to 80% and in general, the lowest efficacy has been found in the countries with the highest incidence of skin test positivity to tuberculin, presumably due to latent TB and exposure to atypical mycobacteria in the environment (Andersen et al., 2005). Today, a consensus has developed that BCG efficiently protects only skin-test negative individuals (primarily children). Many explanations have been suggested but recent studies in animal models have demonstrated that a preexisting immune response against mycobacterial antigens shared between environmental

mycobacteria and BCG prevents the necessary BCG replication and vaccine take (Andersen et al., 2005). BCG is very safe in immunocompetent individuals, but, as it is a live replicating vaccine, it represents a previously unrecognized risk of disseminated BCG disease in immunocompromised individuals. This was recently demonstrated in human immune deficiency virus (HIV)-infected children in a high endemic setting in South Africa where both of these infections rampage uncontrolled (Hesseling et al., 2004). WHO therefore stopped recommending live, attenuated BCG vaccination at birth for HIV-infected infants, even where there is a risk of TB exposure early in life (WHO, 2007). This change of what has been routine childhood health practice for the past 40 years is a very serious development in regions where BCG coverage is of key importance to prevent TB in children and emphasizes the urgent need to develop not only more efficacious, but also safer TB vaccination regimes.

NOVEL INTERVENTION STRATEGIES FOR TUBERCULOSIS

Novel vaccination strategies against TB can be divided based on whether or not the individual is already infected with M. tuberculosis. Both naïve and latently infected individuals will benefit from a new protective vaccine against TB, but the general consensus in the field has been that it is unlikely that a single vaccine will be effective in both situations. As a consequence, there are two major vaccination strategies that are not mutually exclusive: (i) preventive vaccines administered before exposure or postexposure and (ii) vaccines administered after exposure with M. tuberculosis (Fig. 1) (Andersen, 2007). Strategies for preventive vaccines can further be subdivided into two major branches aimed either at substituting BCG or boosting BCG (Fig. 1). It is generally assumed that the ambitious goal of developing a vaccine to substitute for BCG most likely will be reached with a live attenuated vaccine strain. The booster strategy, on the other hand, builds upon BCG vaccination in childhood and is given as a boost to further increase the immune response evoked by BCG, which can best be accomplished with a subunit vaccine. The booster vaccination will most likely be integrated into the childhood vaccination program, followed by a later boost in adolescence before children leave

FIGURE 1 Vaccination strategies. A preventive vaccine (currently BCG) is given at birth to prevent infection and clinical disease. Novel vaccine strategies aim at either boosting BCG in children (early booster), boosting BCG later in adolescents (late booster), or replacing BCG with novel live vaccines such as genetically modified strains of BCG (rBCG), or attenuated strains of M. tuberculosis. The ultimate vaccine strategy may also be based on a combination of both approaches (i.e., a prime– boost vaccination regime composed of priming with the best possible viable vaccine candidate and boosting with the best possible subunit vaccine candidate). A postexposure booster is designed to be effective against latent infection and to prevent reactivation of TB.


school in order to prevent adult manifestations of pulmonary TB (Fig. 1). Recent data have suggested that it may also be possible to vaccinate with BCG and a novel subunit vaccine simultaneously, thereby taking advantage of the built-in adjuvant potential of BCG to boost the subunit vaccine (Dietrich et al., 2007). Because a vaccine administered as a booster to children or adolescents may also be given to individuals who never received the BCG vaccine or who received an ineffective BCG vaccination (e.g., due to presensitization or incorrect administration), a subunit vaccine should also be able to prime an effective immune response. The recent change in WHO guidelines and contraindication of BCG vaccination at birth for HIVinfected infants has emphasized the need to develop subunit vaccines that can be used without the need for prior BCG priming. In this context, it is relevant that recent data from testing some of the leading adjuvanted subunit vaccines in a model of neonatal vaccination suggest that these vaccines also induce strong adultlike immune responses in neonates (Kamath et al., 2009, 2008). Alternatively, a BCG substitute could be developed, which is safer than the current BCG vaccine. In this context, it is interesting to note that substitutes for BCG in clinical trials have been found to be safer in preclinical models using immunodeficient mice (Grode et al., 2005; Horwitz et al., 2006). Further increase in safety could be achieved by introducing genes encoding a suicide or starvation program into the BCG substitute, which allows for a certain degree of persistence of the vaccine, sufficient to stimulate protective immunity. Subsequently, the viable vaccine is forced into death and, in this way, the risk of dissemination is abrogated. Several strategies are currently exploited toward the generation of a safe, viable vaccine substitute for BCG. Both the booster and substitution strategy have strengths and weaknesses, and eventually, the ultimate vaccine strategy may be based on a combination of both approaches (i.e., a heterologous prime–boost vaccination regime composed of priming with the best possible viable vaccine candidate and boosting with the best possible subunit vaccine candidate) (Kaufmann, 2006a).

PREEXPOSURE VACCINATION: REPLACEMENT OF BCG Killed Bacterial Vaccines

Two vaccine candidates are based on killed bacterial vaccines. The first candidate is the heat-killed environmental mycobacterium Mycobacterium vaccae (Dlugovitzky et al., 2006). The second candidate is RUTI, which is composed TABLE 1

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of semipurified components of M. tuberculosis grown under stress conditions (Cardona, 2006). The M. vaccae vaccine has been tested in several trials in Africa with inconsistent results. However, evidence has been gathered that M. vaccae administration can improve the outcome of chemotherapy of patients with active TB. Recently completed phase III clinical trials suggest partial protection by M. vaccae vaccination against active TB in HIV1 BCG-vaccinated individuals with CD4 counts equal to 200/mm3 (Von Reyn et al., 2010) Similarly, RUTI is considered as adjunct to chemotherapy of active TB and has recently completed a phase I clinical trial (Vilaplana et al., 2010).

Recombinant Live BCG

Two recombinant (r) live BCG candidates as substitutes of the conventional BCG vaccine have entered clinical trials (Grode et al., 2005; Horwitz et al., 2006). Both vaccines are based on the general assumption that BCG has its merits because it can protect against childhood TB but needs improvement because it fails to prevent adult TB. The two candidates have been developed from different angles. The rBCG30 aims at improving antigenicity and the rBCG:DureC:hly aims at improved immunogenicity. Both vaccine candidates are considered as preexposure vaccines that replace BCG. Although they will first be tested alone, they will also be considered for heterologous prime– boost vaccination schedules. The basis of the rBCG30 is the overexpression of the immunodominant antigen shared by M. tuberculosis and BCG, antigen 85B (Ag85B). Ag85B is a member of the Ag85 family, a group of three different gene products involved in cell wall biosynthesis (Table 1). Members of the Ag85 family (either Ag85A or Ag85B) are also used in different subunit vaccine candidates. It is of note that Ag85 is expressed by BCG and hence the higher protective efficacy of the rBCG30 is due to higher abundance of this antigen rather than due to reconstitution with an antigen that is present in M. tuberculosis but absent in BCG. Ag85B is a secreted protein which is highly abundant in culture supernatants of BCG and M. tuberculosis. Strong antibody and T-cell responses in BCG-vaccinated and M. tuberculosis-infected individuals demonstrate its immunodominance for both T cells and B cells during M. tuberculosis infection and BCG vaccination (Hoft et al., 2008). The rBCG30 is more efficacious than the parental BCG counterpart in several animal models. Moreover, the vaccine has passed phase I clinical trial, which revealed high immunogenicity without serious adverse effects (Hoft et al., 2008). The vaccine induced higher numbers of Ag85B-specific CD4 and CD8 T cells accompanied by higher IFN-g secretion (Hoft et al., 2008).

Mycobacterial antigens in leading vaccine candidates

Name

Gene

Biological function or relevance

Use in vaccine

Ag85A

Rv3804c

Ag85B

Rv1886c

Involved in cell wall synthesis; required for the biogenesis of trehalose dimycolate (Kremer et al., 2002) Involved in cell wall synthesis; required for the biogenesis of trehalose dimycolate (Kremer et al., 2002)

ESAT-6

Rv3875

MVA-85A, Aeras 402 rBCG30, H1/IC31, H1/CAF01, H4/IC31 H1/IC31

Mtb32 Mtb39a TB10.4

Rv0125 Rv1196 Rv0288

ESX family, virulence factor; have been suggested to be involved in cell-to-cell spread and the dissemination of infection (Gao et al., 2004) Probable serine protease PPE family protein; function unknown ESX family; essential gene (Sassetti et al., 2003) function unknown

MTB72f MTB72f H4/IC31

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The rBCG:DureC:hly (VPM1002) expresses listeriolysin (hly) to achieve pore formation in the phagosomal membrane. Hly is secreted by Listeria monocytogenes at the acidic pH of the early phagosomes. It is critical for egression of this intracellular bacterium from the phagosome into the cytosol. L. monocytogenes efficaciously stimulates CD8 T cells and it has been conclusively shown that this effect depends on hly expression. However, hly has a pH optimum of 5.5. While such an acidic pH is easily achieved in phagosomes harboring L. monocytogenes, BCG neutralizes the phagosomal milieu and hence does not allow formation of an optimum pH for hly. Hence, urease C (ureC) had been deleted from the rBCG DureC hly. Urease neutralizes the pH by ammonia formation and hence deletion of ureC abrogates neutralization. Indeed, rBCG:DureC:hly allows for an acidic phagosome and thus provides the optimum pH for hly. The rBCG:DureC:hly induces profoundly better protection than parental BCG in experimental mice and is safer than parental BCG in immunocompromised mice (Grode et al., 2005). The rBCG:DureC:hly has successfully completed a phase I human trial comprising both PPD2 and PPD1 healthy individuals. No adverse reactions have been observed and the vaccine is highly immunogenic. Even though the exact mechanisms underlying protection of rBCG:DureC:hly have not been fully elucidated, it is most likely that two not mutually exclusive mechanisms are involved, both based on phagosome membrane perforation by hly. First, perforation allows translocation of antigens secreted by the vaccine and thus direct loading onto MHC class I in addition to MHC class II. Second, because of the acidic pH the phagosome can mature leading to phagosome–lysosome fusion. Perforation of the membrane, however, allows release of lysosomal enzymes, including cathepsins, some of which have been shown to induce host cell apoptosis. It is assumed that these apoptotic events promote cross-priming (Winau et al., 2006). Cross-priming, in turn, allows presentation of antigens from BCG by DCs through the different processing/presentation machineries, including MHC I and MHC II. In an attempt to create live BCG replacement vaccine with increased antigenicity and immunogenicity, an rBCG-expressing pfo and overexpressing several antigens including Ag85A, Ag85B and TB10.4 was constructed (Sun et al., 2009). This vaccine candidate more or less replicates data with rBCG 30 and rBCG:DureC:hly. This is not surprising since the strategy combined the two original strategies, namely, utilization of pfo for perforation of phagosomal membranes and overexpression of dominant antigens.

Deletion Mutants of Mycobacterium tuberculosis

Several live vaccine candidates to replace BCG are based on M. tuberculosis. These strategies aim to attenuate the pathogen through deletion of genes essential for its persistence in the host. At least two genes need to be deleted to reduce the risk of reversion to a virulent strain. The MTBVAC01 is deleted in the transcription regulator gene PhoP and in the FadD gene (Soto et al., 2004). PhoP is critically involved in virulence of M. tuberculosis. Mutations in this gene have been found central to the markedly different virulence of the laboratory strain M. tuberculosis H37Rv (v, virulent) and its attenuated counterpart M. tuberculosis H37Ra (a, avirulent) (Lee et al., 2008). Another attenuated candidate MC26020 has double deletions in lysA and panCD. It fails to accumulate lysine and pantothenate and thus cannot survive in the human host (Sambandamurthy et al., 2006). Both the MTBVAC01 and MC26020 have demonstrated protection in mice and guinea pigs and both strains appear safe in preclinical settings.

PROTEIN SUBUNIT VACCINES Antigens for Subunit Vaccines

The first successful attempts to develop a nonviable subunit vaccine against TB were based on complex culture filtrate preparations, and these vaccines were found by several groups to impart considerable protection in various animal models (reviewed in Andersen, 1997). Stimulated by this success, large efforts were invested in many academic and industrial laboratories to identify single protective antigens—a strategy that was greatly accelerated by the sequencing of the M. tuberculosis genome (Cole et al., 1998). Antigens from M. tuberculosis that activate T cells to produce IFN-g in infected but healthy humans and that provide protection in animal models are generally considered promising vaccine candidates and have so far been identified mostly among secreted antigens (Ag85 complex, ESAT6, and the serine protease family) and in the PE/PPE multigene family (Table 1). (For a recent review see Aagaard et al., 2009.) That protective antigens are particularly well represented in these two groups of molecules was confirmed in a recent large scale discovery effort that evaluated approximately 100 selected antigens and identified a number of novel partially protective antigens that were either secreted by the pathogen or belonged to the PE/PPE family (Bertholet et al., 2008). None of these single antigens has so far resulted in more than partial protection against TB and generally at a level which is below the protection caused by BCG. After careful testing of many potential vaccine candidates in animal models, the leading protein subunit vaccine candidates today employ combinations of five selected proteins expressed as recombinantly engineered fusion molecules (Table 1). Ag85BESAT-6 (H1), Ag85B-TB10.4 (H4), and a fusion of domains from Mtb32 and Mtb39 (Mtb72f) are presently the most advanced adjuvanted subunit vaccines and are all reported to protect against TB close to the level obtained with BCG in mice, guinea pigs, and nonhuman primates when formulated in selected adjuvants (Agger et al., 2006; Dietrich et al., 2005; Langermans et al., 2005; Reed et al., 2009) (Table 2). Given that subunit vaccines will most likely be administered as booster vaccines to BCG-vaccinated individuals, all of these vaccines have also proven the ability to boost immunity from prior BCG vaccinations (Dietrich et al., 2006, 2007; Reed et al., 2009; Skeiky et al., 2009). Principally, Mtb72f and H4 should be stronger BCG booster vaccines than H1, because the antigens present in these vaccines are all expressed by BCG. ESAT-6, on the other hand, is strongly recognized after M. tuberculosis infection but, as an RD1 encoded antigen, it is not expressed by BCG (see above), and the H1 fusion protein therefore both boosts preexisting immunity (due to Ag85B also being expressed by BCG) and induces a response to important TB-specific epitopes, thereby expanding the recipient’s immune repertoire. These two vaccines have performed very similarly in animal models so it will most likely take clinical trials to tell whether boosting or expanding the BCG primed T-cell repertoire is more important for protecting humans against TB.

Adjuvants for Subunit Vaccines

Because immunity to TB strongly depends on Th1 immunity, the adjuvant component is obviously of crucial importance. Recent years have witnessed a breakthrough in our understanding of the molecular mechanisms underlying adjuvant activity. Modern adjuvants are based on vehicles that deliver antigen and often function as a depot at the site of injection and as immunomodulator to activate the innate immune system and the APC through TLR or nonTLR (which recognize PAMPs [pathogen associated molecular patterns]) signaling (Dorhoi & Kaufmann, 2009; Ishii &

45. Immune Intervention Strategies Against Tuberculosis TABLE 2

577

The leading TB vaccine candidates in clinical trials

Vaccine name

Vaccine type

Development stage

Institutions/companies

rBCG30

Live, recombinant BCG

UCLA School of Medicine/Aeras

r-BCG DureC:Hly

Live, recombinant BCG

Clinical phase I completed (Horwitz et al., 2006) Clinical phase 1 completed (Grode et al., 2005)

MVA-85A

Modified vaccinia virus

Aeras 402

Replication deficient Adeno35 Adjuvanted subunit

H1/IC31 Mtb72f/ ASO2A H1/CAF01

Adjuvanted subunit

H4/IC31

Adjuvanted subunit

Adjuvanted subunit

Completed and ongoing clinical phases 1 and 2 (McShane et al., 2004) Completed and ongoing clinical phases 1 and 2 (Radosevic et al., 2007) Completed and ongoing clinical phase 1 (Agger et al., 2006) Completed and ongoing clinical phase 1 (Skeiky et al., 2004) Clinical phase 1 ongoing (Langermans et al., 2005; Weinrich Olsen et al., 2001) Clinical phase I completed (Dietrich et al., 2005; Skeiky et al., 2009)

Akira, 2007; Kawai & Akira, 2009). The vehicles that have been evaluated in TB vaccines include ISCOMs (Andersen et al., 2007a), cationic liposomes (Agger et al., 2008), particles (Kirby et al., 2008), and oil emulsions (Skeiky et al., 2004). Aluminium hydroxide, which is widely used as an adjuvant in classical vaccines and stimulates the humoral arm of the immune system, is not suitable for TB vaccines as it drives a strong Th2 response (Lindblad et al., 1997). The PAMP agonists that have been utilized to potentiate the immune response include lipid A-derived molecules such as MPL (monophosphoryl lipid A) (TLR4) (Skeiky et al., 2004), molecules derived from mycobacterial cell wall components such as TDB (trehalose dibehenate) and MMG (monomycoloyl glycerol) (NonTLR) (Andersen et al., 2009; Davidsen et al., 2005) or lipoproteins (TLR2) (Wang et al., 2007), CpG motifs (Hogarth et al., 2003) (TLR9), and poly:IC (polyinosinic:polycytidylic acid) (TLR3) (Lindblad et al., 1997). Although plenty of adjuvants can be found in the published literature, only very few adjuvants with the desired immunogenic profile fulfill safety and regulatory requirements and have a potential for large-scale production. The selection of adjuvants that are currently in clinical TB vaccine trials is therefore much more limited.

CAF01

The aliphatic nitrogenous compound DDA (dimethyldioctadecylammonium bromide) and TDB form the basis of a stable cationic liposome formulation (CAF01) developed by the Statens Serum Institut. The CAF01 formulation was extensively characterized and found to be very stable (Davidsen et al., 2005). CAF01 is known to efficiently target antigens to activated DCs (Kamath et al., 2009; Korsholm et al., 2007), resulting in strong and long-lived polyfunctional CD4 TM cells that are stably maintained more than 1 year postvaccination (Lindenstrom et al., 2009). The formulation efficiently primes responses also in neonates (Kamath et al., 2009) and has just started clinical phase I together with the H1 fusion protein (Table 2).

IC31

The IC31 adjuvant developed by Intercell contains a polycationic KLK peptide and a TLR-9-triggering nonCpG oligonucleotide (ODN1a), and confers protective efficacy in

Max Planck Institute for Infection Biology/ Vakzine Projekt Management Oxford University Aeras and Crucell Statens Serum Institut/Intercell GlaxoSmithKline Statens Serum Institut Statens Serum Institut/Intercell/ Aeras

challenge models of murine TB (Agger et al., 2006). This adjuvant is currently in clinical trials with both the H1 and H4 fusion proteins, and the first clinical data indicate that IC31 is both safe and induces strong and sustained T-cell responses.

AS01 and AS02

In these adjuvants developed by GlaxoSmithKline, MPL is used together with the saponin QS21, vitamin E, and squalene oil in either an oil-in-water emulsion (AS02) or a liposomal formulation (AS01) (Garcon et al., 2007). In combination with the GSK fusion protein, Mtb72F, AS01 was found to confer strong IFN-g responses resulting in prolonged survival of aerosol-infected guinea pigs comparable to that observed after BCG vaccination (Skeiky et al., 2004). Both of these adjuvants have been in clinical trials together with MTB72F, but currently, the interest seems to focus on the more potent AS01 liposomal formulation.

VIRAL VECTORS

Viral vectors such as adenovirus or vaccinia virus bias the response to the expressed M. tuberculosis antigens towards Th1 CD4 and/or CD8 T cells. The first virally vectored TB vaccine to be tested on humans was MVA-85, a recombinant, replication-deficient vaccinia virus expressing Ag85A from M. tuberculosis (McShane et al., 2004). Results from initial human trials found that it was immunogenic but its real potential is best seen in BCG-vaccinated individuals where much higher frequencies of specific T cells are obtained, suggesting that the vaccine boosts preestablished immunity (Beveridge et al., 2007). Side effects are apparently relatively mild and MVA-85 is currently undergoing multiple phase I/II trials in Africa. MVA has the potential to contain large inserts, and recent attempts to improve its efficacy have focused around multivalent, vectored vaccines that express five antigens, ESAT6, Ag85A, Ag85B, HSP65, and Mtb39A of M. tuberculosis in tandem with IL-15 (Perera et al., 2009). Adenovirus (Ad) has also attracted significant interest for the delivery of TB antigens. Adenovirus has a natural tropism for the airway epithelium and, when delivered by the intranasal route, Ad5 vectored vaccines efficiently boost

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a BCG prime (Santosuosso et al., 2006). In agreement with earlier observation from testing recombinant fusion protein antigens as subunit vaccines (Dietrich et al., 2005), a bivalent expression of both Ag85 and TB10.4 in Ad was found to be more efficient than the monovalent expression system (Mu et al., 2009) . Also, as a heterologous boost of H4 subunit prime vaccination, Ad5 expressing these two antigens was recently found to have potential (Elvang et al., 2009). This prime–boost protocol promoted a very broad and balanced CD4/CD8 T-cell response. Subsequent TB challenge predominantly resulted in the expansion of CD4 T cells, supporting a major role for this subset in the early stage of infection. A vaccine based on replication-deficient Ad-35 expressing Ag85A, Ag85B, and TB10.4 (Aeras-402) is currently under clinical evaluation. There are, as yet, limited data on the efficacy or safety of this vaccine in animal models, but the vaccine seems to be immunogenic and promotes detectable levels of protection in the lungs of mice (Radosevic et al., 2007). The vaccine has passed initial phase I testing in the United States and has recently entered a phase II testing in South Africa. An Ad5-based vaccine candidate expressing Ag85A has entered a phase I clinical trial (Santosuosso et al., 2006). Although viral vaccines are not counteracted by prior sensitization to mycobacteria, they still face issues of sensitization to the viral antigens, which may impact their use as booster vaccines. For Ad-based vaccines, there is evidence that prior humoral responses can reduce vaccine efficacy, and this has shifted interest to Ad type 35 as vector—serological responses to Ad type 35 have a relatively low frequency (ranging from 3% to 5% in industrialized countries and 20% in Africa), compared to Ad type 5. In the case of MVA-85, many adults, especially in TB-endemic areas, will have been vaccinated with the vaccinia vaccine, and there is some data to suggest that this can reduce the efficacy of vaccinia-vectored vaccines (Rooney et al., 1988). However, it is not known what effect this will have in a clinical situation where the duration between vaccinations will generally be many years.

reactions, and the protection/pathology caused by these vaccines has been debated extensively elsewhere in the literature (Lowrie, 2006; Orme, 2006). In parallel to the developments in the subunit vaccine field, multivalent DNA vaccine combinations have also been tested. Pools consisting of up to 10 vaccine targets have been assessed and demonstrated to give increased levels of protection compared to the genes individually (Delogu et al., 2002; Morris et al., 2000). Recently, a multicistronic RNA vaccine was developed that allows expression of multiple antigens by single cells. This strategy exploits the self-cleaving peptide (2A) from the foot-and-mouth disease virus for cleavage of single proteins after translation of the polyprotein. Using three different antigens of three different life stages of M. tuberculosis, namely Rv3407, which is expressed during reactivation; Ag85A, which is expressed by metabolically active M. tuberculosis; and HSPX, expressed during dormancy, vaccination studies in mice were performed. The vaccine construct induced protection comparable to that by BCG (Mir et al., 2009). Despite significant progress in the field of DNA vaccine research since its discovery in the early 1990s, the formal acceptance of this novel technology as a realistic new modality of human vaccine delivery has awaited the successful demonstration of its efficacy in clinical trials. By now, the general consensus seems to be that whereas DNA vaccines have an excellent safety profile their immunogenicity in humans has been insufficient. To overcome this obstacle, there has therefore been a major effort to develop optimized strategies to enhance the immunogenicity of DNA vaccines. In the TB field, attempts have centered around optimized delivery of DNA vaccines by cationic lipid formulations (D’Souza et al., 2002), cationic microparticles (Denis-Mize et al., 2003), and PLG microspheres (Oster et al., 2005). In vivo electroporation is a promising technology that increases in vivo expression of genes markedly compared to injection of the plasmid alone. Recently, this strategy has also been evaluated in the TB field, where it was shown to increase the immunogenicity of vaccines based on Ag85B and ESAT6 (Li et al., 2006).

DNA VACCINES

POSTEXPOSURE VACCINATION

DNA vaccines are easy to make and have the obvious advantage that they can be administered without the need for adjuvants. After injection, the DNA is introduced into host cells, which present the antigen in the context of MHC class I molecules, or they release the antigen, which is then engulfed, processed, and presented in the context of MHC class I by macrophages and DCs (so-called cross-priming, see above). By far, the most extensive testing has been with DNA vaccines encoding the Ag85A. These vaccines have been reported to be highly immunogenic and, in mice, gave partial protection against TB challenge (Huygen et al., 1996). Because Ag85 has been used extensively in adjuvanted subunit vaccines or expressed in viral vectors, the use of a DNA vaccine as a prime followed by a heterologous boost has been pursued by several groups and found to increase the overall immunogenicity and protective efficacy of the vaccination scheme (Gilbert et al., 2006; Liang et al., 2008). The Ag85 family is not the only vaccine target that has been evaluated as DNA vaccine. Mycobacterial heat shock proteins (HSP) (Ferraz et al., 2004; Turner et al., 2000), ESAT-6 (Kamath et al., 1999), the PstS family of lipoproteins (Tanghe et al., 1999), and a number of secreted antigens from M. tuberculosis (Delogu et al., 2002) have all been tested as vaccines and resulted in varying levels of protection. In particular, HSP60 has been the subject of conflicting results and ongoing debate. HSPs are highly conserved and can therefore potentially induce autoimmune

The potential of administering vaccines to latently M. tuberculosis-infected individuals to prevent reactivation has been the subject of debate for the last 20 years, and so far, there has been very limited success with prophylactic vaccine candidates as postexposure or therapeutic vaccines. Today, the need to understand the requirement for a postexposure vaccine is emphasized by the fact that a late BCG booster (to be given to adolescents), due to the high prevalence of latent TB in this age group, frequently would be administered postexposure. Over the years, there has been much debate on safety of a postexposure vaccine strategy, stimulated by the original failure (and death of several patients) of Koch’s tuberculin as a therapeutic vaccine (reviewed in Kaufmann & Schaible, 2005; Kaufmann & Winau, 2005; Rook & Stanford, 1996). However, based on recent animal data, this anxiety may be exaggerated. Vaccination with 12 different vaccine preparations in a low bacterial burden mouse model developed to reflect latent TB did not induce any reactivation or worsening of pathology (something that was, in fact, only seen with BCG) (Derrick et al., 2008). However, even if they do not constitute an obvious safety problem, there are significant challenges for postexposure vaccines. As M. tuberculosis adapts to the conditions in the immune host and transforms from a rapidly growing metabolically active state to a state of nonreplicating persistence with low metabolic activity, its gene expression profile is fundamentally changed,


leading to a different antigenic repertoire. This has been confirmed in human studies, showing that the antigens preferentially recognized by the immune system differed in latently M. tuberculosis-infected healthy individuals compared to patients with active TB disease (Demissie et al., 2006; Schuck et al., 2009). Mimicking conditions thought to reflect the environment inside the granuloma in vitro and evaluating the transcriptional response has thus been the subject of intensive research in recent years and include hypoxia (Rustad et al., 2008; Sherman et al., 2001) and nutrient starvation (Betts et al., 2002). What is of key importance is obviously the direct demonstration that the genes up regulated in vitro under these conditions are of similar importance in vivo. Based on the study of transcriptional profiles at various stages of M. tuberculosis infection in the mouse lung, such a pattern is in fact now emerging emphasizing the influence of the host immune status on this change in pathogen gene expression (Shi et al., 2003). The challenge, therefore, is to transform all of these antigen hits into a vaccine that effectively protects against reactivation of the disease. Efforts toward this goal are currently ongoing in several laboratories. Recent data support the idea that the addition of late-stage antigens to the well-established prophylactic vaccines (i.e., the candidates under clinical testing today) can provide the basis for multistage TB vaccines with activity against all stages of infection (reviewed in Andersen, 2007). One of the few vaccines with reported protective activity postinfection is a Hsp60-DNA vaccine. This vaccine was found to markedly reduce both pulmonary and splenic bacterial loads in mice with established TB (Lowrie et al., 1999). However, its therapeutic efficiency and safety was subsequently questioned (Taylor et al., 2003). A few recent reports support the therapeutic potential of DNA vaccines (Ha et al., 2005; Zhu et al., 2005), but Morris and colleagues used a combination of several DNA vaccines (including Ag85A) with demonstrated prophylactic activity and detected no effect against reactivation (Repique et al., 2002). The explanation for these contrasting results is unclear, but it is important to keep in mind that in addition to the induction of antigen-specific responses, the therapeutic effects of DNA vaccines can also be due to short-term, nonspecific stimulation of the innate immune system via CpG sequences. Most animal models for latency represent variations of the classical Cornell model (Scanga et al., 1999), in which animals are treated with antibiotics to reduce the bacterial load down to low levels and vaccines are administered to monitor the prevention of relapse from this artificial state of latency. In one study, a more simplistic angle was chosen by studying the influence of vaccines administered immediately (less than 10 days) postexposure in the guinea pig model. Under these circumstances, a classical preventive vaccine such as Ag85B/ESAT6 was found to have significant influence on bacterial loads but not on the long-term outcome and survival of the animals (Henao-Tamayo et al., 2009). The relevance of this observation (from a model that is clearly not reflecting latent M. tuberculosis infection), for real life postexposure application of vaccines to individuals in different stages of latent infection and subclinical disease remains to be established.

FUTURE VACCINATION STRATEGIES Subdominant Epitopes

The antigen repertoire encountered during infection with a complex pathogen like M. tuberculosis is represented by numerous potential T-cell epitopes but, in reality, the immune response during infection is often focused toward relatively

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few immunodominant epitopes. These naturally occurring T-cell responses are usually directed to the same epitopes as the ones targeted by conventional vaccination. One way of broadening the repertoire is through immune responses to subdominant epitopes that are normally “silent” and neither primed during infection nor primed by conventional immunization. If available during infection, T cells directed to these subdominant epitopes can recognize the infected macrophage and mediate high levels of protection. This was convincingly demonstrated in TB challenge studies in mice, where vaccination with a single subdominant epitope from ESAT6 was demonstrated to provide efficient protection against M. tuberculosis (Olsen et al., 2000). In a recent study, an ESAT-6 vaccine molecule was engineered that lacks the immunodominant epitope localized within the first 15 amino acids of the ESAT-6 sequence. Vaccination with this construct promoted responses to subdominant epitopes that were not detectable after immunization with the full size molecule or during the natural infection. This gave a better protection against M. tuberculosis challenge than the response directed to the immunodominant epitopes promoted by vaccination with full size ESAT6 (Aagaard et al., 2009). Although still in its early stage, these findings clearly illustrate the vaccine potential if future TB vaccines can be designed to “unmask” the full breadth of the epitope repertoire they encode.

Immunomodulation

Current strategies of vaccine design aim at potentiating vaccine-induced protection. Generally, emphasis is given to the mechanism participating in protective immunity. In TB, this mostly includes increased CD4 Th1 and CD8 T-cell responses. However, infection with M. tuberculosis often results in latent infection, and outbreak of TB is a consequence of a weakened immunity. Thus, outbreak of TB disease can be caused by exhaustion or suppression of protective T-cell responses. Recent investigations, notably in the field of viral infections, revealed mechanisms underlying exhausted Tcell immunity (Barber et al., 2006; Day et al., 2006). As a result of persistent antigen stimulation, T cells express PD-1 (programmed death 1), and signaling through this surface receptor causes death of these T cells. PD-1 is stimulated through interactions with its counterreceptor PD ligand (PD-L) expressed on APC, notably DCs. PD-L and PD-1 are members of the B7 family of coreceptors. Recent findings in TB suggest a similar mechanism (Jurado et al., 2008). In tuberculous granulomas, both CD4 and CD8 T cells express PD-1. Moreover, infection with M. tuberculosis causes PD-L expression on DCs. Recently, it was found that PD-L expression is induced by stimulation through DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin). DC-SIGN is a well-known inducer of suppressive mechanisms in TB, and lipoarobinomannan (LAM), an abundant glycolipid of M. tuberculosis, serves as ligand for DC-SIGN (van Kooyk & Geijtenbeek, 2003). Accordingly, DCs stimulated by mycobacterial lipids not only express PD-L, but also suppress T-cell responses, which can be reestablished by blocking the PD-L/PD-1 cross-talk between DCs and T cells (unpublished results). Although, at this stage, highly speculative immune intervention strategies can be envisaged, which block PD-L/PD-1 signaling and thus Tcell exhaustion. This could prevent or at least delay outbreak of TB in latently infected individuals. The second mechanism of immune suppression is mediated by Treg cells. Treg cells have been identified in both human and mouse models. Moreover, experimental models have shown that Treg cells impair protective immunity against TB (Kursar et al., 2007; Scott-Browne et al., 2007). Hence, future vaccination strategies should not only aim at

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activation of effector and memory T-cell responses, but also at inhibition of inhibitory responses, such as T-cell exhaustion and Treg cell stimulation.

Sterile Eradication of M. tuberculosis

Current vaccination strategies are primarily aimed at delaying disease outbreak. The hope behind this strategy is that reactivation, and therefore outbreak, of active TB could be delayed lifelong. However, reemergence of TB is a consequence of the acquired immune deficiency syndrome (AIDS) pandemic. The causative agent, HIV, impairs CD4 T-cell responses, which are critical for containing M. tuberculosis in a state of dormancy. It is obvious that HIV will impair vaccine-induced T-cell responses. Thus, prolonged latent infection under the influence of vaccine-induced immunity will be dramatically shortened by HIV coinfection, leading to disease outbreak despite prior vaccination. At the first encounter with M. tuberculosis—typically in the lung after aerosol infection—the host is faced with very few organisms of M. tuberculosis. Mechanisms that would directly eradicate these pathogens in the lung would prevent infection and, hence, TB disease outbreak. It is conceivable that antibodies, which attack M. tuberculosis as soon as it enters the alveolar space, could prevent stable infection (Kaufmann, 2007). Even though speculative at this stage, antibodies directed at molecules, which are essential for survival of M. tuberculosis in the lung, could cause starvation of the pathogen. For example, M. tuberculosis strongly depends on the availability of sufficient iron and, hence, uses different scavenger molecules for iron accumulation. Blocking of these molecules would reduce the available iron concentration for the microbe, causing death by iron starvation. As an adjunct, these antibodies should possess strong opsonizing activity, by which they could activate an oxidative burst and perhaps also production of reactive nitrogen intermediates. The concerted action of strengthened host effector mechanisms and weakened pathogen survival could prevent stable infection.

CURRENT STATUS OF CLINICAL TRIALS

The primary goal of vaccine phase I trials is to show that a vaccine is safe and immunogenicity is included as a secondary parameter. Most of the TB vaccines in clinical development have started with phase I trials in carefully screened individuals without evidence of previous M. tuberculosis infection, before proceeding to phase I trials in healthy PPD1 individuals. One exception to this general rule was the H4/IC31 vaccine, where clinical testing started directly in BCG vaccinated individuals (Aeras Global TB Vaccine Foundation, 2010). Both Ag85B-ESAT-6 and Mtb72f, the two other recombinant fusion protein vaccines in clinical trials, have been shown to be well tolerated without any adverse reactions and to be highly immunogenic in humans. Ag85B-ESAT-6 was administered in the IC31 adjuvant by intramuscular injection in a phase I trial, whereas the Mtb72f was given in the AS01 or AS02 as described above (Skeiky et al., 2004). For both fusion proteins, expanded phase I studies testing the vaccines in BCG vaccinated and latently infected individuals from TB endemic regions are ongoing. Also, the two virally vectored vaccines, Ad35 and MVA-85, have completed a number of phase I studies. MVA-85 has shown promising safety and immunogenicity and has recently entered proof of concept phase IIB trials in high endemic settings in South Africa (Emergent BioSolutions, 2009). rBCG30 has completed the clinical trial, where it was found to be immunogenic and as safe as BCG. In a similar vein, rBCG:DureC:hly (VPM1002) has completed a phase 1

clinical trial without adverse side effects (L. Grode, personal communication). This includes both tuberculin-negative individuals without previous contact to M. tuberculosis and tuberculin-positive individuals, most likely because of prior BCG vaccination. Although the vaccines will be tested in adults initially, age deescalation will be included in phase II trials since the vaccines are aimed at replacing BCG and thus will be given early in life. All the nonviable vaccines currently in clinical trials will be tested as BCG-booster vaccines in children for two reasons. First, in the absence of efficacy data in humans, it would be ethically difficult to justify replacing BCG with a subunit vaccine. Secondly, it is unclear how long immunity will last, so boosting BCG-induced responses in adolescents may offer longer overall protection. Therefore, the vaccines will be tested in adults (for safety and ethical reasons) but age de-escalation will be included in phase II trials. Studies involving immunogenicity and protection in animal models can indicate which new TB vaccines and vaccination strategies are most promising. However, ultimately protective efficacy can only be measured in phase III trials, and without a clear correlate for protective efficacy, it will likely be impossible to objectively prioritize among candidates that have passed phase I and II trials. As a result of this, despite the cost involved, it is likely that several vaccines will progress to phase III testing. Efficacy trials will use clinical endpoints such as pulmonary TB disease to monitor efficacy. Given the slow development of TB disease, these trials will need large cohort sizes with long-term follow-up periods. However, new diagnostic and potentially even prognostic tests capable of distinguishing between immunity induced by BCG vaccination and M. tuberculosis infection and monitoring the process of developing active TB disease might allow for a surrogate endpoint for future vaccine trials (Andersen et al., 2007b; Parida & Kaufmann, 2009).

BIOMARKERS IN CLINICAL TRIALS

The decisive moment of novel vaccines against TB has to wait for the outcome of phase III clinical trials. Even though these trials will be performed in highly TB-endemic areas, large numbers on the order of several tens of thousands of trial participants, as well as several years of trial duration are expected. Moreover, several vaccine candidates will be ready for testing each of them representing a medical entity by themselves. Hence, combinations of different candidates (e.g., in heterologous prime–boost schemes) will take even longer. Biomarkers could not only shorten clinical trial periods but also provide information about weaknesses and strengths of individual candidates, and thus provide helpful guidelines for the formulation of more efficacious vaccination strategies (Jacobsen et al., 2008; Kaufmann & Parida, 2008; Parida & Kaufmann, 2009). Biomarkers have already become essential elements of the approval process for many new medicines. In the field of vaccine development, biomarkers could serve as a surrogate endpoint of disease outbreak if they can predict the development of active TB in a vaccinated or nonvaccinated study population (Parida & Kaufmann, 2009; Sadoff & Wittes, 2007). It is most likely that in the field of TB vaccines, a single biomarker will not suffice and that a combination of different markers will be required—in short, a bio signature. Because of the profound impact of T cells on protection, the field of TB vaccine development differs fundamentally from conventional vaccines. For example, vaccines against diphtheria induce antibodies that neutralize the diphtheria toxin. Accordingly, neutralizing antibodies of a certain titer can be used as a biomarker and surrogate of protection of diphtheria vaccines. Such simple biomarkers do not exist


for vaccines against TB where protection critically depends on T cells. IFN-g production by CD4 T cells of Th1, in response to specific antigens, can be used for measuring the strength of the immune response induced by subunit vaccines. Yet, it needs to be established if they sufficiently describe the degree of protection afforded by such a vaccine candidate. The situation becomes further complicated for testing of viable vaccine candidates that express a plethora of antigens. Although IFN-g-secreting CD4 T cells with specificity for discriminative antigens of M. tuberculosis are robust indicators of immunity against TB, they are an insufficient readout for protective immunity (i.e., they fail to predict risk of disease outbreak) (Mittrucker et al., 2007). Such T-cell assays could be improved by broadening the spectrum of secreted cytokines and inclusion of different prototypic antigens of active TB disease versus latent infection (Black et al., 2009; Demissie et al., 2006; Lin & Ottenhoff, 2008; Schuck et al., 2009). Further refinements could include phenotypic characterization of the antigen-specific T-cell populations, such as categorization into TEM, TM, and subtyping into Th1, Th2, Th17, and Treg cells. Determination of specific immunologic markers should be complemented by global biomics approaches, including transcriptomics, proteomics, and metabolomics (Idle & Gonzalez, 2007; Jacobsen et al., 2007; Parida & Kaufmann, 2009). Most likely for these studies, peripheral blood will be used—peripheral blood leukocytes for transcriptomics and serum for proteomics and metabolomics. Transcriptomics measures global gene expression of peripheral blood cells, assuming that they reflect the status of immune cells even though TB is focused on granulomatous lesions at distant sites such as the lung (Jacobsen et al., 2008, 2007; Maertzdorf et al., in press; Jacobsen et al., in press; Berry et al., 2010). Metabolomics and proteom-

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ics measure small metabolites or proteins in serum. Both have successfully passed proof of principle by showing that they can reliably differentiate between healthy M. tuberculosis-infected individuals and patients with active disease (Agranoff et al., 2006; Parida & Kaufmann, 2009). Metabolomics also includes analysis of exhaled volatile substances in breath. An easy assay that measures volatile substances in a simple breath test would be highly attractive. Proof of principle has been published recently (Phillips et al., 2007; Syhre et al., 2009). In a proofof-principle studies, transcriptomics proved to be capable of distinguishing between active TB patients and healthy tuberculin-positive individuals (Jacobsen et al., 2007; Maertzdorf et al., in press; Jacobsen et al., in press; Berry et al., 2010). In fact, in these studies, only three different genes were required for reliable differential diagnosis. Future work will be required to combine the different assays and identify the most robust markers for a custom-made biomic signature that will allow assessment of vaccine efficacy in clinical trials.

CONCLUSIONS

Increasing awareness and investment from public or private funds such as the European Union (EU), National Institutes of Health (NIH), and the Bill & Melinda Gates Foundation has, in recent years, accelerated TB vaccine research, development, and evaluation. Although a novel improved vaccination strategy against TB is finally on the horizon, the eventual success will still depend on continued close integration of information from basic research and translational research both from the TB field and from other related areas. Vaccination with a novel vaccine can have many potential outcomes (Fig. 2). Current vaccines prevent

FIGURE 2 Different types of TB vaccination outcomes. The figure describes the course of disease in nonvaccinated individuals (solid line) and different forms of development that the disease may take. The dotted line depicts the outcome of vaccination, which delays TB outbreak. This is the lowest hurdle followed by consistent control of dormant M. tuberculosis to achieve lifelong latent infection, or, in other words, prevention of TB outbreak. A better option would be vaccines that achieve sterilizing immunity. An alternative would be vaccine-induced prevention of stable infection with M. tuberculosis (“vaccine prevents infection” line). The two latter options guarantee protection against TB even if an individual becomes infected with HIV, which causes disease outbreak in latently infected individuals.

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outbreak of TB disease but do not achieve sterilizing immunity. Hence, the labile equilibrium between immune control and dormant M. tuberculosis can be disturbed, leading to TB disease outbreak. Even though a first generation vaccine can delay TB outbreak, it will not prevent it. This, for example, is the most likely scenario in individuals coinfected with M. tuberculosis and HIV, even if they had been vaccinated with a vaccine aimed at preventing TB without achieving sterile pathogen eradication. Thus, vaccines that would achieve sterilizing immunity would be far more desirable. Current vaccination protocols have not achieved sterilizing immunity in experimental models. However, it is hoped that ultimately, a vaccination scheme will be developed that could be crowned with such a result. Current hope lies on a heterologous prime–boost combination with a best replacement vaccine for BCG in combination with the best possible booster vaccine, perhaps supported by additional immunomodulatory interventions. As an alternative, vaccines could be envisaged that prevent infection since they successfully eradicate the pathogen soon after its entry (e.g., in the lung), and before it hides within macrophages and perhaps other host cells. One bottleneck that the TB vaccine field is facing is the very few adjuvants that can be used for clinical trials. Today, only few adjuvants are approved for human use and the access is very restricted due to industrial constraints. Hopefully, a number of important breakthroughs in recent years in our understanding of the requirement and mode of action for efficient adjuvants will continue to benefit the TB field where the number of potentially interesting novel vaccine antigens by far outnumbers the limited number of adjuvants with the desired profile available for clinical testing. Another roadblock in our efforts to develop the best possible TB vaccine strategy is our limited understanding of latent TB. Any new BCG booster vaccine would have maximal impact in TB-endemic regions if administered in adolescence before the highest risk period for the development of pulmonary TB. Because of the high prevalence of latent TB in high endemic regions, a vaccine targeting this age group would frequently be administered postexposure. This emphasizes the need to continue the ongoing efforts to understand the preclinical and clinical requirement for a postexposure vaccine. For the leading candidates, there is no guarantee that they will progress through phase III clinical trials and registration (Brennan et al., 2007). Experiences from HIV and malaria vaccine trials have taught us how important it is to continue preclinical research and keep developing new and even better vaccines strategies for the pipeline.

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46 Immune Intervention in Malaria CAROLE A. LONG AND FIDEL P. ZAVALA

INTRODUCTION

IMMUNITY TO SPOROZOITES AND LIVER STAGES OF PLASMODIUM

In recent years, long overdue attention has been focused on the global problem of malaria, including the infusion of new resources into the area from governments and philanthropic groups. Incremental interventions such as bednets and drug therapies have resulted in significant improvements in child morbidity and mortality in some afflicted areas. However, there is a general consensus that new tools are needed to control and perhaps eventually eliminate this disease. One of these tools is a vaccine, but despite years of effort, progress has been slow. While it is encouraging for vaccine researchers that those living in endemic areas do eventually develop resistance to the clinical symptoms and the high parasitemias of malaria infection, these are complex, eukaryotic organisms with thousands of genes and sophisticated strategies for evading host immune responses. Because of the parasite’s complex life cycle, there are three major targets of vaccines: the infectious sporozoites and the liver cells they invade, the erythrocytic stages responsible for the pathology associated with the disease, and finally the sexual stages which first circulate in the vertebrate host and are responsible for transmission through the mosquito vector (Fig. 1). Overall, each of these stages is antigenically, morphologically, and biochemically distinct so that different researchers have focused on different stages, with different goals: sporozoites for prevention of infection or early liver stage development, erythrocytic stages to limit malaria morbidity and mortality, and the sexual stages to reduce transmission to others. Many have spoken of the need to combine antigens from all three stages as a final vaccine but at present most efforts have focused on a single developmental stage. Therefore, we will review vaccine development efforts separated by stages of infection. (Note that due to space constraints this chapter includes a very selected reference set focusing on reviews. Please consult these for more complete references.)

A number of immunization studies using experimental animal models and humans performed in the 1960s and 1970s demonstrated that protective immunity against preerythrocytic stages of malaria parasites could be induced by immunization with irradiation-attenuated plasmodium sporozoites (Clyde, 1975; Nussenzweig et al., 1967). These early studies indicated that antibodies present in the sera of immunized animals or humans bound to the surface of sporozoites had abolished their capacity to invade hepatocytes, thus preventing subsequent infection of red blood cells. These antibodies induce a distinct reaction in the surface of sporozoites named circumsporozoite precipitation (CSP), which seems to suggest the release or detachment of certain components of the parasite’s surface (Vanderberg et al., 1968). Years later, it was shown that in addition to antibodies, CD81 and CD41 T cells were induced after immunization with sporozoites (Li et al., 1993; Schofield et al., 1987), and it was demonstrated that they also played an important protective role, as antigen-specific T cells efficiently inhibited the intracellular development of parasites infecting hepatocytes (Romero et al., 1989; Tsuji et al., 1990). In view of the strong evidence indicating that protection against malaria infection could be induced by immunization with sporozoites, much of the efforts in the 1980s and 1990s were focused on the characterization of the antigens expressed in sporozoites and liver stages, which were recognized by protective humoral and cellular immune mechanisms. So far, the best-characterized antigen is the circumsporozoite (CS) protein, which is expressed on the surface of sporozoites and also during the early liver stages (Nussenzweig & Nussenzweig, 1989) This protein is present in sporozoites of all species of Plasmodium, and, while its basic structure is conserved, their sequences are, to a large extent, species specific. This protein is characterized by the presence of an internal domain that represents one-third of the total protein and that consists of repeated amino acid sequences. These repeated sequences can be as short as the four amino acids NANP in Plasmodium falciparum CS protein, which is repeated 35 to 40 times depending on the strain, or DRAA/DGQPAG, which is repeated 19 or more times in Plasmodium vivax. While the sequence of the repeats of P. falciparum are conserved in different isolates, those from P. vivax show a restricted polymorphism with

Carole A. Long, Malaria Immunology Section, Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852. Fidel P. Zavala, Department of Molecular Microbiology and Immunity, Bloomberg School of Public Health, Malaria Research Institute, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205.

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FIGURE 1 Life cycle of malaria parasites.

two to three distinct types of repeats having been described in different isolates of this parasite species (Rosenberg et al., 1989). While the central repeats tend to be the dominant B-cell epitopes, CS proteins of human and rodent malaria parasites contain multiple epitopes recognized by CD41 T cells, which are important as helper cells for the activation of B cells and also CD81 T cells. In contrast, this protein appears to contain few epitopes recognized by CD81 T cells. Most of the T-cell epitopes appear to be located in the C-terminal region flanking the repeat domain, while only a few are present in the N-terminal flanking region (Sinnis & Nardin, 2002).

Development of Subunit Vaccines against Preerythrocytic Stages of Plasmodium

The findings described above provided the experimental basis and rationale for the design and development of several subunit vaccines containing selected antigenic domains or the entire CS protein. These included different formulations of synthetic peptides coupled to carrier proteins, synthetic peptide polymers containing B- and T-helper epitopes of the CS proteins, attenuated viruses, DNA constructs, and recombinant proteins. So far, the most promising results in human vaccine trials have been obtained with RTS,S, which is a construct containing the repeat domain and C-terminal flanking regions (amino acids 207 to 395) of the P. falciparum CS protein, which is expressed in a hepatitis B viruslike particle (VLP) (Cohen, et al., 2009). The RTS,S vaccine has been administered with the novel adjuvants AS01B and AS02A, which contain the immunostimulants lipid A and the glycoside QS21. Several vaccine trials were performed with adults, children, and infants living in endemic areas in West and East Africa and the results indicated that this vaccination conferred approximately 30% protection—in one case reaching 60% protection—against parasite infection,

which, in most trials, appeared to last for a limited period of time (Bejon et al., 2008; Bojang et al., 2001). Recent studies clearly show that this vaccine induces mainly antibodies and CD41 T cells and that protection strongly correlated with high titers of anti-CS antibodies and the presence of CSspecific CD41 T cells (Kester et al., 2009). Intriguingly, some of the trials in children and infants revealed that besides protection from infection, this vaccine appeared to have significant antidisease activity, as 50% to 60% of vaccinated children appeared to be protected from developing symptomatic malaria infection (Alonso et al., 2004; Bejon et al., 2008). While there is a consensus that these results are encouraging, it is critical to determine whether this decrease in the prevalence of symptomatic malaria will translate to preventing or significantly decreasing the prevalence of severe malaria, clinically defined by the appearance of serious complications such as respiratory distress, severe anemia, and cerebral malaria, all of which are the main causes of severe morbidity and mortality in children. Currently, a phase III trial that includes thousands of children of the RTS,S vaccine is under way and it is hoped that this study will have enough power to discern the effect of this vaccine on the occurrence of severe malaria. A successful phase III trial confirming previous results should strongly encourage research aimed at designing a new generation of vaccines to further improve protective efficacy.

Enhancing Vaccine Efficacy: Adjuvants, New Construct Design, and Immunization Protocols

While the partial protective effect of the RTS,S vaccine is encouraging, improvements on the immunogenicity of this or similar CS-based vaccine candidates will be necessary to enhance the antiparasite efficacy of the vaccine-induced immune responses. Some possible approaches to increased efficacy are described below.

46. Immune Intervention in Malaria

The identification of better adjuvants for use in human vaccinations is a matter of critical importance and of the highest priority for vaccine research in general. This is being pursued with renewed interest in view of new advances in the identification and characterization of the molecular mechanisms involved in the activation and function of the innate immune system, which critically influences the induction and development of adaptive immune responses. Different ligand-receptor protein families, such as TLRs and NLRs, have been identified as key physiological activators of innate immune responses, which greatly influence the functional properties of professional antigen-presenting cells and, in turn, determine to a significant extent the development and functional properties of antibodies and T-cell responses. This has stimulated intense research aimed at evaluating certain immunomodulators such as TLR ligands (e.g., CpG, Poly:IC, etc.) as well as certain cytokines and chemokines, with the purpose of increasing the immunogenicity of vaccine constructs. While this new area of experimental research has generated an extensive body of information in experimental models, the full translation of this knowledge into a new generation of adjuvants may still be years away, particularly in view of the need for extensive safety data in humans with these formulations. Another significant deficiency of RTS,S is the apparent inability to induce CD81 T-cell responses (Kester et al., 2009). Arguably, the induction of these T cells, together with antibodies and CD41 T cells, will, in all likelihood, significantly increase the protective efficacy of RTS,S. This shortcoming is probably due to the fact that recombinant proteins and VLPs, while quite effective at priming CD41 T cells and inducing antibody responses, appear to be poor inducers of CD81 T-cell responses. This is not entirely surprising as it is well known that the mechanisms of antigen processing and presentation required to prime CD81 T cells are quite different from those involved in priming CD41 T and B cells. This critical biological difference represents a major challenge for the design of single vaccine constructs capable of efficiently inducing both antibody and T-cell-mediated protective responses. It is conceivable that an effective vaccination may require the production of at least two different constructs, which could be administered together but would be processed by different intracellular mechanisms or different antigen-presenting cells. Finally, the immunization protocol is another factor that can greatly impact the magnitude and functional properties of vaccine-induced immune responses. The RTS,S vaccination relies exclusively on using the same construct to prime and boost the anti-CS immune responses. While this approach may be advantageous for its simplicity and cost, prime boosting with different constructs, which only share the CS sequence (i.e., heterologous constructs), may confer significant advantages. A large number of studies have shown that, for reasons that are not well understood, the magnitude of CD81 T cell and antibody responses can be greatly enhanced when primeboosting is done using heterologous constructs such as VLPs, recombinant viruses, and DNA, among others (Daly & Long, 1993; Schneider et al., 1998; Sedegah et al., 2003). There are, however, some limitations to this approach that need to be considered since the sequence of immunizations also appears to be important. As shown for CD81 T cell responses, some immunogens appear to be better at priming T cells while others are much more efficient as boosters, expanding previously established T-cell responses (Zavala et al., 2001).

A Multiantigen Subunit Vaccine

Perhaps a major weakness of the current RTS,S and similar preerythrocytic candidate vaccines is the exclusive reliance on a single parasite antigen, the CS protein. This limited

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antigenic specificity not only restricts the breadth of the immune response but probably also the overall efficacy of immune protection. There is a consensus that the identification of new preerythrocytic stage antigens expressed in sporozoites and liver stages is critical to further improve the efficacy of these vaccines. Importantly, recent studies using transgenic mice that constitutively express the CS protein of Plasmodium yoelii and do not develop T-cell responses to this antigen can, however, develop CD81 T-cell mediated immunity after immunization with sporozoites (Kumar et al., 2006). This clearly demonstrates the existence of protective immune responses against antigens other than the CS. While several immune mechanisms may be able to exert some antiparasite activity NK (natural killer), NKT (natural killer T cells), CD41, and gamma/delta T cells), it can be argued that the search for additional antigens aimed at strengthening the efficacy of a preerythrocytic vaccine should focus on the identification of antigens recognized by the most effective antiparasite immune mechanisms, viz., antibodies and CD81 T cells. The identification of new CD81 T-cell antigens may prove most challenging. Unlike antibody responses that can be induced in most, if not all vaccinees—provided that helper epitopes are present—CD81 T-cell responses are genetically restricted and thus only individuals expressing the MHC (major histocompatibility complex) class I protein with the appropriate specificity can present a defined epitope and induce the development of epitope-specific CD81 T-cell responses. Thus, different individuals with different MHC alleles will recognize different epitopes. This implies that a vaccine construct needs to contain several T-cell epitopes so that a large proportion of vaccinated individuals can develop CD81 T-cell responses, at least to one of the epitopes. In addition, it is also known that, as compared to MHC class II restricted epitopes recognized by CD41 T cells, MHC class I epitopes are relatively uncommon. This is certainly the case for the CS proteins from rodent and human malaria parasites, which have few CD8 epitopes but several epitopes recognized by CD41 T cells (Good et al., 1988; Romero et al., 1988; Sinnis & Nardin, 2002). Taken together, because of the genetic restriction of these T-cell responses as well as the paucity of MHC class I restricted epitopes, it will be necessary to identify many epitopes that might be located in several different proteins expressed in sporozoites and/or liver stages. Although in the last 25 years the search for new epitopes recognized by protective antibodies has not been successful, some success has been achieved on the identification of non-CS antigens involved in T-cell-mediated protection. Immunization with subunit vaccines expressing the rodent P. yoelii Hep-17 and TRAP antigens induces CD41 and CD81 T-cell-mediated protection in mice (Dobano & Doolan, 2007; Doolan et al., 1996; Khusmith et al., 1991). Hopefully, additional candidate antigens will be identified, taking advantage of new advances in the characterization of the transcriptional profile of Plasmodium genes at different stages. The information on gene expression, together with the analysis of the respective protein sequences using available algorithms, should help in the identification of peptide sequences likely to be T-cell epitopes (i.e., likely to be generated after intracellular processing and capable of binding MHC molecules).

A Vaccine Based on Attenuated Parasites

In the last few years, a new school of thought has emerged regarding the development of preerythrocytic vaccines. Since immunization of humans with radiation-attenuated sporozoites has been shown to induce consistently strong protective immunity and considering that this efficacy has not been matched by immunization with available subunit vaccines,

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FIGURE 2 Development of Plasmodium falciparum in human red blood cells. Merozoites attach to and invade mature red blood cells (RBCs) and the parasite develops in a parasitophorous vacuole through ring (0 to 24 hours), trophozoite (24 to 36 hours), and schizont stages (40 to 48 hours). In midcycle, membranous structures appear in the RBC cytoplasm and deformations (knobs) appear on the RBC membrane. These knobs include parasite-encoded proteins in the P. falciparum erythrocyte membrane protein-1 (PfEMP1) family as well as other proteins. After approximately 48 hours, the infected RBC ruptures and releases 16 to 32 daughter merozoites. Taken from Maier and colleagues (2009).

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some researchers have proposed the implementation of a large-scale vaccination program using attenuated sporozoites (Luke & Hoffman, 2003). Until recently, such a possibility has been considered unfeasible due to the major technical obstacles facing the production of purified, vaccine-grade sporozoite preparations that must be obtained by dissecting salivary glands of Plasmodium-infected mosquitoes. Recent technical advances such as greatly increasing the sporozoite load in mosquitoes, standardization of dissection techniques to obtain highly purified parasites, and the development of freezing protocols to store and recover live sporozoites have helped to address some of these practical difficulties. Maximizing the efficiency of these processes could conceivably aid in obtaining the billions of parasites that will be needed to immunize large numbers of individuals. These parasites could be rendered noninfectious by irradiation before freezing. An additional technical advance that could further facilitate the production of these attenuated parasites is the development of techniques for gene transfection. This has allowed the generation of genetically attenuated parasite strains that produce sporozoites that undergo only limited development in hepatocytes and do not generate parasites capable of infecting erythrocytes. It has been shown that these genetically attenuated sporozoites induce immune responses as efficiently as radiation-attenuated wild-type parasites (Mikolajczak et al., 2007). Therefore, the use of these mutant parasites eliminates the irradiation step that is needed for the attenuation of wild-type parasites. In view of the success of the early human vaccine trials with irradiated sporozoites, it is likely that immunization of hundreds or even thousands of individuals with attenuated parasites under this new program is likely to succeed. However, the idea of immunizing hundreds of millions of humans living in endemic areas may still represent an extremely challenging financial and logistical undertaking.

ERYTHROCYTIC-STAGE MALARIA VACCINES

Since they are responsible for the pathology associated with malaria infection, the erythrocytic stages of infection have been a major focus of vaccine researchers (Bejon et al., 2008; Fowkes et al., 2010). There are two principal targets of these studies: (i) antigens that are associated with the merozoite’s surface or its organelles and (ii) proteins that the intracellular parasite exports to the surface of the infected erythrocyte (Fig. 2). The merozoite proteins can elicit immune responses that prevent red cell invasion and development while the red cell surface proteins can induce responses that promote elimination of infected red cells and limit binding to host cells. With respect to the merozoites, the surface proteins are obvious possible candidates due to their exposure and presumed role in red cell invasion. Other merozoite proteins considered to be targets are found primarily in the apical organelles such as the rhoptries and micronemes, since these components are usually discharged during the invasion process and become accessible to serum antibodies. However, the fact that merozoites are likely free for only minutes in the circulation places constraints on the quantity and quality of antibodies required for effector function during this limited window. In contrast, the proteins exported to the red cell surface are displayed for many hours of parasite development. This phenomenon is quite unique to malaria parasites as P. falciparum has evolved elaborate machinery to export between 200 to 400 proteins through the parasitophorous vacuole membrane into the red cell cytosol and some are further localized to the red cell plasma membrane (Maier et al., 2009). Most are part of large, diverse, multigene families that presumably evolved to evade host humoral immune responses. The best


characterized of these protein families is referred to as PfEMP1 (P. falciparum erythrocyte membrane protein-1). These are very large molecules—approximately 250 kDa—that are encoded by about 60 different var (variable) genes in the parasite (Kraemer & Smith, 2006) While each parasitized erythrocyte is thought to express only one PfEMP1 product at a time, parasites are capable of switching from one PfEMP1 to another, adding to the complexity. Also, there is evidence that other gene products such as rifins, stevors, and surfins are also present in or on the red cell membrane (Maier et al., 2009), although their roles are not well understood.

Mechanisms of Immunity to Blood-Stage Parasites

Descriptions of the natural history of the development of immunity in children and adults living in malaria-endemic areas have found that resistance to malaria infection is a function of age, with children under the age of 5 years generally being most susceptible to severe disease and death. Subsequently, children become progressively more resistant to the clinical symptoms associated with malaria and, with age, adults become more successful in controlling parasitemia (Marsh & Kinyanjui, 2006). This naturally acquired immunity does not lead to the complete absence of infection, referred to as “sterilizing immunity,” although malaria-naïve adults become resistant to these parasites more quickly than children. However, the mechanisms responsible for this epidemiologic profile of resistance to malaria are not fully understood. Therefore, there has been an ongoing discussion as to whether a vaccine should be targeted to replicating the type of immunity found in a semi-immune adult or whether the goal of a vaccine should be to elicit responses superior to the partial immunity conferred by long-term and repeated exposure to malaria infection. Many different mechanisms have been implicated in this naturally acquired immunity to malaria in humans (see chapter 24), although most investigators believe it is primarily targeted to the erythrocytic stages of infection. In the 1960s, IgG preparations purified from semi-immune African adults were shown to dramatically reduce parasite counts and symptoms when passively transferred to children (Cohen et al., 1961); however, the sexual stage gametocyte counts were not reduced, confirming the immunologic differences between these stages. Such results have supported the protective role of serum antibodies

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directed to blood stages of infection and provided the rationale for measurements of antibodies elicited after experimental vaccination. Whether these antibodies function in vivo by direct neutralization or agglutination of merozoites or function through binding to Fc-receptor-bearing host cells and promoting parasite clearance or both is not yet clear (Pleass, 2009). In addition, acquisition of antibodies to parasite-encoded antigens on red cell membranes such as PfEMP1 proteins has been reported to be important in the development of resistance to blood-stage infection (Bull et al., 1998). Rodent models of malaria infection as well as measurements in infected humans have also suggested roles for cellular immune responses, although our understanding of these effector mechanisms and their relative importance is rather superficial at this point. Since erythrocytes are class I negative, no role for CD81 T cells has been shown. However, CD41 T cells are required for B-cell antibody production and CD41 cells also produce important cytokines such as interferon gamma (IFN-g). Early studies in the rodent model Plasmodium chabaudi established that acute infections could be controlled by B-cell deficient mice and this control required CD41 T cells (Grun & Weidanz, 1981). Other cell types such as regulatory T cells, gamma/delta T cells, NK cells, and innate immune mechanisms are also under investigation. Future studies will be required to achieve a more integrated view of the contributions of these various effector mechanisms to the acquisition of malaria immunity.

Antigens of Merozoites as Targets of Erythrocytic-Stage Vaccines

As in the case of the pre-erythrocytic antigens, many gene products identified as vaccine candidates were first identified in rodent models of malaria infection. In addition, these models have allowed a dissection of the repertoire of protective immune responses to these antigens in mice but their relevance to humans has not yet been established. To date, it has been easier to elicit protective immune responses in rodents as compared to humans, and the vaccine volunteers have generally produced lower antibody levels than immunized rodents. However, very few P. falciparum antigens have been trialed in humans and our understanding of the required immune responses is currently limited. Some potential targets are illustrated in Fig. 3. Recently, a meta-analysis

FIGURE 3 Illustration of a malaria merozoite as well as major proteins located on the merozoite surface, the rhoptries, and the micronemes. Taken from Richards and Beeson (2009).

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has been published of the various merozoite candidates and their relationship to incidences of malaria in field studies (Fowkes et al., 2010). Detailed tables of malaria antigens and the formulations of these antigens that are in clinical development can be found in the references (Vekemans & Ballou, 2008; Richards & Becson, 2009). Also, an online registry of malaria antigens being tested in human clinical trials is supported by the World Health Organization (www .who.int/vaccine_research/links/Rainbow/en/index.html). The major merozoite surface protein (MSP1) was one of the first to be described and cloned and has been quite extensively studied (Holder, 2009). This large protein (300 kDa) is present in all species of plasmodia, appears to be a required single-copy gene, and in P. falciparum, is proteolytically processed during erythrocyte invasion. While most of the molecule is shed, the carboxy-terminal 19 kDa portion containing two epidermal growth-factor-like domains remains on the surface and is internalized with the merozoite. This 19 kDa region was demonstrated to be the target of a protective monoclonal antibody in the rodent parasite P. yoelii. Subsequently, this 19 kDa region was shown to elicit protective responses in rodent malaria, and the homolog in P. falciparum could similarly protect nonhuman primates (Daly & Long, 1993; Singh et al., 2006). As a result, several groups have pursued this as a vaccine candidate generally using the carboxy-terminal 42 kDa region to add CD41 T-cell helper epitopes. Formulated with alum, this antigen has elicited modest humoral immune responses in humans and a phase 2 clinical trial of this antigen formulated with the adjuvant AS02 showed no protection in Kenyan children (Ogutu et al., 2009). However, MSP1 is encoded by a dimorphic gene and the form used for immunization may not have optimally matched the form most prevalent at the trial site. AMA1, apical membrane antigen-1, is a microneme component that was first identified in the primate parasite Plasmodium knowlesi (Deans et al., 1984; Remarque et al., 2008). It is another single-copy gene that cannot be “knocked out” in P. falciparum. During red cell invasion, this protein appears to participate in the tight junction formed between the merozoite and the red cell. AMA1 proteins from P. knowlesi as well as homologs from rodent parasites have elicited protective immune responses in their respective hosts. P. falciparum AMA1 has been produced in recombinant yeast and Escherichia coli and tested in several clinical trials in the United States, Europe, and Africa using a variety of adjuvant formulations. Immunization of malaria-naïve American volunteers with AMA1 followed by homologous challenge with viable sporozoites did not delay the appearance of blood-stage parasites by microscopy but did show limited retardation of parasite replication by PCR (Kester et al., 2009). Phase 2 efficacy trials with two AMA1 formulations have been conducted in African children. In the first trial using an alum formulation and a mixture of two different allelic forms of the protein, no protection was seen (Sagara et al., 2009). In another, phase 2 trial in Malian children with a single allelic form (3D7), some modest indication for allele-specific protection was obtained (C. Plowe, personal communication). However, the large number of possible polymorphic AMA1 forms seen even in a single village has, to date, proven to be a significant barrier to eliciting antibodies to conserved antigenic determinants. Overall, two findings have limited the potential of this antigen. First, the observation that field isolates of AMA1 are polymorphic in sequence, particularly in the first domain; and second, that vaccine-induced antibodies to this protein are primarily focused on the antigenically variant face of the molecule. Efforts are underway to direct antibodies to the cross-reactive determinants on this molecule, either by mixing AMA1 proteins of different

sequences or by constructing chimeric molecules encompassing multiple variants. MSP3 (merozoite surface protein-3) is being pursued as a vaccine candidate since individuals living in malariaendemic areas develop cytophilic IgG1 and IgG3 antibodies to this protein (Oeuvray et al., 1994). These antibodies have been reported to inhibit blood-stage parasites in an in vitro antibody-dependent cellular inhibition (ADCI) assay with monocytes as the effector cells (Bouharoun-Tayoun et al., 1990). In addition, antibodies to MSP3 and mononuclear cells were able to control parasitemia in immunodeficient SCID (severe combined immunodeficient) mice with circulating P. falciparum parasites. A portion of the MSP3 molecule has been produced as a long synthetic peptide and is undergoing clinical trials in Africa. In addition, a fusion protein of MSP3 and GLURP antigens (designated GMZ2) is being pursued as a vaccine candidate (Chowdhury et al., 2009). Another merozoite protein, EBA-175 (erythrocyte binding antigen of 175 kDa), is found in the micronemes and, in this case, a function has been assigned since it binds to sialic acid residues on erythrocyte glycophorin A (Camus & Hadley, 1985). Antibodies to a cysteine-rich domain of this protein—designated region II—block this binding and a phase 1 clinical trial has been conducted with a recombinant region II protein. However, in P. falciparum, EBA-175 appears to be a member of a larger family of related molecules, affording the parasite a variety of erythrocyte invasion pathways (Adams et al., 1992; Cowman & Crabb, 2006). Therefore, while development of EBA-175 region II is continuing, it is likely that it will require combinations with other partner proteins to achieve efficacy. Many of the merozoite surface proteins are anchored to its surface through glycosylphosphatidylinositol (GPI) moieties, which have been implicated in malaria-induced disease due to triggering proinflammatory host cytokine responses. While efforts are underway to synthesize these compounds and to pursue clinical development (Schofield, 2007), concern remains that antibody responses to GPIs might mask the fever associated with clinical malaria (and retard the seeking of treatment) while not limiting parasite replication. One combination of blood-stage antigens (combination B) has been trialed in Papua New Guinea. This mixture of MSP2 (merozoite surface protein-2), an N-terminal portion of MSP1, and another polypeptide (RESA [ring stage infected erythrocyte surface antigen]) was reported to reduce parasite density in children (Genton et al., 2002). Investigators have pursued the MSP2 component as possibly responsible for this result and early stage clinical testing is in progress. Several research groups have sought to avoid the problems associated with recombinant protein expression using a different platform, viz., replication attenuated adenoviruses that have been genetically modified to incorporate one or more parasite genes encoding proteins such as AMA1 or MSP1 (Limbach & Richie, 2009). Interestingly, vectors such as adenovirus were selected, in part, to elicit strong cellular immunity, although preclinical studies in mice and rabbits with recombinant Ad5 encoding AMA1 or MSP1 have unexpectedly revealed strong antibody responses as well. The majority of studies have used adenovirus 5 (Ad5), and this is complicated by the fact that most individuals in malariaendemic countries have preexisting antibodies to this strain due to previous upper respiratory infections. In addition, a clinical trial with an Ad5-vectored HIV (human immunodeficiency virus) vaccine suggested that those vaccinees with preexisting antiadenovirus antibodies might be more susceptible to HIV infection. Consequently, other adenoviral strains from humans or nonhuman primates are also being pursued as possible vectors, and various combinations of


viral vectors and proteins are being advanced into primeboost development strategies (Bojang et al., 2001). Taken together, major limitations on the development of a merozoite vaccine include the antigenic polymorphism of important targets, the difficulties in producing these molecules in a native conformation, the fact that only a few of the many potential vaccine targets have been investigated, the lack of established in vitro assays that correlate with immunity in vivo, and the limitations in our understanding of the types of immunity that need to be generated.

Parasite-Encoded Antigens Associated with the Infected Red Cell Membrane

As noted above, the PfEMP1 family of molecules is an important component of the infected red cell, particularly because various domains of these proteins have been shown to bind to CD36, ICAM-1, and other molecules on host cell surfaces. As a result of these receptor-ligand interactions, PfEMP1 proteins mediate binding of parasitized cells to host endothelial cells and contribute to pathology (see chapter 29), but their large size and heterogeneity has been exceptionally challenging for vaccine development. However, there is one member of this family, designated VAR2CSA, which is a potential vaccine target. Some time ago, researchers reported that P. falciparum-parasitized erythrocytes could adhere to chondroitin sulfate in human placentas (Fried & Duffy, 1996). It was subsequently shown that sera from African women, but few men, could prevent the adherence of P. falciparum-parasitized erythrocytes to placental tissue. This adherence promotes sequestration of parasitized erythrocytes in the placenta, an event that can elicit an inflammatory process resulting in placental malaria. Among the consequences of placental malaria are low birth weight infants, making this a major maternal– child issue in malaria-endemic areas of the world (Rogerson et al., 2007). The initial findings have evolved to implicate VAR2CSA as the PfEMP1 variant primarily responsible for the adherence, and chondroitin sulfate-A in the placenta as a major target for the binding. Primiparous women are most vulnerable to placental malaria because few have antibodies to VAR2CSA-expressing parasites. In contrast, during the first pregnancy, such exposure results in production of these antibodies and reduction in the likelihood of malaria during subsequent pregnancies. VAR2CSA is present in the genome of all P. falciparum isolates identified and the various domains of VAR2CSA are being pursued as a potential vaccine for pregnancy-associated malaria, based on the premise that antibodies elicited would reduce or eliminate the binding of VAR2CSA-expressing parasites to the placenta and, consequently, reduce pathology (Hviid & Salanti, 2007; Tuikue Ndam & Deloron, 2008).

Whole Cell Vaccine Approaches

Whether a merozoite protein or a red cell surface molecule, most approaches to blood stage vaccination have involved subunit vaccination using antigens made in recombinant expression systems, as outlined above. Given the difficulties associated with the large number of complex antigens appearing in merozoites or on the infected red cell, an alternate approach to a malaria vaccine has been suggested, viz., using whole parasite-infected red cells (McCarthy & Good, 2010). While some studies in rodent and nonhuman primate models of infection have shown that whole blood-stage parasites can elicit protective immunity, the obstacles involved in using human donor blood to produce parasites on a large scale are formidable. Nevertheless, there has been one report of protective cell-mediated immune responses in a limited number of individuals after several immunizations with

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30 P. falciparum-infected red cells (Pombo et al., 2002). However, it appeared that the antimalarial drug treatment used after immunization to prevent infection was not fully cleared prior to challenge with parasites (Edstein et al., 2005).

Vaccines for other Plasmodial Species

Nearly all efforts to date have been focused on P. falciparum, although the extent of P. vivax infection in the world and the accumulating evidence for P. vivax pathogenesis supports stepped-up efforts to address this parasite as well (Galinski & Barnwell, 2008). Only one blood-stage P. vivax candidate has reached clinical development and that is the Duffy binding protein (DBP), a paralog of EBA-175 in P. falciparum. This protein interacts with the Duffy antigen receptor for chemokines (DARC) on red cells and appears to be an essential participant in red cell invasion. Unlike the situation with P. falciparum, there appears to be only a single DARC ligand. This protein and several others are in various stages of preclinical development.

SEXUAL STAGES OF MALARIA INFECTION AND TRANSMISSION-BLOCKING VACCINES

The concept of a vaccine to block transmission from the infected individual to the definitive host, the Anopheline mosquito, is relatively novel in vaccinology, but it may have relevance in malaria since such a vaccine might prevent the spread of a drug-resistant parasite. Moreover, this approach is receiving greater notice during discussion of the possibility of malaria elimination, since, even if morbidity and mortality due to blood stage infections are reduced, there would still be a large population of children and adults with circulating gametocytes capable of transmission. Transmissionblocking vaccines are based on the fact that during blood feeding, the female mosquito ingests not only gametocytes, but also host serum containing antibodies as well (Targett & Greenwood, 2008). If directed to antigens on the gametes, zygotes, or ookinetes, the ingested antibodies may inhibit parasite development within the gut of the mosquito. The other attractive feature of vaccines based on sexual stage antigens is that there is a semiquantitative in vitro assay that appears to replicate the in vivo situation, viz., the mosquito membrane feeding assay. This involves in vitro culture of late-stage gametocytes, mixing with immune serum, and feeding the mixture to mosquitoes through a membrane; parasites are allowed to develop and 7 to 9 days later, the insects are sacrificed and oocysts in the midgut are counted. However, such studies are particularly difficult with P. vivax, since there is no culture system for generating gametocytes so that the parasites must be obtained from infected people or from deliberately infected nonhuman primates. While some targets have been known for decades, identification of additional genes expressed during the sexual stages of development has accelerated in recent years with the completion of the genomic sequences of both P. falciparum and P. vivax (Pradel, 2007). Some of these sexual stage specific gene products are expressed in gametocytes, which develop in the human host over 10 to 14 days. In contrast, other gene products are limited to stages found only in the mosquito and are not present in significant quantity in the human host. This difference may be important for transmission-blocking vaccines since immune responses to gametocyte proteins have the potential to be boosted by natural infection, while those specific to the insect stages of development are not likely to be boosted. Maintaining high levels of antibodies in the latter case then becomes more challenging, although their isolation from the vertebrate immune system may minimize their antigenic diversity.

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Despite the large number of genes now associated with sexual stages and additional insights resulting from knockout parasites and other functional assays, progress in developing transmission-blocking vaccine candidates has been extremely slow. Development of procedures to produce these gene products in recombinant expression systems to validate their role as targets of transmission-blocking immunity has generally proven to be a formidable challenge. To date, most progress has been made with the Pfs25 and Pvs25 proteins that are primarily found on the surface of, respectively, P. falciparum-infected or P. vivax-infected zygotes and ookinetes, with the “s” designating the sexual stages. Both have been produced in recombinant yeast, and antibodies elicited in multiple animal species have shown significant transmission blocking as judged by mosquito membrane feeding assays. Interestingly, Pvs25 has four epidermal growth factor-like domains, which fold into a triangular prism and has been proposed to “tile” the parasite surface. The Pfs25 protein has also been reported to bind to calreticulin, a molecule found in mosquito midgut membranes. To extend studies with these proteins into clinical development, cGMP (current good manufacturing process), Pvs25, and Pfs25 were produced in yeast expression systems and characterized. In one phase 1 trial with an alum formulation, antibodies were elicited to Pvs25, but the immune responses were moderate and the high-titer responses required to block transmission were transient (Malkin et al., 2005). Because of the requirement for long-lasting, high-titer antibodies to block parasite transmission for the entire malaria season, efforts have been made to generate formulations that would elicit longer lasting immunity. One interesting approach has been to conjugate Pfs25 to the outer membrane complex of Neisseria meningitidis (Wu et al., 2006). This large aggregate contains numerous TLR agonists and other immunostimulants and resulted in anti-Pfs25 antibodies, which persisted at high levels for over 18 months in rhesus monkeys. Several other possible transmission-blocking candidates were identified several decades ago as components of zygote surfaces. These include Pfs48/45 and Pfs230, proteins associated with the surfaces of gametocytes and gametes; in P. falciparum, both were subsequently found to be part of a multigene family whose 10 members have a characteristic pattern of conserved cysteine residues (Pradel, 2007). This motif has been extremely difficult to replicate in expression systems, which has hampered the clinical development of these two proteins, although progress has been reported with Pfs48/45 (Chowdhury et al., 2009). Also, despite evidence that anti-Pfs230-specific antibodies will block parasite transmission in membrane feeds, its large size has, to date, compelled researchers to divide it into domains for study. Interestingly, antibodies to this protein are most effective in blocking transmission in the presence of serum complement. Numerous other potential targets have been identified, resulting from the genome databases, including a multigene family of proteins with adhesive-like domains (PfCCP proteins), a thrombospondin-related adhesive protein (CTRP), a secreted micronemal protein (CelTOS [cell-traversal protein for ookinetes and sporozoites]), a large family of proteins sharing a PHIST domain, and a megadalton protein designated Pf11-1. Still another approach has been to immunize with antigens derived from the mosquito itself, such as salivary gland components, in order to generate immune responses that would negatively affect the mosquito rather than the parasite (Dinglasan & Jacobs-Lorena, 2008). Overall, while a number of attractive candidates have been identified and new ones have emerged as a result of the

genome projects, few clinical trials have been conducted and no candidate or formulation has emerged that could be pursued into more advanced clinical development. Several major hurdles remain, including the difficulty in expressing many of these proteins in recombinant systems and the lack of adjuvants and platforms to elicit high-titer, long-lasting antibodies in the human host. In addition, there are challenges in demonstrating efficacy in a field situation with a transmission-blocking vaccine. These issues need to be addressed in order to make progress in this important area.

CONCLUSIONS

It is well established that antiplasmodial adaptive immune responses markedly attenuate or even abolish parasite infections. In fact, epidemiological studies have shown consistently that individuals living in malaria endemic areas develop partial immunity. Children first acquire resistance to severe disease and death, then progressively they develop resistance to symptoms, and, as they continue into adulthood, they develop significantly attenuated or asymptomatic disease. Most importantly, experimental studies in animal models and humans have provided conclusive evidence that passive transfer of parasite-specific antibodies drastically decreases parasitemia, and it has been shown that sterile immunity can be achieved by immunization with attenuated sporozoites. These lines of evidence strongly support the notion that developing an effective malaria vaccine is not only a desirable goal but a feasible one as well. Intense research in the last few decades has achieved significant gains regarding the identification of some parasite antigens recognized by protective T cells and antibodies as well as elucidating a repertoire of possible immune mechanisms. This allowed the design and development of a number of subunit vaccines evaluated in animal models and, in some cases, in human vaccine trials. However, to date, only the RTS,S vaccine candidate has shown partial protection in field studies. These efforts have highlighted a number of significant difficulties facing the field. •

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The existence of abundant polymorphisms in many of the antigens selected for vaccine development, requiring new immunologic strategies for eliciting cross-reactive immune responses or the design of large constructs consisting of multiple alleles of variant antigens. The limitations in our knowledge of the full range of protective immune responses to the various parasite developmental stages, especially possible effector roles for CD41 T cells against preerythrocytic and erythrocytic parasite antigens. The lack of safe, well-defined adjuvants, which can potentiate vaccine-induced immune responses in humans and direct host responses toward specific effector mechanisms. The need to identify new potential vaccine candidates from all parasite developmental stages since less than 1% of the parasite genes have been explored in this context. The lack of rapid and reliable screening methods to select the most promising candidates. The need for recombinant expression systems capable of producing plasmodial proteins in a conformation like that of the native parasite molecule as well as comprehensive structural studies to define the native structures of these antigens. The limited attention to vaccine development for P. vivax, which causes significant worldwide morbidity.


The rationale and experimental basis for the development of malaria vaccines are sound; however, their design and development in humans still represent major challenges. Unlike most vaccines currently in use, the successful development of a fully effective vaccine based on selected parasite-derived antigenic moieties critically depends on advances in basic immunology and modern vaccinology. This work was supported in part by the Intramural Division of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.

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47 Targeting Components in Vector Saliva MARY ANN McDOWELL AND SHADEN KAMHAWI

INTRODUCTION

Hematophagous vectors of disease are not just pathogen transmission vehicles, they dispense pharmacologically active compounds that prevent host hemostasis and thus facilitate the blood-feeding process. Vertebrate hemostasis is highly efficient and involves redundant pathways that result in blood clotting, vasoconstriction, and platelet aggregation; blood-feeding arthropods have evolved numerous activities to combat these processes. Many of these salivary molecules are immunogenic and elicit host immune responses that reduce the feeding efficiency and fecundity of the arthropod vector (Schoeler & Wikel, 2001). In addition, some salivary molecules act as immune effectors that influence the ability of blood-feeding vectors to transmit pathogens (Lal et al., 1994; Ramasamy & Ramasamy, 1990; Ramasamy et al., 1990). Conversely, salivary components can enhance the virulence of some pathogenic organisms, facilitating infection by inhibiting host immune responses (Rohousova & Volf, 2006; Schneider & Higgs, 2008; Schoeler & Wikel, 2001; Titus et al., 2006). Arthropod saliva contains powerful immunogenic molecules that elicit hypersensitivity reactions in repeatedly exposed individuals, and recent studies indicate that a history of vector bites restricts the establishment of some pathogens, leading to attenuated disease (Kamhawi, 2000; Rohousova & Volf, 2006; Titus et al., 2006). These data suggest that targeting vector salivary components may be a viable strategy for the development of vaccines against vector-transmitted pathogens. In this chapter, we outline the major findings supporting the notion that exploiting vector saliva for vaccine development is a viable proposition, focusing on the arthropod vectors most extensively studied: sand flies, mosquitoes, and ticks.

Despite decades of research and multiple initiatives, vectortransmitted diseases remain a major public health threat and have an enormous economic impact. Blood-feeding arthropods transmit some of the most debilitating infections known to mankind, including malaria, lymphatic filariasis, African trypanosomiasis, leishmaniasis, plague, Chagas’ disease, onchocerciasis, Lyme disease, dengue fever, and a multitude of encephalitic diseases. Since the discovery in the early 20th century that insects can transmit pathogens, environmental management of vector populations through habitat modification and pesticides has been utilized to decrease disease incidence in specific areas. Once exalted as the panacea to arthropod-borne disease, insecticidal spraying has proven ineffective in eradicating these infections, primarily due to the development of insecticide resistance in many vector populations and discontinued use because of environmental toxicity and the possibility of human carcinogenesis. Clearly, novel interventions are needed to combat these diseases and innovative strategies targeting the pathogen–vector interface are beginning to emerge. The influence of these vectors on clinical manifestation or progression of the diseases they transmit and the ability to target such interactions for intervention has only recently been appreciated. It is well accepted that human populations inhabiting tropical disease-endemic regions exhibit attenuated infections and are less likely to contract disease compared to newcomers in these areas, this being particularly true for many vector-transmitted diseases. Historically, this phenomenon has been attributed to a gradual onset of immunity against pathogens in endemic individuals. Evidence in animal models has emerged, demonstrating that immune responses to vector components can influence disease outcome for the pathogens they transmit (Rohousova & Volf, 2006; Schneider & Higgs, 2008; Titus et al., 2006), suggesting that these components may be involved in inducing protection in individuals that are repeatedly exposed to vector bites and increased susceptibility in naïve populations.

SAND FLIES

Phlebotomine sand flies serve as vectors for several established, emerging and reemerging infectious diseases, the most devastating of which are the leishmaniases. Diverse pathologies, ranging from self-healing cutaneous lesions (cutaneous leishmaniasis [CL]) to deadly visceral disease (visceral leishmaniasis [VL]), result from Leishmania infection—the clinical presentation primarily depending on the Leishmania species initiating the infection. There are approximately 20 different species and subspecies of Leishmania transmitted by 30 different sand fly species. Phlebotomus is responsible for transmitting leishmaniasis throughout parts of Africa, southwest

Mary Ann McDowell, The Eck Institute for Global Health and Infectious Diseases, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556. Shaden Kamhawi, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, 12735 Twinbrook Parkway, Rockville, MD 20852.

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Asia, the Middle East, and the Mediterranean. Lutzomyia species are indigenous vectors throughout South and Central America. There is a close evolutionary relationship between Leishmania species and their sand fly vectors such that, in an endemic area, a sand fly species only transmits a single Leishmania species (Sacks & Kamhawi, 2001). Leishmania spp. have a digenetic life cycle, alternating between flagellated promastigotes in sand flies, to obligate intracellular amastigotes that multiply primarily within macrophages of the vertebrate host. Murine models injecting Leishmania parasites have generated extensive information about immunity to infection. Generally, mouse strains that are genetically resistant express a Th1 polarized response (i.e., nitric oxide [NO], interferon g [IFN-g] and interleukin 12 [IL-12] are all required for the resistant phenotype to remain intact). Conversely, susceptible mice either induce Th2-mediated responses or just fail to activate Th1 cytokines (Sacks & Noben-Trauth, 2002). Recently, there has been considerable interest in the potential use of sand fly salivary proteins as components of anti-Leishmania vaccines. Primarily utilizing animal models, efforts have focused on two approaches: (i) neutralizing the Leishmania-enhancing effects of salivary components and (ii) identifying immunogenic salivary proteins that shift the host immune response towards Th1-mediated immunity. Here, we provide an account of the studies that have impacted this field and debate the relevance of their findings.

Exacerbation of Leishmanial Disease by Sand Fly Saliva

Sand fly saliva unquestionably enhances infection, as coinoculation of salivary components with Leishmania parasites has exacerbated disease and increased parasite numbers in virtually every model analyzed, including Lutzomyia longipalpis/Leishmania amazonensis, Ph. Lu. longipalpis/L. braziliensis, Lu. whitmani/L. braziliensis, P. papatasi/L. major, and Lu. longipalpis/L. major (Kamhawi, 2000; Rohousova & Volf, 2006; Titus et al., 2006). For most studies, salivary gland homogenates (SGH) have been introduced by needle inoculation; however, natural introduction of saliva by sand fly bite followed by needle inoculation of Leishmania also exacerbates infection (Theodos et al., 1991). The exacerbation of infection is evident even when parasites are introduced 4 hours after saliva exposure (Theodos & Titus, 1993) indicating that the virulence enhancement effect is long-lasting. It is generally thought that the enhancement of Leishmania infection by sand fly saliva is due to its nonspecific immunomodulatory activities. For example, SGH exposure inhibits CD41 T-cell proliferation to concanavalin-A in vitro (Rohousova et al., 2005; Titus, 1998) and in the response of mice to sheep red blood cells in vivo (Titus, 1998). The exacerbating effect of SGH is correlated with increased levels of the Th2 cytokine IL-4 (Belkaid et al., 1998; Lima & Titus, 1996; Mbow et al., 1998). Furthermore, the Th1 cytokines IFN-g and IL-12 are inhibited in the presence of SGH (Mbow et al., 1998). Therefore, in a naïve individual (i.e., one that has never been exposed to sand fly saliva), it is suggested that the presence of saliva during Leishmania infection will induce a Th2 cytokine environment, resulting in increased susceptibility. Salivary glands of phlebotomine sand flies contain a complex array of biologically active molecules that are both conserved and divergent among sand fly species; many of these molecules have immunosuppressive effects. For example, maxadilan (MAX), found only in some New World sand flies, is the most potent vasodilatory polypeptide known to date (Ribeiro et al., 1989) and mediates, at least in part, the enhancement of Leishmania infection by Lu. longipalpis saliva (Qureshi et al., 1996). The effects of Lu. longipalpis saliva, of

which many were later ascribed to MAX (Titus, 1998), promote the up regulation of Th2-type cytokines (IL-6, TGF-b, and IL-13) and IL-10 and down regulate Th1-associated cytokines including IFN-g, TNF-a, and IL-12, affecting both macrophages and dendritic cells (Brodie et al., 2007; Titus et al., 2006; Wheat et al., 2008). MAX, specifically, exhibits a range of immunomodulatory activities including inhibition of macrophage killing and proinflammatory functions (Bozza et al., 1998; Qureshi et al., 1996; Soares et al., 1998) and Tcell proliferation and delayed-type hypersensitivity (DTH) in mice (Qureshi et al., 1996). In addition, this peptide stimulates host hematopoiesis (Guilpin et al., 2002), potentially increasing the amount of cells that Leishmania species infect. MAX differs genetically by as much as 23% from different sibling species of the Lu. longipalpis complex. This genetic variability has no effect on their vasodilatory activities (Lanzaro et al., 1999); however, significant differences in the amount of MAX mRNA in these species have been reported (Yin et al., 2000). Interestingly, the difference in MAX expression in sibling species collected from Central and South America was associated with different sized erythemas at the bite site and to the atypical cutaneous disease caused by L. infantum chagasi in Costa Rica compared with visceral disease caused by L. infantum chagasi in Brazil (Yin et al., 2000). Phlebotomus spp. utilize other vasodilatory substances to facilitate blood-feeding. Ph. papatasi (Ribeiro et al., 1999) and Ph. argentipes (Ribeiro & Modi, 2001) secrete adenosine and AMP following a blood meal. It is worth noting that other properties of adenosine and AMP differ from those of MAX. The treatment of murine macrophages with P. papatasi, and not Lu. longipalpis, saliva inhibits the production of bacterial lipopolysaccharide (LPS) induced nitric oxide production (Katz et al., 2000). This effect has been attributed to adenosine present only in P. papatasi salivary glands, suggesting that different sand fly vectors enhance Leishmania infection by different mechanisms. The vast majority of studies exploring the influence of sand fly saliva on Leishmania infection have utilized labcolonized sand flies, thus limiting the genetic diversity of the saliva. Most recently, comparisons have been made between colonized and wild-caught sand flies (Laurenti et al., 2009). While wild-caught Lu. longipalpis SGH enhanced L. amazonensis infection compared to infection alone, lesions were significantly smaller than those injected with SGH from labreared Lu. longipalpis flies. The exact mechanism that mediates this difference remains to be completely elucidated.

Neutralizing the Exacerbative Effects of Sand Fly Saliva

In the section above we discussed salivary proteins as immunomodulators and enhancers of Leishmania infection. Therefore, it is logical to conclude that neutralizing such properties would contribute toward protection from leishmaniasis. Using Ph. papatasi and L. major, a natural vector-parasite pair, Belkaid and colleagues demonstrated that mice immunized with SGH of Ph. papatasi and challenged in the ear with a dose of 1,000 purified infectious stage parasites and SGH were protected from infection. SGH-immunized mice exhibited a significant reduction in lesion size and a 75-fold decrease in parasite burden compared to naïve animals following challenge with L. major and SGH (Belkaid et al., 1998). The parasite burden was comparable in naïve mice challenged with a parasite alone, suggesting that the observed protection in SGH-immunized mice resulted from neutralization of the enhancing effect of saliva. This protection was mediated by antisaliva antibodies believed to have neutralized components promoting a Th2-type immune response and IL-4 production (Belkaid et al., 1998). The rationale for this study was based on the observation that

47. Targeting Components in Vector Saliva

saliva of Ph. papatasi exacerbates L. major infection by promoting a Th2-type immune response typified by the presence of IL-4 (Belkaid et al., 1998; Mbow et al., 1998; Titus et al., 2006); the Ph. papatasi salivary components responsible for the IL-4 production have not been identified to date. This impediment is not the case for the New World sand fly Lu. longipalpis, where MAX was identified as the major exacerbative component in this species’ saliva (Morris et al., 2001). Previous studies have demonstrated that Lu. longipalpis saliva exacerbates infection with L. major through the immunomodulatory properties of MAX (Titus et al., 2006). Certainly, immunization of mice with synthetic MAX abrogated its Leishmania-enhancing effects and protected animals co-inoculated with L. major and SGH from disease (Morris et al., 2001). Following footpad challenge with 105 stationary phase L. major promastigotes and SGH, immunized mice developed significantly smaller lesions and over a 13,000-fold decrease in the number of parasites compared to sham-vaccinated mice (Morris et al., 2001). This protection was mediated by a Th1-type immune response orchestrated by CD4 T cells and characterized by substantial production of IFN-g and NO. In addition, elevated levels of anti-MAX antibodies were observed in these mice; however, their contribution to protection was not directly investigated. Interestingly, immunized mice challenged with Leishmania alone also displayed significant protection as determined by both lesion size and parasite burden, the latter being reduced 26-fold compared to sham-vaccinated mice (Morris et al., 2001). Unfortunately, the protective effect of immunization with MAX on protection from the VL causing agent, L. i. chagasi, the natural parasite this vector sand fly transmits in nature, was never explored.

Driving a Leishmania-Protective Immune Response Using Saliva

In addition to reports of exacerbative effects of Ph. papatasi saliva on L. major infection (Belkaid et al., 1998; Mbow et al., 1998; Titus et al., 2006), studies aimed towards finding immunogenic salivary molecules that can drive a Th1-type immune response considered protective against Leishmania infection were undertaken. These efforts were spurred by the observation that mice preexposed to the bites of uninfected Ph. papatasi and challenged with L. major-infected Ph. papatasi bites produced a delayed-type hypersensitivity response (DTH-R), characterized by IFN-g production at the bite site (Kamhawi et al., 2000) (Color Plate 13). Moreover, in this study, there was no clear role for IL-4 in naïve or preexposed mice challenged by infected fly bites (Kamhawi et al., 2000). Seminal research soon followed identifying a Ph. papatasi salivary protein, PpSP15, that alone protects mice from L. major infection in both wild-type and B-cell deficient mice, thus demonstrating that antibodies are not essential for protection in this system (Valenzuela et al., 2001). These findings initiated a new line of research focused on the identification of Th1-inducing salivary molecules of vector species and investigating their potential in protecting a variety of animal models from CL and VL (Collin et al., 2009; Gomes et al., 2008; Oliveira et al., 2009, 2006, 2008). In a summary of these studies, a high-throughput transcriptomic approach was fine tuned to identify secreted salivary proteins from as little as 30 pairs of sand fly salivary glands (Oliveira et al., 2006; Valenzuela, 2002, Valenzuela et al., 2004); DNA vaccination in appropriate rodent models (Gomes et al., 2008; Oliveira et al., 2006, 2008) and dogs (Collin et al., 2009) has been performed with the identified targets, revealing important aspects of salivary proteins pertinent to their status as vaccine candidates. Oliveira and colleagues demonstrated that immunization of mice with distinct salivary molecules can generate two functional types of

601

DTH-R associated with different cytokine patterns (Oliveira et al., 2008). Immunization of mice with PpSP15 validated earlier findings and induced a protective Th1-type DTH-R associated with IFN-g; parallel immunization with another P. papatasi salivary cDNA, encoding PpSP44, resulted in a Th2type DTH-R that was associated with immunopathology and disease exacerbation (Oliveira et al., 2008). Indeed, immunization with whole saliva usually produces a mixed Th1/Th2 response (Collin et al., 2009; Oliveira et al., 2006, 2008). Another significant observation from this study is that antiPpSP15 immunity induces a Th1-type immune response as early as 2 hours following challenge with L. major infectious stage parasites and SGH, potentially driving specific protective anti-Leishmania immunity soon after parasite codeposition with saliva by the sand fly (Oliveira et al., 2008). Evidence of early priming for protective anti-Leishmania immunity by an existing immune response directed at a salivary protein accounts for the protection observed in a hamster model of VL (Gomes et al., 2008). Hamsters immunized with a cDNA encoding LJM19, a salivary protein from Lu. longipalpis, survived the fatal outcome of VL observed in all the other test groups following challenge with L. i. chagasi and SGH (Gomes et al., 2008). LJM19-immunized hamsters were protected for the duration of the study and displayed a Th1-type response in the spleen and liver with a high IFN-g/ TGF-b ratio and expression of inducible nitric oxide synthase up to 5 months postinfection. Demonstration that a Th1-type DTH-R in the skin of immunized animals 2 hours (Gomes et al., 2008) and 48 hours (Oliveira et al., 2008) following sand fly bites definitively establishes that immunity to saliva is efficiently induced at the bite site. Recently, the significance of salivary components in anti-Leishmania vaccines was taken to a higher level by the demonstration that immunization of dogs with distinct salivary molecules (Collin et al., 2009) or repeated exposure of humans to sand fly bites (Vinhas et al., 2007) induces specific Th1-type immune responses including IFN-g production. Dogs, natural reservoirs of L. infantum and L. i. chagasi, develop a DTH-R to sand fly bites characterized by marked cellular infiltration of T cells, macrophages, and eosinophils 48 hours postbite (Collin et al., 2009). DNA immunization of dogs with cDNA encoding either LJL143 or LJM17, two salivary proteins from Lu. longipalpis, resulted in a powerful Th1-type immune response both locally at the bite site and systemically (Collin et al., 2009). Importantly, immunized dogs did not develop an allergic reaction when challenged by a total of 35 sand flies, an important consideration for salivary-based vaccines. As there is no established experimental model for dog infection induced by sand fly exposure, the only anti-Leishmania effect explored in this study was carried out in vitro. Macrophages differentiated from PBMC collected from cDNA-immunized dogs and infected in vitro with L. i. chagasi showed over a 70% reduction in infection upon the addition of autologous T cells and SGH compared to cells from sham-immunized dogs (Collin et al., 2009). This observation suggests that Leishmania are indirectly killed by an immune response to a salivary protein and brings back into focus the initial interpretation that antisaliva immunity results in in situ killing of Leishmania parasites at the bite site (Kamhawi et al., 2000). Much needed evidence to validate the potential for immunogenic salivary molecules in protection against leishmaniasis was recently provided (Vinhas et al., 2007). The authors demonstrated that humans experimentally exposed to repeated bites of Lu. longipalpis produced saliva-specific IFN-g in five of six exposed individuals upon stimulation with SGH (Vinhas et al., 2007). Additionally, PBMC from these individuals controlled parasite growth in vitro in a macrophage-lymphocyte autologous system (Vinhas et al.,

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2007). Even more encouraging was the demonstration that 1 year after the last exposure to bites, a robust recall response was observed in all tested individuals after getting a booster exposure. This memory response was characterized by IFN-g production that controlled parasite survival in vitro (Vinhas et al., 2007). The protective influence of prolonged exposure to sand fly bites may not be a universal phenomenon. Vaccination of BALB/c mice with Lu. intermedia SGH, a primary vector of L. braziliensis, results in a delay of cutaneous lesion development, but ultimately prolonged disease and increased parasite burden (de Moura et al., 2007). Whether disease exacerbation in this model is due to specific immunomodulation or to immunosuppressive properties specific to Lu. intermedia remains unclear, nevertheless, this study does highlight the potential complexity of developing a pan-sand fly vaccine. The addition of appropriate adjuvants in such vaccines may overcome such difficulties.

Saliva and Neutrophils

The effect of saliva on neutrophils is worth separate consideration for two reasons: (i) the central role attributed to neutrophils in the establishment of Leishmania infection by sand fly transmission (John & Hunter, 2008; Peters et al., 2008, 2009), and (ii) the disparate reports on the effect of saliva on neutrophil recruitment (Carregaro et al., 2008; Monteiro et al., 2007, 2005; Teixeira et al., 2005). A leukocyte chemoattractant effect was first described for Lu. longipalpis SGH using a mouse air pouch model of inflammation (Teixeira et al., 2005). Compared to saline alone, the injection of SGH into an air pouch in both BALB/c and C57BL/6 mice resulted in a significant influx of neutrophils 12 hours following challenge (Teixeira et al., 2005). Interestingly, incubation of SGH with anti-SGH serum did not affect the number of recruited neutrophils, rather, it reduced the number of macrophages and eosinophils (Teixeira et al., 2005). These data suggest that antisaliva antibodies do not neutralize the neutrophil attracting components of saliva. The neutrophil attracting property of sand fly saliva was supported by recent findings investigating leukocyte recruitment into the peritoneal cavity of BALB/c mice (Monteiro et al., 2007). This study demonstrated that SGH of Lu. longipalpis promotes marked recruitment of leukocytes, including neutrophils, by 6 hours following challenge, the effect subsiding by 48 hours post challenge. In both studies, neutrophil recruitment was enhanced in the presence of L. major (Monteiro et al., 2007; Teixeira et al., 2005). At the other end of the immune spectrum and focusing on the anti-inflammatory properties of saliva, a model of immune inflammation demonstrated that pretreatment of ovalbumin (OVA)-immunized mice with SGH of either Lu. longipalpis (Monteiro et al., 2005), Ph. papatasi, or Ph. duboscqi (Carregaro et al., 2008) inhibited OVA-induced neutrophil migration through a mechanism involving dendritic cell production of PGE2 and IL-10. The authors suggested that components in saliva may represent promising therapeutic molecules to target immune inflammatory diseases. The seemingly opposing effects of SGH on neutrophil recruitment may be explained by preexisting conditions that determine the prevailing inflammation at the time of SGH exposure. In light of the importance of neutrophils in the establishment of sand fly transmitted-Leishmania infection (John & Hunter, 2008; Peters et al., 2008, 2009), the effect of saliva on neutrophil recruitment at the inflammatory site of sand fly bites should be investigated. Sand fly transmission, in sharp contrast to needle challenge, results in a sustained neutrophilic infiltration localized to the bite sites. This influx is significant as neutrophils engulf parasites that may otherwise perish and deliver them live to their final host cell, the macrophage (Peters et al., 2008). This feature

of sand fly transmission was intimately associated with virulence and the failure of vaccines highly effective against needle challenge in experimental animal models (Peters et al., 2009). The cause of this sustained neurophilic recruitment has not been clearly established and the potential contribution of saliva to this scenario needs to be elucidated. This observation is relevant particularly in light of the fact that the initial influx of neutrophils in response to tissue damage inflicted by bite or needle is comparable and probably overrides Leishmania-specific signals (Peters et al., 2008).

Conclusion

Saliva is an integral part of every natural Leishmania-transmission event. The proteins in saliva are unrelated to Leishmania, but they affect parasite survival and development by their inherent proximity to Leishmania parasites at the bite site. Experimental evidence provided by independent investigators has convincingly demonstrated that an immune response specific to a salivary protein can have a prolonged adverse effect on Leishmania parasites. Despite significant improvement in our understanding of these remarkable molecules and their promising potential as vaccine candidates, several aspects challenging the utility of salivary-based Leishmania vaccines remain to be explored. Most of these challenges are global, applying to most arthropod vectors and are addressed at the end of the chapter.

MOSQUITOES

As a group, mosquitoes transmit the most devastating of the arthropod-borne pathogens. In addition to malaria and lymphatic filariasis, these insects serve as vectors for a plethora of arboviruses, including dengue, yellow fever, and other encephalitis viruses. Combined, these diseases account for nearly 14% of the infectious and parasitic disease global morbidity, leading to greater than 40 million healthy living years lost (World Health Organization, 2008). The major vaccine efforts aimed at mosquito components have focused on malaria transmission blocking targets in the mosquito midgut (Dinglasan et al., 2008); however, it is clear that mosquito feeding has a substantial immunomodulatory effect on their vertebrate hosts that can influence pathogen transmission (Schneider & Higgs, 2008), thus making mosquito salivary proteins attractive candidates for vaccine development. Studies assessing modulation of host immune responses to mosquito saliva mainly have focused on single exposures to bites or SGH. Combined these studies engender a model where mosquito saliva is immunosuppressive, potentiating infectivity of a variety of mosquito-borne pathogens (Schneider & Higgs, 2008) including Plasmodium berghei (Alger et al., 1972; Vaughan et al., 1999), Cache Valley virus, La Crosse virus, vesicular stomatitis virus, and West Nile virus (Schneider & Higgs, 2008). In vitro systems evaluating systemic cytokine responses indicate that for the majority of cases saliva inhibits Th1 and antiviral cytokines, while Th2 cytokine production is either unaffected or up regulated (Schneider & Higgs, 2008). In the presence of a strong inducer of Th2 immunity, however, mosquito saliva appears to inhibit Th2 cytokine production as well (Gillan & Devaney, 2004). While a systemic shift in cytokine balance certainly has the potential to influence disease outcome, these pathogens are predominantly deposited in extravascular sites in the skin (Kebaier et al., 2009, Schneider & Higgs, 2008) where the cytokine milieu could influence initial infectivity. Possibly due to the difficulty of measurement accuracy in tissues, few studies have assessed the influence of mosquito saliva in the local environment (Depinay et al., 2006; Donovan et al., 2007; Schneider et al., 2004). In conglomerate, these studies reveal that single exposures to mosquito bites alone do

47. Targeting Components in Vector Saliva

not profoundly affect cytokine production at the bite site, although MIP-2 is selectively induced by Anopheles stephensi bites (Depinay et al., 2006) and Aedes aegypti SGH up regulates IL-4 (Schneider et al., 2004). Inoculation of Ae. aegypti SGH, in the presence of Sindbis virus infection, dramatically influences the local immune response, inhibiting both type I and II IFNs and enhancing IL-4, IL-10, and IL-12 production compared to virus alone controls (Schneider et al., 2004). Even in the highest areas of transmission, less than 10% of mosquitoes are infected with a given pathogen, yet in some areas, a single individual can receive over 10,000 bites per year (Gil et al., 2003). The immunological climate induced by this repeated exposure likely contributes to priming of antipathogen immune responses. Unlike research investigating sand flies, data exploring the effect of multiple exposures to mosquito bites are sparse and evoke discordant mechanistic models. Similar to what is observed for sand files, repeated exposure to An. stephensi bites induces a Th1 profile that leads to increased resistance to P. yoelii infection (Donovan et al., 2007). Although cytokine production was not assessed and the mechanism not identified, presensitization to mosquito saliva also conferred partial protection to P. berghei infection in mice (Alger & Harant, 1976; Alger et al., 1972) and P. gallinaceum infection in chickens (Rocha et al., 2004). Most recently, these studies have been expanded into P. vivax-infected human populations, where higher An. darlingi-specific antibody levels and reduced serum IFN-g/IL-10 ratios correlated with asymptomatic infections (Andrade et al., 2009), suggesting that increased IL-10, rather than Th1 cytokine production is associated with decreased disease. In contrast to studies demonstrating saliva induced protection to pathogen assault, sensitization to A. aegypti bites was associated with increased IL-10 production at the bite site and exacerbated West Nile virus infections (Schneider et al., 2007), suggesting that preexposure to this species may not skew responses towards Th1 immunity or protection. The role that different host and mosquito species have on modulating cytokine responses remains to be completely elucidated, although these seemingly contradictory observations imply that the mechanisms are complex and may be species specific.

TICKS

There are two families of ticks, the hard ticks (Ixodidae) and the soft ticks (Argasidae). Each possess strikingly different feeding behaviors. Ixodids feed for prolonged periods lasting a few days to over 1 week with adult females feeding only once; argasids typically feed for less than 1 hour and their adults will feed multiple times (Francischetti et al., 2009). In addition, the nymphs secrete a different repertoire of salivary molecules compared to adults. This modulation results in a complex interplay between the multitude of salivary molecules expressed by different tick stages at different times postfeeding, the pathogens they harbor and the hosts they feast upon. Francischetti and colleagues (Francischetti et al., 2009) give a comprehensive account of tick salivary molecules and provide a link to a catalogue of over 3,500 putative salivary proteins from various species. Ticks are vectors of several human diseases including Lyme disease, tick-borne encephalitis, and hemorrhagic fever. Similar to other hematophagus arthropods, tick saliva contains a multitude of potent anti-inflammatory and antihemostatic molecules that facilitate blood feeding (Francischetti et al., 2009; Hovius et al., 2008; Titus et al., 2006). Many of these molecules are also immunomodulatory and affect both innate and adaptive immune responses, promoting pathogen survival and contributing to disease enhancement (Hovius et al., 2008; Titus et al., 2006). Here, we present a brief account of molecules recently identified from tick saliva, their contribution

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towards disease exacerbation, and their potential as vaccine candidates against human disease. Contrary to most experimental studies involving sand fly saliva vaccines, where the focus has been on modulation of cell-mediated immune responses, the purpose of developing vaccines incorporating tick salivary components has mainly centered on eliciting antibodies that neutralize saliva’s immunomodulatory activities.

Salp15 from Ixodes scapularis

Salp15, a salivary protein isolated from Ixodes scapularis but also present in Ixodes ricinus, proved to be central to the establishment of Borrelia burgdorferi (the etiological agent of Lyme disease) in mice, directly binding to the spirochete and protecting it from antibody-mediated lysis (Hovius et al., 2008; Ramamoorthi et al., 2005). This protein has immunosuppressive properties that inhibit adaptive immune responses against B. burgdorferi impairing dendritic cell function and T-cell proliferation (Hovius et al., 2008). Immunization with Salp15 represents an attractive strategy against B. burgdorferi. Anti-Salp15 antibodies could potentially neutralize its immunosuppressive properties or bind to Salp15 already bound to the spirochete forming immunocomplexes that would clear the infection (Hovius et al., 2008).

Salp 25 from Ixodes scapularis

Salp 25D, another salivary protein identified from I. scapularis has a fundamentally different effect on disease transmission. A homolog of peroxiredoxins, Salp 25D seems to enhance the acquisition of B. burgdorferi by I. scapularis through detoxification of reactive oxygen species at the vector–pathogen–host interface (Narasimhan et al., 2007). Ticks in which Salp 25D was silenced were impaired in their ability to acquire the spirochete upon feeding on B. burgdorferi-infected mice (Narasimhan et al., 2007). Additionally, immunizing mice against SaIp25D decreased Borrelia acquisition by the ticks (Narasimhan et al., 2007). This result demonstrates that B. burgdorferi exploits tick salivary molecules to improve its chances of being acquired by the vector.

Sialostatin from Ixodes scapularis

Sialostatin L2 was identified as an immunomodulator that impaired the feeding ability and increased the rejection rate of I. scapularis nymphs in vaccinated guinea pigs (Kotsyfakis et al., 2008). Named a “silent” antigen, this salivary protein did not induce humoral immunity following repeated exposure of guinea pigs to ticks; instead, it required a supra-physiological dose of sialostatin L2 to induce immunity, via inhibition of cathepsins, illustrating how normally nonimmunogenic antigens can also be useful as vaccine candidates.

Prostaglandin E2 (PGE2) from Ixodes scapularis

PGE2 is the first identified dendritic cell inhibitor from arthropod saliva (Sa-Nunes et al., 2007). It is responsible for the inhibition of IL-12 and TNF-a production by dendritic cells from C57BL/6 mice stimulated by several toll receptor ligands (Sa-Nunes et al., 2007). In addition, other observed effects of I. scapularis saliva on dendritic cell maturation and function, including the suppression of their ability to stimulate CD4 T cell proliferation and IL-2 production, were attributed to PGE2 because these effects were replicated by standard PGE2 (Sa-Nunes et al., 2007).

Sphingomyelinase-Like Enzyme (IsSMase) from Ixodes scapularis

Another relevant salivary molecule from I. scapularis is IsSMase, a sphingomyelinase-like enzyme associated with driving a host immune response towards a Th2 profile (Alarcon-Chaidez et al., 2009). In a mouse model of infestation, this enzyme acted directly on CD4 T cells driving the expression of IL-4, a cytokine

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associated with Th2 responses. Additionally, it was shown to superimpose this Th2 response onto a virally primed Th1 response (Alarcon-Chaidez et al., 2009).

ISL 929 and ISL 1373 from Ixodes scapularis

Two other proteins, ISL 929 and ISL 1373, recently identified from I. scapularis saliva, were shown to inhibit neutrophil function and promote B. burgdorferi survival (Guo et al., 2009). Immunization of mice with a mixture of these proteins increased the numbers of neutrophils recruited to the site of tick attachment as well as reduced spirochete burden in the skin and joints (Guo et al., 2009).

Iris from Ixodes ricinus

Serpins are serine protease inhibitors that have antihemostatic and immunomodulatory properties. Iris, an elastase specific serpin from the saliva of I. ricinus, is a salivary protein whose vaccine potential lies in its effect on the ticks themselves (Prevot et al., 2007). Iris acts on CD4 T cells promoting Th2-type immune responses (Hovius et al., 2008). Immunization with Iris produced antibodies that neutralized the proteasic activity of Iris and induced a Th1 response that protected rabbits from tick infestation (Prevot et al., 2007).

B-Cell Inhibitory Protein (BIP) from Ixodes ricinus

BIP was identified as the salivary protein responsible for the B-cell inhibitory property of I. ricinus saliva (Hannier et al., 2004). Inhibition of B-cell proliferation may facilitate the transmission of B. burgdorferi to the mammalian host.

Cement Protein 64TRP from Rhipicephalus appendiculatus

Ticks need to engorge for several days and have evolved a cement protein secreted in saliva to form an attachment cone to their host. Recombinant 64TRP corresponds to a cement protein from the saliva of Rhipicephalus appendiculatus and has been used as a transmission blocking vaccine that affects tick fitness (Labuda et al., 2006). Mice vaccinated once with 64TRP were protected from a lethal challenge of viral encephalitis by infected ticks (Labuda et al., 2006). It is worth noting that anti-64TRP antibodies disrupt the feeding site and recognize tick midgut epitopes, resulting in tick death (Labuda et al., 2006). To avoid host rejection, this protein shares similarities with host antigens and must be investigated for the risk of autoimmunity before it can be considered as a safe vaccine candidate.

Secretory Phospholipase A2 (sPLA2) from Amblyomma americanum

sPLA2, an enzyme from the saliva of Amblyomma americanum exhibits borreliacidal activity (Zeidner et al., 2009). sPLA2s are part of a small family of molecules that hydrolyze membrane phospholipids into smaller bioactive molecules that can be converted into eicosanoids including immune mediators such as prostaglandins, leukotrienes, hepoxilins, and lipoxins (Zeidner et al., 2009). Amblyomma sPLA2 is not present in I. scapularis and may explain why A. americanum is refractory to infection with B. burgdorferi. Phospholipase A2 may have a therapeutic potential against the Lyme disease spirochete and others. In conclusion, ticks pose a challenge to scientists looking for anti-tick vaccines and anti-tick pathogen vaccines. These arthropods have a complex life cycle involving nymphs and adults that differ in their ability to transmit diseases and possess a multitude of differentially expressed salivary proteins. In addition, a single tick species transmits several diseases and can influence the establishment of more than one pathogen. On the other hand, tick saliva represents a rich source of antigens with specific and potent physiological, pharmaceuti-

cal, and immunomodulatory activities. New technologies are accelerating the screening and identification of pertinent tick salivary proteins as demonstrated by the number of recently characterized molecules listed above. Table 1 summarizes the salivary molecules recently identified from sand flies and ticks that show promise as components of vaccines. Although the sialomes of several mosquito species have been characterized (Calvo et al., 2007, 2006, 2009; Holt et al., 2002; Ribeiro et al., 2004; Valenzuela et al., 2003, 2002), investigation of the vaccine potential of these proteins is only in its infancy and is mostly in the earlier phase of exploring the effects of SGH. Hopefully, the findings mentioned above will kindle further interest towards the identification of the molecules in mosquito saliva responsible for any pertinent effects.

CONCLUSIONS

Although targeting arthropod salivary proteins in multicomponent, anti-pathogen vaccines holds great promise, much work remains before these vaccines are to be realized. Below we list some advantages and drawbacks to consider:

Salivary Proteins Represent a Novel and Rich Source of Antigens for Vector-Transmitted Disease Vaccines

Apart from being unique in other ways (many have unknown functions and are present only in arthropods), the fact that these proteins are unrelated to pathogen antigens significantly increases the repertoire of novel molecules to be tested as vaccine candidates for vector-transmitted diseases. On the other hand, this divergence narrows the window by which they must exert their protective effect. Therefore, demonstration that such antigens initiate a protective immune response under simulated physiological conditions, most importantly the natural antigen dose an immunized subject would receive, is a prerequisite to demonstrate their usefulness.

Immunological Memory of Salivary Vaccines

Despite the fact the immunological memory to salivary proteins has not been thoroughly addressed, natural boosting by vector bites in endemic areas potentially minimizes the requirement for the generation of long-lasting immunity following vaccination, a major hurdle faced in vaccine development. The downside of prolonged exposure is the potential development of tolerance, a phenomenon that would eliminate the efficacy of an antisaliva vaccine component. This aspect will require exploration when salivary antigens are being considered for vaccine development.

Polymorphism of Salivary Proteins

A molecule conserved between arthropod vectors that transmit the same pathogen would be the ideal vaccine candidate. So far, such a salivary molecule has not been identified. For example, in sand flies, MAX is only present in Lu. longipalpis and a small fraction of other Lutzomyia species. Additionally, it is highly polymorphic within Lu. longipalpis (Titus et al., 2006). These features of MAX represent a significant stumbling block to its practical value in a Leishmania vaccine. In contrast (Elnaiem et al., 2005), showed that PpSP15 was not under a diversifying selection pressure in Ph. papatasi populations. Similarly, salivary proteins from Ph. duboscqi, a sister species of P. papatasi and the vector of L. major in sub-Saharan Africa, are highly conserved in populations from Mali and Kenya, at opposite ends of the geographical distribution of this species (Kato et al., 2006). Comparative transcriptomic analysis of salivary proteins of vectors of visceral leishmaniasis revealed several expressed genes that were genus-specific, species-specific, and common among the different sand fly species investigated (Anderson et al., 2006). This approach may prove to be a promising tool for identification of pan-arthropod antigens.

47. Targeting Components in Vector Saliva TABLE 1 Salivary vaccine candidate

605

A summary of recent salivary vaccine candidates from sand flies and ticks Source

Animal model

Target species

Disease

Predicted/Potential mechanism of protection

Reference(s)

Sand flies PpSP15

Phlebotomus papatasi

Mice

Human

CL

Polarization towards a Th1 response

Maxadilan

Lutzomyia longipalpis

Mice

Human

VL

LJM19

Lu. longipalpis

Hamster

Human/Dog

VL

LJL143 LJM17

Lu. longipalpis

Dog

Human/Dog VL

Neutralization of immunosuppressive properties of maxadilan Polarization towards a Th1 response Polarization towards a Th1 response

Ticks Salp 15

Ixodes scapularis

Mice

Human

Salp 25D

I. scapularis

Mice

Human

Sialostatin L2 I. scapularis

Guinea pig

Human

PGE2

I. scapularis

Mice

Human

IsSMase

I. scapularis

Mice

Human

ISL 92 ISL 1373

I. scapularis

Mice

Human

Iris

I. ricinus

Rabbit

Human

BIP

I. ricinus

Mice

Human

64TRP

Rhipicephalus appendiculatus

Mice

Human

sPLA2

Amblyomma americanum

In vitro

Human

Lyme disease

Neutralization of immunosuppressive properties of Salp 15 Lyme disease Neutralization of properties of Salp 25D that detoxify reactive oxygen radicals impairing tick colonization Lyme disease Neutralization of sialostatin L2 inhibition of cathepsins leading to tick rejection and feeding impairment Lyme disease Neutralization of the immunosuppressive effects of prostaglandin E2 on dendritic cell maturation and function Lyme disease Neutralization of the Th2 polarizing properties of IsMase Lyme disease Neutralization of ISL 929 and ISL 1373 neutrophil inhibitory properties Lyme disease Neutralization of the antiproteasic activity of Iris and induction of a Th1 immune response Lyme disease Neutralization of the B cell inhibitory activity of BIP viral encephalitis Disruption of the feeding site and tick midgut epitopes, resulting in impaired feeding and death of the tick Lyme disease sPLA2 posses direct borreliacidal activity

Apart from genetic diversity, the expression level of targeted salivary proteins and how it is influenced by age, diet, and seasonal related differences also require evaluation.

Specificity of Host Immune Responses to Salivary Proteins

Another challenge to the identification of appropriate salivary vaccines is the narrow specificity of their recognition by different animal models. For example, salivary proteins of Lu. longipalpis identified as immunogenic in dogs were different from those reported for hamsters. This host specificity

Oliveira et al., 2008 Valenzuela et al., 2001 Morris et al., 2001 Titus et al., 2006 Gomes et al., 2008 Collin et al., 2009

Francischetti et al., 2009 Hovius et al., 2008 Ramamoorthi et al., 2005

Narasimhan et al., 2007

Kotsyfakis et al., 2008

Sa-Nunes et al., 2007 Alarcon-Chaidez et al., 2009 Francischetti et al., 2009 Guo et al., 2009 Prevot et al., 2007 Hannier et al., 2004

Labuda et al., 2006

likely holds true for other salivary proteins and is not surprising considering that recognition is dependent on major histocompatibility complex class II molecules that can be restrictively specific. Much care will be required in identifying the salivary components important for influencing human disease.

Immunization with Salivary Proteins

Ideally a vaccine would include both a vector salivary antigen and a pathogen antigen, but how would such a vaccine work? Most of the studies discussed in this chapter established

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antisaliva immunity first to influence and direct subsequent antipathogen immunity. Comparing the outcome of initiating two independent arms of the immune system (antisaliva and antipathogen) through simultaneous vaccinations to the effectiveness of establishing antisaliva immunity first then boosting with a pathogen antigen requires investigation.

Hypersensitivity Reactions

Insect bites cause hypersensitivity reactions in their hosts; this phenomenon is the fundamental basis of exploring vector saliva as a vaccine component. However, in some instances, these immune responses can be severe, resulting in necrotic, local reactions, or sometimes acute systemic responses, including asthma and anaphylaxis (Peng et al., 2007). Considering the exposure intensity to vector bites in some endemic regions, these adverse responses require consideration during vaccine development particularly when testing conserved, ubiquitously expressed salivary proteins.

Cross-Reactivity and Potential Disease Exacerbation

The development of a pan-arthropod vaccine targeting multiple vector/disease combinations is an attractive aspiration and certainly cross-reactive responses have been identified among vector species. Mounting evidence suggests, however, that immunity to saliva may provide protection for some diseases but cause exacerbation in others. Clearly, identification of key protective antigens in each specific system is the first priority. Once this goal has been achieved, how vaccination with these antigens may influence other vector/disease systems should also be addressed. If vector salivary proteins as vaccine components are to be realized, it will be necessary to undertake studies of natural vector populations to determine pertinent features of their biology. The rapid spread of vector-borne diseases, attributed partly to global warming and insecticide resistance, emphasizes the need to use all available arsenals at our disposal to develop protective vaccines. We therefore cannot afford to ignore the potential that vector salivary proteins present in this regard. We thank Drs. Fabiano Oliveira and Jennifer Anderson for critical review of the manuscript.

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X



48 AIDS Vaccines: the Unfolding Story STEPHEN NORLEY

INTRODUCTION

limitations and obvious ethical (and financial) concerns, AIDS-vaccine research involving these animals has virtually ceased and despite continuing efforts to genetically engineer mice to render them susceptible to HIV-1 infection, the most commonly used model remains infection of macaques with the relevant simian immunodeficiency virus (e.g. SIVmac). SIVmac, like HIV-2, is derived from SIVsm (Hirsch et al., 1989), a naturally occurring infection of African sooty mangabeys (a monkey species that, like all natural hosts of SIV, do not develop disease). In Asian macaques, SIVmac replicates and induces a disease very similar to that caused by HIV-1 in humans (Simon et al., 1992). However, SIVmac is not HIV-1 and in order to facilitate the testing of vaccines based, for example, on the HIV envelope glycoprotein, a range of chimeric SIVs in which the env gene has been replaced by that of HIV-1 to form a SHIV (simian/human immunodeficiency virus) have been generated (Reimann et al., 1996). Although such SHIVs have been used extensively to evaluate candidate vaccines in macaques, it has become increasingly clear that a successful outcome using the SHIV/macaque model does not necessarily translate to protective efficacy in humans, and the gold-standard for efficacy has switched back to protection against robust challenge with a pathogenic strain of SIV itself. It is therefore important to keep the relevance and vigor of the challenge virus in mind while reviewing the results of preclinical vaccine trials.

Due to the very large number of people infected globally and the very high mortality rate (close to 100% if untreated), the HIV (human immunodeficiency virus)/AIDS (acquired immune deficiency syndrome) pandemic has justifiably been described as the modern plague. An AIDS Epidemic Update (Joint United Nations Programme on HIV/AIDS [UNAIDS] & World Health Organization [WHO], 2009) estimates that over 33 million humans are currently living with HIV/AIDS, a number that, after decades of annual increase, is now relatively stable because new infections each year are approximately matched by AIDS-related deaths (2.7 million and 2.0 million, respectively, for 2008). Despite the alarming rise in the rate of new infections in Latin America, eastern Europe, and Asia, two-thirds of HIV-infected individuals are living in Sub-Saharan Africa. Because HIV is most prevalent in young to middle-aged adults, the social strain (e.g., high numbers of orphans) and the economic impact of losing huge segments of the workforce are particularly devastating in developing countries. Although antiviral drugs able to limit the replication of HIV were first developed in the 1990s, economic, logistical, and political barriers can still prevent such therapies being made available to those in most need. It is generally accepted that the only realistic way to stop the pandemic is to develop an effective and affordable vaccine. Despite decades of intense research, such a vaccine remains elusive and, in this chapter, we will first briefly describe the history of AIDS vaccine development (with a discussion of why “traditional” forms of vaccine have failed) before concentrating on the pros and cons of the various modern forms of vaccine currently being developed and tested in both the animal model and in human clinical trials (Table 1).

TRADITIONAL VACCINES

Historically, the most successful forms of antiviral vaccines have been those based on whole killed virus (e.g., Salk polio vaccine, influenza vaccines), or live but harmless variants of the pathogen (e.g., Sabin’s oral polio vaccine and the smallpox vaccine). It is therefore not surprising that one of the first forms of vaccine to be developed and tested in the SIV/macaque animal model was whole inactivated SIV. Indeed, a number of laboratories succeeded in the late 1980s and early 1990s in inducing what appeared to be a state of sterilizing immunity to SIV challenge using such vaccines (Carlson et al., 1990; Hartung et al., 1992). It was optimistically assumed that, based on such a resounding success, it would only be a matter of time before the protective components of the inactivated virus vaccine would be identified and safely produced using recombinant DNA technology. However, this whole approach was nullified by

ANIMAL MODELS FOR AIDS

The preclinical evaluation of vaccine efficacy requires the use of animals that can be vaccinated and challenged with the live virus. Unfortunately, the only nonhuman animals in which HIV-1 is known to productively replicate are chimpanzees and gibbon apes and even in these viral loads are low and infection results in little or no disease. Due to these Stephen Norley, Robert Koch Institute, 13353 Berlin, Germany.

611

612 TABLE 1

THE MAJOR KILLERS (CLINICS, EPIDEMIOLOGY, AND IMMUNE PARAMETERS) Types of AIDS vaccines

Vaccine Type Whole inactivated virus

Live attenuated virus

Virus-like particles/ Single round infectious virus

Purified recombinant protein

Plasmid DNA

Recombinant vectors

Pros

Cons

Simple production. Induction of neutralizing antibodies. Proven record as a vaccine (influenza). Simple production. Infection mimics that of real virus. Induction of both antibody and cellular immune responses. Mimic the natural structure and/ or early life cycle of the virus but are devoid of infectious genetic material. Induction of both antibody and cellular immune responses. Extremely safe. Easily produced in high quantity.

Production involves live HIV. Risk of incomplete inactivation. No induction of cellular immune response. Vaccine virus induces rapid AIDS in newborn macaques and eventual AIDS in adult macaques.

None. Initial success shown to be due to immune responses to human cell proteins, not viral proteins.

Difficult to produce in quantity. Potential safety issues.

Limited. There is some suppression of viral load after challenge in the primate model.

Induction of type-specific neutralizing antibodies only. No induction of cellular immune response. Generally requires modifications (delivery systems, immunomodulatory cytokine genes, etc.) to boost immunogenicity.

Mixed. Failed when used alone as a human vaccine. Some evidence for success in combination with poxvirus vector.

Very safe. Easy to produce in quantity. Induction of both antibody and cellular immune responses. Replication-defective vectors generally safe. Induction of both antibody and cellular immune responses. Wide range of vectors available.

Can be difficult to produce in bulk. Preexisting immunity can limit immunogenicity. Some evidence for enhancement of HIV infection.

the demonstration that the protection being achieved was mediated not by the induction of an immune response to one or more viral proteins, but to human cell proteins incorporated into both the vaccine and challenge virus preparations as a result of production in transformed human T-cell lines (Stott, 1991). When the human cell proteins (later identified to be MHC molecules) in the challenge virus were replaced with the simian equivalent by a single passage through rhesus cells, no protection could be achieved (Norley et al., 1998). These initial, promising results were therefore simply an antixenogeneic artifact of the model system that has so far not been translatable to the HIV-1 infection of humans. This story of high initial optimism followed by devastating disappointment was unfortunately repeated for the second form of traditional vaccine. By removing one or more regulatory genes (such as nef) that are not essential for productive replication, Desrosiers’s laboratory produced an attenuated form of SIVmac that was not only apparently nonpathogenic when replicating in macaques but was also able to fully protect vaccinated macaques from subsequent challenge with the full-length, pathogenic wild-type virus (Daniel et al., 1992). The case for using such a variant of HIV as a vaccine in humans was

Success?

None. Total protection achieved but vaccine too dangerous for use in humans.

Mixed. Some success alone or as part of a prime-boost schedule in primates. Disappointing immunogenicity in humans. Mixed. Many vectors shown to be effective in animal models, particularly if replication competent. Adenoviral vectors failed to protect humans but marginal protection was achieved with poxvirus vectors plus protein.

strengthened by the unusual case of a cohort of patients who had been tragically infected from a common blood donation with a naturally occurring nef-deletion mutant of HIV-1 but who had remained free of AIDS for at least a decade (Deacon et al., 1995). However, during the course of experiments designed to assess the suitability of using a live attenuated virus in newborns, it was discovered that the vaccine virus itself caused rapid and severe AIDS in neonatal macaques (Baba et al., 1999). Furthermore, adult macaques inoculated with the live attenuated vaccine virus were found to eventually progress to AIDS, as did (a final nail in the coffin) those humans infected with the naturally occurring nef-deleted HIV-1 (Learmont et al., 1999). It was therefore clear that the induction of disease by these engineered attenuated viruses was only delayed, not abrogated. Although clearly unsuitable for use as a vaccine, the fact remains that live attenuated variants of SIV did induce total protection against challenge with the fully pathogenic wild-type virus, and identifying the underlying immunological mechanism for this protection remains a high priority in the AIDS vaccine field. It is hoped that, once understood, a similar state of immunity may by induced using a safe form of vaccine. Unfortunately, there is reason to believe that the failure

48. AIDS Vaccines: the Unfolding Story

of the challenge virus to establish a productive infection in animals previously infected with the attenuated virus is due more to competition for available, activated target cells than to a strong antiviral immune response. If so, even this promising avenue of vaccine research will be closed.

SUBUNIT VACCINES

Soon after the identification of HIV as the causative agent of AIDS, its outer envelope glycoprotein (gp120)— responsible for recognizing and binding to the cellular receptors on the surface of its target cells—was cloned, sequenced, and produced in large amounts as a potential vaccine. Based on experiences with other viruses, it was assumed that antibodies induced to this protein would be able to bind to any virus entering the body and neutralize its infectivity. Indeed, antibodies produced in animals immunized with the HIV-1 envelope glycoprotein were able to neutralize the available laboratory isolates of HIV-1 and immunized chimpanzees appeared to resist infection with the virus (Girard et al., 1991). It was during the course of clinical trials in humans that it became clear that the neutralizing antibodies being induced by these vaccines, although fully capable of neutralizing the corresponding and related isolates of HIV, were ineffective against primary isolates of the virus in circulation. Furthermore, as the number of primary isolates available increased, it became apparent just how restricted neutralizing antibodies generally are with regard to their breadth of neutralization. In other words, a subunit gp120-based vaccine might be able to protect against infection with viruses closely related to the isolate upon which the vaccine was based, but it is unlikely to be effective against even slight variants in circulation. The enormous variability of HIV (the variation in one HIV-infected individual exceeds that of influenza

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viruses globally) would therefore render a vaccine virtually ineffective, no matter how able it was to induce neutralizing antibodies. However, the fact that (in rare cases) HIV-infected individuals develop antibodies able to neutralize a wide range of HIV isolates continues to drive research and development in this area. For example, some human monoclonal antibodies specific for highly conserved regions of the HIV-1 transmembrane glycoprotein gp41 are broadly specific (Muster et al., 1993) and, more importantly, are able to reduce or inhibit infection when delivered passively (Mascola et al., 1999). Unfortunately, all efforts to induce such antibodies by vaccination have so far failed, possibly because the neutralizing monoclonals appear to have unusually long CDR H3 domains (Zwick et al., 2004), allowing them access to epitopes that are, due to steric hindrance, protected from the “normal” antibodies induced by immunization. However, the recent identification of an alternative target epitope for broadly specific neutralizing antibodies that is present on an exposed region of the HIV-1 gp120 has recently rejuvenated these efforts (Walker et al., 2009). Much effort is being put into reproducing in the immunogen the natural folding structure and trimeric form of the HIV envelope glycoprotein (Dey et al., 2009), preferably one “frozen” in the temporary state that occurs during interaction with the cellular receptors when hidden regions of the protein may transiently become accessible to antibodies.

GENETIC VACCINES

The failure of traditional vaccines and the difficulties in inducing a broadly reactive antibody response by immunization (Table 2) have led most laboratories to develop and evaluate so-called “genetic vaccines” (i.e., vaccines that deliver the genes coding for HIV proteins rather than the

TABLE 2 Problems facing the development of an AIDS vaccine Theoretical problems • Correlates of protective immunity remain unclear, giving no definitive target for vaccine development. • The immune response to active infection cannot clear the virus. How should a vaccine do better?

Special problems • HIV infects and eliminates the very cells of the immune system (CD41 T cells) needed to coordinate the antiviral response. • Antibody targets on the HIV envelope glycoprotein are heavily shielded by conformation or by glycosylation. • HIV down regulates expression of MHC-I molecules on the cells it infects, rendering them invisible to cytotoxic T cells. • Broadly specific neutralizing antibodies have proven to be very difficult to induce by vaccination.

The problem of variability • There is a staggering degree of variability in the sequence of HIV proteins, making the development of a universal vaccine difficult. • Even within a single infected human, there is continuous expansion of the HIV quasispecies, which contains mutants able to escape immunological control.

Practical problems • SIV infection of macaques, the only practical animal system for challenge studies, is extremely expensive, limited, and is based on an HIV-2 related virus, not HIV-1. • There are enormous organizational, financial, and ethical problems in carrying out large-scale AIDS vaccine trials in humans. • Most major pharmaceutical companies are reluctant to participate in AIDS vaccine development. • The success of antiretroviral drugs in industrialized nations has reduced the public and political will to fund vaccine development

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proteins themselves). The rationale behind this approach is that de novo expression of the gene(s) of interest in the recipient’s cells will lead not only to the production of proteins able to induce an antibody response, but also to endogenous processing of these proteins, resulting in presentation of protein fragments at the cell surface by MHC-I molecules and the induction of a specific cytotoxic T-lymphocyte (CTL) response. CTLs are perhaps the primary mechanism for combating virus infections and there is a known association between a host’s MHC-haplotype and the time to the development of AIDS in the patient, an observation that implicates CTLs in active control of HIV. Furthermore, studies in both humans and macaques indicate that there is severe selective pressure on the virus to mutate, if possible, the target epitopes recognized by CTLs, and that such escape mutants can replicate better (in the presence of the immune response) than the original virus. In macaques, the dramatic reduction from peak viral load that occurs a few weeks after infection coincides with the development of antiviral CTLs, and prior removal of all CD81 T cells (including CTLs) prevents this reduction from occurring (Schmitz et al., 1999). There is, therefore, ample evidence that a preexisting anti-HIV CTL response—despite, by definition, being unable to prevent infection per se—may help limit the spread and establishment of infection upon exposure to the virus.

DNA Vaccines

Perhaps the simplest method of introducing isolated HIV genes to the body is to immunize with plasmid DNA. Margaret Lui and colleagues demonstrated in the early 1990s that injection of mice with plasmid DNA coding for influenza genes induced a CTL response able to protect from overt infection and disease upon challenge (Montgomery et al., 1993). Countless immunogenicity studies in mice have shown the ability of “naked” DNA to induce both humoral and cellular immune responses to HIV proteins, and a number of early experiments in chimpanzees and macaques demonstrated immunogenicity and even some degree of protective efficacy against challenge with HIV-1 and SHIVs, respectively (Barouch et al., 2000; Boyer et al., 1997). However, in primates (including humans), the immune responses tended to be relatively weak, and a number of modifications have since been developed to enhance the immunogenicity of DNA vaccines. First, for certain genes such as gag, expression (and therefore immune response) can be increased by orders of magnitude if the DNA is synthesized with codons optimized (Gao et al., 2003) for use in eukaryotic cells (without changing the resulting protein sequence). Improved methods of DNA administration, including bioballistic delivery of DNA-coated microscopic gold particles into cells of the skin (Haynes et al., 1994), in vivo electroporation (Hirao et al., 2008) and even DNA tattooing (Pokorna et al., 2008), can also dramatically enhance efficacy. Furthermore, the co-delivery of DNA coding for cytokines, such as IL-2 and GM-CSF, resulting in production of these immunomodulators at the site of vaccine gene expression, has been shown to significantly enhance the resulting immune response (Siegismund et al., 2009). Despite the early successes and the advances made in optimizing the immunogenicity of DNA vaccines, it soon became clear that administration of DNA alone was generally insufficient to induce a vigorous and prolonged immune response. However, DNA-based vaccines are considered to be an excellent method of priming the immune response for further boosting by a second form of vaccine, and many of the vaccine strategies currently being developed use this approach.

Recombinant Viral Vectors

Most genetic vaccines are based on the delivery of the HIV/ SIV gene(s) of choice by a recombinant viral vector. For safety reasons, these are usually engineered to be replication incompetent (i.e., the viruses can infect their target cells, deliver the HIV genes for protein synthesis and induction of the immune response, but are unable to form new progeny viruses). It appears that if a virus is able to infect human cells, is amenable to insertion of foreign genes, and can be rendered safe, then it will almost certainly have been developed as a putative AIDS vaccine by one or more laboratories. Each of these vectors has its pros and cons and until we know which immune responses against which proteins at which sites are needed to give protection against infection, the strategy remains to try everything possible and hope for the best.

Poxvirus Vectors

Superficially, it makes sense to base a vaccine vector on a virus that already has a long track record of use in humans as a normal vaccine. The most successful vaccine known is vaccinia virus, used to eradicate the scourge of smallpox from the world. Unfortunately, despite early attempts to use recombinant vaccinia virus as an AIDS vaccine (Zagury et al., 1987), administration to people already immunocompromised (e.g., by an unrecognized HIV infection) can lead to systemic infection and even death. However, safer forms of vaccinia virus do exist, and one of the most commonly used vaccine vectors is that based on modified vaccinia Ankara, a variant of the virus that, by repeated passage through avian cells, has lost a significant proportion of its genome (Meyer et al., 1991) and is incapable of productive replication in (nearly all) mammalian cells. This virus has been used to immunize 150,000 Europeans against smallpox infection, so its safety (but not its efficacy) is well established. MVA, together with other replication-deficient poxviruses (in mammals) such as NYVAC and ALVAC (an avian poxvirus) have been used extensively as vectors in monkeys, chimpanzees, and humans, either alone or in combination with a DNA vaccine prime (Sutter & Staib, 2003). Indeed, over one-third of all ongoing clinical trials in humans involve a poxvirus component.

Adenovirus Vectors

For a number of reasons, human adenoviruses have been considered to be one of the most promising choices for a vaccine vector. Much of the initial work in this system used replication-competent viruses as a basis for vector development, and a number of successful vaccination/challenge experiments in both macaques and in chimpanzees were reported (Buge et al., 1997; Lubeck et al., 1997). However, being replication competent, these viruses were found to be shed from the recipient animals, a situation unlikely to be acceptable to licensing authorities, and focus shifted to the use of replication-defective versions of adenovirus. Like replication-defective MVAs, these adenovirus-based vectors were able to deliver the HIV gene of interest to the cells of the vaccinee and to stimulate a vigorous immune response. Of particular interest was the induction of mucosal immunity, an important characteristic for a vaccine designed to prevent infection with a virus whose major port of entry is the mucosal surface (Baig et al., 2002). Head-to-head comparisons of DNA-, MVA- and adenovirus-based vaccines for immunogenicity and efficacy in the animal model clearly showed the superiority of the recombinant adenoviruses in terms of virus load reduction following SHIV challenge (Shiver et al., 2002), and some degree of vaccine efficacy (albeit with a replicating vector) was even seen against the


more rigorous SIVmac challenge (Patterson et al., 2004). Based on these and other reports, large-scale clinical trials of adenovirus-based vaccines were soon initiated in humans, as will be discussed in some detail later. One of the drawbacks of using viruses that are either common pathogens in humans (like adenoviruses) or have been extensively used as vaccines in humans (such as poxviruses) is the problem of preexisting immunity to the vector itself. A large proportion of the population has already been exposed to the strains of adenovirus commonly used for vector production, and the resulting antiviral immune response can severely limit the efficacy of the vaccine virus. One approach to circumvent this problem is to base a vaccine on a serotype of adenovirus not generally in circulation in the target population (Vogels et al., 2007), or to pseudotype the vector to express the surface receptors of a rare serotype (Xin et al., 2007). With poxvirus vectors, the problem is less acute because most people at risk for HIV infection are nowadays too young to have received the smallpox vaccine. However, if, as seems likely, an effective AIDS vaccine will require more than one inoculation, the problem of antivector immunity will appear with the second and subsequent shots. Although some studies have suggested that the problem of antivector immunity may not be as severe as first supposed, it is possible to avoid the problem entirely through a vaccine regime that uses the same HIV genes expressed in a range of different vectors (i.e., a heterologous prime-boost). This ensures that each inoculation boosts the immune response to the gene-product of interest without influencing the antivector responses.

Adeno-Associated Virus Vectors

Adeno-associated virus (AAV) is a small parvovirus that lacks the capacity for autonomous replication. Although the vast majority of humans have been infected at some point in their lives with one of the many serotypes of AAV, it is not known to be associated with any disease. AAV is attractive as a vaccine vector because (i) it can induce long-term gene expression in even terminally differentiated and nondividing cells, (ii) it has long been used as a vector for gene therapy studies and has an established safety record, and (iii) it is possible to replace virtually the entire viral genome with the gene(s) of choice and still generate complete (albeit replication incompetent) virus particles. Recombinant AAV constructs carrying HIV genes, when delivered either systemically or orally, are able to induce strong humoral and cellular immune responses in mice (Xin et al., 2002) and a single immunization of macaques with an AAV vector expressing SIV genes resulted in a reduction of viral load upon challenge (Johnson et al., 2005). However, clinical trials in humans have failed so far to match the promising results seen in the animal models and there is some evidence (in mice at least) that the CD81 CTLs induced in response to HIV proteins expressed by AAV vectors are functionally impaired with regard to efficacy (Lin et al., 2007). As mentioned previously, efforts to stimulate antibodies with properties similar to the known broadly specific human monoclonal neutralizing antibodies have so far failed, probably due to the unusual structure of such antibodies. The properties of AAV that make it so attractive as a vector in gene therapy have recently been exploited in a novel fashion in an attempt to overcome this problem. Rather than try to stimulate the induction of antibodies in the standard way by inoculation with an HIV protein or gene, recombinant AAVs were generated that carry the genetic information coding for the antibodies themselves. Macaques inoculated with such constructs were found to maintain high levels of the expressed neutralizing antibodies in their serum for

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many months and to resist infection with SIV (Johnson et al., 2009). This approach, while in its infancy, holds obvious promise to overcome the HIV vaccine problem by simply supplying the necessary antibodies independently of the host immune system.

Other Viral Vectors

As mentioned earlier, there is hardly a vector system that has not been exploited to develop a candidate AIDS vaccine. In addition to the poxvirus, adenovirus, and AAV-based vectors, these include different members of the herpesvirus family, various alphaviruses, measles virus, rhabdoviruses (including rabies virus), and poliovirus. Replication-defective herpes simplex virus (HSV)-1, being developed as an antiHSV vaccine itself, has been used to deliver multiple SIV genes to macaques and to reduce viral load upon challenge (Kaur et al., 2007). However, the situation with another herpesvirus vector, varicella-zoster virus (VZV) serves as a warning that the outcome of a vaccination may sometimes be the opposite of that desired. Macaques immunized with recombinant VZV vectors expressing the SIV env gene were found, upon challenge with SIV, to experience viral loads orders of magnitude higher than those receiving the wild-type vector or no vaccine at all (Staprans et al., 2004). This was accompanied, as one would expect, with a dramatic loss of CD41 T cells and a rapid progression to disease. It seems possible that any immunization that primes the body to respond with a rapid expansion of activated CD41 cells (a favored target cell for HIV) without inducing a concomitant antiviral effector mechanism such as CTLs will have this effect, as will be discussed later in the section on clinical trials.

Single-Round Infectious Viruses

If one assumes that the very vigorous protection induced by live attenuated versions of SIV is indeed a result of the immune response (an assumption that still remains unjustified), then an obvious approach to vaccine development is to make such vaccines safe without compromising their immunogenicity. This can theoretically be achieved by rendering the virus incapable of producing infectious progeny virus. For example, nucleocapsid deletion mutants of SIV have been produced that, upon inoculation, are able to produce intact viral particles that lack the viral RNA (Gorelick et al., 2000). Although, unlike replicating live attenuated SIVs, such constructs could not protect macaques against infection with the wild-type virus, they were able to limit the viremia for at least 2 years post-challenge. Single-round viruses have also been produced by deletions in the vif gene (Kuate et al., 2003), by mutating the gag-pol frameshift site and providing gag-pol in trans (Evans et al., 2004) and by combining defects in vif, nef, and env with a pseudotyped VSV-G protein (Tang & Swanstrom, 2008). Furthermore, mutating the catalytic domain of the integrase gene produced a virus unable to integrate into the host cell’s genome or produce progeny virus (Zheng et al., 2008).

Bacterial Vectors

Most of the genetic vaccines mentioned so far have been based on viruses able to deliver the HIV/SIV gene(s) of choice by infecting the host cell. There is, however, an active field of research aimed at exploiting the properties of nonpathogenic or attenuated bacteria to achieve the same goal. Salmonella, for example, is particularly attractive because it is known to induce solid mucosal immunity and wellcharacterized vaccine strains of the bacteria are available. So far, the results using recombinant Salmonella carrying various HIV and SIV genes, including synthetic polyepitope strings, have been disappointing, both in macaques and in humans

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(Kotton et al., 2006), but a number of approaches for improving efficacy (such as codon-optimization for expression in bacterial cells) are being pursued (Tsunetsugu-Yokota et al., 2007). Two other enteric bacteria that are being developed as potential vaccine vectors are Shigella and Listeria (Shata & Hone, 2001; Neeson et al., 2006). Of particular interest is the listeriolysin O protein used by Listeria monocytogenes to escape the phagolysosome and enter the cytoplasm to replicate, a characteristic that makes it very efficient at inducing the CD81 T-cell response. Indeed, incorporating the gene coding for listeriolysin O into bacteria that normally remain within the phagolysosome has been shown to enhance their ability to induce CTLs (Bu et al., 2003). One potential bacterial vector whose safety record is unquestionable is BCG, an avirulent strain of Mycobacterium bovis that has been used to vaccinate billions of humans against tuberculosis. It is therefore no surprise that the first recombinant bacteria expressing HIV genes were developed as early as 1991 (Fuerst et al., 1991) and many recombinant BCGs expressing a variety of HIV and SIV genes have been produced and evaluated since. However, despite being immunogenic, the results of challenge experiments in macaques have been largely disappointing and work is ongoing to further improve efficacy. Indeed, in an effort to overcome the potential problem of much of the world’s population having preexisting immunity to BCG through tuberculosis vaccination, recombinant vectors have been produced based on the related bacteria Mycobacterium smegmatis (Cayabyab et al., 2006).

CLINICAL TRIALS

Despite the enormous amount of work carried out to evaluate the immunogenicity and efficacy of putative AIDS vaccines in animal models, the only real measure of a vaccine’s success is its ability to protect humans from infection or disease under field conditions. Dozens of vaccine candidates have been tested for safety and immunogenicity in small-scale phase I and II clinical trials in humans, but to date, only three clinical trials have been carried out with the power to address protective efficacy. The first used purified recombinant HIV envelope glycoprotein (AIDSVAX B/E) to immunize thousands of volunteers at different locations throughout the world. The result was that no significant protective effect could be demonstrated (i.e., there was no discernible difference between the number of volunteers who became infected with HIV after receiving the vaccine and those who became infected after receiving the placebo) (Pitisuttithum et al., 2006). This outcome, disappointing though it was, was not entirely unexpected based on the results of earlier trials in animals and on the outcome of a phase II clinical trial of the vaccine. However, there was great hope for the later Merck STEP trial in which 3,000 volunteers at risk for HIV infection received either a mixture of replication incompetent adenovirus constructs expressing the gag, pol, and nef genes of HIV-1 or a placebo. These hopes were dashed in late 2007 when it became clear that vaccinated volunteers with a strong preexisting immune response to adenovirus were becoming infected at a far higher rate than adenovirus-seropositive volunteers in the placebo group (Sekaly, 2008). The inference was that the immune response, already primed by a prior infection with adenovirus, reacted to the vaccine by the activation and proliferation of CD41 T cells specific for the vector and that these cells provided an ideal target cell for HIV infection. This conclusion, which has been both contradicted (O’Brien et al., 2009) and supported (Benlahrech et al., 2009) by subsequent analyses, has severe implica-

tions for not only AIDS vaccines based on adenovirus vectors (or indeed, any virus against which there is a significant degree of preexisting immunity in the population), but also for similar vaccines against other human pathogens being evaluated in humans. Even if the fears of enhancing infection by vaccination turn out to be unfounded or the effect can be abrogated, the fact remains that this most promising of vaccine candidates failed to show any protective efficacy whatsoever. Following such high-profile failures, the AIDS vaccine field was in great need of a positive clinical trial, and this was delivered in late 2009 when the results of the RV144 phase III clinical trial carried out in Thailand were published (Rerks-Ngarm et al., 2009). In this trial, over 16,000 volunteers at risk for infection were recruited, screened, and, in a double-blinded, randomized, placebocontrolled protocol (an organizational tour de force) were inoculated with a combination of a replication-deficient poxvirus vectors (canarypox vCP1521) expressing the HIV-1 env, gag, and protease genes plus booster inoculations with a recombinant envelope glycoprotein mix (AIDSVAX B/E). The outcome, which prompted a tidal wave of optimism both in the press and in the AIDS vaccine field, was that there appeared to be a slight but significant (31%) reduction in the risk for infection for the vaccine group compared to the placebo group. Although the effect was clearly insufficient to justify using the vaccine in the general population, the results were widely heralded as a breakthrough giving researchers a basis upon which to refine and improve the vaccine. However, this euphoria should be tempered by caution because there are a number of aspects to this study to suggest that the positive outcome may turn out to be simply a statistical anomaly. One curious point is that the virus loads in the recipients of the vaccine who became infected during the follow-up period were similar to those in the volunteers who were infected subsequent to receiving the placebo. This observation, which implies an “all-or-nothing” protection, is in direct contrast to results in the animal model where a reduction in viral load, but no sterilizing immunity, is usually seen with candidate vaccines of this nature. The second point is that, as widely acknowledged, the beneficial effect of the vaccine only appeared to operate during the first 6 months of follow up with infection rates being virtually identical over the ensuing 3 years. However, a closer look at the data reveals that this initial difference was due to an unusually high number of infections in the placebo group rather than an unusually low number of infections in the vaccine group, a phenomenon difficult to explain biologically. Hopefully, these concerns will be eliminated by the analysis of stored samples and clinical trials in areas where the general rate of infection is much higher. Until then, it will be important to maintain an attitude of cautious optimism and avoid investing all resources in following what may well turn out to be a red herring.

THERAPEUTIC VACCINES

Most AIDS vaccine research concentrates on the development of prophylactic vaccines (i.e., vaccines given to uninfected recipients that induce an immune response capable of preventing subsequent infection upon exposure to HIV). However, given the very large numbers of people already infected with HIV, particularly in regions of the world with (at best) limited access to antiretroviral drugs, a therapeutic vaccine able to delay or prevent the onset of disease would have an enormous impact at both the personal and the economic level. Unlike some dormant viruses (such as VSV), whose reappearance can be inhibited by therapeutic vaccination, HIV is a continuously ongoing, acute infection


characterized by a state of hyperimmune activation. Given that the immune system is being massively exposed to viral antigen all the time, it is difficult to see how immunization of HIV-infected individuals would be likely to help. However, attempts have been made to immunize infected volunteers with immunogens alone or in combination with cytokines to steer the immune response toward a more effective antiviral state (Gotch et al., 2001). Such attempts have unfortunately been unsuccessful in the long term. One potentially promising approach has been to simulate immunization during a state of viral latency (à la VSV) by administering the vaccine to HIV-infected patients in which viral replication is suppressed by antiretroviral drugs (Moss et al., 2000). The hope is that stimulating an immune system not consistently subjected to the onslaught of acute HIV infection will allow the development of an effective immune response able to control viral replication when drug therapy stops. A variation of this approach is to give the patient a series of “drug holidays,” in which therapy is briefly withdrawn to allow a temporary, controlled burst of virus replication to stimulate the immune response (Ruiz et al., 2000). Although some benefit has sporadically been seen using these approaches, the results have generally been disappointing and therapeutic vaccination remains a minor avenue of AIDS vaccine development. As mentioned above, HIV infection is characterized by continuous high-level replication and a state of hyperimmune activation that appears to drive the immune system to exhaustion. Apart from the infection and/or elimination of activated CD41 cells, there is clear evidence that CD81 T cells, particularly HIV-specific CTLs, lose their functional capacity to attack and eliminate infected cells as disease progresses. Efforts to modulate and enhance the functional immune response by administering appropriate cytokines have been disappointing. However, more recently, this loss of T-cell function has been shown to be associated with the expression of the PD-1 receptor, normally used to turn off activated T cells after a pathogen has been eliminated. Indeed, the level of PD-1 expression appears to be the most reliable marker for disease progression (Day et al., 2006). Interestingly, blocking the interaction between PD-1 and its ligand can restore the antiviral activity of “exhausted” HIV-specific T cells in vitro. Although there are a number of obstacles to overcome, including the potential for inducing a state of autoimmunity by interfering with this negative regulator of immune responses, approaches such as these may eventually allow HIV-infected individuals to maintain immunologic control of the infection and prevent or delay the onset of AIDS (Simone et al., 2009).

REPLICATING VECTORS

As pointed out by others, there appears to be an almost inverse relationship between the safety of a candidate AIDS vaccine and its efficacy. Immunogens such as “naked” DNA or recombinant protein are very safe but have so far been mostly ineffective in animal and clinical trials whereas, at the other end of the scale, live attenuated viruses are highly effective but eventually cause AIDS. The vast majority of research currently concentrates on the middle ground (i.e., replication-defective vectors that are engineered to be as safe as possible). In these modern times, neither vaccine producers nor licensing authorities will even consider using a vaccine with a known risk of severe side effects, no matter how small they may be. It is generally acknowledged that the two most successful vaccines ever made (smallpox and polio), vaccines that have been used to immunize virtually everyone on

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the planet, would not get licensing approval in today’s risk-free climate. However, as safe AIDS vaccine candidates continue to prove ineffective, some research teams are returning to the development of live, replicating vectors, arguing that a prolonged and active stimulation of the immune response may be necessary to induce a state of protective immunity. Safety issues could be addressed if and when efficacy has been demonstrated. Vaccines based on the attenuated measles virus vaccine expressing the HIV envelope gene have been developed and shown to be immunogenic in mice and macaques (Lorin et al., 2005). Even more promising results were obtained using engineered versions of attenuated vesicular stomatitis virus (VSV) expressing HIV and SIV genes. Macaques inoculated with such constructs experienced very low or undetectable viral loads and no disease for over a year after challenge with a corresponding SHIV, while control animals progressed to AIDS within 6 months (Rose et al., 2001). Concerns about the inherent neurovirulence of VSV have lead to the development of modified vectors with dramatically reduced pathogenicity (after intracranial inoculation into the brains of mice) but enhanced immunogenicity (Cooper et al., 2008). More recently, replicating recombinant vectors based on human cytomegalovirus have been shown to give an impressive degree of protection against SIV infection, with most vaccinated animals having undetectable or only sporadically detectable viral loads after challenge (Hansen et al., 2009). Some groups, like our own, are even developing recombinant replicationcompetent retroviruses as a proof-of-principle for vaccine efficacy. Although safety remains the priority in AIDS vaccine development, it is possible that an effective vaccine that is 100% safe may never be developed. If an AIDS vaccine were to be developed with high efficacy but significant risk like, for example, the smallpox vaccine, it would almost certainly not be considered for use in industrialized countries where the risks of HIV infection are relatively low and where there is easy access to antiretroviral drugs should the worst happen. However, in some countries of the African continent where the attack rate of HIV among young people is in double figures and antiretroviral therapy is severely limited, the benefits of immunity to HIV infection would drastically outweigh the possible risk of severe side effects from vaccination. Whether or not the use of such a vaccine would be acceptable in such regions of the world would then become a social and political, rather than a scientific, question.

GROUNDS FOR OPTIMISM

This short overview of the unfolding AIDS vaccine story makes, on the surface, somewhat depressing reading. It terms of success (i.e., the general availability of an effective AIDS vaccine), we appear to be no closer today than we were in the early 1980s when HIV was first identified as the causative agent of AIDS. Indeed, one could argue that the situation is now even worse, because many of the major routes of vaccine development (e.g., whole inactivated virus, live attenuated virus, recombinant envelope glycoprotein) have since run into dead-ends. Critics of the AIDS vaccine field often suggest that a vaccine will never become a reality and that efforts and funding should be directed elsewhere. However, the fact remains that the only realistic option for eventually overcoming the horrors of the AIDS pandemic is to develop a vaccine. The response to the many setbacks should therefore be a redoubling of efforts, not frustrated capitulation in the face of adversity.

618 TABLE 3

THE MAJOR KILLERS (CLINICS, EPIDEMIOLOGY, AND IMMUNE PARAMETERS) Reasons for optimism

• Our understanding of HIV and the way in which it interacts with its host continues to expand rapidly, opening up novel, innovative avenues of vaccine development. • Despite failures, recent types of vaccine have been shown to induce impressive levels of protective immunity against vigorous challenge in the animal model. • New target epitopes for broadly neutralizing antibodies are being discovered, which, together with detailed knowledge of the structure of the HIV envelope glycoprotein, gives new directions for vaccine development. • Strategies for inducing protective immune responses at the mucosal level, rather than systemically, are being developed and tested. • Innovative techniques for providing a protective immune response through gene therapy rather than vaccination have been developed and successfully tested in the animal model. • Despite a number of caveats that need to be addressed, the most recent phase III trial of an AIDS vaccine showed, for the first time, a modest but significant protective effect. • Cohorts of highly exposed persistently sero-negative individuals demonstrate that protection against infection despite frequent high-risk behavior is possible under real-life conditions.

There are, in fact, a number of reasons to be optimistic about the future success of the AIDS vaccine effort (Table 3). First, thanks to the decades of intense research on a global scale, we now have a deeper knowledge of HIV and its interaction with its host than we have for most, if not all, other viruses. This provides a solid basis upon which to continue building vaccines and other intervention strategies in a targeted and informed manner. Second, there are enough positive results coming from the field of preclinical testing in the macaque animal model to show that solid protection against even highly rigorous SIVmac challenge can be achieved by prophylactic vaccination. For example, replication-competent CMV vectors, while not protecting 100% of vaccinees, gave very impressive, robust protection in a majority of vaccine recipients (Hansen et al., 2009) and further work should allow vaccine refinement and identification of the elusive correlates of protective immunity. We may end up having to relax our requirement for a zero-risk vaccine if it transpires that live, replicating vectors are indeed required for protective immunity, but it is likely that objections would fade if such an effective vaccine became available. Third, novel targets for broadly neutralizing antibodies are being identified, including a target that, unlike those protected epitopes in the transmembrane gp41, is present in an exposed region of the outer envelope gp120 (Walker et al., 2009). If such antibodies can be induced by a vaccine, protection against infection may well become a reality. Fourth, progress is being made in stimulating and maintaining antiviral immune responses at the mucosal surfaces used by HIV to enter the body (Demberg & Robert-Guroff, 2009). If localized immune responses able to prevent the establishment of infection can be stimulated by (for example) combination microbicide/vaccine formulations, the induction of systemic immunity by conventional vaccination that is proving so difficult may not be necessary. Fifth, novel approaches to impart a state of protective immunity are being developed. For example, even if broadly neutralizing antibodies (which are known to protect against infection when delivered passively) continue to resist

induction by vaccination, the use of gene therapy to deliver DNA coding for such antibodies could potentially be used to overcome this obstacle (Hansen et al., 2009). Finally, the one observation that shows resistance to HIV infection is possible under field conditions is the existence of so-called highly exposed, persistently seronegative (HEPS) individuals. Perhaps the best example of the HEPS phenomenon is the cohort of commercial sex-workers in Nairobi that, despite having frequent unprotected sex with men whose average prevalence of HIV infection is in double-digit figures, continue to remain free of overt HIV infection. There is evidence that this protection is associated with a systemic and mucosal T-cell response (Kaul et al., 2000) and/or the production of HIV-specific IgA at the vaginal mucosal surface (Devito et al., 2000), possibly resulting from an earlier abortive infection with HIV. If the precise biological mechanism for this state of protective immunity can be precisely identified and reproduced artificially, we would have a vaccine able to protect against infection under the most challenging of conditions.

CONCLUSIONS

It would be an understatement to state that the AIDS vaccine story has not unfolded in the way that many at the beginning expected or hoped it would. It was initially assumed that bringing the full might of modern science to bear on the problem would rapidly result in the production of a safe and effective vaccine. Unfortunately, traditional approaches to vaccine development turned out to be either ineffective or too dangerous for use in humans, and the dozens of modern forms of vaccines, most of which are based on recombinant replication-deficient viruses, have generally been shown to be very safe but relatively impotent. However, results from preclinical studies in macaques, particularly those appearing recently, do suggest that protection against a rigorous challenge can be achieved and such approaches are now being translated into human clinical trials. HIV has now revealed some of the chinks in its armor and we are better armed today than ever before to push home the attack. Although it is almost a cliché to say so in the field of AIDS vaccine development, the breakthrough may indeed be just around the corner.

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49 Tuberculosis GERHARD WALZL, PAUL VAN HELDEN, AND PHILIP R. BOTHA

Active infection with Mycobacterium tuberculosis is termed tuberculosis (TB) and is the leading cause of death due to bacterial infection. Approximately one-third of the world’s population is thought to harbor latent forms of this infection and the resultant huge reservoir for future disease, the recent increase in multidrug resistant disease and a dangerous interplay between tuberculosis and human immunodeficiency virus (HIV) infection will continue to pose serious challenges for health care in the years to come (Young et al., 2009).

at 142 cases per 100,000 of the global population, whereas the corresponding figure was 139 cases per 100,000 people in 2007 (World Health Organization [WHO], 2009).

Trends in Tuberculosis Mortality

The global tuberculosis mortality peaked around 1990 and has seen a small but steady decline since then. Current statistics from the 13th annual report on global control of tuberculosis published by the WHO demonstrated a decrease in mortality in all six WHO regions. The Americas, Southeast Asia, and the eastern Mediterranean are currently on track to halve the tuberculosis prevalence rate by 2015—a target set out by the Stop TB Partnership and WHO Global plan to stop TB 2006–2015 (Stop TB Partnership and World Health Organization [WHO], 2006). Although this is encouraging, it is unlikely that the target to reduce mortality will be achieved in these regions. In addition, the prevalence and mortality targets set out by the WHO for 2015 will not be met by Europe and Africa, and, as a result, overall targets with respect to prevalence and mortality rates will not be halved by 2015. There were an estimated 2 million deaths attributed to tuberculosis in 2007.

EPIDEMIOLOGY OF TUBERCULOSIS

Tuberculosis has evolved with humans for thousands of years. The first reports of this disease originated from ancient Egyptian illustrations, depicting individuals with spinal tuberculosis. However, as far as can be ascertained, it was not until the start of the Industrial Revolution that tuberculosis became a major public health problem with rapid urbanization and overcrowding fueling its spread. Since then, the epidemic has expanded and is currently only surpassed by HIV as the global leading cause of death due to an infectious agent (Fitzgerald & Haas, 2005). The World Health Organization (WHO) declared tuberculosis a global public health emergency in 1993. One-third of the global population is estimated to be infected with tuberculosis and, in 2007, there were 9.27 million new tuberculosis cases worldwide, with Asia and Africa being the areas worst affected. India, China, Indonesia, Nigeria, and South Africa are the five countries most severely affected, and 55% of the total number of tuberculosis cases occurs in Asia and 31% in Africa. This is an increase from 6.6 million cases in 1990 and 8.3 million cases in 2000, respectively. However, there is a gradual decline in the number of cases per capita, but this is not reflected in the total number of incident cases due to the global population growth. Tuberculosis rates peaked in 2004

Epidemiology of HIV-Associated Tuberculosis

There were 1.3 million new tuberculosis cases in HIV coinfected individuals in 2008 (WHO, 2009) accounting for 456,000 deaths, which represented one-third of all tuberculosis associated deaths (Lawn & Churchyard, 2009). The sub-Saharan region has been worst hit (79% of all TB and HIV coinfected individuals) with Southern African countries disproportionally affected due to the concentration of the HIV epidemic in this region (WHO, 2009). For example, in a Cape Town (South Africa) informal settlement, the rate of tuberculosis was in excess of 2,000 per 100,000 people, which is largely driven by the high prevalence of undiagnosed tuberculosis (Middelkoop et al., 2008). It is clear that the current WHO strategies, including the directly observed therapy– short course (DOTS) are inadequate for control of the HIV and tuberculosis epidemic in this region, or alternatively, proper implementation has not occurred (Lawn et al., 2006). This has dire consequences for HIV infected individuals, with tuberculosis being the number one cause of death in this population. There does not seem to be an increased risk

Gerhard Walzl and Paul van Helden, DST/NRF Centre of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa. Philip R. Botha, Division of Infectious Diseases, Department of Medicine Tygerberg Academic Hospital, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa.

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of tuberculosis during the HIV seroconversion period, but the risk of acquiring tuberculosis increases threefold in the first 2 years after seroconversion (Lawn et al., 2006). In addition, HIV coinfected individuals are also at increased risk of recurrent tuberculosis (Charalambous et al., 2008), which is increasingly problematic as the CD4 count declines. HIV infection is well-known to predispose to reactivation of endogenous infection, but molecular epidemiological data from South Africa demonstrated that most recurrent episodes may be the result of exogenous reinfections rather than reactivation of latent M. tuberculosis infection in those uninfected with HIV (van Rie et al., 1999) and in HIV infected patients (Charalambous et al., 2008). Multiple studies have shown a substantial survival benefit from antiretroviral therapy. There is a 54% to 74% reduction in tuberculosis rates associated with antiretroviral therapy; however, active tuberculosis still occurs commonly before the diagnosis of HIV infection (Muga et al., 2007). A resurgence of tuberculosis occurred in the United States from 1985 to 1992 due to the HIV epidemic. This increase was also partly due to a breakdown of the national tuberculosis control program, as well as an increase in homelessness and i.v. (intravenous) drug use in urban areas (American Thoracic Society, 1992). Foreign-born residents only make up a small proportion of the population in the United States, yet they accounted for 50% of the cases due to a concentration of risk factors in their communities. However, the rate of HIV–TB coinfection has declined threefold in the United States from 1993 to 2004 due to improved diagnosis and treatment of HIV, as well as restoration of the national tuberculosis control program (Fitzgerald & Haas, 2005). A growing concern is the effect of HIV on the multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis epidemics (also see below). HIV has lead to an increase in the global tuberculosis incidence and is likely to contribute to the increased number of MDR and XDR cases. The combination of a large population of HIV-infected susceptible hosts with poor tuberculosis treatment success rates, a lack of airborne infection control measures in resourcepoor high incidence settings, inadequate drug-sensitivity testing, and an overburdened MDR-TB treatment program could lead to a MDR-TB and XDR-TB epidemic of unprecedented scale (Andrews et al., 2007).

Epidemiology of Drug-Resistant Tuberculosis

The first antituberculosis drug, streptomycin, was developed in 1944 but only months after its use became widespread, the first resistant strains were isolated, signifying the onset of the problem of drug resistance. Currently, MDR-TB is characterized as resistant to the two most effective first line agents (rifampicin and isoniazid), and XDR-TB is defined as infection with an MDR strain that is also resistant to any fluoroquinolone as well as one or more of the second-line injectable agents (amikacin, capreomycin, and kanamycin) (Jassal & Bishai, 2009). Globally, there were approximately 500,000 MDR cases in 2007 alone: 85% of these were in 27 countries with India, China, the Russian Federation, South Africa, and Bangladesh being hardest hit (WHO, 2009). Management of MDR-TB is complicated by long treatment durations, frequent adverse drug effects, exorbitant costs, and limited efficacy. From the increase in MDR-TB cases, it is inferred that there will be a concomitant rise in the number of XDR-TB cases. Initial hopes that XDR-TB is less transmissible than susceptible TB were dashed with the description of an XDR-TB outbreak in a hospital in Tugela Ferry, in the Kwazulu-Natal province on the east coast of South Africa in 2006. In this outbreak, 53 patients were affected, the majority of whom

were HIV coinfected. Of the patients, 52 died, with a median survival of only 16 days. Molecular genotyping demonstrated that 85% of the strains were similar, raising fears about nosocomial transmission (Gandhi et al., 2006). By the end of 2008, XDR-TB cases had been reported in 55 countries or regions, with the worst affected areas being Eastern Europe and the former Soviet republics in central Asia. Of all the MDR cases reported to the WHO, 7% met the criteria for XDR-TB. Of great concern is that the reported nosocomial transmission clusters of XDR-TB may evolve into community-based epidemics under the public health conditions that are common in many developing countries (Basua et al., 2009).

The Spread of Tuberculosis and Risk of Progression to Active Disease

M. tuberculosis is transmitted by the inhalation of small droplet nuclei, which are aerosolized infectious particles generated by coughing, talking, and sneezing. Once airborne, the particles dry out and can remain suspended for long periods. When inhaled, the particles are deposited in the terminal airways where viable bacilli can cause infection. A single cough can generate up to 3,000 infectious droplets; sneezing can generate many more (Bates & Stead, 1993). Large respiratory droplets and fomites are not important sources of M. tuberculosis transmission. Transmission most commonly occurs indoors in the setting of prolonged exposure and multiple inocula. The risk of infection is determined by the closeness of the contact (often household or institutional), as well as the infectiousness of the source case (Morrison et al., 2008). Individuals who are sputum smear positive are generally more infectious than those who are culture positive but smear negative, as the former group have a higher number of bacilli in the sputum. In HIV-uninfected individuals, transmission occurs almost exclusively in the setting of cavitating disease. Patients with HIV infection or AIDS have been shown to transmit the organisms even in the absence of lung cavitation or with normal chest radiographs (Pepper et al., 2008). HIVinfected patients, however, are not more infectious than HIV-negative individuals (Klausner et al., 1993). There are particular concerns about the high risk of tuberculosis outbreaks among patients with AIDS, which is compounded by the high incidence of tuberculosis in this population and the frequent delayed diagnosis and treatment due to atypical presentations of the disease. The institutional spread of tuberculosis has been a concern for many years (Bock et al., 2007). Advances in genotyping have helped to confirm nosocomial transmission of tuberculosis between patients as well as from patients to healthcare workers (Daley et al., 1992). Other high-risk settings include correctional facilities as well as shelters for homeless individuals; overcrowding, drug abuse, malnutrition, and high rates of HIV coinfection often contribute to this (Fitzgerald & Haas, 2005). While an infectious tuberculosis case may transmit the organism to many contacts, not every contact will be infected or will progress to active disease. Active tuberculosis will develop in 3% to 5% of infected patients within the first 12 months after infection. The lifetime risk of developing active disease ranges from 5% to 15%. There are a number of factors that influence this risk; for example, host genetic factors can increase or decrease the risk of developing active disease. Impaired immunity plays a major role in the risk of progression, with HIV/AIDS, malnutrition, alcoholism, renal failure, immunosuppressive therapy, and diabetes mellitus all contributing significantly to infection or progression of the disease (Fitzgerald & Haas, 2005).

49. Tuberculosis

PATHOGENESIS OF TUBERCULOSIS AND CLINICAL PRESENTATION Latent Tuberculosis Infection

Latent infection is asymptomatic and is identified based on the presence of a T-cell response directed against mycobacterial antigens, as evidenced by a positive tuberculin skin test (TST) or interferon gamma (IFN-g) release assay (IGRA). However, the clinical classification of tuberculosis as being either latent or active is an oversimplification of the spectrum of infection or disease caused by M. tuberculosis (Young et al., 2009). Young proposed that M. tuberculosis infection is classified according to four response spectra based on the host immune reaction to the organism. In the innate immune stage, the host innate immune response is able to eradicate the infection without the need for acquired immune responses and with a subsequent absence of detectable immunological memory against the organism. In the acquired immune stage, both the innate and acquired immune systems operate to eliminate the infection. In the quiescent infection stage, the host immune system can still control the infection although there are viable (but nonreplicating) organisms. In the active infection stage, bacteria are not only viable, but also able to replicate. At this stage, the host immune response is still able to control the infection up to the point where there are no symptoms or signs of infection. Progression of the infection beyond this point will lead to clinically apparent infection or to active disease. Examples of clinical scenarios that support such a classification include individuals (such as healthcare workers), who have multiple exposures to tuberculosis, yet never go on to develop a positive TST, supposedly because the innate immune system is able to eradicate the infection (innate immune stage). An interesting group of people are those who have a positive IGRA, but remain TST negative. The mechanism for this could be a low level of infection that is eliminated by the host acquired immune response that is insufficient to trigger a positive TST (acquired immune stage), yet is sufficient to elicit a measurable IFN-g response in the IGRA assays (Young et sal., 2009). In vitro culture studies have yielded interesting results with regard to the microbiology of latent tuberculosis infection. Under certain environmental conditions, an adaptation of M. tuberculosis takes place that enables the Mycobacteria to survive under physiologically stressful conditions. For example, in the presence of hypoxia, Mycobacteria develop a nonreplicative persistent state that enables the organism to survive for prolonged periods (Wayne & Lin, 1982). These adaptive processes have important implications for the treatment of latent M. tuberculosis infection. Isoniazid (INH) monotherapy (IMT) remains the mainstay of treatment for latent tuberculosis infection and can reduce the risk of active disease by as much as 50% (Woldehanna & Volmink, 2004). The mechanism of INH action is the inhibition of mycobacterial cell wall synthesis, which should leave nonreplicating organisms relatively resistant to its actions. However, IMT is still effective, raising interesting theories as to its mechanism of action in latent tuberculosis infection. It is proposed that mycobacterial colonies are in a constant cycle between replicating and nonreplicating phases, and that the replicating stage leaves the colonies susceptible to the effects of isoniazid. In addition, even though Mycobacteria do not replicate actively, it is thought that there is still a need for ongoing production of cell wall components in order to maintain and repair existing cell walls (Young et al., 2009).

Pathogenesis

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Following exposure to infectious droplets, approximately 30% of patients will develop a primary infection. Due to higher airflow, bacilli are deposited preferentially in the lower parts of the upper lobes and upper parts of the middle and lower lobes. The first line of defense in the terminal airways is the alveolar macrophage, and, in many cases, they contain the infection, resulting in the development of focal pneumonitis. Thereafter, infected macrophages transport viable bacilli to regional lymph nodes. In the nonimmune host, widespread hematogenous dissemination can take place, and the most common metastatic sites are the posterior apical segments of the lungs, lymph nodes, kidneys, vertebrae, long bones, and meninges (Fitzgerald & Haas, 2005). Of these sites, dissemination to the lung apices is the most frequent. Three to eight weeks after the initial infection, cell mediated immunity can develop and this can manifest through a delayed-type hypersensitivity reaction as seen in the TST. Diverse clinical manifestations, such as erythema nodosum and phlyctenular keratoconjunctivitis, can occur (Fitzgerald & Haas, 2005). In the majority of patients (90%), the infection is controlled with the development of latent tuberculosis infection, but in a small percentage of patients, the primary complex (consisting of the initial primary pulmonary focus and draining regional lymph nodes) undergoes enlargement and develops necrosis. Patients who develop progression of the primary complexes may develop enlargement of the hilar and mediastinal lymph nodes that can cause obstruction of a bronchus with distal airway collapse and atelectasis. Erosion of a bronchus by an enlarging lymph node will lead to distal spread of the infection and lobar consolidation. Rupture of a subpleural focus into the pleural space may lead to the formation of a pleural effusion (Fitzgerald & Haas, 2005). In children and immune compromised adults (such as in HIV infection), progression of the primary complex can lead to the development of pneumonia, so called primary progressive tuberculosis. Widespread hematogenous dissemination (from a primary complex or metastatic focus in a pulmonary vein) can lead to miliary tuberculosis, often manifesting as tuberculous meningitis in children (Hussey et al., 1991). However, infected individuals who do not progress rapidly to active disease may experience reactivation months or years after the primary infection as an endogenous focus (typically in the lung apices) undergoes reactivation, causing cavitary lung disease, coined post-primary tuberculosis. A previously infected patient may develop active infection due to either endogenous reactivation or exogenous reinfection. Age plays an important role in the presentation of tuberculosis. In infants, infection often results in progression to active disease, and, in children under 5 years of age, there is an increased risk of disseminated disease (miliary and meningeal tuberculosis) (Sharma et al., 2005). After 5 years of age, patients are more resistant to disseminated disease and usually present with pulmonary infection with prominent hilar and mediastinal lymph node involvement. Tuberculosis in adolescents and young adults may resemble childhood infections, but often presents as upper lobe cavitary disease that is typical of post-primary tuberculosis. Elderly patients are at high risk of developing active disease, which may be related to multiple exposures to M. tuberculosis as well as age-related decline in cellular immunity. Comorbid conditions (including the use of immunosuppressive therapies) are common in this population and contribute to the high risk. In elderly patients, upper lobe cavitary disease is less common and patients present with nonresolving pneumonitis involving the middle and lower lobes and, occasionally, the

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anterior segments of the upper lobes. This resembles childhood disease, however, lymph node involvement is usually absent (Stead, 1989).

Tuberculosis and HIV Coinfection

A chest radiograph of a HIV co-infected patient with active tuberculosis is shown in Fig. 1. In addition to increased nosocomial and community exposure to M. tuberculosis, there are several immunological mechanisms that lead to a dramatic increase in susceptibility of HIV-infected people to tuberculosis. Pulmonary innate immune defenses are impaired, cellular recruitment to the site of infection is affected, which impacts on the cell-mediated granulomatous response, and there is functional impairment of established granulomas that need to contain latent infection (Lawn et al., 2002). Color plate 14 illustrates a strong granulomatouse response during active tuberculosis, which is often impaired in HIV infected people. There are also altered clinicopathological manifestations, which can contribute to delayed diagnosis, including cutaneous anergy (manifesting as negative TST); extrapulmonary and miliary disease; altered radiological features (less frequent cavitation, upper lobe disease, and fibrosis); and histological features, including defective granuloma formation (Lawn et al., 2002). A minority of patients (8% to 13%) with tuberculosis, who are started on highly active antiretroviral therapy (HAART) will experience immune reconstitution disease (IRD) due to rapid restoration of the immune system with an increased response to mycobacterial antigens. IRD can manifest as either paradoxical worsening (i.e., clinical deterioration of tuberculosis after initiation of HAART in a patient on effective antituberculosis therapy) or unmasking (i.e., initiation of HAART triggers the clinical presentation of an untreated subclinical tuberculosis infection).

FIGURE 1 Anteroposterior chest radiograph of an adult female with sputum culture confirmed tuberculosis with HIV coinfection (i.e., CD41 T-cell count of 274 3 106/L and not on antiretroviral treatment). There are bilateral infiltrates, right upper lobe cavitation, and right-sided pleural involvement. Courtesy of the Division of Radiology, Tygerberg Academic Hospital.

Risk factors for tuberculosis IRD include disseminated tuberculosis, a low CD4 count, rapid increase in CD4 count coupled with a decline in HIV viral load after the initiation of HAART, and initiation of HAART shortly after treatment is begun for tuberculosis. In a South African cohort, patients who were initiated on HAART in the first month of tuberculosis treatment were at a 70 times greater risk of developing IRD compared to patients who were initiated on HAART after 3 months of tuberculosis treatment (Lawn et al., 2007). The clinical manifestation of tuberculosis IRD is variable, but the most frequent manifestations include tuberculous lymphadenitis and pulmonary and intra-abdominal disease with hepatic involvement. The optimal timing of HAART remains a controversial and unresolved issue. Antiretroviral treatment programs in resource-limited settings, where HAART is initiated at advanced stages of HIV infection, have been plagued by high early mortality rates, thus making a case for early initiation of HAART. This will have to be weighed against the mortality risk of tuberculosis-related IRD in patients who are started on HAART shortly after antituberculosis therapy is initiated (Lawn et al., 2007).

DIAGNOSTIC METHODS FOR INFECTION AND DISEASE DUE TO M. tuberculosis Diagnosis of Latent Tuberculosis Infection (LTBI)

Identifying patients with LTBI remains a major challenge for clinicians. The gold standard for detecting LTBI in highrisk populations remains the TST, which detects delayed hypersensitivity due to previous M. tuberculosis infection. Five units of purified protein derivative (PPD) are injected intradermally and the skin induration is measured after 48 to 72 hours. The TST is technically difficult and labor-intensive as it requires two visits to a healthcare worker. False positive results can occur due to exposure to environmental nontuberculous Mycobacteria and in individuals who received childhood BCG. The rate of false negative results approaches 20% and may be the result of disseminated tuberculosis or immunocompromised states such as HIV/AIDS, malnutrition, malignancies, and long-term immunosuppressive therapy, including corticosteroids (Nash & Douglass, 1980). An alternative to the TST are the recently developed IGRAs. These tests detect the release of IFN-g by sensitized CD4 T-helper cells in response to mycobacterial antigens. Antigens that are specific to M. tuberculosis (ESAT-6, CFP-10, and TB7.7) are incubated with whole blood in the QuantiFERON test or with PMBCs (peripheral blood mononuclear cells) in the T-SPOT.TB test (without TB7.7). The antigens are taken up by antigen-presenting cells and presented to M. tuberculosis-specific memory T cells. This leads to the production of IFN-g, which is detected by an ELISA (QuantiFERON tests) or ELISPOT (T-SPOT.TB) assay. The advantage of IGRAs is increased specificity compared to the TST in patients with nontuberculous mycobacterial infections and those who have received childhood BCG vaccination (Davies & Pai, 2008). However, IGRAs have certain limitations, including the requirement of rapid processing of samples, a short window period for incubating mycobacterial antigens, the need for trained laboratory staff, and the additional expense compared to the TST. The performance of IGRAs in HIV infection, extrapulmonary tuberculosis, and the pediatric population requires further validation. Their applicability in high burden settings has also been questioned (Dheda et al., 2009) as TST correlates at least as well with measures of exposure in these settings and because the treatment of latent M. tuberculosis infection is not a priority in such countries, except in children and in HIV coinfected people.

49. Tuberculosis

Diagnosis of Active Tuberculosis Disease

Sputum smear microscopy has been the mainstay of diagnosing tuberculosis for many decades. Previously, specimens were stained with Carbol-Fuchsin (a component of the Ziehl-Neelsen stain, also known as the acid-fast stain), but newer techniques make use of fluorochrome stains (Phenol-Auramine or Auramine-Rhodamine) in conjunction with the use of fluorescent (or LED) microscopes. Although this technique has improved the sensitivity of smear microscopy by as much as 10%, increased costs have prevented the widespread application of this method, specifically in resource-poor areas where tuberculosis is highly endemic. Smear microscopy can be performed on a variety of clinical specimens but requires a high number of organisms ( 10,000 bacilli per milliliter) to yield a positive result. Patients who are coinfected with HIV typically have pauci-bacillary infections and thus higher rates of smearnegative sputa (Davies & Pai, 2008). Color plate 15 shows a typical Ziehl Neelsen stain of a sputum sample that is positive for acid fast bacilli. Culture of M. tuberculosis still remains the gold standard of diagnosing tuberculosis. Selective growth media are used and can detect 10 to 100 bacilli per milliliter. Solid media such as the Lowenstein-Jensen egg based medium takes 3 to 8 weeks to yield positive results. Liquid broths are a significant improvement on the older solid media and can yield positive results within days. However, tests can only be considered negative when no growth is detected after 42 days. Automated liquid broth media such as the BACTEC mycobacterial growth indicator tube (MGIT) systems have been developed to increase the number of specimens that can be processed by laboratories. Detection of mycobacterial growth is facilitated by radiometric or fluorochrome methods (Perkins & Cunningham, 2007). Detection of mycobacterial DNA is one of the recent additions to the laboratory tools used in the diagnosis of tuberculosis. Nucleic acid amplification techniques (NAAT) have sensitivity and specificity of 96% and 85% in detecting M. tuberculosis complex in smear positive sputum samples, and 66% and 98% in smear negative samples (Greco et al., 2006). NAAT have been used in the detection of extrapulmonary tuberculosis, but sensitivities remain moderate and the cost and technical problems can be prohibitive in many regions of the world where tuberculosis is highly endemic. In tuberculous pleural effusions, NAAT have variable sensitivity and should not replace conventional tests (Pai et al., 2004). Another drawback of NAAT is its inability to distinguish killed Mycobacteria from viable organisms and, therefore, it cannot be used to assess treatment response.

Drug Susceptibility Testing (DST)

Drug resistant tuberculosis became a problem shortly after the introduction of antituberculous chemotherapy. The increase of MDR-TB and XDR-TB is threatening global tuberculosis control programs and a need exists to diagnose drug resistance rapidly. The gold standard for tuberculosis DST is the agar proportion method by which growth on a drug-containing medium is compared with growth on a drug-free medium. This method can take up to 8 weeks, which delays appropriate therapy and the institution of infection control measures necessary to curb the spread of this devastating infection. Most of the culture-based methods of tuberculosis detection can also be applied to test drug susceptibility. Several methods have been successfully adapted for DST. Automated liquid culture systems for DST with both first- and second-line tuberculosis drugs can provide results within 2 to 4 weeks. A further reduction in delay of results is possible

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if liquid culture is performed directly from smear-positive specimens and results can be available in 1 to 3 weeks. Direct solid media inoculation and readout via colony inspection or by the addition of colorimetric substrates, as in the nitrate reductase assay, or Griess method may also be useful. Newer, rapid culture-based tests have been developed, which can be used in resource-poor settings (e.g., microscopic-observed drug susceptibility assay [MODS]). This new technique allows the direct inoculation of sputum in a liquid broth containing antituberculosis drugs and is a potentially suitable method of rapid DST in resource-limited settings, but takes several days to yield a result because it is culture-based (Perkins & Cunningham, 2007). Resistance against antituberculosis drugs is the result of genetic mutations. Mutations in the mycobacterial rpoB gene are responsible for 96% of all rifampicin resistant strains, and the katG and inhA genes are the dominant genes in selecting for isoniazid resistance. NAAT is used to detect these genetic mutations that select for resistant strains and can fulfill the role of a rapid DST. New line-probe assays not only detect infection with M. tuberculosis, but also identify resistance to the main first-line drugs, rifampicin and isoniazid. Of the strains that are resistant to rifampicin, 90% will have concomitant isoniazid resistance. This technology is better at detecting rifampicin resistance than isoniazid resistance and, due to the high rate of dual resistance, assays that only detect rifampicin resistance are suitable for the prediction of multidrug resistant tuberculosis (i.e., resistance to both rifampicin and isoniazid) (Johnson et al., 2006; Moore et al., 2006).

Extensively Drug Resistant Tuberculosis

Since the introduction of antituberculous drugs 60 years ago, it has become apparent that combination therapy will be required to curb the emergence of drug resistant strains. Never before has the global tuberculosis control program been under so much threat due to the rising epidemic of MDR-TB and XDR-TB. The development of drug resistance starts with spontaneous mutations during mycobacterial replication. Selection pressure from antimycobacterial drugs aid in the establishment of resistant strains (acquired resistance). Inappropriate therapy will amplify the resistance (amplification resistance), and transmission to other individuals will spread the epidemic (transmitted resistance). Not surprisingly, a history of previous tuberculosis is the most important risk factor for drug resistant tuberculosis and suboptimally treated MDR-TB leads to XDR-TB. Transmission of XDR-TB is variable with certain mutations, causing decreased mycobacterial fitness, but this can be overcome by compensatory mutations. XDR-TB is difficult to treat and necessitates the use of multiple and potentially toxic drugs. Treatment failures are common and the mortality is high, especially in HIV coinfected patients (Jassal & Bishai, 2009; Donald & van Helden, 2009).

THE GOAL OF STOPPING TUBERCULOSIS

The Stop TB Partnership is a global movement to accelerate social and political action to stop the spread of tuberculosis around the world. It is a network of international organizations, countries, donors from the public and private sectors, governmental and nongovernmental organizations, and individuals with an interest in working together to achieve the goal of eliminating tuberculosis as a public health problem. The partnership formulated The Global Plan to Stop TB. The 2005 target was to detect 70% of new sputum smear positive cases and cure at least 85% of these cases, followed by the 2015 target to halve the tuberculosis prevalence and deaths compared to 1990 levels. This ambitious plan, at an

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estimated cost of $56 billion, would require tremendous improvements in health systems, rapid reduction in HIV incidence, and the early availability of new tools to increase diagnostic capacity, substantially shorten treatment, and effectively prevent tuberculosis transmission (Stop TB Partnership & WHO, 2006). New diagnostics, shortened treatment regimens through new drugs and effective vaccines are urgently needed and the search for these tools would be significantly enhanced through the discovery and validation of appropriate biomarkers.

THE ROLE OF BIOMARKERS IN ACHIEVING THE GOAL OF STOPPING TUBERCULOSIS

Biomarkers are measurable biological characteristics that characterize normal biological processes, pathogenic processes, or responses to therapeutic interventions (Biomarkers Working Group, 2001). They may, therefore, find new application as tuberculosis diagnostic markers and may represent surrogates for clinical endpoints in trials of new vaccines or new chemotherapeutics. To be valuable as a surrogate endpoint, a biomarker should measure an event that is directly involved in pathogenesis or protection and should change early during treatment. Biomarkers may allow for earlier “go/no go” decisions in developing treatments, help to reduce the duration of trials by years, and reduce cost significantly by providing a clearer indication of treatment efficacy, dosage, or safety. In the field of HIV treatment there has been significant progress due to the availability of CD4 counts and viral load measurements as biomarkers for disease severity and response to antiretroviral treatment (Graham, 1996). However, biomarkers have disappointed, especially in the fields of antiarrhythmic (Zumla et al., 2008) and hypoglycemic therapy (Nissen & Wolski, 2007), where increased mortality and myocardial infarction, respectively, followed apparently effective responses of biomarker behavior. The determinants of the different outcomes of interactions between M. tuberculosis and its human host are not clear. Exposure may lead to infection or no infection; 90% of people are naturally protected against progression; in some people vaccines induce protection; and antibiotic treatment is effective in the majority of patients, but some fail to respond or develop recurrence of disease after initial cure. It is well documented that there are different populations of bacteria in the same patient, some fast growing (more sensitive to antibiotics), and some slow growing (more resistant to treatment) or even dormant (Wallis et al., 2009). The human host presents many poorly understood layers of complexity. Nearly half of the leading causes of death in our society are attributable to behavioral risk factors (Gruman & Follick, 1998). This may also apply to tuberculosis, where smoking; alcohol abuse; delayed health-seeking behavior; poor treatment adherence; and system failures, like diagnostic delays and inadequate drug availability at the point of care, impact the epidemic. Individual responses against Mycobacteria depend on incompletely understood immune responses that are affected by genetic, nutritional, and environmental factors, including coinfection with organisms like HIV, but probably also others like helminths and environmental (mostly nonpathogenic) Mycobacteria. Response to treatment may depend on host genetic factors, drug absorption, pharmacokinetics, extent of disease prior to treatment, and other illnesses (like diabetes). Taken together, these complex interactions may suggest that simple biomarkers for tuberculosis infection outcome will be difficult to find.

Biomarkers for Protective Immunity

The natural protective immune response during infection with M. tuberculosis is poorly understood. It is still unclear

which mycobacterial antigens are targeted by the human immune system during the different phases of infection and disease. Such knowledge, however, will be essential to allow the design of new vaccines, and to test such vaccines through the use of surrogate endpoints as the clinical endpoints (i.e., the development of active disease in vaccine recipients) remain relatively rare events in the context of clinical trials and may take years or decades to manifest, thereby posing significant logistical and budgetary challenges for the tuberculosis vaccine field. Approximately 90% of M. tuberculosis infected individuals are naturally protected against the development of active tuberculosis, and targeting of CD41 T cells, monocytes, macrophages, and dendritic cells by HIV infection substantially increases susceptibility. Both CD41 and CD81 T cells are important to control tuberculosis (Flynn & Chan, 2003) and the essential role of the type I cytokine axis (IL-12 [interleukin 12]/IL-23/IFN-g) is apparent from genetic deficiencies (Ottenhoff et al., 2005) although the IFN-g cytokine has not been found to be a suitable correlate of protection (Fletcher, 2007). Multiparameter flow cytometric analysis of M. tuberculosis specific T cells that simultaneously produce IFN-g, tumor necrosis factor a (TNFa), and IL-2, so called polyfunctional T cells, may have increased protective potential compared to T cells that produce only one cytokine (Seder et al., 2008), as these cells may be associated with long-term memory. More human data involving longitudinal follow-up of individuals exposed to M. tuberculosis is required to support this concept.

Diagnostic Markers for Active Tuberculosis

The diagnosis of active tuberculosis, and (until recently) latent tuberculosis infection, relies on tests that are approximately 100 years old. Sputum smear examination by light microscopy for acid-fast bacilli remains the most widely used test in high incidence settings but has low sensitivity and cannot discriminate between M. tuberculosis and nontuberculous Mycobacteria. Sputum culture for M. tuberculosis is sensitive but takes several days to weeks to yield a result, and it is not available in many areas where it would be most needed. Sputum smear- and culture-negative tuberculosis occurs frequently and these tests are of limited value in extrapulmonary disease. Similarly, the test for latent infection with M. tuberculosis most widely used in resource-limited settings is the TST, which lacks specificity in BCG-vaccinated populations and in those exposed to environmental Mycobacteria. While IGRAs are useful in the diagnosis of M. tuberculosis infection, an important limitation of these assays is their inability to discriminate between LTBI and active tuberculosis. These assays are therefore of little value in high TB incidence areas with a very high LTBI burden. Discovery of biomarkers that can rapidly and reliably differentiate between the two infection states would be a major advance.

Tuberculosis Treatment Biomarkers The Need for Biomarkers for Relapse

A shortened tuberculosis drug regimen of 1 to 2 months in duration is a key component of The Global Plan to Stop TB. The reason for the current 6-month regimen is that unacceptably high recurrence rates accompany shorter (3- to 4-month) regimens. Clearly, more effective drug regimens are needed to allow treatment shortening. Recurrence usually occurs within the first 2 years of initially successful treatment, and clinical trials of shortened drug regimens would therefore have to include a lengthy follow-up time for each participant, resulting in lengthy, logistically challenging, and expensive clinical trials for new drugs. Recurrence

49. Tuberculosis

is further complicated by the fact that both relapse (recurrence due to the same bacterial strain) and reinfection with a new strain occur and that the underlying mechanisms for these forms of recurrence may be very different. Biomarkers for relapse that are measurable early on during tuberculosis treatment would constitute a major advance to facilitate new tuberculosis drug development by shortening clinical trials and by allowing stratification of patients with different risks for poor outcome into appropriate study groups. Additionally, such markers would find clinical use, as high-risk patients identified by such markers could be followed up more closely or could be treated for longer periods.

The Need for Biomarkers of Treatment Effect

The earliest measure of treatment efficacy in current use is sputum smear or culture conversion after 8 weeks of therapy (Walzl et al., 2008). Furthermore, smear or culture conversion may by only 50% to 75% after 2 months of treatment on first-line antibiotics (Carroll et al., 2008), and prediction of response based on these microbiological parameters is no greater than 75%. Additionally, approximately 40% to 50% of tuberculosis cases are smear negative from the onset, especially in HIV-infected people. This marker, therefore, has limited clinical utility. Cultures are more sensitive than smear tests but are not widely available in high prevalence areas, are expensive, and have an even longer lag time of weeks before the result becomes available. This long lag time to obtain a measure of treatment efficacy may have serious implications for infection with drug-resistant strains, where such delays may lead to progression of lung destruction in the affected individual and spread of this dangerous form of the disease to others. New biomarkers of early treatment effect that are measurable very early during treatment would therefore be very valuable.

The Need for Biomarkers for the Extent of Disease

Baseline differences in chest X-ray characteristics, like cavitation and more extensive lung involvement, are linked to higher recurrence rates (Zumla et al., 2008). Chest radiology, however, is not widely available in the developing world as radiology facilities are mainly concentrated at referral centers. The ability to classify tuberculosis patients at diagnosis or early during chemotherapy into risk groups requiring different durations of treatment might improve adherence and thus treatment outcome by allowing shorter treatment regimens in the majority of patients. Healthcare providers in resource-limited settings may then be able to focus more attention on patients that have a high risk of poor treatment outcomes. Such baseline biomarkers would not only improve therapeutic strategies, but also be crucial in the validation of novel tuberculosis drug candidates by allowing the stratification into different treatment arms with subsequent reduction in required participant numbers.

Candidate Microbiological Markers Sputum Microbiology and Treatment Response

Sputum culture status after 8 weeks of therapy is currently the best validated marker for a relapse-free cure. Although failed conversion is related to relapse in study populations this marker lacks adequate positive predictive value for relapse in individual patients (Wallis et al., 2009). Liquid culture speeds up detection of Mycobacteria in culture and can be used in the place of colony counts on agar. It may be more sensitive to demonstrate viable bacteria but there are also drawbacks, including the need to take readings at intervals during culture, resulting in a discontinuous variable with a restricted range of responses and, subsequently,

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a possible loss of sensitivity as a measure of the culture conversion rate (Perrin et al., 2007). Early bactericidal activity (EBA) is used in the early evaluation of new tuberculosis drugs and is based on the rate of fall of colony-forming unit counts of M. tuberculosis (log10cfu/day) in overnight sputum samples that are collected before and on alternate days of treatment, up to day 14. EBA provides a good indication of bactericidal activity of a drug, but fails to identify sterilizing activity, as seen with pyrazinamide (Perrin et al., 2007). Sequential colony counts over 28 days, time to culture conversion, and whole blood bactericidal assays have also been proposed as markers for treatment response but further validation is required.

Mycobacterial Antigens and Antibodies

A decrease in the levels of mycobacterial antigen 85 protein and mRNA in sputum shortly after initiation of tuberculosis therapy is associated with treatment success, whereas sustained expression is associated with relapse (Wallis et al., 2009). This work has not been repeatable in other laboratories, as the technique is technically complex. Generally, antibodies against mycobacterial targets have been disappointing as accurate biomarkers. However, the level of antibodies against ESAT-6, Rv2626c, LAM (lipoarabinomannan), and 38kDa antigen are higher in active tuberculosis patients than in healthy household contacts of cases. Antituberculous chemotherapy reduces anti-ESAT-6 and anti-RV2626c antibodies, but anti-LAM and anti-38kDa antibodies increase. Additionally, severity of tuberculosis disease correlates with antibody levels and antibody titers to two mycobacterial enzymes (alanine dehydrogenase and malate synthetase) and correlate with treatment failure. Titers of antibodies against some mycobacterial proteins in serum increase well before the diagnosis of tuberculosis can be made on bacteriological or clinical grounds (Zumla et al., 2008). These antibodies should therefore be further evaluated as predictive biomarkers in future studies.

Mycobacterial Markers in Urine

Small mycobacterial DNA fragments caused by apoptosis of mammalian cells have been found in urine and have been termed trans-renal (tr) DNA, which can be detected by nested polymerase chain reaction amplification. Although this approach is potentially useful in situations where sputum cannot be obtained or in extrapulmonary tuberculosis, further work is needed to define the performance of this test. Urinary mycobacterial LAM has been detected in tuberculosis patients, but the clearance of LAM during treatment and the relationship to clinical outcome needs to be investigated (Zumla et al., 2008).

Breath Markers

Exhaled breath contents, including volatile metabolites from M. tuberculosis, hold promise as diagnostic markers. Solid phase microextraction and gas chromatography followed by mass spectrometry enable detection of such volatile substances (Zumla et al., 2008), and studies are ongoing to define the role of these analytes in the diagnosis and evaluation of treatment response.

Candidate Host Markers Cytokines

IFN-g is an essential cytokine for protection against tuberculosis. Although levels in peripheral blood decrease during active disease and recover after successfully completed therapy, its use as a biomarker has been disappointing. Immunosuppression during active disease and compartmentalization

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of immune responses complicate the utility of this marker. Additionally, altered kinetics of INF-g production during disease and a lack of standardization of IFN-g assays make interpretation of the existing literature difficult (Veenstra et al., 2007). High levels of IFN-g released in response to ESAT-6, however, may identify those M. tuberculosis contacts with the highest risk of progressive disease (Doherty et al., 2009), analogous to a strong TST response that is correlated with a higher risk of subsequent disease (Watkins et al., 2000). The magnitude of ESAT-6-specific IFN-g production by circulating PBMCs (peripheral blood mononuclear cells) may indeed reflect bacterial load, but previously generated T-cell memory responses make interpretation of IFN-g levels as a biomarker for infection outcome difficult. Unstimulated IFN-g levels in pleural, cerebrospinal, and bronchoalveolar lavage fluid may also hold promise as a marker of active tuberculosis (Dheda et al., 2009). Elevated IL-4 mRNA expression predicts subsequent development of active disease in tuberculosis contacts and decreases during tuberculosis treatment as IFN-g levels increase. The expression of mRNA for the IL-4 antagonistic splice variant, IL-4d2, increases over time in treated tuberculosis patients, similarly to IFN-g. IL-4d2 mRNA levels are also elevated in those with LTBI who mount a protective immune response. The IL-4/IL-4 splice variant ratio correlates with the extent of disease and changes during treatment, and may reflect the subsequent outcome of treatment, suggesting that this ratio should be evaluated further as a potential marker (Wallis et al., 2009).

Nonspecific Markers of Inflammation and Shedding of Cell Surface Receptors

A host of nonspecific markers have been described that appear promising as biomarkers for tuberculosis. The nonspecific nature of these markers, usually measured ex vivo in serum, does not necessarily detract from their potential value as surrogates for clinical outcomes. In many clinical situations the expression of markers has to be interpreted within a relatively narrow clinical context as coexisting conditions can significantly affect levels. C-reactive protein (CRP), soluble urokinase plasminogen activator receptor (suPAR), soluble intercellular adhesion molecule 1 (sICAM-1), granzyme B, LAG-3, neopterin, E-selectin, soluble IL-2 receptors, and soluble TNF receptors have been correlated to disease and/or treatment response, but further studies are needed (Walzl et al., 2008; Zumla et al., 2008; Doherty et al., 2009; Wallis et al., 2009).

Novel Technological Platforms to Identify Biomarkers

A complex disease like tuberculosis will, in all likelihood, require more than a single biomarker to allow for accurate definition of infection, disease, and treatment response status. Biosignatures that are comprised of several molecular markers may be needed, and the marker panels will have to be identified with the use of global analyses—the so-called “omics” approaches—including transcriptomics, proteomics, lipidomics, and metabolomics (Jacobsen et al., 2008). Careful attention to the study design and statistical aspects is required to address the issues of technical and biological variance and multiple testing with resultant false-positive discovery. Classification tools have been developed, which require proper cross-validation, including validation with external data. It is also probable that data from different “omics” techniques will have to be combined, requiring integrative bioinformatics.

CONCLUSIONS

The extent of the tuberculosis problem that exists today represents an affront to modern society. It is caused by a

combination of lack of funding commitment (both for operational aspects of tuberculosis care and research), lack of political will to implement proven interventions, and the failure of healthcare systems in the developing world. New tools are urgently needed and only multidisciplinary approaches that include all sectors of healthcare providers and researchers stand a chance to address the problem effectively.

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50 Malaria: Clinical and Epidemiological Aspects ANDREA A. BERRY, MYAING M. NYUNT, AND CHRISTOPHER V. PLOWE

INTRODUCTION

stories have stimulated a renewed sense of optimism about the prospects for global eradication (Roberts & Enserink, 2007). If it is to succeed, this nascent drive toward countryby-country elimination and possible eventual eradication of malaria will require powerful new tools, including malaria vaccines that produce protective immune responses that surpass those acquired through natural exposure to malaria (Plowe et al., 2009).

For millennia prior the advent of the AIDS pandemic, malaria was indisputably the single biggest consistent killer of human beings. Today, along with AIDS and tuberculosis, malaria remains one of the “big three” infectious diseases, every year sickening hundreds of millions, killing millions, and impeding social and economic well-being. Although significant progress toward malaria control (defined as reducing the burden of malaria to a level at which it is no longer a public health problem) has been achieved in some places where the burden of malaria was modest (Color Plate 16), malaria continues to exact a heavy toll on human life and health in parts of Central and South America, large regions of Asia, and throughout most of sub-Saharan Africa, where up to 90% of malaria deaths occur (World Health Organization [WHO], 2008). In the 1950s, the availability of the long-acting insecticide dichlorodiphenyltrichloroethane (DDT) and the highly efficacious and safe drug chloroquine provided the basis for optimism that worldwide eradication was possible. Although malaria was eliminated in several countries on the margins of the malaria map during a global malaria eradication campaign (Color Plate 16), resistance to insecticides and to antimalarial drugs (Color Plate 17), as well as waning economic and political support, led the campaign to stall by the late 1960s, resulting in the rapid resurgence to previous disease levels in most locations (Rieckmann, 2006). For the next 30 years, the focus shifted from eradication to control, with an emphasis on case management. During this period, the emergence and spread of drug-resistant malaria, donor fatigue, and other factors contributed to an overall lack of progress against the disease. But, starting in the late 1990s, new tools, including long-lasting insecticideimpregnated nets and highly efficacious combination drug therapies, led to a wave of successes, including dramatic reductions in disease burden in some areas and complete elimination of malaria in others (WHO, 2007). These success

MALARIA AND ITS LIFE CYCLE

Malaria is a potentially fatal parasitic disease transmitted to humans and other animals by female Anopheline mosquitoes when they take a blood meal. Four species of Plasmodium cause malaria disease strictly in humans: P. falciparum, P. vivax, P. ovale, and P. malariae. A fifth species, P. knowlesi, infects mainly nonhuman primates but was recently found to also infect and sicken humans (Cox-Singh et al., 2008). Hundreds of other Plasmodia species infect other mammals, reptiles, and birds, and generally do so in a species-restricted fashion. To complete its life cycle (see Fig. 1 in chapter 46) the parasite requires two hosts, the female Anopheline mosquito vector and the vertebrate host. The infection is usually transmitted by the bite of infected mosquitoes, and rarely by transfusion of infected blood, sharing needles, and congenitally. The malaria life cycle in humans begins when sporozoites are injected from the female mosquito’s salivary gland as she takes a blood meal. Vermiform sporozoites rapidly enter the circulation and invade hepatocytes, where the parasites develop and multiply. Over the next 5 to 15 days, depending on the Plasmodium species, the infected hepatocyte develops into a tissue schizont containing as many as 10,000 to 30,000 tiny merozoites that have developed from a single sporozoite. The infected hepatocytes rupture, each releasing a shower of merozoites into the circulation, where they initiate the blood stage of the infection, which is responsible for disease. The liver stage of malaria is asymptomatic. In P. vivax and P. ovale infection, some tissue parasites can develop into latent forms called hypnozoites, which persist in the liver for as long as 3 to 5 years. These “sleeping parasites,” which do not produce any clinical manifestations, can be reactivated and cause relapses long after the disease-causing blood stage parasites have been eliminated by drug cure. Merozoites released from hepatocytes enter the bloodstream where they invade erythrocytes and undergo asexual

Andrea A. Berry, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201. Myaing M. Nyunt, Department of International Health, Global Disease Epidemiology and Control Program, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205. Christopher V. Plowe, Center for Vaccine Development, Howard Hughes Medical Institute and University of Maryland School of Medicine, Baltimore, MD 21201.

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multiplication. Most develop from compact ring forms into larger but still unicellular trophozoites, and then multiply to become multicellular blood schizonts. Once mature, the schizonts rupture, releasing 6 to 32 merozoites, which, in turn, invade more erythrocytes. This process continues in a periodic cycle, until it is interrupted by host immunity, drug treatment, or the death of the host. Clinical symptoms can develop during this blood stage of infection. The cycle of invasion, multiplication, and reinvasion takes place over 48 hours for P. falciparum, P. vivax, and P. ovale; 72 hours for P. malariae; and 24 hours for P. knowlesi. When the infecting parasites cycle in a synchronized fashion, the classic malaria symptoms of fever and chills followed by a period of relative relief can follow the same periodicity. A more chaotic fever pattern is typical of P. falciparum, which tends not to be synchronized, especially in endemic populations that are exposed to infection frequently and therefore likely to have multiple infecting “broods.” During erythrocytic development in humans, some parasites develop into sexual forms, male and female gametocytes. Gametogenesis is increased when the parasite is stressed by drug treatment or clinical deterioration of the host. These sexual forms are taken up by the mosquito vector, emerge from the erythrocyte in the mosquito midgut, and fertilize to form diploid zygotes. The zygotes differentiate into ookinetes that burrow across the wall of the midgut and develop into oocysts, where haploid asexual reproduction occurs until each oocyst contains up to 1,000 sporozoites. The sporozoites, carried by the mosquito hemolymph, invade the salivary glands from which they are injected into the human host to complete the parasite life cycle. This extrinsic incubation period takes about 1 to 2 weeks depending on ambient temperature and humidity.

MALARIA PATHOGENESIS AND DISEASE Pathogenesis

Malaria is a set of complex clinical syndromes involving several of the body’s vital systems. Depending on the infecting species and on the immunologic status of the human host, the severity of clinical manifestations varies widely, ranging from a complete lack of clinical symptoms, to a mild febrile illness, to severe disease and death. Because uncomplicated clinical malaria is similar for all four human malaria species and most manifestations of severe malaria are associated only with P. falciparum, the reader should assume discussion of malaria pathogenesis and disease refers to falciparum malaria unless otherwise specified. The clinical syndrome of falciparum malaria originates with changes in the infected erythrocytes. After they invade, malaria parasites effectively hijack the host cell and its machinery, and can express their own proteins on the surface of the erythrocyte. Several strain-specific, antigenically variant large protein receptors have been described; the best characterized of these is the P. falciparum erythrocyte membrane protein (PfEMP1). Encoded by a large, diverse family of 50 to 60 var genes, PfEMP1 are expressed on the surface of infected red blood cells in clumps known as knobs, which are visible by electron microscopy on the surface of the infected cells (Fig. 1). These sticky knobs are responsible for the adherence of parasitized erythrocytes to the vascular endothelium (cytoadherence), as well as to other infected red cells (agglutination) and to uninfected red cells (rosetting). The ability of falciparum malaria to sequester plays a critical role in disease severity; the other human malarias do not appear to sequester, and therefore are not associated with most of the severe manifestations seen with falciparum malaria.

FIGURE 1 Scanning electron micrographs showing a normal uninfected erythrocyte (top), a Plasmodium falciparum trophozoite-infected erythrocyte with knobs expressing cytoadherent P. falciparum erythrocyte membrane protein-1 (center), and abnormally large and widely separated knobs on a similarly parasitized erythrocyte from an individual with hemoglobin AC, which is associated with protection from severe malaria (bottom). Reprinted by kind permission from the author and Macmillan Publishers Ltd.: Nature 435:1119, copyright 2005.

Cytoadherence and agglutination result in sequestration of infected red cells in the microcirculatory compartments of organs, most notably in the brain and placenta, leading to disease, and most abundantly in the spleen, causing splenomegaly. Sequestered infected red cells not only interfere with microcirculatory blood flow (and thus with critical metabolic functions and tissue perfusion, and maternal support to the fetus in the case of placental malaria) but also hide outside the reach of host defense mechanisms. Infected red cells lose their deformability and compromise blood flow in small capillaries and venules. The protection against severe malaria associated with certain hemoglobinopathies appears to result from abnormal formation of knobs in heterozygous erythrocytes (Fig. 1) (Fairhurst et al., 2005). The binding of infected erythrocytes to uninfected erythrocytes is termed rosetting, and is mediated in part by PfEMP1 variants. The presence of rosetting parasites has been associated with severe P. falciparum infection (Mercereau-Puijalon et al., 2008). Rosetting appears to facilitate erythrocyte invasion, leading to higher parasite levels. In addition, the clumps of erythrocytes exacerbate disease by obstructing blood flow to small vessels (Rowe et al., 2009). Blood group A and B antigens appear to be the receptors for rosetting on uninfected erythrocytes, and a decreased risk of severe malaria has been observed in patients with blood group O, presumably because their erythrocytes lack A and B antigens. The presence of variant surface antigens leads to a robust immune response that appears both to harm the human host as well as to lead to the eventual development of protective immunity (see chapters 24 and 29). For example, up regulation of tumor necrosis factor alpha (TNF-a) likely plays a

50. Malaria: Clinical and Epidemiological Aspects

role in the sequestration of parasites in multiple organ systems, including the brain in cerebral malaria (Haldar et al., 2007). In addition to sequestration, proinflammatory cascades leading to up regulation of ICAM-1, an indicator of endothelial cell activation, also likely contributes to cerebral edema, a hallmark of cerebral malaria (Brown et al., 2001). These observations raise the possibility of therapeutic benefit from anti-inflammatory treatment. High dose steroids have no benefit in Asian adults with severe malaria (Hoffman et al., 1988), but have not been assessed in African children, whose clinical presentation is somewhat different. The presence of sequestered infected erythrocytes in brain biopsies at autopsy and the observation that differential var genes are expressed in cerebral malaria as compared with hyperparasitemic patients support a role for variant surface antigens in the pathogenesis of this syndrome (Kyriacou et al., 2006). Immunity to severe malaria has been observed to develop comparatively rapidly, after only a few infections (Gupta et al, 1999), possibly due to antibody responses that protect against a relatively conserved subset of PfEMP1 variants that are associated with severe malaria. In contrast, the slow acquisition of immune protection against uncomplicated malaria over years of repeated exposure to malaria is thought to represent the accumulation of protective immune responses to a repertoire of diverse antigens (Hviid, 2005). Although some degree of anemia resulting from hemolysis of infected red blood cells is an expected consequence of symptomatic malaria infections, the pathogenesis of severe malarial anemia, seen most commonly in P. falciparum disease, and also present with P. vivax, is multifactorial. Loss of uninfected erythrocytes due to uptake by monocytes and macrophages, increased clearance, complement-mediated phagocytosis, and suppression of erythropoiesis have all been implicated (Milner et al., 2008).

Uncomplicated Malaria

Uncomplicated malaria refers to clinically symptomatic malaria without signs of severe malaria disease. The clinical presentation of uncomplicated malaria tends to be nonspecific, resembling the symptoms of a viral syndrome, and is similar for all four species of Plasmodium that cause malaria primarily in humans. A classic pattern of cyclical fevers punctuated by periods of relative relief (known as malaria paroxysms), corresponding to the waves of merozoites released into the bloodstream during schizogony, is characteristic of malaria, but continuous and chaotic fever patterns are also seen (Church et al., 1997). Besides fever, common symptoms include headache, body aches, chills, and rigors. Gastrointestinal symptoms such as abdominal pain, nausea, vomiting, and diarrhea can be prominent, and respiratory symptoms may also be present. Clinical signs can include anemia, jaundice, and hepatosplenomegaly. Thrombocytopenia is common, but rarely associated with bleeding even when profound. Leukocytosis is not caused by malaria and its presence suggests alternative sources of fever or concurrent bacterial infection, which is common. The typical time from exposure to disease is 8 to 25 days, although the incubation period can be prolonged by incomplete prophylaxis or treatment, or by partial immunity. The diagnosis is classically made by microscopic examination of stained peripheral blood smears to determine the presence of parasites, the species (which dictates choice of treatment drug), and the degree of parasitemia, expressed as a percentage of erythrocytes that contain asexual parasites or the number of parasites per microliter of blood. Rapid diagnostic tests that detect malaria-specific antigens on a dipstick format are also increasingly used because they require less expertise and equipment, although they do not permit quantification of parasitemia, an important prognostic indicator that guides treatment decisions.

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In malaria endemic countries, local languages often have only one word to connote both “malaria” and “fever,” and fevers are routinely treated with antimalarial drugs, usually without any other diagnostic evaluation. If treated promptly with effective drugs, uncomplicated malaria can be effectively prevented from progressing to severe or fatal malaria. Cure is rapid, with clearance of parasites and fever in 1 to 3 days. Uncomplicated malaria should be diagnosed and treated without delay, especially in vulnerable young children, pregnant women, and other nonimmune individuals such as tourists, military troops, and other travelers from nonendemic areas. Knowledge of regional patterns of antimalarial drug resistance is important in determining which treatment to use. Where malaria remains sensitive to chloroquine (mainly Central America north of the Panama Canal) (Color Plate 17), this old drug remains the first choice for treating uncomplicated malaria because of its high efficacy, rapid action, and safety. In most of the rest of the world, combination therapies based on artemisinins (very rapidly acting drugs based on an ancient Chinese herbal remedy) are now recommended for treating uncomplicated malaria. The increasingly widespread availability of these artemisinin-based combination therapies (ACTs) has contributed to the renewed hope that malaria can eventually be eliminated, but concerning reports of apparent artemisinin-resistant falciparum malaria have recently emerged in Southeast Asia (Color Plate 17) (Dondorp et al., 2009; Noedl et al., 2008), the same region where chloroquine-resistant and multi-drug resistant falciparum malaria emerged previously.

Severe Malaria

While serious complications and death are rare in nonfalciparum malaria, P. falciparum, the most common species in Africa, carries a high risk of morbidity and mortality. The two most common manifestations of severe malaria are severe malarial anemia and cerebral malaria, which is characterized by seizures and impaired consciousness leading to coma and death. Hypoglycemia, acidosis, respiratory failure, and liver and kidney failure also can occur with severe malaria. Disturbances in immune mediators are associated with the pathogenic changes seen in the vital organs of patients with severe falciparum malaria, including endothelial damage, massive vasodilatation, and increased vascular permeability. Pathological hemodynamic consequences include hypovolemia, late hypotension, and subsequent cardiac failure. These hemodynamic derangements are more common in adults in low transmission areas than in African children, who tend to have primary respiratory failure not related to cardiovascular compromise. Hyperparasitemia (variously defined as more than 3% to 10% of erythrocytes harboring parasites on microscopic examination of peripheral blood) alone is less worrisome than other signs of severe malaria, but still warrants hospitalization and aggressive treatment, especially in nonimmune patients. The progression of falciparum malaria from uncomplicated to severe disease can be extremely rapid, and death may occur within hours of presentation. The case fatality rate for untreated severe malaria is estimated at 50% and is about 5% to 15% with appropriate treatment. Severe malaria is a medical emergency, and the major objective of treatment is to prevent death. Hospitalization for intravenous administration of antimalarial drugs with aggressive supportive care and monitoring in an intensive care setting is ideal, but often not available in malaria-endemic areas. Severe malaria can be treated with parenteral quinine or quinidine, but these older drugs have serious toxicities and intravenous artesunate is now preferred because of its very rapid action, high efficacy, good tolerance, and safety.

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Malaria in Pregnancy

Pregnant women are at a higher risk of acquiring malaria infection, and of developing symptomatic and severe disease, particularly severe anemia. Malaria during pregnancy also carries a risk of adverse birth outcomes such as spontaneous abortion, stillbirth, and premature birth or intrauterine growth restriction, both of which contribute to low birth weight, which is the single most important indicator of infant mortality. Physiologic alteration or suppression of immune functions essential to maintain stability and growth of the fetus may be partially responsible for this heightened vulnerability during pregnancy, especially during the first pregnancy. The pathophysiology of malaria in pregnancy involves a massive sequestration of parasitized red blood cells in the placenta. The clinical presentation of malaria in pregnancy depends on the degree of malaria transmission. In areas of low transmission, acute symptomatic cases are common and the development of severe disease is rapid. Once severe malaria develops, the case fatality rate of falciparum malaria in pregnancy approaches 50%, even with appropriate treatment (WHO, 2000). In areas of moderate-to-high transmission, acute clinical episodes and severe malaria are relatively uncommon in semi-immune pregnant women; instead, the infection tends to be chronic, leading to maternal anemia and complications to the fetus. The epidemiological clue of more adverse outcomes in primigravid women, with relatively fewer complications of malaria in subsequent pregnancies, led researchers to hypothesize that there may be a local placental immune response that could be acquired over time and that would result in protection for subsequent pregnancies. A specific PfEMP1, VAR2CSA, was found to be selectively expressed during pregnancy. Like other proteins in the PfEMP1 family, VAR2CSA is expressed on infected erythrocytes. However, VAR2CSA uniquely binds to chondroitin sulfate A, which is concentrated in the intervillous spaces of the placenta, and allows parasites to attach selectively to the placenta, causing placental sequestration (Rogerson et al., 2007).

CLINICAL MANIFESTATIONS OF NON-FALCIPARUM SPECIES Relapsing Malaria: P. vivax and P. ovale

Relapsing malaria is seen with P. vivax and P. ovale due to some parasites transitioning to the hypnozoite phase in the liver, with subsequent reemergence up to 3 to 5 years later. Since the drugs used to treat acute malaria do not kill hypnozoites, treatment of blood stage parasites with schizonticidal drugs like chloroquine or ACTs must be followed with an antirelapse treatment of primaquine, which also has rare transmissionblocking activity against gametocytes. Because primaquine can precipitate hemolysis in persons with glucose-6-phosphate dehydrogenase (G6PD) deficiency, testing for this condition should precede this antirelapse treatment.

P. vivax

P. vivax has been increasingly associated with severe disease and has the ability to cause relapsing malaria through the reactivation of hypnozoites. There is only limited evidence of cytoadherence and sequestration in P. vivax; rather, the manifestations of severe vivax disease appear to occur in an acuteon-chronic fashion with recurrent parasitemia from relapsing disease playing a major role, perhaps by increasing the risk for severe malarial anemia (Price et al., 2009). This may explain why severe vivax malaria is uncommon in malaria-naïve individuals and in temperate regions but is being more widely recognized in endemic tropical regions with limited access to health care, where antirelapse treatment to eliminate hypnozoites

may be inconsistent. In regions where it has been studied, infants are particularly affected, with higher hospitalization rates and an increased risk of severe anemia compared with P. falciparum infections. Acute lung injury is common in both adults and children with severe vivax disease. Coma may occur but is uncommon. Chloroquine-resistant P. vivax was recognized decades after the first descriptions of P. falciparum chloroquine resistance, but is now prevalent in parts of Asia and Oceania. Splenic rupture is a rare but notable complication of P. vivax, and less frequently, of P. falciparum infection. Splenic rupture tends to occur in nonimmune adults. Many mechanisms have been proposed, including an increase in tension due to hyperplasia and engorgement (Hershey & Lubitz, 1948).

P. malariae

Unique features of P. malariae, which typically presents with “mild” malaria disease, include the following. The prepatent period (time between infectious bite and detectable parasites in the blood) is longer and more varied (ranging from 16 to 59 days). In untreated infections, parasite levels can drop to undetectable levels for a prolonged period of time but can then recrudesce to levels high enough to cause symptomatic disease. Recrudescence can occur as many as 50 years after the original exposure, and may be exacerbated by stress in the human host (e.g., as caused by another infection or by suppression of the immune system). P. malariae is also distinguishable by an association with nephrotic syndrome, which has pathological features associated with immune complexmediated disease (Collins & Jeffery, 2007)

P. knowlesi

P. knowlesi, which is a well-studied pathogen of nonhuman primates, was only recently recognized as causing disease in humans, and is now considered “the fifth human malaria parasite” (White, 2008). Its natural hosts are macaques in Southeast Asia. Blood stage forms have morphologies similar to P. malariae and P. falciparum, which has led to misdiagnosis, usually as P. malariae. However, unlike P. malariae, P. knowlesi has a 24-hour replication cycle. In addition, hyperparasitemia associated with severe disease—similar to that seen with P. falciparum—has been observed, leading to the recommendation that malaria that has been diagnosed by microscopy as P. malariae but with hyperparasitemia and a clinical presentation more severe than expected be treated aggressively and considered a possible case of P. knowlesi infection (CoxSingh et al., 2008). The identification of this malaria zoonosis has led to speculation that there may be other yet unidentified malaria zoonoses (Baird, 2009), which has implications for vaccine design and could complicate malaria control efforts.

EPIDEMIOLOGY AND IMMUNOLOGY

Malaria is endemic in most of the tropics and subtropics. With 109 malaria-endemic countries in 2008, 3.3 billion people live at risk of acquiring malaria, which causes hundreds of millions of cases of illness and nearly one million deaths every year (WHO, 2008). Of the four Plasmodium species that cause disease primarily in humans, P. falciparum is responsible for the most disease and death and is the most common cause of malaria in Africa, Haiti, Papua, and Indonesia. P. vivax is the most prevalent species in Southeast Asia and Central America. Both P. vivax and P. falciparum are commonly found in South America, East Asia, and Oceania, and can occur together in the same individual, complicating both diagnosis and treatment. P. ovale is seen mostly in Africa, where it is relatively uncommon. P. malariae can be found in all endemic regions but is less common than other species. The nonhuman primate malaria P. knowlesi, which was recently found also to infect humans, is mainly found in Southeast Asia. Approximately 60%


of all clinical malaria cases and 80% to 90% of deaths occur in Africa. Most of the disease burden in Africa is borne by young children and pregnant women. Severe disease is also common in nonimmune adults, such as travelers and those living in areas of unstable malaria transmission.

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Naturally Acquired Immunity and Malaria Exposure

The epidemiology of malaria is determined primarily by the patterns and intensity of malaria transmission, which, in turn, drives the prevalence of malaria infection and the incidence of different forms of malaria disease (Fig. 2). Malaria transmission

FIGURE 2 Schematic representation of infection, disease, and death rates by transmission intensity. Protective immunity to malaria is acquired more rapidly in high and moderate transmission areas than in low transmission areas, and disease patterns also vary with transmission intensity. (A) In high transmission areas (entomological inoculation rate [EIR] .100 infected mosquito bites per person per year), the incidence of uncomplicated malaria falls rapidly after childhood despite the high prevalence of asymptomatic parasitemia. Severe anemia occurs in the youngest children, while cerebral malaria is less common and occurs in slightly older children, reflecting the contribution of acquired immunity to pathogenesis of this syndrome. (B) In areas of low but stable transmission (EIR 5 1–10), the incidence of uncomplicated malaria declines later and more slowly than in higher transmission settings, and the prevalence of asymptomatic parasitemia also declines with age. Severe anemia and cerebral malaria are equally common, with cerebral malaria again occurring in slightly older children. (C) In areas of unstable or epidemic malaria transmission (e.g., EIR , 1 and R0 , 1), asymptomatic infection rates are low, but people of all ages remain susceptible to both uncomplicated and severe disease. Severe anemia remains limited mainly to young children but the risk of cerebral malaria extends into adulthood and is compounded by risk of multiorgan disease in adults. Adapted from Immunological Reviews (Struik & Riley, 2004) with kind permission of the author and John Wiley & Sons, Inc.

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can be measured by the entomological inoculation rate (EIR), an estimate of the number of infected mosquito bites per person per unit of time, usually years. Annual EIRs range from less than 1 in parts of Latin America and Asia to more than 300 in some parts of Africa. Areas with annual EIRs of less than 10 are generally considered to be low malaria transmission areas, with medium transmission characterized by annual EIRs of 10 to 100, and high transmission by EIRs of more than 100. Transmission patterns can also be categorized as stable versus unstable, and described by the basic reproduction rate (R0, pronounced “R naught”), which refers to the number of new cases arising from a single case of malaria. Where R0 , 1, malaria transmission is unstable and would be expected to fall to zero if R0 remains ,1. Where R0 . 1, malaria transmission is stable and can continue indefinitely as long as infected humans and vectors remain in contact with each other (Smith et al., 2009; Smith, 2007). The level of malaria endemicity can also be categorized as hyperendemic, holoendemic, mesoendemic, or hypoendemic based on the “spleen rate” or prevalence of splenomegaly, but this classification system has fallen out of use. In low transmission settings with unstable malaria, there is a potential for epidemic disease when transmission recurs or increases as a result of reintroduction to a population not recently exposed to malaria, or to changing climactic or environmental conditions that favor contact between humans and Anopheles mosquitoes. Outbreaks can occur when malaria-naïve populations such as transmigrants, miners, or soldiers are exposed to malaria, causing high rates of disease. Depending largely on the degree of host immunity, the manifestations of malaria infection can range from completely asymptomatic parasitemia, to mild disease that can be treated on an outpatient basis with oral drugs, to acute catastrophic life-threatening illness requiring intensive care. Very young infants are thought to be protected from malaria

disease by maternal antibodies and possibly by other factors such as persistent hemoglobin F. While they may be infected congenitally or by mosquito bites in the first days or weeks of life, infants do not experience clinical disease until they are a few months old. Following this brief period of relative insusceptibility in early infancy, protective immunity against malaria disease is acquired through repeated exposure to infection and is therefore related to transmission intensity. Where malaria transmission is moderate or high, the risk period for death from malaria is highest in infants and young children who are in the process of developing acquired immunity. In a typical moderate or high transmission setting in subSaharan Africa, most severe malaria is experienced by children less than 5 years of age; children aged 10 to 12 years experience frequent episodes of uncomplicated malaria; and older teenagers and adults, while still often infected, will rarely experience symptoms of malaria illness (Fig. 2, panels A and B). In such settings, the youngest infants suffer from severe anemia, whereas cerebral malaria tends to peak in children aged 3 to 4 years who have experienced previous malaria episodes, suggesting the hypothesis—now supported by autopsy evidence of cerebral inflammation (Haldar et al., 2007)—that an overly exuberant immune response contributes to the pathogenesis of cerebral malaria. In contrast, in low transmission settings, persons of all ages have a similar risk of infection and uncomplicated malaria, and most who are infected become sick. In addition, the risk for cerebral malaria and severe malaria with multiorgan involvement, but not severe anemia, persists throughout life (Fig. 2, panel C) (Okiro et al., 2009). The relationship between transmission intensity and acquired protective immunity is illustrated by studies showing that at higher altitudes with lower malaria transmission, the age distribution of hospitalizations for severe malaria shifts progressively to the right (Fig. 3). There may be other age-related factors that affect

FIGURE 3 Age distribution of hospital admissions with severe malaria and case-fatality rate at various altitudes in Tanzania, showing concentration of severe disease in younger age groups in lowaltitude areas with higher malaria transmission and relatively stronger and more rapidly acquired natural immunity. At higher altitudes, there is less malaria transmission, less immunity, and an extension of disease risk into older age groups. Adapted with kind permission of the author and the publisher from the Journal of the American Medical Association, 2005, 293:1465. Copyright © 2005 American Medical Association. All rights reserved.


the risk of severe malarial anemia independent of transmission intensity, resulting in the observation that severe malarial anemia is uncommon after 2 years of age, regardless of transmission intensity (Reyburn et al., 2005). Protective immunity was also noted to develop more quickly in older individuals among malaria-naïve transmigrants to a malaria-endemic part of Indonesia, suggesting that age-dependent factors unrelated to cumulative exposure to infection also contribute to acquired immunity (Baird, 1998). Semi-immune adults, although they remain susceptible to asymptomatic parasitemia, are protected against clinical malaria disease, rarely becoming ill even when persistently infected. This protective immunity is lost in the absence of exposure; in a typical scenario, an African scientist returning home and being exposed to malaria infection after spending 4 to 5 years getting a Ph.D. in the United States or Europe will fall ill with malaria for the first time since childhood. Acquired immunity is also diminished in pregnancy, in that women pregnant with their first child are susceptible to severe P. falciparum disease from placental malaria because they lack immunity to placenta-specific cytoadherence proteins, as described above. As placental immunity develops in subsequent pregnancies, there is a reduced risk of adverse effects of malaria in pregnancy (Duffy, 2007).

Innate Immunity and Human Genetics

Host genetic factors that play important roles in malaria risk and epidemiology are described in other chapters and summarized briefly here. The presence of several red blood cell polymorphisms, including sickle cell trait, both homozygous and heterozygous expression of hemoglobin C, thalassemia trait, and G6PD deficiency are protective against severe malaria (Verra et al., 2009). The Duffy-negative blood type is protective against both P. vivax and P. knowlesi because the Duffy antigen is an obligate receptor for the parasite to invade erythrocytes, explaining the absence of P. vivax in West and Central Africa, where most of the population is Duffy-negative (Miller et al., 1976). Blood group O appears to be protective against severe malaria (Rowe et al., 2009), and certain HLA types may also be associated with protection from specific disease patterns (Ghosh, 2008).

Coinfections

Coinfection can modulate the epidemiological and clinical manifestations of malaria in that the immune response to one infectious agent may predispose one toward increased disease severity or protection against another pathogen. This is best exemplified by the interactions between HIV and malaria (Hewitt et al., 2006; Khoo et al., 2005; Laufer & Plowe, 2007). During pregnancy, mothers with HIV-associated immunosuppression have higher rates of malaria infection and disease, and deliver babies with lower birth weights. Nonpregnant adults living with HIV in high malaria transmission settings have some increased risk of malaria infection and more frequent episodes of fever in the presence of malaria infection, although some of the apparent increase in clinical malaria is likely due to misattribution of nonmalaria febrile illnesses to malaria. This is because where rates of asymptomatic malaria infection are high, fever of any cause is likely to be accompanied by incidental parasitemia that is unrelated to the fever. Malaria does not behave like typical AIDS-related opportunistic infections in the sense that while adults who have developed immunity to malaria prior to HIV infection do have some increased risk of clinical malaria disease, they do not return to the life-threatening risks of malaria experienced by nonimmune children. Where malaria transmission is low or unstable and naturally acquired immunity is low, HIV infection is associated with an increased risk of severe malaria disease. The immunological mechanisms underlying these epidemiological observations are not well understood.

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An example of a coinfection exerting a protective effect on clinical malaria disease is seen in Schistosoma hematobium infection. Among Malian children, asymptomatic S. hematobium was associated with a longer time until first clinical malaria infection. This protective effect was associated with blunting of the IL-6 and IL-10 responses to malaria infection, suggesting that a well-controlled immune response to chronic schistosomiasis elicits a Th2 response that protects against acquisition of a subsequent malaria infection (Lyke et al., 2006, 2005). Invasive bacterial infections and severe malaria occur together more often than would be expected by chance, and the presence of both carries a higher mortality rate than does severe malaria alone. Nontyphoid Salmonella and other gram-negative invasive bacteria are more common in malaria patients with the highest parasitemia levels. As with HIV, the immune mechanisms explaining malaria–bacterial interactions are not well understood (Berkley et al., 2009).

Malaria Control and Elimination

Current malaria control interventions include treatment with artemisinin-based combination therapies (ACTs); indoor residual spraying (IRS); insecticide treated nets (ITN); and intermittent preventive therapy in infants, children, and pregnant women (IPTi, IPTc, and IPTp, respectively). With the recent call for malaria elimination, more tools, including vaccines, can be anticipated in the near future. When these tools are applied successfully, transmission levels decrease and the level of acquired immunity in the population subsequently diminishes as well, increasing susceptibility to malaria in the population even as the risk of exposure to malaria declines. The deployment of ITNs was accompanied by concerns about the possible loss of immunity and shifting of the burden of severe disease from younger to older children, or from severe anemia to cerebral malaria. Long-term follow-up studies of ITN use have shown significant and persistent reductions in overall childhood mortality, suggesting that any such shift of disease patterns is outweighed by the benefits of reducing malaria exposure. However, “rebound effects” have been observed following mass chemoprophylaxis campaigns in the form of increased risk of malaria infection and/or disease following a period of preventive drug treatment (Geerligs et al., 2003; von Seidlein & Greenwood, 2003). The extent of such rebound effects following short courses of IPT appear to be small compared to the benefits of the intervention. As malaria control and elimination programs are rolled out, patterns of malaria transmission, epidemiology, and immune protection, and the populations at risk of malaria, will change profoundly. In particular, after centuries of being an extremely common disease of infants, children, and pregnant women in most of Africa, malaria in Africa can be expected to become a less common disease that afflicts people of all ages as control proceeds toward elimination. If elimination efforts are highly effective for years or decades but not sustained to the point of elimination, raging epidemics can be expected as malaria is reintroduced into newly nonimmune populations.

MALARIA VACCINES

Vaccines could be useful as a means of controlling and possibly eliminating malaria. In high transmission areas where there is only limited application of other interventions, even a modestly efficacious vaccine may reduce the burden of disease significantly (Greenwood & Targett, 2009). The new goal of using vaccines for malaria elimination places an increased emphasis on vaccines that would completely prevent infection and thus transmission by targeting the preerythrocytic stages of the parasite life cycle or that block transmission directly by generating immunity against the sexual and mosquito stages (Plowe et al., 2009).

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Despite major advances in understanding the molecular and cellular biology of malaria parasites, an effective malaria vaccine has remained elusive. Chief among the obstacles to developing malaria vaccines is the partial and temporary nature of natural immunity, which means that vaccines must do better than nature, or at least achieve more quickly the clinical protection that takes years to develop naturally. Parasite biology also presents challenges. The extreme efficiency of parasite amplification means that a vaccine that allows even a few parasites to escape and establish infection may only delay but not prevent disease. Compared to other organisms against which effective vaccines exist, malaria parasites are monstrously large and complex. For example, the polio genome consists of 0.8 Mb of DNA; Haemophilus influenzae, 1.8 Mb; but the P. falciparum genome contains 30 Mb of DNA spread across 14 chromosomes containing 5,000 genes that control a highly complex set of morphologically distinct stages. Furthermore, P. falciparum maintains extensive genetic polymorphism through both mutation and sexual recombination, and has the ability to switch expression of the cytoadherence antigens it expresses on the surface of infected erythrocytes, thus evading memory immune responses. This extreme genetic diversity likely accounts for the failure to date of vaccines targeting polymorphic blood stage antigens to show good efficacy in field trials, and raises the possibility that initially efficacious vaccines might fail after rapid emergence of “vaccine-resistant malaria” (Takala, 2009). Clear correlates of immune protection have yet to be identified, making it difficult to assess likely efficacy of vaccine candidates short of expensive field trials. Finally, the key role host immune responses play in pathogenesis raises a concern than a potent vaccine could actually worsen disease. Renewed attention to malaria as a global problem has resulted in a surge of support for vaccine development activity in recent years, resulting in more than 45 malaria vaccine candidates being in various, mostly early, stages of development. Most malaria vaccines are based on recombinant versions of antigenic targets from specific stages of the parasite life cycle, although DNA vaccines and viral vector expression approaches have been used with limited success to date (Ballou et al., 2004). The most advanced malaria vaccine is based on the circumsporozoite protein that coats the surface of the infectious sporozoites. This pre-erythrocytic vaccine, RTS,S, appears to reduce malaria disease mainly by delaying parasite amplification, but it does not completely block infection. Efforts to improve its efficacy by adding additional antigens and improving the adjuvant are underway (Heppner et al., 2005), even as this modestly effective vaccine moves toward licensure in its present form. Blood stage vaccines, several of which are being evaluated in clinical trials in Africa, are intended to prevent disease by blocking parasite amplification in the blood. Transmissionblocking vaccines would inhibit development of the sexual stages of the parasite in the mosquito, but would not directly prevent vaccinated individuals from becoming infected or ill with malaria. The only highly effective malaria vaccines have been live, radiation-attenuated whole sporozoite vaccines, delivered by infected mosquitoes in experimental challenge studies first done in the 1970s. This seemingly impractical approach has recently been resurrected in the form of a metabolically active, nonreplicating whole P. falciparum sporozoite vaccine that is manufactured by mass-producing sporozoites in mosquitoes raised in aseptic conditions, irradiating the mosquitoes, and harvesting the attenuated sporozoites by manual dissection for freezing, thawing, and injection (Hoffman et al., 2010). An attenuated sporozoite vaccine is currently being evaluated in a clinical trial

in the United States. There is great hope that vaccines can be added to the tool kit for malaria eradication, but all predictions of when an effective malaria vaccine will be ready have been proven overly optimistic.

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51 The Epidemiology and Immunology of Influenza Viruses RAFAEL A. MEDINA, IRENE RAMOS, AND ANA FERNANDEZ-SESMA

INTRODUCTION

or antigenic shift” occurs when segments of different virus strains reassort in the host cells during coinfections, which leads to a new combination of RNA segments carrying a novel HA or NA subtype. In addition, since the influenza virus RNA-dependent RNA polymerase (RdRp) is highly error prone, “genetic or antigenic drift” results from selection pressure, usually due to preexisting immunity in the population, which leads to the accumulation of nucleotide mutations that result in amino acid changes in the surface glycoproteins. All the pandemic viruses of recent history have arisen due to significant changes in virus antigenicity as a result of reassortment. The origins and epidemiological information, as well as the characteristics of these viruses are detailed below. Although there are other viruses, such as HIV (human immunodeficiency virus), Ebola, or SARS (severe acute respiratory syndrome) virus, that present higher mortality if untreated, influenza A is currently the greatest pandemic disease threat, mainly due to its mode of transmission, together with the high mutation frequency of its genome. The airborne transmission of the virus and its widespread seasonal distribution result in potential infection of a high percentage of the world’s population within a matter of months. Several virulence factors have been associated with the pathogenicity of the influenza viruses (Table 1). Studies of past pandemic viruses have allowed the identification of specific virulence determinants, although it is widely accepted that pathogenicity is multigenic and that the determinants may vary among species. The HA glycoprotein plays an important role in influenza pathogenesis, given that it mediates the binding of the virus to its receptor on host cells, allowing subsequent fusion and endocytosis of the viral particle. The HA binds receptors that contain terminal sialic acids (SA) linked to galactose residues (Gal) of carbohydrate chains in the cell surface. Two major linkages between SA and Gal are found in nature, namely a2,3 and a2,6 linkages, and their localization seems to be species specific. Experiments with linkagespecific lectins have shown an abundance of a2,6-linked SA in human lungs, and of a2,3-linked SA in bird intestines. On the other hand, in pigs, both linkages were detected in the respiratory tract cells. Interestingly, in humans and pigs, influenza is a respiratory infection; in birds, it is an enteric infection. Preference of the HA for a2,3 or a2,6 linkages determines a host specificity, since avian viruses

Influenza viruses cause yearly, seasonal outbreaks, as well as frequent epidemics and occasional pandemics in humans (Cox et al., 2005; Neumann & Kawaoka, 2006; Palese & Shaw, 2007). In the 20th century, the influenza virus caused three pandemics in humans: the 1918 “Spanish influenza,” the 1957 “Asian influenza,” and the 1968 “Hong Kong influenza” (Fig. 1). In addition, other sporadic epidemics have occurred throughout the century, which aid in the understanding of the genesis of these pandemics and the 2009 swine-origin influenza virus (SOIV) pandemic. In this chapter, we will give an overview of past and present pandemics and epidemics of influenza virus and the most significant findings about how those viruses evade immunity in humans. Influenza viruses (IV) belong to the family Orthomyxoviridae and are negative sense RNA viruses containing eight segments of single-stranded RNA. These eight segments encode for 10 or 11 proteins, including the polymerase proteins (PB1, PB2, and PA); the nucleoprotein (NP); the receptor binding hemagglutinin protein (HA); the receptordestroying neuraminidase protein (NA); the matrix protein (M1); the ion channel M2 protein; the interferon (IFN) antagonist NS1 protein; the nuclear export protein (NEP); and, in some strains, the PB1-F2 protein. Influenza viruses are classified based on the type of HA and NA proteins for which they encode. Currently, 16 HA and 9 NA antigens have been described for type A influenza, which have the potential of combining to give rise to distinct virus subtypes (e.g., H1N1, H3N2, H5N1, etc.). Thus far, some subtypes appear to be restricted to specific animal hosts; however, other subtypes have been detected in numerous species, such as the H1N1 and the H3N2 viruses, which are endemic in birds, swine, and humans. Seroarcheological (retrospective) analysis of human sera have indicated that only subtypes H1, H2, and H3, and N1, N2, and N8 have become established in humans, namely H1N1, H2N2, H3N3, H3N8, and H1N2 (Wright et al., 2007) . One particular ability of influenza viruses is that they undergo genetic changes through shift and drift. “Genetic Rafael A. Medina, Irene Ramos, and Ana Fernandez-Sesma, Department of Microbiology and the Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, NY 10029.

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FIGURE 1 Circulation of human and swine influenza A viruses. To date, influenza A viruses containing three hemagglutinin subtypes (H1, H2, and H3) and two neuraminidase subtypes (N1 and N2) have been identified in humans. The introduction of new subtypes through reassortment has resulted in antigenic shift leading to the origin of four pandemics in 1918, 1957, 1968, and 2009. H1N1 viruses, descendents of the 1918 Spanish influenza, were introduced in the swine population sometime after 1918. The detection of three other swine origin viruses in pigs that provided the genetic pool for the genesis of the 2009 H1N1 pandemic virus are also included. Broken lines denote the lack of virus isolates from that particular time, and question marks highlight the uncertainty of the date of circulation and/or the origin of the subtype. Solid lines demonstrate circulation of influenza strains, which undergo antigenic drift overtime during interpandemic years. Modified with permission from Palese & García-Sastre, 2002.

are known to bind preferentially a2,3-linked sialyl receptors, while human isolates bind to receptors containing a26-linked sialyl-galactosyl residues (Nobusawa et al., 1991). These differences are important in limiting the transmission of viruses between birds and humans. However, since pigs present both types of receptors in their trachea, it is possible for them to host human and avian influenza viruses, thus providing a milieu conductive to viral replication and genetic reassortment. This supports the idea of swine as a potential “mixing vessel” in which new pandemics might emerge as a consequence of reassortment, a phenomenon that might be responsible for the genesis of the 2009 H1N1 pandemic virus. Another virulence factor of the influenza A virus is the PB1-F2 protein. This small viral encoded protein (of 87 or 90 amino acids) has been recently described and is transcribed from the 11 reading frame of PB1. PB1-F2 localizes to mitochondria in infected cells, inducing dissipation of mitochondrial membrane potential, resulting in subsequent cell death, with a higher effect in human monocytic cells (Chen et al., 2001). Further studies revealed that the PB1-

F2-induced apoptosis proceeds through a mechanism involving its interaction with two mitochondrial proteins, ANT3 and VDAC1 (Zamarin et al., 2005). Most of the influenza A viruses isolated from numerous mammalian and avian species present the PB1-F2 open reading frame. Nevertheless, in some viruses, particularly swine isolates, stop codons can be generated, leading to the expression of a truncated protein (Chen et al., 2001). The NS1 protein from influenza can also contribute to the virulence of the virus. This protein allows evasion of the immune response by inhibiting IFN-mediated antiviral host responses (García-Sastre, 2002). This protein binds to double-stranded RNA (dsRNA) preventing the activation of 2–5 oligo(A) synthetase and the later activation of RNase L, which plays an important role in the innate immune responses. In addition, NS1 has been shown to block the RIG-I signaling induced during viral infection and it specifically suppresses the maturation, migration, and T-cell stimulatory activity of dendritic cells (DCs) (Fernandez-Sesma et al., 2006). Thus, this NS1 has the ability of modulating both innate and adaptive immune responses.

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51. The Epidemiology and Immunology of Influenza Viruses TABLE 1

Determinants of pathogenicity of the 20th century pandemic and highly pathogenic avian influenza viruses Determinant by function HA (binding to the host cell and fusion to the membrane)

Virus

1918 H1N1 1957 H2N2 1968 H3N2 2009 H1N1 H5N1

Sialic acid linkage specificity

HP

LP

Multibasic cleavage site

Single aa cleavage site

a2,6 a2,6 a2,6 a2,6 a2,3

x x x x x

PB1-F2 (induction of apoptosis)

NS1 (suppression of the host antiviral response)

PB2 (replication)

HP

LP

HP

LP

HP

LP

66S

66N/not expressed

Residues in the C terminus KSEV/ESEV/ EPEV

Residues in the C terminus RSKV

L627

E627

x

KSEV x x Not expressed

x

RSKV RSKV Truncated ESEV/EPEV

x x x x x

HP, high pathogenicity; LP, low pathogenicity.

PB2, an essential component of the polymerase complex of influenza has also been identified as a determinant of pathogenicity. Analysis of H5N1 high pathogenic and low pathogenic virus sequences allowed the identification of a lysine at position 627 that had a strong effect in virulence. Low pathogenic viruses have a glutamic acid at this position (Hatta et al., 2001). The lysine at position 627 has been shown to be a host restriction factor, since it is present mainly in human isolates, including H1N1, H2N2, and H3N2 viruses from 1933 to 1975, whereas in avian isolates, glutamic acid is the residue observed at this position (Subbarao et al., 1993).

THE 1918 PANDEMIC

In 1918, the introduction of an avian-like H1N1 virus into the human population developed into the most deadly influenza pandemic to date, the Spanish 1918 influenza. A mild first wave of the disease occurred during the spring of 1918, and was followed by a second wave in the autumn that resulted in an estimated 50 million deaths worldwide (Johnson & Mueller, 2002). A hallmark of the 1918 influenza was the high mortality rate observed in the young adult population (18 to 30 years of age) (Ahmed et al., 2007), a rate not usually seen in other influenza epidemics and outbreaks, in which children and the elderly show the highest susceptibility and mortality rates. It is believed that previous influenza viruses that circulated in the human population prior to 1889 could have been of the H1 subtype (Langford, 2002), thus offering a possible explanation that a portion of the population (those over 30 years old) could have had some degree of preexisting immunity to the 1918 pandemic virus. Nevertheless, the 1918 influenza had a high rate of attack in children (5 to 14 years of age), but this population was largely spared from death. This phenomenon has been described extensively and remains not well understood (Ahmed et al., 2007). It has been suggested that a differential regulation of immune responses in children and adults could account for this effect, in which the balance between the beneficial and harmful effects of this response favors recovery in children (Ahmed et al., 2007). The virus that caused the 1918 pandemic is likely to have emerged from a reassortment event resulting in antigenic shift (Tumpey et al., 2005), and in the acquisition of virulence factors from its predecessors (Fig. 1). Indeed, determination of the genomic sequence and reconstruction of the

1918 pandemic (Tumpey et al., 2005) has allowed the understanding of several aspects of the influenza pathogenicity and the identification of virulence determinants that provide information about the pathogenic and pandemic potential of this and new viruses introduced in the human population. In addition, these studies revealed that the genome of the 1918 virus, an avian-like H1N1, contained human signatures in some of its proteins, and that the combination of different virulence factors was critical for the unique pathogenic characteristics of the virus (Tumpey et al., 2005). Studies with recombinant viruses bearing the 1918 HA have revealed that this gene is responsible for the increased virulence seen in mice. Viruses expressing the 1918 HA protein induce high levels of cytokines and chemokines (Kobasa et al., 2004), which induce inflammatory cell infiltration and hemorrhage. Nevertheless, the HA protein of the 1918 virus, unlike other high pathogenic viruses, does not present a multibasic cleavage site, suggesting that this feature is not responsible for the high pathogenicity observed with this virus. In addition, closer sequence and structural analysis have shown that this protein, although avian in origin, contains an Asp residue at position 190, which confers it with the ability to bind to human cell-surface receptors (Gamblin et al., 2004; Glaser et al., 2005). The NS1 protein of the 1918 pandemic virus has been shown to be more effective at suppressing the expression of IFN-inducible genes than the A/WSN/33 strain of influenza virus (Geiss et al., 2002). Four amino acids in the C terminus of this protein have been shown to be related to the pathogenicity of the virus. This was demonstrated by infecting mice with viruses engineered with NS1 proteins containing the last four C-terminal residues of the 1918 virus (amino acids AspSer-Glu-Val) or those from avian high pathogenic viruses H5N1 (Glu-Ser-Glu-Val or Glu-Pro-Glu-Val) into the NS1 sequence of the low pathogenic virus A/WSN/33. Human low pathogenic viruses usually present a different sequence (Arg-Ser-Gku-Val) (Obenauer et al., 2006). In the case of PB1-F2, a single amino acid mutation at position 66 has been associated with an increase of virulence. While the majority of the influenza A viruses present an asparagine at this position, a change to a serine was identified in the 1918 pandemic virus (Conenello et al., 2007). Generation of a 1918 virus containing this amino acid mutated from serine to asparagine, results in significant attenuation of the virus, suggesting that this mutation contributes to the high lethality observed with the 1918 pandemic.

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Similarly, the PB2 protein of 1918 contains a lysine residue at position 627. This amino acid has been related with a high virulent phenotype. Experiments conducted in ferrets showed that mutating the 1918-PB2 627 residue from lysine to glutamic acid resulted in reduced transmission by the aerosol route, but it did not affect the ability of the virus to transmit in direct contact experiments (Van Hoeven et al., 2009). In addition, a virus containing this mutation was inefficient at forming plaques at 33°C. These studies suggest that a human adapted PB2 gene is required for efficient virus transmission in order to support virus replication in the mammalian airway at lower temperatures (Van Hoeven et al., 2009).

OTHER PANDEMICS OF THE 20TH CENTURY

Descendents of 1918 virus (1918-like viruses) circulated in humans with considerable antigenic drift in HA until they were replaced by the 1957-H2N2 pandemic (Fig. 1) (Gillim-Ross & Subbarao, 2006). Several years later, in 1968, an antigenically different subtype was introduced into the human population, replacing the previously established H2N2. In the 1970s, an H1N1 virus resembling the one circulating in the late 1950s reemerged in the human population (Nakajima et al., 1978). Currently, both H3N2 and H1N1 viruses cocirculate in humans. In addition, in 1976 in the United States, a swine H1N1-IAV (classical H1N1) epidemic outbreak was reported among soldiers at Fort Dix Army base in New Jersey (Gaydos et al., 2006; Sencer & Millar, 2006). Because of its novel HA antigenicity and the concerns of potentially becoming a pandemic virus, this outbreak led to first mass vaccination of people at historical proportions, with nearly 40 million people being immunized with an inactivated A/NJ/76 vaccine in the United States. Fortunately, the 1976 swine H1N1 virus did not cause a pandemic and no infections were reported outside of Fort Dix. Of interest, however, several interspecies transmissions have been reported between humans and pigs as early as 1938.

The 1957 Pandemic

The Asian influenza pandemic of 1957 arose due to the reassortment of human and avian viruses. This virus contained the PB1, HA, and NA segments from an avian H2N2 virus and the rest of the genes (PB2, PA, NP, M, and NS) came from a human H1N1 virus (Neumann et al., 2009). This led to the introduction of an H2N2 virus in the human population that caused an initial outbreak in Southern China in early 1957. In subsequent months the virus spread to other parts of Asia and eventually to Europe and the United States by October of that year (Neumann & Kawaoka, 2006; Neumann et al., 2009). A second wave of infections was detected during January of 1958. This pandemic is thought to have caused approximately 70,000 deaths in the United States alone and approximately 1 million deaths throughout the world. This novel strain replaced the circulating H1N1 viruses of those years, but it did not appear to be as pathogenic as the 1918 virus. Indeed, sequence analysis of this virus did not reveal some of the pathogenic features observed in the 1918 virus (Table 1), and thus the higher mortality rate was mainly attributed due to the lack of preexisting immunity of the human population to this novel HA subtype.

The 1968 Pandemic

The pandemic virus, referred to as the Hong Kong influenza, appeared in 1968 and caused a pandemic during the winters of 1968–1969 and 1969–1970. This virus also originated through reassortment of an avian virus containing an H3

HA and a human H2N2 virus. As a result, the HA and the PB1 genes were derived from an avian virus and the reminder genes from a human H2N2 virus (Neumann et al., 2009). This novel H3N2 virus completely replaced the H2N2 subtype circulating in humans since the 1957 pandemic. The first outbreak of this pandemic virus was detected in the summer of 1968 in Southern Asia and the virus was isolated in July 1968 in Hong Kong. As with the two previous pandemics, the attack rates were higher in children of 10 to 14 years of age. The estimated mortality in the United States was of 33,800 people. Previous immunity to the N2 protein in the human population has been suggested as an explanation of the rather moderate severity of this pandemic virus as compared to the previous two pandemics. The pandemic H2N2 and H3N2 viruses of 1957 and 1968 had a “low pathogenic” profile according to their virulence factors (Table 1). First, they did not present multibasic cleavage sites in the HA, so it was unlikely to produce systemic infections. The PB1-F2 of both viruses had an asparagine at position 66, and the NS1 had the Asp-Ser-Glu-Val motif, both of which were associated with low virulence. Nevertheless, H2N2 and H3N2 of the 1957 and 1968 pandemics acquired their PB2 genes from a human influenza A virus parent, and thus, they possess a lysine residue at position 627 on this protein. It is noteworthy to mention that all the previous pandemic viruses contained the PB1-F2 coding capacity, and therefore, this has also been suggestive as a signature of influenza virus pathogenesis in humans.

The 1977 Pandemic

Reemergence of the H1N1 serotype viruses occurred in May 1977 in China. Once isolated, the virus that circulated during the winter of 1977–1978 was found have great similarity to the one that had circulated in the early 1950s. This outbreak was characterized by morbidity observed almost exclusively in the population younger than 25 years of age, leading to the idea that the older population was protected by preexisting immunity. There was no significant pathogenicity associated with this virus and therefore this was not a full scale pandemic. This phenotype and the lack of mutations of this virus as compared with the previous circulating H1N1 of the 1950s has led to the widely accepted view that this could have resulted from an accidental release of this virus. Of interest, in contrast to the previous pandemics, the reemergence of H1N1 viruses in 1977 did not result in the replacement of the H3N2 viruses. As a consequence, both virus subtypes continue to circulate worldwide in the human population.

THE H5N1 EPIDEMIC

Wild aquatic birds are a natural reservoir of influenza A virus. Commonly, these viruses do not cause disease in birds and, hence, provide a source of viruses that is unlikely to be controlled. Nevertheless, since 1997, high pathogenic avian influenza (HPAI) H5N1 viruses have caused several outbreaks in birds with a high mortality rate that are accompanied by occasional transmission to humans. Infection of humans by these viruses usually results in a severe and rapidly progressive pneumonia and subsequent systemic disease, with a fatal outcome rate of approximately 50%. The first cases of transmission to humans took place in Hong Kong in 1997, resulting in 6 deaths out of 18 people infected. There were no more cases of transmission to humans until early 2003, when two other cases were reported in Hong Kong, one of them with a fatal outcome. In November 2003, another case was reported in China. In a matter of months,

51. The Epidemiology and Immunology of Influenza Viruses

the virus was reported to have spread to Vietnam and Thailand, where sporadic human cases have occurred. From 2004 to the writing of this chapter, more cases have been confirmed in Cambodia, Indonesia, Turkey, Iraq, Azerbaijan, Egypt, Djibouti, Nigeria, Lao People’s Democratic Republic, Pakistan, and Bangladesh (World Health Organization [WHO], 2009c). Collectively, as of September 24, 2009, 442 human infections with HPAI H5N1 viruses and 262 deaths were reported to the WHO (WHO, 2009b). But fortunately, at present, no sustained human-to-human transmissions for these viruses have been detected. Humans infected by H5N1 viruses have been described to have unusually high serum concentration of chemokines and proinflammatory cytokines, also referred to hypercytokinemia (Cheung et al., 2002; de Jong et al., 2006). It is thought, as different studies suggest, that this cytokine dysregulation induced by HPAI viruses may contribute to disease severity (Cheung et al., 2002). Interestingly, despite their abundance in nature, the avian influenza viruses cross the species barrier only rarely. The HA of the avian viruses have specificity for a2,3-linked sialic acids (Table 1), and thus, they are usually unable to infect humans due to the low amount of these receptors in the human respiratory tract. Specific mutations in the HA have been observed to result in a change of the receptor specificity (Stevens et al., 2006), and might allow for better transmission of the virus from human to human. The HA of the HPAI viruses is an important determinant of its virulence. The influenza virus HA is synthesized as a precursor (HA0), which needs to be cleaved into HA1 and HA2 in order to promote fusion of the viral and host membrane, allowing for the uncoating of the viral RNA segments into the host cell. The N terminus of HA2 is required for membrane fusion and infectivity. In most influenza viruses, the cleavage site is limited to a single amino acid (an arginine) that is cleaved by proteases located in the intestinal and respiratory tract, so the replication of the virus is restricted to these tissues. However, some highly pathogenic viruses, such as H5N1 HPAIV viruses, have several basic amino acids in its cleavage site (Bosch et al., 1981), which can be cleaved by ubiquitous proteases distributed in other tissues, such as furin or PC6 (Horimoto et al., 1994) that generally leads to a systemic infection. Other proteins that account for the high virulence of the H5N1 viruses are the NS1, the PB1-F2, and the PB2. These three factors present similar characteristics to those observed in the 1918 pandemic influenza virus. First, the NS1 possesses the four amino acids that are known to confer increased virulence (Jackson et al., 2008). Second, the PB1-F2 has been described to have a serine at position 66 (Conenello et al., 2007). And finally, the PB2 protein presents a lysine at position 627, which was shown to have a strong effect in virulence (Hatta et al., 2001). Although H5N1 viruses are highly pathogenic, resulting in a high mortality rate in humans, they do not transmit efficiently. Host restriction factors thus far have prevented adaptation and efficient human-to-human transmission of these viruses. This is in clear contrast to the current SOIVs that appear to transmit very efficiently among humans, but infection results in a limited number of fatal cases. Due to its high virulence, and its constant circulation in wild birds, the H5N1 viruses present an imminent threat to humans, and therefore, a good and coordinated system of control and surveillance is necessary. Ongoing epidemiological surveillance has already proved crucial at the global level to anticipate the impact of a potential H5N1 pandemic, and in the international coordination of the responses that should be adopted should a H5N1 pandemic evolve.

THE NOVEL (SOIV) H1N1 PANDEMIC

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Apart from humans and wild birds, IAV can infect a variety of other species, including poultry, horses, pigs, and seals, among others (Neumann & Kawaoka, 2006; Neumann et al., 2009). As with aquatic birds, swine are also natural reservoirs of different subtypes of IAV. Soon after the 1918 pandemic, from 1918–1930, H1N1 viruses resembling the 1918 virus (or a descendant of it) were introduced in the American domestic swine population (Nelson et al., 2008; Taubenberger & Morens, 2006). As a result, H1N1 viruses have circulated since then in the swine population with relatively modest antigenic changes in the HA (Fig. 1) (Vincent et al., 2008). These swine H1N1 viruses are referred as the “classical” swine H1N1 viruses and are antigenically distinct from currently circulating seasonal human H1N1 viruses. Pigs can be infected experimentally or naturally by both human and avian viruses, probably as a consequence of the expression of both a2,3 and a2,6 linked SA in their trachea (detailed above). In addition, several human infections with swine viruses of different subtypes have been documented in the last 35 years (Myers et al., 2007) Thus, pigs have been hypothesized to serve as “mixing vessels” in which reassortment of viral segments may occur. In April 2009, the Centers for Disease Control and Prevention (CDC) of the United States announced the detection of a novel strain of influenza virus. Further investigation revealed that this novel virus was derived from a swine influenza H1N1 virus that had spilled over to humans (Fig. 2). Due to sustained human-to-human transmission of this novel virus throughout the world, on June 11th, 2009, the World Health Organization (WHO) raised the worldwide pandemic alert level to phase 6 (e.g., ongoing global spread and community level outbreaks in multiple parts of world). All of the past pandemics and the recent 2009 swine-origin IAV H1N1 (2009 SOIV H1N1) pandemic have been caused by IAV strains carrying an antigenically novel HA segment to which the populations is immunologically naïve. Phylogenetic analyses have shown that the novel SOIV H1N1 contains six gene segments (PB2, PB1, PA, HA, NP, and NS) that were similar to ones previously found in triple reassortant swine influenza viruses circulating in pigs in North America since 1997–1998 (Garten et al., 2009), indicating that this virus probably resulted from the reassortment of these viruses with Eurasian avian-like swine viruses (NA and M). Overall, the 2009 H1N1 pandemic virus is a triple reassortant that possesses the PB2 and PA genes of North American avian virus origin; the PB1 from human virus H3N2 origin; the HA (H1), NP, and NS genes of classical swine virus origin; and NA (N1) and M genes of Eurasian avian-like swine virus origin (Smith et al., 2009). Although several direct infections with swine IAV have been reported in humans, such infections are usually rare and are restricted to individual persons and close contacts (Myers et al., 2007). Surprisingly, the 2009 SOIV H1N1 pandemic has proved to be very efficient in human-to-human transmission compared to other swine H1N1 infections. As of November 8, 2009, over 503,536 laboratory confirmed cases of the 2009 pandemic influenza H1N1 in more than 206 countries have been reported worldwide to WHO. Of these, 6,260 cases have resulted in death. The 2009 H1N1 pandemic virus continues to be the dominant influenza virus in circulation in the world, particularly in the Northern Hemisphere, where over 89% of the cases correspond to the pandemic strain (for further updates, see http://www.who .int/csr/disease/swineflu/updates/en/index.html). Most of the cases reported of persons infected with this novel H1N1 virus develop an uncomplicated influenza-like illness in healthy individuals, with full recovery within a week, even without

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FIGURE 2 Origin of the 2009 H1N1 pandemic influenza virus. The 2009 pandemic virus originated through reassortment of a Eurasian swine H1N1 virus with a triple reassortant North American swine H1N2 virus that arose in 1998. This novel virus therefore contains the PB2 and PA genes of North American avian virus origin, the PB1 gene of human H3N2 virus origin, the HA (H1), NP and NS genes of classical swine virus origin, and NA (N1) and M genes of Eurasian “avian-like” swine virus origin. Predecessors leading to the triple reassortant viruses are depicted. Arrows denote the sequential reassortment events. Years of emergence are in parentheses. Segments within virions from top to bottom are: PB2, PB1, PA HA, NP, NA, M and NS.

medical treatment. However, concern is mainly focused on the clinical course and management of small subsets of patients who rapidly develop very severe progressive pneumonia, which is often associated with failure of other organs, or marked worsening of underlying asthma or chronic obstructive airway disease. It seems that the risk of severe or fatal illness is highest in three groups: pregnant women, especially during the third trimester of pregnancy; children younger than 2 years of age; and people with chronic lung disease, including asthma (WHO, 2009a). Apart from lack of immunity against this virus in most humans, it is still unclear what viral genetic factors contribute to this higher transmission rate. It is interesting to note that recent epidemiological data indicate a skewed higher rate of infection with 2009 H1N1 in children and adults younger than 18 years of age (Dawood et al., 2009). Seasonal influenza viruses predominantly infect children and the older population. However, infections with the 2009 SOIV H1N1 are considerably lower in people 65 years or older, likely due to immunity from prior exposure and/or vaccination to a virus antigenically similar to 2009 H1N1. Nevertheless, it is currently unknown which specific virus or viruses that circulated in the past and during what years might be responsible for this apparent serum cross-reactivity reported in the older population (above 65 years of age).

In 1976, a H1N1 virus antigenically closer to swine H1N1 isolates from the 1930s caused an influenza outbreak at Fort Dix. After the 1976 swine H1N1 outbreak, nearly 40 million people in the United States were immunized with an A/NJ/76 inactivated vaccine (Sencer & Millar, 2006). Interestingly, sequence analysis of this virus as compared to the current SOIV H1N1 demonstrates a high degree of identity of the HA sequences. Recent human serology studies have shown a high prevalence of neutralizing antibodies against 2009 SOIV H1N1 in people born prior to 1930 (Itoh et al., 2009) and sera from adults previously primed by a natural H1N1 infection and immunized with A/NJ/76 vaccine show cross-reactivity against 2009 H1N1. In general, the 2009 H1N1 pandemic virus is a low virulent virus. Unprecedented mass sequence analysis of its genome provided early and precise information about the potential virulence determinants of this new virus. First, the HA has a single amino acid in the cleavage site, which is associated with infection limited to the respiratory tract in humans. In addition, analysis of the sialyl receptor specificity of the HA of the 2009 H1N1 virus, determined using carbohydrate microarrays analysis (glycan arrays), demonstrated that while previous H1N1 seasonal influenza viruses present a2,6-linked SA specificity, the novel pandemic H1N1 virus has a2,6-linked SA affinity, but is also able to


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PERSPECTIVES ON VACCINATION AND OTHER INTERVENTIONS FOR INFLUENZA

fluenza virus is able to induce longer and broader immunity, as shown in animal models. Another vaccine alternative is the use of live attenuated influenza virus vaccines, and the most accepted one is the cold adapted nasal spray vaccine (flu mist) that is generated by reassortment of recombinant viruses containing mutations that confer cold adapted properties and the HA and NA from the new strains of influenza virus A, H1N1 and H3N2, and influenza B virus. Thus, these live attenuated vaccines contain viruses that replicate in the upper respiratory tract and have been shown to induce better mucosal immunity. These vaccines better mimic the early stages of influenza virus infection and are proposed to induce longer lasting and more protective immunity, since they generate not only antibody responses but also T-cell mediated immunity against the vaccine viruses. Recently, live attenuated influenza viruses with truncations in the NS1 protein have been tested in different systems for their efficiency as vaccine candidates (Solorzano et al., 2005). These viruses show high attenuation phenotype associated to a suboptimal NS1 protein due to truncations. These types of vaccines may prove to be very efficient due to the high immunogenicity associated with them as well as their protective potential as tested in animal models and primary cell systems. The development of universal influenza virus vaccines will be highly beneficial, since it would eliminate the need for annual updates in the seasonal vaccine. By using conserved epitopes in the HA protein that are not subjected to high selective pressure in the host, it might be possible to generate such vaccines. Several groups are currently exploring this approach.

Influenza Virus Vaccines

Antiviral Treatments for Influenza Virus

bind a2,3 linkages. Interestingly, the novel H1N1 SOIV showed similar specificity to those observed in a triple reassortant H1N1 and a classical swine H1N1 isolated in 2006 and 1976, respectively (Childs et al., 2009), indicating that no major changes in the receptor-binding specificity were necessary for the virus to become established in humans, and therefore, other factors likely play an important role in its successful “spillover” into humans. One of these factors could have been the acquisition of a novel NA by genetic reassortment (Childs et al., 2009). Second, the 2009 H1N1 pandemic virus does not contain a functional PB1-F2, suggestive of a mild disease in humans compared to the previous pandemic viruses. Third, the PB2 protein contains a glutamic acid at position 627 that is related to its avian origin, and is associated with a low pathogenic phenotype. Lastly, the NS1 segment, the novel influenza H1N1, has a truncated protein with a deletion of the 11 C-terminal amino acids (Neumann et al., 2009), which is also associated with low pathogenic phenotype. Thus, early on during the pandemic, many experts predicted a relative mild pathogenesis potential of this novel virus (Wang & Palese, 2009) . In conclusion, the novel SOIV does not present any of the known high pathogenic phenotypes in terms of virulence factors. But the threat will arise if the virus mutates, turning more virulent, since the direct human-to-human transmission is already highly efficient.

Vaccination has been one of the most effective means of protection against IAV. Vaccine-induced production of antibodies against the viral surface glycoproteins HA and NA are crucial for immune protection (Gillim-Ross & Subbarao, 2006). These two glycoproteins play a critical role in the virus life cycle: HA mediates virus binding and entry into host cells surface, and NA facilitates viral spreading by cleaving cell surface sialic acid moieties, thereby preventing the aggregation of progeny virions on the host cell surface (Palese & Shaw, 2007; Skehel & Wiley, 2000). HA-specific antibodies have been demonstrated to block the IAV infection by preventing receptor binding and/or fusion and NAspecific antibodies prevent virion release from infected cells. However, HA and NA proteins, due to antibody-mediated immune selection pressure, undergo antigenic evolution by accumulation of mutations (“antigenic drift”). As a result, trivalent inactivated influenza virus vaccines are administered yearly to the human population. The seasonal trivalent vaccine, or “flu shot” is normally manufactured from killed influenza viruses. The three major strains circulating in humans (H1N1, H3N2, and B influenza viruses) are included and updated in the vaccine every year for both the southern and northern hemispheres based on primary isolates from the previous season (Palese & García-Sastre, 2002) (http:// www.cdc.gov). These vaccines are based on reassortant viruses using the six internal genes from the influenza virus strain A/PR8/34 (PR8) and the HA and NA genes from the newly identified circulating strain (Palese & GarcíaSastre, 2002). While a number of vaccinated individuals elicit strong antibody responses against the inactivated virus vaccines, there is a lack of CD4 T-cell memory response in those individuals, which makes it necessary to upgrade the vaccine most years. The existence of antibody responses in humans against the inactivated vaccine indicates that the virus strains are sufficiently recognized by B cells in vivo. Inactivated influenza virus vaccines do not induce longlasting, broad cross-protection, whereas infection with in-

Currently, there are two types of antiviral drugs available for treatment of IAV: the so-called neuraminidase inhibitors (oseltamivir and zanamivir) and the adamantanes (rimantadine and amantadine), which interfere with the M2 protein of the virus, thereby preventing the release of infectious viral nucleic acid into the host cell (uncoating) by interfering with the function of the transmembrane domain of the viral M2 protein. Almost all circulating human influenza A H1N1 viruses tested during the 2008–2009 season showed resistance to neuraminidase inhibitors and sensitivity to adamantanes. However, genetic and phenotypic studies have shown that the novel 2009 H1N1 pandemic strain is susceptible to oseltamivir and zanamivir, but resistant to adamantanes (Centers for Disease Control and Prevention, 2009). Since the appearance of resistance to these antiviral treatments is quite common, other proteins of influenza virus are being explored as targets for antiviral treatment. One example is the NS1 protein, since its IFN antagonist function is essential for evasion of immunity by influenza virus, thus antivirals targeting this protein are likely to have an important effect in controlling infection in vivo. This will increase the options available to treat influenza virus infections because the high rate of mutations gives rise to viruses that are resistant to one or more antivirals. A combined antiviral therapy is more likely to be more efficient for pandemic preparedness since it is difficult to predict the degree of antiviral resistance that may occur for new circulating viruses.

INNATE IMMUNITY AND INFLUENZA VIRUS

Protection against reinfection with influenza viruses relies on the presence of neutralizing antibodies in the host, while clearance of infection is mediated by cellular cytotoxic Tcell immunity. In order to clear influenza virus infection from the lungs, it is important to generate Th1 immunity against the virus (Graham et al., 1994). The optimal Th1 response consists of virus-specific IFN-g-secreting CD4 T

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FIGURE 3 Effects of influenza virus on the initiation of immunity by dendritic cells. Influenza virus can block dendritic cell activation and function and that affects both innate and adaptive immunity. By blocking cytokine and chemokine production by dendritic cells (DCs), including IFN-a/b, influenza virus has an inhibitory effect on innate immunity. Blocking DC activation and up regulation of MHC class II and costimulatory molecules, allows influenza virus inhibition of adaptive immunity.

cells and cytotoxic CD8 T cells that lyse virus-infected cells. Dendritic cells (DCs), the most efficient antigen-presenting cells able to initiate primary immune responses, survey the body and upon contact with particular pathogens such as viruses or bacteria undergo maturation and migrate to lymph nodes where they present pathogen-specific antigens to T cells. The phenotypic changes that occur in maturation include the up regulation of MHC class II and costimulatory molecules and the release of proinflammatory cytokines and chemokines, which enhance their ability to stimulate T cells, and thus leading to the initiation of adaptive immune responses specific for the infecting pathogen (Banchereau et al., 2000). In addition to their critical role in initiating adaptive immune responses, DCs contribute to the antiviral innate immune system by secreting IFN-a/b, a powerful antiviral cytokine, in response to viral infection. The NS1 protein of influenza virus has been shown to inhibit IFN-a/b production from infected cells (García-Sastre, 2002), including human DCs (Fernandez-Sesma et al., 2006). We also found that the NS1 protein of human influenza virus inhibits human DC maturation, thus influenza virus can modulate both innate and adaptive immunity (Fig. 3) (FernandezSesma et al., 2006). The ability of a specific virus to evade important elements of innate immunity in the host may contribute to the establishment of infection by that virus in the host. The NS1 protein of influenza virus inhibits type I IFN production in several mammalian systems, including human DCs (Fernandez-Sesma et al., 2006; Kochs et al., 2007; Solorzano et al., 2005). By inhibiting the IFN response in infected cells, influenza viruses are able to escape innate immunity and establish infection in the host. Also, we have shown that the NS1 protein is able to inhibit DC activation and the ability of DCs to induce Th1 CD4 T-cell responses in

a human system (Fernandez-Sesma et al., 2006). This is a double immunomodulatory strategy of influenza virus by which both innate and adaptive immunity are affected in the host, which allows the virus to more efficiently establish infection. Additionally, the PB1-F2 protein of influenza virus has been shown to play a role in the regulation of apoptosis in infected cells (Chen et al., 2001; Zamarin et al., 2005). In particular, it has been shown that immune cells are more sensitive to the proapoptotic phenotype induced by this protein of influenza viruses than other nonimmune cells (Chen et al., 2001).

CONCLUSIONS

There are several features that a particular influenza virus must possess in order to establish itself in the human population. From previous pandemics and the most recent 2009 H1N1 pandemic, several determinants of viral pathogenesis and replication have been elucidated. Although the H5N1 viruses have shown to be highly pathogenic, human-tohuman transmission has not been established efficiently due to host restrictions. Nevertheless, the unprecedented level of surveillance and worldwide response to the current pandemic has been a consequence of years of coordinated international efforts preparing for a potential H5N1 pandemic. The massive amount of data arising in real time early on during the outbreak in Mexico and the United States allowed the rapid analysis of its sequence, the determination of its origin, and the characterization of its antigenicity and virulence by a number of laboratories around the world. Thus, it was predicted that this virus did not contain the molecular signatures that would confer to it a highly pathogenic phenotype. In addition, the rapid spread of the virus around the world was efficiently monitored due to the high


level of reporting through the WHO. This permitted the evaluation of transmission and mortality rates in little less than a month after the beginning of the outbreak. Although great concern has arisen due to the possibility of an H5N1 pandemic, the 2009 H1N1 pandemic has shown that there is considerable infrastructure and that the advances of modern medicine have substantially aided in the control of the modern pandemic. It is noteworthy to mention that in contrast to the settings of the 1918 pandemic, we now have several tools that would allow us to better combat a more virulent pandemic: (i) wide use of antibiotics, (ii) established and safe vaccination programs, (iii) the possibility of using antivirals as prophylactics and as a treatment measure early on during the course of disease, and (iv) an extensive surveillance network that would allow for the early detection of a sustained influenza outbreak. Luckily, H1N1 has not resulted in a highly pathogenic virus, and thus, it is likely to continue to spread throughout the world. However, due to the high rate of infection and the massive vaccination programs in place around the world, it is likely that this strain will undergo antigenic drift in the next few years due to preexisting immunity. Of interest, however, is the low level of seasonal H1N1 and H3N2 seen at his time, suggesting that the 2009 H1N1 virus will become the predominant, if not the only, circulating influenza A virus worldwide. Finally, the current nomenclature and pandemic phase category of the WHO does not allow for an assessment of the severity of the pandemic virus. Therefore, the high public alert once a pandemic is declared is, at times, due to a misunderstanding of the term pandemic. Nevertheless, the declaration of a phase 6 pandemic level due to the great and sustained spread of the virus relatively early on during the 2009 H1N1 pandemic permitted the rapid release of resources to address the situation. Thus, it would be beneficial to incorporate a measure for severity or pathogenicity of a virus into the current system in order to more accurately and more clearly inform the public of the status of a pandemic strain.

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Index

AAV. See Adeno-associated virus AAVV. See Adeno-associated virus vectors ABC. See ATP-binding cascade Acanthocheilonema viteae, 527 N-Acetylmuramic acid, 9 Acinetobacter lwoffi, chronic inflammatory infections and, 527t Acquired cellular responses, 283–284 Acquired immunity acute bacterial infections, 269–276 acute infection and, 274–276 antibacterial, 274–276 in gastrointestinal tract, 314–317 Helicobacter pylori, 342–344 to helminths, 313–321 immunogenetics, 486t induction of, 274 innate immunity and, 239–240, 272, 571–572 to intracellular protozoa, 301–308 Mycobacterium leprae, 285–286 Mycobacterium tuberculosis, 284–285 Mycobacterium ulcerans, 285 TB and, 572–573 against viral infection, 239–251 Acquired immunodeficiency syndrome (AIDS), 1, 134, 580 animal models for, 611 clinical trials, 616 DNA vaccines, 614 genetic vaccines, 613–614 reasons for optimism, 617–618 recombinant viral vectors, 614 replicating vectors, 617 subunit vaccines, 613 therapeutic vaccines, 616–617 vaccines, 611–618 Acrocephalus anundinaceus, 50 Activation-induced cytidine deaminase (AID), 30, 124 Active tuberculosis disease, 627 diagnostic markers for, 628 ACTs. See Artemisinin-based combination therapies Acute infection acquired immunity, 274–276 immune responses following, 256–258 Acute phase reactants, 403 Acute rheumatic fever (ARF), 524 Entries followed by an f indicate a figure; those followed by a t indicate a table.

Acute viruses, immunity to, 404–409 Adaptive immune system evolutionary origins of, 41–52 innate immune system linking with, 32 in jawed invertebrates, 42–45 in jawed vertebrates, 42–45 lymphocytes in, 21 MHC and, 51–52 ontogeny of cells of, 21–33 parasitic infections and, 231–232 viral immunity and, 404 ADCC. See Antibody-dependent cellular cytotoxicity ADCI. See Antibody-dependent cellular inhibition Adeno-associated virus (AAV), 239 Adeno-associated virus vectors (AAVV), 615 Adenovirus, 577–578 vectors, 614 Adhesion defects, 477–479 Adjuvants pathogens as, 515 for protein subunit vaccines, 576–577 synthetic, 552 Treg, 529 Aedes aegypti, 15 Ag85, 576 Aging animal models of, 419t bacterial immunity and, 413–419 CD8 T-cell memory and, 405–406 CMV and, 408–409 De Novo infections and, 406 EBV and, 407–408 HCV and, 409 HIV and, 409, 421 HSV and, 409 human studies of, 420t immune function and, 413 peripheral memory T-cells and, 406 respiratory infections and, 405 viral immunity and, 403–410 VZV and, 407 Agnathans, 17 AHR. See Allergic airway hypersensitivity response AID. See Activation-induced cytidine deaminase AIDS. See Acquired immunodeficiency syndrome AIDS-associated retrovirus (ARV), 493 AIM2, 190

653

AIM2-ASC inflammasome, 191 AKT, Theileria and, 541t Alarmins, 66 Alkyl hydroperoxide reductase (AphC), 431 Allergic airway hypersensitivity response (AHR), 385 Allergic response, 384t Alternative activated macrophages, 317–318 Alternative complement pathway, 88 deficiencies, 90 Alum, 552–553 ALVAC, 565, 566 AMA1, 592 Amblyomma americanum, 604 Ambystoma mexicanum, 50 Amphimedon queenslandica, 16 Amphioxus, 44 AMPs. See Antimicrobial peptides Ancient antiviral defense mechanisms, 191 Ancylostoma duodenales, 146 Anemia, Plasmodium vivax and, 372 Annelids, 16 Anopheles gambiae, 15, 638 ANT3, 644 Anthrax, 3 Antibacterial-acquired immunity, 274–276 Antibacterial host defenses, 473–480 barrier defects, 473 cutaneous defenses, 473–474 immune defects, 474–477 pulmonary defenses, 474–477 Antibiotics, 529 Antibodies mycobacterial, 629 usefulness of, 250–251 viral infection control by, 249–250 Antibody-dependent cellular cytotoxicity (ADCC), 104, 250, 272 Antibody-dependent cellular inhibition (ADCI), 592 Antibody-dependent enhancement of infection, 384t, 385 Antibody-mediated immune effector mechanisms, 307–308 Antibody-mediated immunity, fungal infections, 292–295 Antibody-mediated protection, 269–270 Antibody responses importance of, 247 kinetics, 247–248 Antibody-secreting cells (ASC), 125–126, 247

654

Index

Antigenic drift, 647 Antigenic variation, 427–429 Neisseria gonorrhoeae, 427, 428f trypanosome, 454–455 Antigen presentation, 75 viral immunity and, 242–243 Antigen-presenting cells (APCs), 111, 212, 282, 348, 515, 571 alterations in, 457–459 costimulatory molecules and, 243–244 cytokine secretion, 199–201 signal 3 cytokines and, 244 virus detection by, 199–201 Antigen receptor-expressing lymphocyte repertoires, 26–28 Antigens barriers to, 98–99 delivery, 174–175 maintenance, 176–177 at mucosal surfaces, 98–101 mycobacterial, 629 Antigen sampling, 99 exploitation of, 101 at mucosal effector sites, 100 at mucosal inductive sites, 100 Antigen-specific B-cells, 126–127 Antigen-specific receptor, 45–46 Antigen-specific serum antibody responses, 125–126 Anti-inflammatory mediators fungal diseases and, 297t helminths and, 353 Antimicrobial activity BPI, 64 cathelicidins, 60 defensins, 58 iNOS, 77–78 lysozyme, 65 mechanisms of, 62 Antimicrobial immunity, 109–117 Antimicrobial peptides (AMPs), 7, 213, 220, 328, 342 Antimitochondrial antibodies, 526 Antineutrophil cytoplasmic antibodies, 526 Antiprion therapy, 179 Anti-Rv2626c, 629 Anti-TNF therapy, 331 Antiviral B-cell responses, 249 Antiviral cytokines, 191–194, 245 Antiviral response, Drosophila, 13 Antiviral vaccination strategies, 387–388 APCs. See Antigen-presenting cells AphC. See Alkyl hydroperoxide reductase Apicomplexians, 143–145 Apis melliera, 15 aPKC. See Atypical protein kinase C APOBEC3G, 201 APOBEC family, 194, 498 Apoptosis, 384t, 393–396 viral immune evasion of, 399–400 APRIL, 31, 99, 247, 271 ARF. See Acute rheumatic fever l-Arginine, 78, 353, 354f Arg1, 304 schistosomiasis and, 349–350 Arginase-1, 318 helminths and, 350–351 Artemisinin-based combination therapies (ACTs), 635 Arthroderma benhamiae, 169 ARV. See AIDS-associated retrovirus ASC. See Antibody-secreting cells Ascaris lumbricoides, 146 GLWS, 487t ASO1, 577

ASO2, 577 Aspartic acid, 50 Aspergillus fumigatus, 166, 289, 291 Aspergillus nidulans, 166 Asthma, 524 Asymptomatic parasitemia, 363 Atherosclerosis, 523–524 ATP-binding cascade (ABC), 474 Attacins, 8 Attenuated parasite vaccines, 589–590 Atypical protein kinase C (aPKC), 10 Auramine-Rhodamine, 627 Autoantigen-presenting cells, in primary lymphoid organs, 29 Autoantigen reactivities, 28–29 Autoimmune disorders, infections as promoters of, 521–526 Autoimmune inflammation bacteria as inhibitors of, 526–531 helminths as inhibitors of, 526–531 molecular mimicry during, 525t Autoimmune vasculitis syndromes, 526 Autoimmunity infectious etiologies for, 511–512 murine models of, 512t viruses and cancer and, 511–519 virus-induced, 387–388 babA2. See Blood group antigen-binding gene A2 Bacillus anthracis, 58, 65, 155 Bacillus Calmette-Guerin (BCG), 476, 575–576 Bacillus subtilis, 514 Bacteria-induced shock syndromes, 329–330 Bacterial immunity, aging and, 413–419 Bacterial infection acquired immunity to, 269–276 acute, 269–276 adhesion, 159 autoimmune inflammatory damage induced by, 334 chronic, 279–286 complement system in, 90–93 course of, 158 in elderly, 413 extracellular, 327–330 growth, 160 innate recognition receptors in, 214–219 invasion, 159–160 pathogenesis of, 327–334 pathology of, 327–334 transmission, 158–159 Bacterial pathogens, 155–162. See also specific pathogens construction of, 161–162 of elderly, 413 general aspects of, 211–212 host interactions, 156 as inflammation inhibitors, 526–531 innate immunity and, 209–222 survival of, 161 transmission of, 156–157 virulence factors, 159–161 Bacterial survival, 425–437 Antigenic variation, 427–429 biofilm development and, 433–434 complement evasion, 429–430 cytokines and, 434–435 immune adaptation and, 430–433 immune subversion and, 434–435 immune suppression and, 435 molecular disguise, 425–427

stealth, 425–427 stress resistance and, 430–433 Bacterial vectors, 615–616 Bactericidal permeability-increasing protein (BPI), 63 antimicrobial activity, 64 immunomodulation, 64–65 therapeutic potential, 65 Bacteroides fragilis, chronic inflammatory infections and, 527t Bacteroides thetaiotamicron, 161 Bacteroidetes, 156, 157 Baculoviral IAP repeat (BIR), 395 BAFF, 99 BAFF-receptor, 31 BALB/c mice, 303, 304 BALT. See Bronchus-associated lymphoid tissue Barrier defects, 473 Bartonella henselae, 157 Basophils, 210, 316f Bax-Bak, 395 B-cell(s), 29–30, 260 antigen-specific, 126–127 central tolerance failure, 33 development, 27 differentiation, 103 GALT-associated compartments, 32 ignorance of, 31 immature, 29–30 memory, 125–127 peripheral, without IG, 31–32 positive selection of, 30–31 responses, 247–250, 261–262, 264 subsets, 249 viral antigen recognition by, 247 B-cell differentiations, 26 B-cell inhibitory protein (BIP), from Ixodes ricinus, 604 B-cell receptors (BCR), 21, 26, 247 in immature compartments, 28 B-cells, regulatory, 530 BCG. See Bacillus Calmette-Guerin BCMA, 31 BcR. See B-cell receptors BcR complex, 32 BCV. See Brucella-containing vacuole Beauveria bassiana, 15 Bifidobacter, chronic inflammatory disorders and, 527t Bifidobacterium infantis, 529 Big Bang, 44 Biofilm development bacterial survival and, 433–434 extracellular bacterial infection, 329 Biomarkers breath, 629 candidate, 629 cytokines and, 629–630 for extent of disease, 629 identifying, 630 mycobacterial, 629 nonspecific, 630 for protective immunity, 628 TB, 628–630 of treatment effect, 629 Biomphalaria glabrata, 16, 43 BIP. See B-cell inhibitory protein BIR. See Baculoviral IAP repeat BL3, 539 BL20, 539 B-lineage commitment, 25 Blood and marrow transplantation (BMT), 165 Blood group antigen-binding gene A2 (babA2), 340

Index Blood-stage parasites, 591 Bloom, Barry R., 4 bmi-1, 23 BMP-4. See Bone morphogenic protein 4 BMT. See Blood and marrow transplantation BoMAC cells, 543 Bombyx mori, 15 Bone morphogenic protein 4 (BMP-4), 23 Bordet, Jules, 3 Bordetella pertussis, 60, 111, 112, 156, 159, 271, 447 Borrelia burgdorferi, 93 IL-10 and, 435 Bovine spongiform encephalopathy (BSE), 173, 178 BPI. See Bactericidal permeabilityincreasing protein Branchiostoma floridae, 42 Breath markers, 629 Bronchus-associated lymphoid tissue (BALT), 283 Brucella, 213 Brucella abortus, 217 Brucella-containing vacuole (BCV), 217 Brugia malayi, 146, 227, 320, 350 Brugia pahangi, 146 BSE. See Bovine spongiform encephalopathy BUNV. See Bunyamwera virus Bunyamwera virus (BUNV), 395 Burkholderia cepacia, 474 Burkholderia gladioli, 474 Bystander activation, 515, 522 C. diphtheriae, 162 C. trachomatis, 162 C1 complex, 85–86 C1r, 86 C1s, 86 C3, 429 C4 binding protein (C4BP), 429 C4BP. See C4 binding protein C57BL, 263, 332 Cache Valley virus, 602 Caenorhabditis elegans, 12, 17, 42, 145 CAF01, 577 Calnexin, 49 Calreticulin, 49 cAMP, 117 synthesis, 436 Campylobacter jejuni, 157, 334, 436 Cancer EBV and, 518 HBV and, 518 HCV and, 518–519 HHV-8, 519 HTLV-1 and, 519 polyomavirus and, 519 viruses and, 515–519 viruses autoimmunity and, 511–519 Candida albicans, 60, 93, 113, 165–166, 295 Candida dubliniensis, 165 Candida parapsilosis, 165 Candidate host markers, 629–630 Carbohydrates, 212 Carcinogenesis, 516–517 Carcinoscorpius rotundiacauda, 15 CARD. See Caspase-recruitment domain CARD domains, 189, 213, 219 Carrier protein (CRM), 274f Caspase-1, 190, 219 Caspase-8, 394 Caspase-recruitment domain (CARD), 72

CAT2, 350 Catalase, 71 Cathelicidins, 60–62, 64, 66 antimicrobial activity, 61 immunomodulation, 61–62 localization, 60–61 regulation, 60–61 Cathepsin, 213 Cationic HDP, 57 Caveolae-vesicle complexes, 376 Plasmodium vivax, 373 CCC. See Chronic Chagas cardiomyopathy CCL1, 100 CCL2, 317, 328 CCL9, 100 CCL20, 99, 100 CCL23, 100 CCL25, 99, 102 CCL28, 102, 271 CCR5, 101, 204, 493 CCR6, 102 CCR7, 122, 123, 551 CCR10, 102, 271 CCR62L, 123 CD1 molecules, 47, 215 CD41 T cells, 260, 261 CMV and, 264 CD81 T cells, 260–261, 304 CMV and, 263–264 memory, 405–406 viral immune evasion of, 400 CD14, 216 CD21, 175–176 CD23, 51 CD25, 114, 117, 333 CD35, 175–176, 221, 368 CD36, 167, 215, 306 CD38, 551 CD45R, 333 CD45RA, 551 CD45RO, 100, 333 CD55, 368 CD59, 227 CD62L, 122, 551 CD80, 116 CD89, 272 CD94, 229 CD95, 205, 540 CD103, 103 CD127, 551 CD161, 263 CDK. See Cyclin-dependent kinase CDRs. See Complementary determining regions CDT. See Cytolethal-distending toxin CEACAM1, 492 Cecropins, 8 Cell-mediated cytotoxicity, 245–246 Cell-mediated immunity fungal infections, 292–295 humoral immunity and, 272–273 Cell surface receptors, 630 Cellular activation, 459–460 Cellular immunity, vaccines and, 564–565 Cellular responses, 14–15 Cement protein 64TRP, from Rhipicephalus appendiculatus, 604 Central interacting domain (CID), 193 Central tolerance failure, 33 Cerebral malaria, 366–367 Cervarix, 553 CFP-10, 626 CGD. See Chronic granulomatous disease Chagas’ disease, 145 immune suppression and, 446

655

Chelicerata, 71 Chemokines, 65–66, 403 in extracellular bacterial infection, 328 Chitinase family, 351–352 Chlamydia pneumoniae, 414, 418, 523–524 Chlamydia trachomatis, 155, 270 Cholera, 3 Chronic bacterial infections, 279–286 causes of, 279–280 immune responses to, 280–282 T-cell immunity to, 282 Chronic Chagas cardiomyopathy (CCC), 525–526 Chronic granulomatous disease (CGD), 75–76, 476–477 Chronic inflammatory disorders, 521–531 bacteria as inhibitors of, 526–531 Bifidobacter and, 527t Faecalibacterium prausnitzii and, 527t firmicutes and, 527t flarial nematode, 527t Heligmosomoides polygyrus and, 527t helminths as inhibitors of, 526–531 implications for therapy, 531 intestinal microbiota and, 531 Lactobacillus and, 527t persistent infections, 522–523 regulatory T-cells in, 529 Schistosoma mansoni and, 527t Trichinella spiralis and, 527t Trichuris suis and, 527t Chronic inflammatory infections Acinetobacter lwoffi and, 527t Bacteroides fragilis and, 527t Helicobacter pylori and, 527t Mycobacterium vaccae and, 527t Salmonella and, 527t Chronicity, of parasitic infections, 149 Chronic wasting disease (CWD), 173, 178 CID. See Central interacting domain Ciona intestinalis, 42, 51 CIqR, 30 Circumsporozoite protein (CSP), 143, 306, 587 CJD. See Creutzfeldt-Jakob disease CK2, Theileria and, 541t CL. See Cutaneous leishmaniasis Classical complement pathway, 85–86 deficiencies, 90 specific serine proteases, 87–88 structure of, 87 ClfA. See Clumping factor A Clofazamine, 523 Clonal selection hypothesis, 21–22 Clostridium difficile, 156 Clostridium perfringens, 159 Clostridium tetani, 155, 329 Clostridium trachomatis, 157, 160 CLRs. See C-type lectin-like receptors Clumping factor A (ClfA), 430 CMV. See Cytomegalovirus Cmv1, 499–500 Cmv3, 500 c-Myc, Theileria and, 541t Cnidaria, 7, 16–17 Coinfections, 639 Collagenase, 213 Colonization, extracellular bacterial infection, 329 Colony-stimulating factor (CSF), 464 Complement, 327–328 evasion, 429–430 Complementary determining regions (CDRs), 27, 45 Complement inhibitors, Staphylococcus aureus, 430f

656

Index

Complement-like opsonins, 14–15 Complement receptor 1 (CR1), 30, 89, 228, 364, 371, 464 Complement regulator acquiring surface proteins (CRASP), 169 Complement system, 85–93, 168 activation pathway, 86 alternative pathway, 88 in bacterial infection, 90–93 biological effects of, 89–90 classical pathway, 85–86 composition of, 85 control of, 89 deficiencies, 90 innate immunity and, 221–222 lectin pathway, 87 organization of, 85 Streptococcus pneumoniae and, 90–92 in viral infection, 93 Complexity, 483–484 Concomitant immunity, 148 Coordinated gene expression, 136 Correlates of protection, 549–552 Corynebacterium, 159 Costimulatory molecules, APCs and, 243–244 COX-2, 341 Coxiella burnetii, 157 Coxsackievirus B3 (CVB3), 529 CpG. See Cytosine phosphate guanosine CpG motifs, 495 CR1. See Complement receptor 1 CR3, 89, 169, 214, 228 CRAMP, 62 CRASP. See Complement regulator acquiring surface proteins C-reactive protein (CRP), 92, 221 Creutzfeldt-Jakob disease (CJD), 173 CRM. See Carrier protein Crohn’s disease, 60, 522–523 incidence of, 523 CRP. See C-reactive protein Cryptosporidia, 101 CSF. See Colony-stimulating factor CSP. See Circumsporozoite protein CTLA4, 114 C-type lectin-like receptors (CLRs), 552 CUB domain, 87 Cutaneous defenses, 473–474 Cutaneous leishmaniasis (CL), 599 CVB3. See Coxsackievirus B3 CWD. See Chronic wasting disease CXCL8, 328 CXCL10, 319 CXCL13, 283 CXCR1, 328 CXCR3, 115 CXCR4, 493 Cyclin-dependent kinase (CDK), 539 Cytoadherence, 634 of parasitized red blood cells, 364–365 Cytochrome c, 394 Cytokine receptors, 202–203 Cytokines, 403 bacterial survival and, 434–435 biomarkers and, 629–630 inflammatory, 201–202 secretion by APCs, 199–201 signal 3, 244 Cytokine storm, 384t Cytolethal-distending toxin (CDT), 436 Cytolysis, 384t T-cell mediated, 384–385 Cytolytic T lymphocytes (CTL), 573 Cytomegalovirus (CMV), 123, 263–264, 404 aging and, 408–409 CD41 T-cells and, 264

CD81 T-cells and, 263–264 susceptibility loci, 504t Cytosine phosphate guanosine (CpG), 230 Cytosol, 135 Cytosolic DNA sensors, 191 Cytotoxicity, cell-mediated, 245–246 Cytotoxic T cells (CTLs), 49, 105, 197, 549, 614 D25, 333 DAF. See Decay-accelerating factor DAP. See Diaminopimelic acid DAP12-associated NK cell receptor, 499 DARC. See Duffy antigen receptor for chemokines David, John R., 4 DBP. See Duffy-binding protein DC. See Dendritic cell DC-SIGN, 168, 216 DDT. See Dichlorodiphenyltrichloroethane Death effector domain (DED), 12 Death-inducing signaling complex (DISC), 394 Decay-accelerating factor (DAF), 226 Dectin 1, 167, 168, 216 Dectin 2, 216 DED. See Death effector domain Defensins, 57, 64 antimicrobial activity, 58 in human disease, 60 immunomodulation, 59–60 localization, 57–59 regulation, 57–59 Delayed type hypersensitivity (DTH), 246, 416, 600 Leishmania and, 601–602 TB and, 573–574 Deltex, 25 Dendritic cell (DC), 29, 211, 302, 315, 341, 347, 403, 528 activation of, 303–304 antigen presentation and, 79 epithelial cells, 99–100 functional defects, 442t in fungal infections, 292 Helicobacter pylori and, 341 Leishmania donovani, 442t Leishmania major, 442t malaria and, 442t production of, 434 suppression of function, 441–443 Treg and, 446–448 Trypanosoma cruzi and, 442t Dengue, 134 Dengue fever (DF), 385 Dengue hemorrhagic fever (DHF), 385–386 Dengue shock syndrome (DSS), 385 De novo infections, aging and, 406 DEREG mice, 443 DF. See Dengue fever DHF. See Dengue hemorrhagic fever Diaminopimelic acid (DAP), 8 Diarrhea, 134 Dichlorodiphenyltrichloroethane (DDT), 633 Difficult viruses, 559–568 characteristics of, 560t Dimyristoylglycerol (DMG), 454 Diphtheria, 550 Diphtheria toxin (DT), 443 Diptericins, 8 DISC. See Death-inducing signaling complex DMG. See Dimyristoylglycerol

DN3, 29 DNA vaccines, 578 for AIDS, 614 DOCK8 deficiency, 480 Domeless, 12 Double-stranded RNA (dsRNA), 644 Down’s syndrome, 43 Downstream regulatory events, 464 Drosocins, 8 Drosophila, 17, 42, 191 antiviral response in, 13 blood cell lineages, 14 epithelial defense reactions, 13–14 host defense of, 7–13 IMD pathway, 11–12 immune effectors, 8 innate immune system, 7–13 JAK/STAT pathway, 12–13 JNK pathway, 12 phagocytosis, 14 sensing and signaling, 8 systemic immune response, 7–8 Drug-resistant tuberculosis, 624 extensively, 627–628 Drug susceptibility testing (DST), 627 Dscam, 43 dsRNA. See Double-stranded RNA DSS. See Dengue shock syndrome DST. See Drug susceptibility testing DT. See Diphtheria toxin DTH. See Delayed type hypersensitivity Dual oxidase (DUOX), 13 DUBA, 189 Duffy antigen receptor for chemokines (DARC), 372, 374, 593 Duffy-binding protein (DBP), 593 Duodenal ulcer promoting gene A (dupA), 340 DUOX. See Dual oxidase dupA. See Duodenal ulcer promoting gene A E1B-19K, 394 E2A, 25 E2F, Theileria and, 541t EAE. See Experimental autoimmune encephalomyelitis EBF, 25 EBNA1. See EBV-encoded nuclear antigen EBV. See Epstein-Barr virus EBV-encoded nuclear antigen (EBNA1), 514 Echinococcus, 146, 147, 320 Echinococcus granulosus, 227 ECP. See Eosinophil granule protein ECs, nonhuman primate model for, 503 Effector T-cells, 105 EGFR. See Epidermal growth factor receptor Ehrlichia chaffeensis, 78 Eicosanoids, 604 EIR. See Entomological inoculation rate Elastase, 213 Elderly. See also Aging bacterial pathogens of, 413 foodborne infection in, 417–418 immune function in, 413 Listeria monocytogenes, 417–418 ocular infection in, 418–419 Salmonella in, 418 septicemia in, 419 Streptococcus pneumoniae in, 414–415 ELISA, 247, 550, 626 ELISPOT assay, 126, 247, 626 Embryonic stem (ES) cells, 22, 23 hematopoietic cells developing from, 23

Index Endocarditis, 414t in animal models of aging, 419t human studies of, 420t Endogenous Treg, 115 Endoplasmic reticulum (ER), 134 ENL. See Erythema nodosum leprosum Entamoeba histolytica, 226 Enterobacteriaceae, 215, 222 Entomological inoculation rate (EIR), 638 Entry strategies, 135–136 ENU. See Ethyl-nitrous urea Eosinophil granule protein (ECP), 221 Eosinophils, 210, 316f Epidermal growth factor receptor (EGFR), 63 Epidermodysplasia verruciformis (EV), 518 Epithelial cells, 99 dendritic cells and, 99–100 Epithelial defense reactions, Drosophila, 13–14 Epitope spreading, 515 EPS. See Extracellular polymetric substances Epstein-Barr virus (EBV), 31, 93, 123, 251, 395, 498, 512 aging and, 407–408 cancer and, 518 Eptatretus stoutii, 42 ER. See Endoplasmic reticulum ERAAP, 42 ERK-1/2, 63 ERp57, 49 Erythema nodosum leprosum (ENL), 333 Erythrocytic-stage malaria vaccines, 590–593 ESAT6, 576, 626 ES cells. See Embryonic stem cells Escherichia coli, 14, 58, 101, 133 uropathogenic, 433–434 Ethyl-nitrous urea (ENU), 492 Euprymna scolopes, 16 EV. See Epidermodysplasia verruciformis Evasion, of parasitic infections, 149 Evolutionary origins, of adaptive immune system, 41–52 Experimental autoimmune encephalomyelitis (EAE), 515 Extracellular bacterial infection, 327–330 biofilm development, 329 chemokines in, 328 colonization, 329 immediate inflammatory response to, 327–328 inflammation in, 328 neutrophils in, 328–329 pathological outcomes of, 329 Extracellular control mechanisms, 23–24 Extracellular persistence, 429–430 Extracellular polymetric substances (EPS), 433 Factor C, 15 Factor H-like protein (FHL-1), 89, 169 FAD. See Flavin adenine dinucleotide FADD. See Fas-activation death domains FAE, 100 Faecalibacterium prausnitzii, chronic inflammatory disorders and, 527t Fas-activation death domains (FADD), 246 FasL. See Fas ligand Fas ligand (FasL), 205 Fatal familial insomnia (FFI), 173 FcR epsilon, 216 FcR gamma, 216 FcRm, 30 FcRn, 105 FDC. See Follicular dendritic cells FFI. See Fatal familial insomnia FHA. See Filamentous hemagglutinin (FHA)

FHL-1. See Factor H-like protein Fibrinogen-related proteins (FREPs), 16 Fibrogen-related proteins (FREPs), 43 L-Ficolin, 89 Filamentous hemagglutinin (FHA), 111, 112, 434 Filoviruses, 561 Fimbriated bacteria, 526 Firmicutes, 156 chronic inflammatory disorders and, 527t FIZZ molecule, 351 Flagellin, 212 Flarial nematode, chronic inflammatory disorders and, 527t Flavin adenine dinucleotide (FAD), 74 Flavin mononucleotide (FMN), 74 Flaviviridae, 193, 385 FLICE protein, 395 FLT3L, 33 FMDV. See Foot and mouth disease FMN. See Flavin mononucleotide FNGN. See Focal necrotizing glomerulonephritis Focal necrotizing glomerulonephritis (FNGN), 526 Follicular dendritic cells (FDC), 127, 174, 175, 176, 177 Foodborne infection, 414t in animal models of aging, 419t in elderly, 417–418 human studies of, 420t Foot and mouth disease (FMDV), 395 Foxp3, 109, 112, 117, 444, 447 FPRL-1, 63 Fracastoro, Girolamo, 1 Francisella tularensis, 271 FREPs. See Fibrinogen-related proteins (FREPs) FTY720, 117 Functional signatures, 549–552 vaccine development and, 555 Fungal immune modulators, 170 Fungal infections acquired immunity, 289–297 antibody-mediated immunity, 292–295 anti-inflammatory strategies in, 296t cell-mediated immunity, 292–295 dendritic cells and, 292 IDO, 296 immunomodulation, 297 inducing tolerance to, 295–296 regulatory T-cells in, 295t resistance to, 291 Th1 in, 293–295 Th2 in, 293–295 Th17 in, 293–295 tolerance to, 291 Treg and, 295–296 vaccine development, 296–297 Fungal pathogens, 165–170. See also specific pathogens complement escape of, 168–169 immune recognition, 167 immune system, 166–167 innate immune system and, 167 Fungal recognition, 291–292 Fungi, interactions with mammalian hosts, 289–290 FUT2, 494–495 G6PC3. See Glucose 6 phosphatase catalytic subunit 3 G6PD deficiency, malaria and, 370–371 Gadus morhua, 50 Galactin 3, 167, 168

657

Gallus gallus, 48 GALT. See Gut-associated lymphoid tissue GALT-associated B-cell compartments, 32 GAS. See Group A streptococci Gastritis, patterns of, 338f Gastrointestinal tract, acquired immunity in, 314–317 GATA, 241 GATA-3, 282, 316 GBS. See Guillain-Barré syndrome GCSFR, 475 GDI. See GDP-dissociation inhibitor GDP-dissociation inhibitor (GDI), 72 Genetic diversity, 560–561 Genetic effects, measuring, 484–485 Genetic vaccines, AIDS, 613–614 Gene transcription, iNOS, 77 Genome-wide linkage scans (GWLS), 484, 487t Leishmania, 487t Germinal centers antigen maintenance in, 176–177 PrP maintenance in, 177 Gerstmann-Straussler-Scheinker syndrome (GSS), 173 GF1. See Growth factor independent-1 Giardia lamblia, 78 GIL4, 495 Ginglymostoma cirratum, 48, 50 GIP. See Glycosylinositolphosphate GIPL. See Glycoinositolphospholipid GlcNac, 212 Glucan binding proteins (GNBPs), 8 Glucocorticoid receptor (GR), 348 Glucose-6-phosphatase catalytic subunit 3 (G6PC3), 475 Glucose phosphate isomerase (GPI), 538 GLURP antigens, 592 Glutamic acid, 50 Glycoinositolphospholipid (GIPL), 443 Glycophosphatidylinositols (GPI), 230, 302, 305, 372, 592 Glycoprotein 160 (gp160), 105 Glycosylinositolphosphate (GIP), 459 GM-CSF. See Granulocyte-macrophage colony-stimulating factor GNBPs. See Glucan binding proteins GPI. See Glucose phosphate isomerase; Glycophosphatidylinositols G-protein coupled receptors, 63 GR. See Glucocorticoid receptor Gram-negative cell wall, 158f Gram-positive cell wall, 159 Granular hemocytes, 15 Granular proteins, 219–220 Granulocyte-macrophage colony-stimulating factor (GM-CSF), 538, 573 Granulomatous inflammation, helminths and, 348 Granzyme, 205 Group A streptococci (GAS), 193, 524 Growing old. See Aging Growth factor independent-1 (GF1), 475 GSS. See Gerstmann-Straussler-Scheinker syndrome GTPase, 228 Guillain-Barré syndrome (GBS), 334, 526 Campylobacter jejuni and, 526 Gut-associated lymphoid tissue (GALT), 20, 31, 528 GWLS. See Genome-wide linkage scans H1N1 virus, 645 origin of, 648f H3K4me3, 115

658

Index

H3K27me3, 115 H3N2 virus, 651 H5N1 epidemic, 646–647, 651 H-60, 397 HAART. See Highly active antiretroviral therapy Haemophilus influenzae, 60, 159, 274, 414, 640 Hantavirus pulmonary syndrome (HPS), 385 Haplotype polymorphism, 48 HAV. See Hepatitis A virus HAX1, 475 HbC. See Hemoglobin C hBD. See Human beta defensins hBD2, 58 hBD3, 58 HBV. See Hepatitis B virus hCAP18, 60 in human disease, 62 hCAP18/LL37, 58t HCC. See Hepatocellular carcinoma Hck, Theileria and, 541t HCMV. See Human cytomegalovirus HCV. See Hepatitis C virus HDP. See Host defense peptides Helicobacter hepaticus, 436 Helicobacter pylori, 65, 337–344, 437, 522, 555 acquired immunity, 342–344 chronic inflammatory infections and, 527t dendritic cells and, 341 disease risk, 341 epithelial cells, 339 host defenses, 339–344 inflammatory factors, 341 innate immunity, 339–342 macrophages and, 341 mediated diseases, 337–338 NK cells and, 341 PRRs and, 341–342 T-cell responses, 342–343 Heligmosomoides polygyrus, 316, 318, 350 chronic inflammatory disorders and, 527t Helminths, 145–146 acquired immunity to, 313–321 animal models of infection, 314 anti-inflammatory mediators and, 353 Arginase-1 and, 350–351 granulomatous inflammation and, 348 gut damage and, 353 immunity to, in humans, 313–314 as inflammation inhibitors, 526–531 lectin pathway and, 351–352 lung damage and, 353 macrophages and, 348–349 pathogenesis, 347–354 recognition of, 230–231 Th1 and, 352 Th2-associated immune responses and, 347–348 Th17 and, 352 as therapeutic agents, 531 tissue-dwelling, 319–320 Helobdella, 16 Hemagglutinin, 643 Hematopoiesis malaria and, 368 suppression of, 368 waves of, 22 Hematopoietic cells adult development of, 22–23 development of, 22–23 embryonic development of, 22–23 ES cell development of, 23 extracellular control mechanisms, 23–24 intracellular control mechanisms, 23 self-renewal, 23–24

Hemoglobin C (HbC), 370 Hemoglobinopathies, 369 Hemolymph, 15 Hep-17, 589 Hepatitis A virus (HAV), 527 Hepatitis B virus (HBV), 114, 134, 140, 239, 245, 394, 491, 516 cancer and, 518 Hepatitis C virus (HCV), 111, 134, 239, 246, 251, 262–263 aging and, 409 cancer and, 518–519 susceptibility loci, 504t Hepatocellular carcinoma (HCC), 518 Hepoxilins, 604 HERC5, 194, 554 Heritability, 483–488 Herpes B virus, 139, 199 Herpes simplex virus (HSV), 58, 93, 110, 117, 202, 615 aging and, 409 Herpes simplex virus-induced stromal keratitis (HSK), 384 Herpesvirus entry mediator (HVEM), 112, 395 Heterodimeric antigen receptors, 28 Heterologous immunity, in viral infections, 251 HHV-6. See Human herpesvirus 6 HHV-8. See Human herpesvirus 8 Hib, 274 Highly active antiretroviral therapy (HAART), 626 High mobility group (HMG), 543 Hippocrates, 1 HIV. See Human immunodeficiency virus HLA. See Human leukocyte antigen HLA-B*08-bound peptides, 503 HLA-B27, 260, 503, 564 HLA-B57, 564 HMG. See High mobility group Hodgkin’s disease, 408 Horseshoe crab, 15–16 Host defense, against Streptococcus pneumoniae, 91–92 Host defense molecules secreted, 219–221 soluble, 219–221 Host defense peptides (HDP), 62 cationic, 57 in immune response, 58 Host defense proteins, 63 Host genes, 137 Host immunity, 199 Host markers, 629–630 HOX B4, 23 Hox gene clusters, 44 HPS. See Hantavirus pulmonary syndrome HPV. See Human papillomavirus HPV16. See Human papillomavirus 16 HPV18. See Human papillomavirus 18 HSE. See HSV-1 encephalitis HSK. See Herpes simplex virus-induced stromal keratitis HSP70, 46 HSV. See Herpes simplex virus HSV-1 encephalitis (HSE), 495 HTLV-1. See Human T-cell leukemia virus type 1 Human beta defensins (hBD), 342 Human cytomegalovirus (HCMV), 203, 239, 395 Human herpesvirus 6 (HHV-6), 514 Human herpesvirus 8 (HHV-8), 516 cancer and, 519

Human immunodeficiency virus (HIV), 58, 105, 110, 123, 139, 155, 260–261, 286, 399 aging and, 409, 421 animal models, 561–562 controlling, 559–568 empiricism and, 567 genetic diversity and, 560–561 immune evasion and, 560–561 malaria and, 367 natural immunity and, 559–560 replication, 503 resistance to, 493 susceptibility loci, 504t TB and, 623–624 T-cell response in, 114 tuberculosis, 626 vaccine development for, 562–565 Human leukocyte antigen (HLA), 561 Human papillomavirus (HPV) immune response after, 517–518 life cycle, 517 pathogenesis, 517 Human papillomavirus 16 (HPV16), 516 Human papillomavirus 18 (HPV18), 396, 516 Human T-cell leukemia virus type 1 (HTLV-1), 395 Human T-lymphotropic retrovirus type 1, 516 cancer and, 519 Humoral immunity cell-mediated immunity and, 272–273 long-lived, 127–128 T-cell-dependent, 124–125 vaccines and, 565–566 Humoral innate immunity, 221–222 Humors, 1 HVEM. See Herpesvirus entry mediator Hydra, 16 Hyper-IgE syndrome, 479, 497 Hypersensitivity reactions, 606 Hypoxia, 333 IAPs. See Inhibitor of apoptosis proteins (IAPs) IAV. See Influenza A virus IBD. See Inflammatory bowel disease IBS. See Irritable bowel syndrome IC31, 577 ICAM-3, 167 IDO, fungal infections and, 296 IECs. See Intestinal epithelial cells IFN. See Interferons IFNAR1, 202 IFNAR2, 202 IFN-g. See Interferon g IFNGR1, 204 IFNGR2, 204 IFN-stimulated genes (ISG), 498 IFN-stimulus response elements (ISRE), 497 IgA, 30, 102, 103, 272, 274f IgG versus, 270–271 regulation of production of, 271–272 IgD, 272 IgE, 347, 384 elevation, 479–480 IgG, 105, 247, 274f IgA versus, 270–271 mucosal, 105 IgG1, 308 IgG2a, 282 IgG3, 282, 308 IgH, 31 IgL, 28 IgM, 20, 29, 103 IgSF. See Immunoglobulin superfamily

Index IL-1. See Interleukin 1 IL-1b. See Interleukin 1b IL-1R. See Interleukin 1 receptor IL-1 receptor-associated kinase (IRAK), 9, 74, 249 IL-2. See Interleukin 2 IL-4. See Interleukin 4 IL-5. See Interleukin 5 IL-6. See Interleukin 6 IL-7. See Interleukin 7 IL-8. See Interleukin 8 IL-9. See Interleukin 9 IL-10. See Interleukin 10 IL-12. See Interleukin 12 IL-13. See Interleukin 13 IL-15. See Interleukin 15 IL-17. See Interleukin 17 IL-23. See Interleukin 23 IL-27. See Interleukin 27 IL-33. See Interleukin 33 IL-IR/TLR. See Interleukin IR/Toll-like receptor IMD pathway, 7 Drosophila, 11–12 PGN and, 9 Immune adaptation, bacterial survival and, 430–433 Immune cells, differentiation of, 75 Immune complex formation, 384t Immune complex-mediated pathology, 330 Immune defects, 474–477 adhesion, 477–479 neutrophil granule formation, 476 neutrophil oxidative metabolism, 476 Immune effectors, Drosophila, 8 Immune evasion, 453–465 cellular activation and, 459–460 HIV, 560–561 Leishmania, 461–465 T-cell, 398–399 Immune modulation, mechanisms of, 461 Immune reconstitution disease (IRD), 626 Immune regulated catalase (IRC), 13 Immune stromal keratitis (ISK), 384–385 Immune subversion, bacterial survival and, 434–435 Immune suppression bacterial survival and, 435–437 Chagas’ disease and, 446 of dendritic cells, 441–443 IL-10 and, 443–446 leishmaniasis and, 445–446 protozoa and, 441–449 regulatory T-cells and, 443–446 Immunity. See specific types Immunogenetics acquired immunity, 486t innate immunity, 486t nonimmune related, 486t of parasite host response, 483–488 of virus pathogenesis, 491–506 Immunoglobulin-like repeat proteins, 14 Immunoglobulin superfamily (IgSF), 16 Immunology, birth of, 3 Immunomodulation, 59–60 BPI, 64–65 cathelicidins, 61–62 defensins, 59–60 fungal infections, 297 iNOS, 79 mechanisms of, 62–63 ROI, 74 Immunoreceptor tyrosine-based activation motif (ITAM), 14, 32, 272, 329 Immunoreceptor tyrosine-based inhibitory motif (ITIM), 46, 329, 492

Immunoregulation, microorganism exposure and, 528–529 IMT. See Isoniazid monotherapy Indoleamine 2,3-dioxygenase, 289 Inducible regulatory cells, 110–112 Infected red blood cells (iRBCs), 306 Infections. See specific types Infiltrating leukocytes, 340 Inflammasomes, 200 activation of, 190 NLRP3-ASC, 190–191 Inflammation in extracellular bacterial infection, 328 in malaria, 365 Inflammatory bowel disease (IBD), 476 Influenza, 404–405, 643–651, 650f circulation of, 644f effects of, 650 innate immunity, 649–650 1918 pandemic, 645–646 1957 pandemic, 646 1977 pandemic, 646 1968 pandemic, 646 pathogenicity of, 645t susceptibility loci, 504t 2009 pandemic, 648f vaccines, 649 Influenza A virus (IAV), 239 Inhibitor of apoptosis proteins (IAPs), 395 Innate B lymphocytes, 211 Innate cellular responses, 229–230 Innate cytokines, 395–396 Innate immune system adaptive immune system linking with, 32 in annelids, 16 bacterial pathogens and, 209–222 comparative analysis of, 15–16 complement system and, 221–222 Drosophila, 7–13 fungal pathogens and, 167 general aspects of, 209–212 humoral, 221–222, 226–227 invertebrate, 7–17 modulation of, 213 in mollusks, 16 ontogeny of cells of, 21–33 origin of, 16–17 parasitic infections and, 225–232 viruses and, 185–194 Innate immunity, 485–486 acquired immunity and, 239–240, 272, 571–572 immunogenetics, 486t influenza, 649–650 malaria, 639 Innate recognition receptors, in bacterial infection, 214–215 iNOS, 303, 331, 351, 431 antimicrobial activity, 77–78 conceptual framework, 77 cytotoxic effects of, 77–78 enzyme activity, 77 functions of, 77 gene transcription, 77 immunomodulation and, 79 mRNA, 77 negative regulators of, 76 positive regulators of, 76 protein, 77 subcellular localization of, 76 Interferon g (IFN-g), 228, 282, 284–285, 286, 307, 321, 456 Interferon-mediated immunity, 495–502 effectors, 498 signaling, 497–498 viral escape, 498

659

Interferon receptors, 201 Interferons (IFNs), 185, 200 amplification of, 191 antiviral effectors induced by, 193 induction, 192 NK cells, 201–202 type 1, 191–194 Interferon stimulated response element (ISRE), 193 Interleukin 1 (IL-1), 89, 306, 337 Borrelia burgdorferi and, 435 Interleukin 1b (IL-1b), 58 Interleukin 1 receptor (IL-1R), 8 Interleukin 2 (IL-2), 122 Interleukin 4 (IL-4), 284, 315, 317 Interleukin 5 (IL-5), 229, 315, 320 Interleukin 6 (IL-6), 60, 61, 306 Interleukin 7 (IL-7), 33, 122 receptors, 259 Interleukin 8 (IL-8), 427 Interleukin 9 (IL-9), 315 Interleukin 10 (IL-10), 109, 149–150, 206, 241, 283, 303, 307, 415, 446, 447, 448 production of, 434 subsets of, 444t suppression of immune responses by, 443–446 Interleukin 12 (IL-12), 201, 228–229, 283, 302, 441, 447 type 1, 202 Interleukin 13 (IL-13), 314, 315, 317 Interleukin 15 (IL-15), 122, 202 receptors, 259 Interleukin 17 (IL-17), 303, 474 Interleukin 23 (IL-23), 283 Interleukin 27 (IL-27), 447 Interleukin 33 (IL-33), 316 Interleukin IR/Toll-like receptor (IL-IR/TLR), 7 Intestinal epithelial cells (IECs), 315 Intracellular bacteria pathology of, 330–334 rapidly growing, 331 Intracellular control mechanisms, 23 Intracellular protozoa acquired immunity to, 301–308 of phagocytic cells, 302–304 T-cell dependent control of, 302–303 Intraepithelial lymphocytes, 98 Invertebrate innate immune defenses, 7–17 IPS-1, 200 IRAK. See IL-1 receptor-associated kinase IRAK-1, 462 rapid inactivation of, 463f IRAK-4, 475–476 iRBCs. See Infected red blood cells IRC. See Immune regulated catalase IRD. See Immune reconstitution disease IRF3, 192, 200, 495 IRF7, 192 IRF9, 202 Iris, from Ixodes ricinus, 604 Irritable bowel syndrome (IBS), 529 ISG. See IFN-stimulated genes ISG15, 194 ISK. See Immune stromal keratitis Isoniazid monotherapy (IMT), 625 ISRE. See IFN-stimulus response elements; Interferon-stimulated response element IsSMase. See Sphingomyelinase-like enzyme ITAM. See Immunoreceptor tyrosine-based activation motif ITIM. See Immunoreceptor tyrosine-based inhibitory motif

660

Index

Ixodes ricinus, 604 B-cell inhibitory protein from, 604 iris from, 604 Ixodes scapularis, 603–604 PGE2, 603 Salp15 from, 603 Salp25 from, 603 sialostatin from, 603 JAK. See Janus kinase JAK1, 396, 496f, 497 Theileria and, 541t JAK2, 462f JAK/STAT pathway, 202 Drosophila, 12–13 Janeway, Charles A., 4 Janus kinase (JAK), 74, 202, 395 Japanese encephalitis virus, 134 Jawed invertebrates, 42–45 Jawed vertebrates, 42–45 Jenner, Edward, 3 JNK. See Jun N-terminal kinase JNK pathway Drosophila, 12 Theileria and, 541t Job’s syndrome, 479–480 Jun N-terminal kinase (JNK), 75 Kaposi’s sarcoma (KS), 516 Kaposi’s sarcoma herpesvirus (KSHV), 395, 407–408, 516, 519 Killed bacterial vaccines, 575 Killer cell lectin-like receptor G1 (KLRG1), 122, 206, 246, 406 Killer inhibitory receptor (KIR), 48, 49, 500–502 KINDLIN3, 478 Kinetoplastids, 145 KIR. See Killer inhibitory receptor KIR2DL1, 397 KIR3DL1, 199 KIR3DS1, 502 Kircher, Athanasius, 1 Klebsiella pneumoniae, 60, 161, 222, 329 KLRG1. See Killer cell lectin-like receptor G1 Koch, Robert, 3 KS. See Kaposi’s sarcoma KSHV. See Kaposi’s sarcoma herpesvirus Kupffer cells, 328 Kuru, 173 Laboratory of genetics and physiology 2 (LGP2), 189 La Cross virus, 602 Lactobacillus, chronic inflammatory disorders and, 527t Lactobacillus reuteri, 530 Lactoferrin, 64, 65 LAD1. See Leukocyte adhesion deficiency, type 1 LAD2. See Leukocyte adhesion deficiency, type 2 LAD3. See Leukocyte adhesion deficiency, type 3 LAM. See Lipoarabinomannan Lamellocytes, 14 Lamina propria, 30 LAMP 1, 217, 526 LAMP 2, 526 Lassa fever, 139 Latent membrane proteins (LMP), 31–32 Latent persistent viruses, 407–409

Latent tuberculosis infection, 625 L-chain loci, 31 LCMV. See Lymphocytic choriomeningitis virus Lectin-like receptors, 216 Lectin pathway, 87 deficiencies, 90 helminths and, 351–352 recognition molecules, 87 specific serine proteases, 87–88 structure of, 87 Leeuwenhoek, Antonius van, 2 Legionella in animal models of aging, 419t human studies of, 420t Legionella pneumophila, 155, 213, 218, 219, 414, 418, 431 Leishmania downstream regulatory events, 464 DTH and, 601–602 GLWS, 487t immune evasion by, 461–465 protective immune response, 601–602 Leishmania brasiliensis, 445 Leishmania donovani, 78, 113, 147, 149, 226, 227, 228, 301, 304 DC and, 442t Leishmania major, 282, 284 DC and, 442t Leishmania mexicana, 464 Leishmaniasis cutaneous, 599 immune suppression and, 445–446 sand flies, 600 visceral, 485 LEKT1. See Lymphoepithelial Kazal-type trypsin inhibitor Leprosy, pathology of, 333–334 Leucine rich repeats (LRRs), 8, 44, 219, 495 Leukocyte adhesion deficiency, type 1 (LAD1), 477–478 Leukocyte adhesion deficiency, type 2 (LAD2), 478 Leukocyte adhesion deficiency, type 3 (LAD3), 478 Leukocytes, innate, 210 Leukotrienes, 604 LGP2. See Laboratory of genetics and physiology 2 Lipid A, 157 Lipids, 212 Lipoarabinomannan (LAM), 212, 435, 579 Lipophosphoglycan (LPG), 226, 302 Lipopolysaccharides (LPSs), 15, 157, 212, 350, 419, 425, 552, 600 Lipoproteins, 212 Lipoteichoic acid (LTA), 58, 212, 214 Lipoxins, 604 Lister, Joseph, 3 Listeria, 14, 61, 273 Listeria monocytogenes, 78, 124, 159, 218, 330, 331 in elderly, 417–418 replication of, 426f Litomosoides sigmodontis, 149, 321, 352 Live attenuated virus vaccines, 612t Liver stage specific antigen-1 (LSA-1), 306 LJM19, 600 LL-37, 61 in human disease, 62 LMP. See Latent membrane proteins LMP-2, 463 LMP-7, 463 LouTat1, 460f LOX-1, 222 LPG. See Lipophosphoglycan

LPSs. See Lipopolysaccharides LRC. See Lymphocyte receptor region LRRs. See Leucine rich repeats LRS. See Lymphoreticular system LSA-1. See Liver stage specific antigen-1 LTA. See Lipoteichoic acid Lunatic Fringe, 25 Lutzomyia, 600 Ly49H-m157 axis, 499–500 Ly49P-H2-Dk-m04 axis, 500 Lymphocyte receptor region (LRC), 51 Lymphocytes in adaptive immune system, 21 adult development of, 22–23 development of, 22–23 embryonic development of, 22–23 selection of immature, 28–30 transfer of, 32–33 Lymphocytic choriomeningitis virus (LCMV), 124, 242, 245, 256, 257, 260, 264, 529 Lymphoepithelial Kazal-type trypsin inhibitor (LEKT1), 473 Lymphoid development, 24–26 environmental influences, 24 Lymphoid organs, 174 Lymphoreticular system (LRS), 174 Lymphotoxin a, 286 Lysozyme, 64, 65 antimicrobial activity, 65 MAb, 115 Mackaness, George B., 4 Macrolides, 523 Macrophages, 210, 341 activation of, 303–304 alternative activated, 317–318 Helicobacter pylori and, 341 helminth immunity and, 348–349 regulatory, 530 suppressor, 460 Macropus eugenii, 50 MADCAM1, 102, 271 Major histocompatibility complex (MHC), 41, 134, 135, 216, 398f, 499f, 517 adaptive immune system and, 51–52 coevolution of genes, 48–51 evasion, 399 evolution of, 47 primordial, 46–47 viral escape, 505 in viral pathogenesis, 502–505 Malaria, 305–308. See also Plasmodium blood-stage parasites, 501 cerebral, 366–367 coinfections, 639 control, 639 DC and, 442t elimination, 639 epidemiology, 636–639 erythrocytic-stage, vaccines, 590–593 G6PD deficiency, 370–371 hematopoiesis and, 368 HIV, 367 immune intervention in, 587–595 immunology, 636–639 inflammation in, 365 initiation, 305–306 innate immunity, 639 integrated view of, 375–376 life cycle of, 588f, 633–634 metabolic acidosis and, 368 microvascular obstruction in, 365 mortality, 638f non-falciparum, 636

Index pathogenesis, 634–636 pathology, 361–363 pregnancy-associated, 363, 368–369, 636 relapsing, 636 respiratory distress and, 368 severe, 362–363, 635–636 sexual stages of, 593–594 transmission-blocking vaccines, 593–594 uncomplicated, 362, 635 vaccines, 590–594, 639–640 whole cell vaccine approaches, 593 Malassezia, 291 MALT. See Mucosa-associated lymphoid tissue MAMPs. See Microbe-associated molecular patterns ManLAM. See Mannosylated lipoarabinomannan Mannan-binding lectin (MBL), 226 Mannan-binding lectin-associated serine proteases (MASPs), 88 Mannose-binding lectin (MBL), 327 Mannose R, 216 Mannosylated lipoarabinomannan (ManLAM), 435 MAP3K, 12 MAPK. See Mitogen activated protein kinase MAP-kinase, 63 MAPKKK. See Mitogen activated protein kinase kinase kinase Marburg virus, 139 MARCO, 276 Marshall, Barry, 337 MASPs. See Mannan-binding lectinassociated serine proteases Mast cells, 210, 316f, 317 Matrix metalloproteinases (MMPs), 539 MAVS. See Mitochondria antiviral signaling protein MAX. See Maxadilan Maxadilan (MAX), 600 MBL. See Mannan-binding lectin; Mannose-binding lectin MBP. See Myelin basic protein MCMV. See Mouse cytomegalovirus MD2, 330 MDA-5. See Melanoma-differentiationassociated gene 5 MDP. See Muramyl di-peptide Measles, 125–126, 134 MECL-1, 463 MEK5, 161 Melanization, 14 Melanoma-differentiation-associated gene 5 (MDA-5), 189, 495 Membrane attack complex, 88, 221, 226, 429 Memory, 121–128 lineage, 122–123 Memory B-cells, 125 lifespan of, 127 Memory T-cells, 105 differentiation, 258–260 heterogeneity, 123 longevity of, 123–125 maintenance of, 123–125 peripheral, 406 properties of, 257 recall of, 246–247 Merozoites, 308, 591–593, 633 Merozoite surface protein (MSP), 306, 592 Mesocestoides cortii, 147 Mesocricetus auratus, 49 Metabolic acidosis, malaria and, 368 Metabolomics, 581 Metchnikoff, Elie, 3 Methemoglobin, 71

Methionyl-leucyl-phenylalanine, 72 Mf. See Microfilariae MGIT. See Mycobacterial growth indicator tube MHC. See Major histocompatibility complex MHC class II, 32 MHV68, 399 Miasma, 1 Microbe-associated molecular patterns (MAMPs), 521, 552 Microbe-induced inflammation, 521–522 Microbes, 1 Micrococcus lysodeikticus, 65 Microfilariae (Mf), 320 Microvascular endothelial cells (MVECs), 361 MIF. See Migration-inhibitory factor Migration-inhibitory factor (MIF), 247 Miller, J.F.A.P., 4 Mitochondria antiviral signaling protein (MAVS), 188, 200 Mitogen activated protein kinase (MAPK), 16, 59, 74, 185 Mitogen activated protein kinase kinase kinase (MAPKKK), 10 MMCP1, 317 MMPs. See Matrix metalloproteinases Molecular mimicry, 513–514 during autoimmune inflammation, 525t by microbial structures, 524–526 Mollusks, 16 Monocytes, 210 Monophosphoryl lipid A (MPL), 553 Mortality malaria, 638f tuberculosis, 623 Mosquitoes, 602–603 Mouse coronavirus, 492–493 Mouse cytomegalovirus (MCMV), 197, 203, 205 susceptibility loci, 504t, 505t MPL. See Monophosphoryl lipid A MPO. See Myeloperoxidase mRNA, iNOS, 77 MS. See Multiple sclerosis MSH. See Mycothiol MSP. See Merozoite surface protein Mucosa-associated lymphoid tissue (MALT), 97–98, 338 local inductive sites, 97–98 T-cell dependent responses in, 101–102 Mucosal IgG, 105 Mucosal immune responses, 104f induction of, 101–102 regulation of, 103 Mucosal tissues antigens at, 98–101 immune defense at, 97–105 as immune effector sites, 98 Mucosal vaccines, 105 MULT-1, 397 Multiantigen subunit vaccine, 589 MULTI family, 198 Multiple cystein clusters, 11 Multiple sclerosis (MS), 385 Mumps, 125–126 Muramidases, 65 Muramyl di-peptide (MDP), 212, 213 Murine hepatitis virus, susceptibility loci, 504t Murine models, 61 of autoimmune disease, 512t MVECs. See Microvascular endothelial cells Mycobacteria, 14 Mycobacterial antibodies, 629 Mycobacterial antigens, 629

661

Mycobacterial catalase-peroxidase, 524 Mycobacterial growth indicator tube (MGIT), 627 Mycobacterial susceptibility, 478–479 Mycobacteria tuberculosis, infections, 331–333 Mycobacterium abscessus, 474 Mycobacterium avium, 93, 414 paratuberculosis, 523 Mycobacterium bovis, 573 Mycobacterium leprae, 162 acquired immunity to, 285–286 as intracellular pathogen, 155, 162 pathology of, 333–334 Mycobacterium marinum, 285 Mycobacterium paratuberculosis, 101 Mycobacterium tuberculosis, 113, 213, 217, 279–280, 283, 514, 522–523, 524 acquired immunity to, 284–285 chronic inflammatory infections, 527t deletion mutants of, 576 diagnostic methods, 626–627 in elderly, 415–417 sterile eradication of, 580 Mycobacterium ulcerans, 157 acquired immunity to, 285 pathology of, 333–334 Mycobacterium vaccae, chronic inflammatory infections and, 527t Mycolic acid, 212 Mycoplasma arthritidis, 330 Mycothiol (MSH), 431 MyD88 complex, 186, 213, 230, 497 MyD88 deficiency, 475–476 Myelin basic protein (MBP), 514 Myeloid development, 24–26 environmental influences, 24 Myeloid-lymphoid progenitors, 24 Myeloperoxidase (MPO), 69, 71, 477 Myxomatosis virus (MYXV), 395, 396 MYXV. See Myxomatosis virus NAAT. See Nucleic acid amplification techniques NADPH, 69, 73, 303, 476 NALP3, 230, 291 NAP1, 188 NAPlr. See Nephritis-associated streptococcal plasmin receptor Natural killer (NK) cells, 21, 51, 57, 197–206, 211, 316 activation, 398 direct recognition of viruses by, 203 direct viral control by, 198–199 effector functions of, 203–204 evasion of, 396–398 Helicobacter pylori and, 341 in host immunity, 199 inflammatory cytokines and, 201–202 interferon activation of, 201–202 regulation of, 206 viral inhibition of, 397f Naturally acquired immunity, 637–639 Necator americanus, 146 Necrotizing stromal keratitis (NSK), 384–385 Negative feedback loops, 448f Neglected tropical diseases (NTDs), 145 Neisseria gonorrhoeae, 156, 159, 161 antigenic variation of, 427, 428f phase variation of, 427, 428f Neisseria meningitidis, 215, 427, 594 Nematodes, 146 Nematostella, 16 NEMO, 478

662

Index

NEP. See Nuclear export protein Nephritis-associated streptococcal plasmin receptor (NAPlr), 330 Netherton syndrome, 480 NETs. See Neutrophil extracellular traps Neuraminidase, 643 Neutrophil extracellular traps (NETs), 72 Neutrophil granule formation defects, 476 Neutrophil oxidative metabolism defects, 476 Neutrophils, 210 in extracellular bacterial infection, 328–329 saliva and, 602 Neutrophil serine proteases, 219–220 NFAT. See Nuclear factor of activated T-cells 1918 pandemic, 645–646 1957 pandemic, 646 1977 pandemic, 646 1968 pandemic, 646 Nippostrongylus brasiliensis, 227, 353 Nitric oxide (NO), 69, 204, 460 protective immune response by, 78–79 resistance mechanisms, 78 Nitric oxide synthase (NOS), 65 NK cells. See Natural killer cells NKG2A, 229 NKG2D, 198, 204 NK receptors, 499–502 models, 501f viral escape, 500 NKT cells, 28 NLR. See Nod-like receptors NLRC4, 218 Nlrc4, 219 NO. See Nitric oxide NOD. See Nucleotide-binding and oligomerization domain Nod-like receptors (NLR), 99, 157, 189–190, 213, 219, 291, 552 Nonparasitized red blood cells, 367–368 Nonspecific biomarkers, 630 Norwalk virus, resistance to, 494–495 NOS. See Nitric oxide synthase Nos2, schistosomiasis and, 349–350 NOTCH-1, 25 NOX2, 71, 431 NOX/DUOX family, ROIs and, 72 NP. See Nucleoprotein Nramp1, 304, 485 NRG1, 165 NSK. See Necrotizing stromal keratitis NTDs. See Neglected tropical diseases Nuclear export protein (NEP), 643 Nuclear factor of activated T-cells (NFAT), 436 Nucleic acid amplification techniques (NAAT), 627 Nucleic acids, 212 Nucleoprotein (NP), 250, 643 Nucleotide-binding and oligomerization domain (NOD), 515 NZM2410/NZW, 33 OAS1, 498 OAT. See Ornithine aminotransferase Obesity, 529 Ocular infection, in elderly, 418–419 Ocular keratitis, 414t in animal models of aging, 419t human studies of, 420t ODC. See Ornithine decarboxylase Odds ratios (ORs), 485 ODN. See Oligodeoxynucleotides Oligoadenylate synthetase (OAS), 193, 201

Oligodeoxynucleotides (ODN), 447 olpA. See Outer inflammatory protein gene A OmpA, 212 Onchocerca volvulus, 146 Ornithine aminotransferase (OAT), 351 Ornithine decarboxylase (ODC), 351 ORs. See Odds ratios Orthomyxoviridae, 193 Orthomyxovirus resistance gene proteins, 193–194 Outer inflammatory protein gene A (olpA), 340 OVA. See Ovalbumin Ovalbumin (OVA), 149, 602 OX40, 243 P2X7, 63 P13-K, Theileria and, 541t p53, 394f p202, 191 P554S, 497 PA, epitope, 405 PACRG, 286 PaI. See Pathogenicity islands PAMP. See Pathogen-associated molecular patterns PAMP agonists, 577 PANDAS. See Pediatric autoimmune neuropsychiatric disorders associated with streptococci Paneth cells, 211 Paralogs, 44 Paramyxoviridae, 193 Parasite-encoded antigens, 593 Parasitemia, asymptomatic, 363 Parasite-targeted oncogenic pathways, 540–541 Parasitic infections adaptive immunity and, 231–232 chronicity, 149 evasion, 149 host response to, 483–488 immune evasion by, 453–465 innate immunity to, 225–232 regulatory responses in, 232 Parasitic pathogens, 143–151 chronicity, 149 diseases caused by, 143–147 evasion, 149 immunity to, 147–148 persistence, 149 relationship between immunity and disease in, 149–150 Parasitophorous vacuole (PV), 143 PARC2, 286 Parvovirus B19 resistance to, 493–494 susceptibility loci, 504t Pasteur, Louis, 2 Pathogen-associated molecular patterns (PAMP), 110, 157, 158, 219, 552 Pathogenicity islands (PaI), 339–340 Pattern recognition molecules (PRM), 157, 395, 515 Pattern recognition receptors (PRR), 230, 291, 305, 425 Helicobacter pylori and, 341–342 PAX5, 25 expression of, 26 PB1, 405 P blood group antigens, 494 PBMCs. See Peripheral blood mononuclear cells PCD. See Primary ciliary dyskinesia

PD-1, 259 pDC. See Plasmacytoid dendritic cells PDC-E2, 526 PDI. See Protein disulfide isomerase Pediatric autoimmune neuropsychiatric disorders associated with streptococci (PANDAS), 525 Peptide-loading complex (PLC), 42 Peptidoglycan (PGN), 8, 158, 159, 212 IMD pathway and, 9 Toll pathway and, 9 Peptidoglycan recognition proteins (PGRPs), 8, 213, 220 Perforin, 205 Peripheral blood mononuclear cells (PBMCs), 364, 419, 497, 538 Peripheral memory T-cells, 406 Peripheral tissue, T-cell invasion of, 246 Peroxidase, 64 Persistence, of parasitic infections, 149 Persistent infections, 522–523 response to, 524 Persistent viruses, 255–265, 407–410 latent, 407–409 memory T-cell differentiation during, 258–260 pathology, 255–256 strategies of, 256 Peyer’s patch, 175, 530 PfEMP1, 144, 364, 365–366, 442, 591, 634 Pfs25, 594 Pfs230, 594 PG. See Phosphatidyl glycerol PGE2. See Prostaglandin E2 PGN. See Peptidoglycan PGRPs. See Peptidoglycan recognition proteins Phagocyte, 69 antimicrobial effector mechanisms, 70 innate defenses mediated by, 227–228 Phagocytosis, 15 bacterial pathogens, 213 Drosophila, 14 responses to, 213 Phase variation, 427 Neisseria gonorrhoeae, 427, 428f Phenol-Auramine, 627 Phenoloxidase (PO), 14 ROI, 70–71 PHIST domain, 594 Phlebotomus papatasi, 600 Phosphatidyl glycerol (PG), 433 Phosphatidylinositol-3-kinase (PI3-K), 63 Phosphatidylinositol-3-kinase AKT-mTOR pathway, 33 Phosphotyrosine kinase (PTK), 461 Phox, 72 pHSCs. See Pluripotent hematopoietic stem cells Phylatoxin, 93 PI3-K. See Phosphatidylinositol-3-kinase Picornaviridae, 193 PKA, Theileria and, 541t PKC. See Protein kinase C PKDL. See Post-kala-atar dermal leishmaniasis PKR. See Protein kinase R Plagues, 1 Plasma cells, 125, 241 Plasmacytoid dendritic cells (pDC), 211 Plasmodium, 301, 464, 589 GLWS, 487t life cycle of, 362f preerythrocytic stages of, 588 Plasmodium berghei, 15, 148, 226

Index Plasmodium chabaudi, 442, 591 Plasmodium falciparum, 229, 308, 441 development of, in human red blood cells, 590f human genetic resistance to, 369–370 liver stages of, 587–590 multiplication rate, 364 pathogenesis, 363–369, 376f sporozoites, 587–590 Plasmodium knowlesi, 636 Plasmodium ovale, 636 Plasmodium vivax, 371–375, 587, 636 anemia and, 372 caveolae-vesicle complexes, 373 severe infections, 374 thrombocytopenia and, 372 Plasmodium yoelii, 113 PLC. See Peptide-loading complex Pluripotent hematopoietic stem cells (pHSCs), 23 PLZF, 28 PMCA. See Protein kinase R PMN, 210 Pneumococcal cell-surface proteins, 91 Pneumococcal cell wall, 91 Pneumococcal surface protein A (PspA), 92 Pneumocystis carinii, 169 Pneumolysin, 91 Pneumonia, 134 in animal models of aging, 419t human studies of, 420t PO. See Phenoloxidase Polymeric peptidoglycan, 212 Polymicrobial peritonitis, 92–93 Polyomavirus and cancer, 519 Polysaccharide, 275 Porifera, 7, 16–17 Porphyromonas gingivalis, 552 Postexposure vaccination, 578–579 Post-kala-atar dermal leishmaniasis (PKDL), 445 Post-streptococcal glomerulonephritis (PSGN), 330 Poxvirus vectors, 614 PPAR gamma, 99, 348 PPD. See Purified protein derivative Pre-BCRs, 27 Pregnancy-associated malaria, 363, 368–369, 636 Primary ciliary dyskinesia (PCD), 474 Principles of Virology, 133 Prionoses, 173–178 blood cells in dissemination of, 178 diagnostics, 179 immune system in, 174–178 replication of, 174 treatment, 178–179 vaccination, 178–179 Prion protein, 173 lymphoid tropism of, 177–178 maintenance in germinal centers, 177 PRM. See Pattern recognition molecules Progenitor cells, 26 Prokaryotic ubiquitin-like protein (PUP), 432 Prostaglandin E2 (PGE2), from Ixodes scapularis, 603 Protein disulfide isomerase (PDI), 42 Protein kinase C (PKC), 230 Protein kinase R (PKR), 193, 396 Protein misfolding amplification assay (PMCA), 179 Protein subunit vaccines, 576–577 adjuvants for, 576–577 Proteobacteria, 156, 157 Proteomics, 581 Protists, 143

Protozoa immune suppression and, 441–449 infections, 144 intracellular, 301–308 recognition of, 230 PRR. See Pattern recognition receptors P-selectin, 367 Pseudocommensals, 526, 528f Pseudomonas aeruginosa, 58, 65, 71, 78, 90, 156, 219, 222, 329, 433, 474 Pseudomonas entomophila, 13 PSGN. See Post-streptococcal glomerulonephritis Psoriasin, 64 PspA. See Pneumococcal surface protein A PTK. See Phosphotyrosine kinase PTX3, 222 Pulmonary defenses, 474–477 Pulmonary infections, in elderly, 413–417 PUP. See Prokaryotic ubiquitin-like protein Purified protein derivative (PPD), 573 Purified recombinant protein vaccines, 612t Putrefaction, 2 PV. See Parasitophorous vacuole Pvs25, 594 pX protein, 394 PYD-containing subfamily, 189 Quiescent intracellular reservoirs (QIRs), 434 RA. See Rheumatoid arthritis Rab7, 217 RAC2 deficiency, 478 RAE-1, 397 RAG, 25, 31, 45 RAG1, 27, 41 RAG2, 25, 27, 41 RALDH2, 530 RANTES, 500 Rapidly growing intracellular bacteria, 331 RBC. See Red blood cell rBCG30, 580 Reactive nitrogen intermediates (RNI), 69, 75–79 generation of, 75–76 perspective, 79 Reactive oxygen intermediates (ROI), 69–75 enzymatic systems for, 69–70 function of, 74 generation of, 70–71 immunomodulation, 74 NOX/DUOX family and, 72 perspective, 79 phenoloxidases, 70–71 signaling, 74 Reactive oxygen species (ROS), 8, 432f Receptor interacting protein 1 (RIP1), 11 Recombinant vector vaccines, 612t for AIDS, 614 Recombination signal sequences (RSS), 45 Red blood cell (RBC), 301 polymorphisms, 375 Ref2, 10 Regulatory B-cells, 530 Regulatory macrophages, 530 Regulatory T-cells, 343–344 in chronic inflammatory disorders, 529 in fungal infections, 295t immune suppression and, 443–446 subsets of, 444t Relapsing malaria, 636 REL factors, 15 RELM, 351 Reoviridae, 193

663

Replicating vectors, 617 RER. See Rough endoplasmic reticulum Respiratory distress, malaria and, 368 Respiratory syncytial virus (RSV), 112, 385, 559 vaccine development, 567 Respiratory viruses, 404–405 aging and, 405 Restriction fragment length polymorphism (RFLP), 538 Retinoic acid-induced protein 1 (RIG-1), 495, 521 Retroviridae, 193 RFLP. See Restriction fragment length polymorphism Rhazes, 1 RHD. See Rheumatic heart disease Rheumatic heart disease (RHD), 334 Rheumatoid arthritis (RA), 30 Rhipicephalus appendiculatus, cement protein 64TRP from, 604 Rickettsia prowazekii, 155 Rifabutin, 523 Rifampicin, 627 RIG-1. See Retinoic acid-induced protein 1 RIG-I, 189 Rig I-like (RLR) receptors, 188, 213, 219 RIP1. See Receptor interacting protein 1 RISC. See RNA-induced silencing complex RLR. See Rig I-like receptors RNA-induced silencing complex (RISC), 191 RNA interference (RNAi), 12, 150, 191 RNase A, 221 RNase L, 193 RNI. See Reactive nitrogen intermediates ROI. See Reactive oxygen intermediates RORgT, 303 ROS. See Reactive oxygen species Rough endoplasmic reticulum (RER), 217 RSS. See Recombination signal sequences RSV. See Respiratory syncytial virus RTS,S, 588 Rubella, 125–126 RUT1, 575 Rv3407, 578 Saliva. See Vector saliva Salivary proteins, 604 immunization with, 604–605 Salivary vaccines, 604 candidates, 605t Salmonella, 101, 157, 159, 161, 273 chronic inflammatory infections and, 527t in elderly, 418 serotypes, 426 Salmonella enterica, 219 Salmonella enterica serovar Typhi, 428 Salmonella enterica serovar Typhimurium, 428 Salmo salar, 50 Salp15, from Ixodes scapularis, 603 Salp25, from Ixodes scapularis, 603 Sample size, 485 Sand flies, 599–602 leishmaniasis, 600 neutralizing effects of, saliva, 600–601 saliva, 599–602 SAP. See Secreted aspartyl proteinases; Serum amyloid protein Sarcoidosis, 524 SARS. See Severe acute respiratory syndrome SCARF1, 167 Scavenger receptors, 14, 212, 215, 216, 459

664

Index

SCF. See Stem cell factors Schistosoma haematobium, 146 Schistosoma japonicum, 146 Schistosoma mansoni, 78, 111, 146, 227, 232 chronic inflammatory disorders and, 527t GLWS, 487t Schistosomiasis Arg1 and, 349–350 Nos2 and, 349–350 SCID. See Severe combined immune deficiency SCID mice, 229 Secreted aspartyl proteinases (SAP), 170 Secreted phospholipase A2 (sPLA2), 213, 604 Secretor gene, 494 Secretory leukoprotease inhibitor (SLPI), 64 SED. See Subepithelial dome Selectivity index (SI), 364 Semliki Forest virus (SFV), 513 Semmelweis, Ignaz, 2 Sendai virus (SeV), 186 Septicemia, 414t in animal models of aging, 419t in elderly, 419 human studies of, 420t Serine protease inhibitor (SERPIN), 89 SERPIN. See Serine protease inhibitor Serprocidins, 64 Serratia marcescens, 13 Serum amyloid protein (SAP), 92 Serum resistance associated (SRA) gene, 227 SeV. See Sendai virus Severe acute respiratory syndrome (SARS), 492 Severe combined immune deficiency (SCID), 474 Severe malaria, 362–363, 635–636 Severe malaria anemia (SMA), 366, 367 SFV. See Semliki Forest virus Shigella, 61, 158, 160 Shigella flexneri, 218, 219 SI. See Selectivity index Sialic acids, 212 Sialostatin, from Ixodes scapularis, 603 Siculus, Diodorus, 1 S-IgA, 104 Siglec 1, 216 Siglec H, 216 Signal 3 cytokines, 244 Signal transducers and activators of transcription (STAT), 74 SIGN-R1, 328 SIL. See Squamous intraepithelial lesions Simian immunodeficiency virus (SIV), 101, 113, 262, 563–564 susceptibility loci, 504t Simian varicella virus (SVV), 407 Simian virus 40 (SV40), 394, 516 SINBAD, 188 Single cysteine cluster, 11 Single nucleotide polymorphism (SNP), 90 Single-round infectious viruses, 615 siRNA. See Small interfering RNA SIV. See Simian immunodeficiency virus Skin infection, 414t in animal models of aging, 419t human studies of, 420t SLC11A1, 485–486 Slc11A1, 304 SLE. See Systemic lupus erythematosus SLP-65, 27 SLPI. See Secretory leukoprotease inhibitor Sm16, 231 SMA. See Severe malaria anemia

Small interfering RNA (siRNA), 13 SmOmega-1, 148 Snow, John, 3 SNP. See Single nucleotide polymorphism SOCE. See Store-operated calcium entry SOCS1, 188 SOD. See Superoxide dismutase SOIV. See Swine-origin influenza virus Southeast Asian ovalocytosis (SAO), 375 Spallantani, Lazzaro, 2 Sphingomyelinase-like enzyme (IsSMase), 603–604 SPI-7, 426 SPINK5, 473, 480 sPLA2. See Secreted aspartyl proteinases Sputum microbiology, 629 Squamous intraepithelial lesions (SIL), 518 SRA gene. See Serum resistance associated (SRA) gene SREC-1, 222 Staphylococcus aureus, 14, 58, 60, 71, 161, 213, 275, 327, 418 complement inhibitors, 430f Staphylococcus epidermidis, 433 Staphylokinase, 430f Stargardt’s disease, 487 STAT. See Signal transducers and activators of transcription STAT-1, 63, 77, 396, 443, 447, 464 STAT-2, 77, 443 STAT-3, 63, 447, 474 deficiency, 479–480 Theileria and, 541t STAT-6, 316, 320 Stealth, bacterial survival and, 425–427 Stem cell factors (SCF), 25, 317 Store-operated calcium entry (SOCE), 72 Streptococcus agalactiae, 157 Streptococcus mutans, 159 Streptococcus pneumoniae, 90, 161, 215, 270, 272, 275, 327, 413 capsular polysaccharides of, 91 complement system and, 90–92 in elderly, 414–415 host defense against, 91–92 pneumococcal cell wall components, 91 Streptococcus pyogenes, 155, 327, 330 Streptolysin O, 327 Stress resistance, bacterial survival and, 430–433 Stromal cell-clonal precursors, 33 Strongylocentrotus purpuratus, 42 Strongyloides stercoralis, 146, 316 SuAT1, 543 Subdominant epitopes, TB and, 579 Subepithelial dome (SED), 530 Suberites domuncula, 16 Subunit vaccines, AIDS, 613 Superoxide dismutase (SOD), 69, 431 Suppressor macrophages, 460 SURFIN-like sequences, 373 Susceptibility loci, 504t SV40. See Simian virus 40 SVV. See Simian varicella virus Swine-origin influenza virus (SOIV), 643, 647–649 Synthetic adjuvants, 552 Systemic inflammatory response syndrome, 385–386 Systemic lupus erythematosus (SLE), 30, 90 Systems vaccinology, 554 T3SS. See Type 3 secretion system T4SS, 340 TAB2, 12

TACI, 31 Taenia crassiceps, 352 Taenia solium, 146 Taeniopygia guttata, 50 TAK1, 12, 16 TANK, 188 T antigen, 516 TAP, 41–42 TAP1, 48 TAP2, 48 Tapasin, 42, 51 TashAt, 542, 543 TashHN, 542, 543 TB. See Tuberculosis T-bet, 115 TBMs. See Tingible body macrophages T-cell(s), 21, 60, 260. See also Regulatory T-cells central tolerance failure, 33 chronic pathogens and, 282 development, 27 effector, 105 evasion of, 457 exhaustion, 259, 260 expansion, 505 function, 75 Helicobacter pylori and, 342–343 in HIV, 114 immune evasion, 398–399 invasion of peripheral tissue, 246 kinetics of, 244 memory, 105, 123–125, 246–247, 258–260, 406 peripheral memory, 406 phenotypes, 282–283 responses, 240–247 survival, 75 VacA and, 436 VSG specific, 456–457 T-cell dependent (TD), 249 control of intracellular protozoa, 302–303 T-cell-dependent humoral immunity, 124–125 T-cell effector systems, 245 T-cell-mediated protection, 272 T-cell receptors, 21, 26, 41, 46, 198, 503, 549 in immature compartments, 28 secondary rearrangements at, 28 stimulation, 110 T-cells, MALT and, 101–102 TCM, 123 TD. See T-cell dependent TDT. See Transmission disequilibrium test TdT. See Terminal deoxynucleotidyl transferase TEMRA cells, 408 Tenebrio molitor, 15 Terminal deoxynucleotidyl transferase (TdT), 25 Terminal mannose, 212 Tetanus, 550 Tetherin, 194 TFF3. See Trefoil factor 3 TGF-b. See Transforming growth factor beta Th1, 112, 115, 225, 282, 342, 456, 521 in fungal infections, 293–295 helminths and, 352 initiation of, responses, 231 Th2 and, 530 Th2, 112, 225, 282, 284, 285, 314, 343 cytokine-dependent immune effector mechanisms, 317 in fungal infections, 293–295 helminths and, 347–348 initiation of, responses, 231–232 Th1 and, 530

Index Th17, 103, 112, 148, 241, 329, 343, 384t, 522 in fungal infections, 293–295 helminths and, 352 Tregs and, 529–530 viral pathology and, 385 Theileria, 537–544 activated signaling pathways in, 542f AKT and, 541t AP-1 and, 541t CK2 and, 541t c-Myc and, 541t E2F and, 541t Hck and, 541t host and parasite interplay, 537–538 host cell information tracking, 541–544 immunization against, 538 JAK and, 541t JNK and, 541t NOTCH-CSL and, 541t P13-K and, 541t PKA and, 541t sporozoites, 537 STAT-3 and, 541t Theiler’s murine encephalomyelitis virus (TMEV), 513 Therapeutic vaccines, AIDS, 616–617 Thrombocytopenia, Plasmodium vivax and, 372 Thrombospondin-related adhesive protein (TRAP), 143, 306, 308, 589 Thymic stromal lymphoprotein (TSLP), 315, 316f, 348 Thymopoiesis, 22 Thymus, Treg cells in, 29 Ticks, 603–604 TIM-3, 117 Tingible body macrophages (TBMs), 175 Tissue-dwelling helminths, 319–320 Tissue tropism, 137 kinetic aspects of infection, 137 Tissue type plasminogen activator (tPA), 169 TiterMax, 179 TLF1. See Trypanosome lysis factor T-lineage commitment, 25–26 by external controls, 26 by internal controls, 25 TLR. See Toll-like receptor T lymphocytes, 4, 79 TME. See Transmissible mink encephalopathy TMEV. See Theiler’s murine encephalomyelitis virus TNF. See Tumor necrosis factor TNF1R. See Tumor necrosis factor-receptor 1 TNFR. See Tumor necrosis factor-receptor TNFR1, 74 TNF-receptor family, 31 TNFRSF17, 554 Togaviridae, 193 Toll 2 mutants, 11 Toll 8 mutants, 11 Toll 9 mutants, 11 Toll-like receptor (TLR), 8, 43, 74, 178, 185–188, 209, 212, 225, 230, 328, 521, 552 agonists, 448 associated molecules, 214 endosomal, 187, 188 expression, 185, 187 pattern recognition, 185–188 role of, 435 signaling, 185, 186 Toll pathway, 7 activation of, 10 PGN and, 9 Toxic shock syndrome (TSS), 330

Toxocara canis, 229 Toxoplasma, 144, 147, 301, 304 Toxoplasma gondii, 105, 228, 231, 487 tPA. See Tissue type plasminogen activator TprK, 428 Tr1 cells, induction of, 110–111 TRAF, 16 TRAF2, 10 TRAF3, 186 TRAF6, 186 TRAIL, 203, 205 Transcription controls, 25 Transforming growth factor beta (TGF-b), 109–110, 317 Transmissible mink encephalopathy (TME), 173 Transmissible spongiform encephalopathies (TSEs), 173 Transmission disequilibrium test (TDT), 484 TRAP. See Thrombospondin-related adhesive protein Trefoil factor 3 (TFF3), 319 Treg, 109–110, 149, 344 during acute infections, 112–113 adjuvants, 529 boosting, 116 during chronic infections, 113–114 dendritic cells and, 446–448 endogenous, 115 fungal infections and, 295–296 manipulating, 115–117 modulating responses, 111 negative control of, 115 plasticity, 115 Th17 and, 529–530 Treponema pallidum, 155, 427–428 Triakis scyllium, 48, 50 Tribolium castaneum, 15 Trichinella, 229 Trichinella spiralis, 320 chronic inflammatory disorders and, 527t Trichostrongylus orientalis, 147 Trichuris muris, 229, 352 Trichuris suis, chronic inflammatory disorders and, 527t Trichuris trichiura, 146, 229 GLWS, 487t TRIF, 231, 563 TRIM5a, 194 TRIM genes, 48 Trypanolytic factor, 227 Trypanosoma, 464 Trypanosoma brucei, 226–227 Trypanosoma cruzi, 525–526 DC and, 442t Trypanosome antigenic variation, 454–455 Trypanosome lysis factor (TLF1), 227 Trypanosome virulence, 460–461 TSEs. See Transmissible spongiform encephalopathies TSLP. See Thymic stromal lymphoprotein TSS. See Toxic shock syndrome TST. See Tuberculin skin test Tuberculin skin test (TST), 625 Tuberculosis (TB), 1, 414t, 418, 483 acquired immunity and, 572–573 active, 627 in animal models of aging, 419t attenuated parasite vaccines, 589–590 biomarkers, 628–630 clinical trials, 580–581 drug-resistant, 624 DTH and, 573–574 epidemiology of, 623–624 extensively drug resistant, 627–628 future vaccination strategies, 579–580

665

HIV-associated, 623–624 human studies of, 420t immune intervention strategies against, 571–582 latent, 625 memory and, 573 mortality, 623 pathogenesis of, 625–626 pathology of, 332–333 postexposure vaccination, 578–579 progression of, 624 spread of, 624 stopping, 627–628 strategies against, 574–575 subdominant epitopes, 579 suppression, 573 vaccine candidates, 577t vaccine construct design, 588–589 Tumor necrosis factor (TNF), 74, 89, 174, 201, 209, 394, 456, 628, 634–635 Tumor necrosis factor-receptor (TNFR), 7 Tumor necrosis factor-receptor 1 (TNF1R), 11 Turandot protein family, 12 2R hypothesis, 44 2009 pandemic, 648f TYK2. See Tyrosine kinase 2 Tyk2 deficiency, 480 Type 3 secretion system (T3SS), 158 Type O blood group antigen, 371 Tyrosine kinase 2 (TYK2), 202 UBE1L, 194 UL16, 398 UL18, 397 ULBP, 398 UNC93B, 186 Uncomplicated malaria, 362, 635 UniProt, 60 uPA. See Urokinase plasminogen activator UPEC. See Uropathogenic Escherichia coli Urinary tract infection (UTI), 413, 414t in animal models of aging, 420t human studies of, 420t Urokinase plasminogen activator (uPA), 169 Uropathogenic Escherichia coli (UPEC), 433–434 UTI. See Urinary tract infection V-Ab immune complex formation. See Virus-antibody immune complex formation Vaccine(s) AIDS, 611–618 attenuated parasite, 589–590 candidates for TB, 577t cellular immunity and, 564 chips, 554 development of, for HIV, 562–565 development of, for RSV, 567 DNA, 578, 614 empiricism and, 567 erythrocytic-stage malaria, 590–593 evolution of, 568f functional signatures and, 555 fungal infections, 296–297 future, strategies for TB, 579–580 genetic, 613–614 humoral immunity and, 565–566 influenza, 649 killed bacterial, 575 live attenuated virus, 612t malaria, 590–594, 639–640 mucosal, 105 multiantigen subunit, 589

666

Index

Vaccine(s) (continued) new construct design, 588–589 postexposure, 578–579 preexposure, 575–576 prion, 178–179 protein subunit, 576–577 purified recombinant protein, 612t recombinant vector, 612t, 614 RSV, 567 salivary, 604, 605t signaling networks, 552–554 specialized tissue and, 566–567 subunit, 613 therapeutic, 616–617 traditional, 611–612 transmission-blocking, 593–594 virus-like particle, 612t whole cell, 593 whole inactivated virus, 612t Vaccinia, 125–126 Vaccinia virus (VV), 239 Vaccinology, 549–555 systems, 554 Vacuolating cytotoxin (VacA), 340 T-cell function and, 436–437 VAR2CSA, 368, 369, 593 var genes, 365–366 Variable lymphocytes receptor (VLR), 17, 44 Variable region-containing chitin-binding protein (VCBP), 46 Variant antigenic type (VAT), 454 Variant surface glycoprotein (VSG), 145, 454, 455–456, 460 specific T cells, 456–457 Varicella zoster virus (VZV), 125–126, 199, 239 aging and, 407 VariVax, 407 Varro, Marcus Terentius, 1 VAT. See Variant antigenic type V-ATPase, 431 VCBP. See Variable region-containing chitin-binding protein VDAC1, 644 VDJ, 27 VDR. See Vitamin D receptor Vectors. See specific vectors Vector saliva immunological memory and, 604 neutrophils and, 602 polymorphism, 604 targeting components in, 599–606 Vesicular stomatitis virus (VSV), 186, 497, 602

V genes, 45 Vibrio cholerae, 101, 162 Viperine, 194 vir gene, 373 Viral immune evasion, 393–400 of apoptosis, 399–400 of CD81 T cells, 400 evolutionary considerations, 399–400 of IFN, 399 Viral immunity adaptive, 404 age and, 403–410 innate, 403–404 LCMV, 407 Viral infection. See also Persistent viruses acquired immunity against, 239–251 antigen presentation and, 242–243 complement system in, 93 control of, by antibodies, 249–250 heterologous immunity in, 251 pathology of, 383–388 Viral pathogens. See also specific pathogens classification of, 134 detection by APCs, 199–201 difficult, 559–568 diseases associated with, 134 entry strategies, 135–136 genetic instability of, 136–137 immune attack and, 140–141 infection sites, 138–139 innate immunity of, 185–194 outcomes of infection by, 136 pathogenicity, 140 species specificity, 137–140 structural properties, 133–135 tissue tropism, 137 Viral vectors, 577–578 Virulence factors, 159–161 interactions between, 340 transmission, 159 Virus-antibody (V-Ab) immune complex formation, 385 Virus entry, genetic control of, 492–495 Virus-induced autoimmunity, 387–388 Virus-induced immunopathology, 384–385 Virus-induced tissue damage, 383 Virus-like particle vaccines, 612t Virus pathogenesis immunogenetics of, 491–506 mouse genetics, 492 susceptibility loci, 504t Virus-specific T-cell epitopes, 242 activation of, 244–245

Visceral leishmaniasis (VL), 485, 599 Vitamin D3, 61 Vitamin D receptor (VDR), 332 Vivax malaria genetic resistance to, 375 pathogenesis of, 371 reticulocytes and, 371–372 severe, 374 VL. See Visceral leishmaniasis VLR. See Variable lymphocytes receptor von Behring, Emil, 3 VSG. See Variant surface glycoprotein VSV. See Vesicular stomatitis virus V to J rearrangements, 28 VV. See Vaccinia virus VZV. See Vesicular stomatitis virus Warren, Robin, 337 Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM), 475 West Nile virus (WNV), 239, 406–407, 493, 602 susceptibility loci, 504t WHIM. See Warts, hypogammaglobulinemia, infections, and myelokathexis Whole cell vaccines, 593 Whole inactivated virus vaccines, 612t Wnt-signaling pathway, 23 WNV. See West Nile virus Wound healing, 75 Wright, Almroth, 4 Wuchereria bancrofti, 146 Xenophagy, 191 Xenopus laevis, 50 Xenopus tropicalis, 48, 50 X-linked severe congenital neutropenia (XLN), 475 XLN. See X-linked severe congenital neutropenia Yellow fever virus (YFV), 553 susceptibility loci, 504t Yersinia enterocolitica, 159, 161 Yersinia pestis, 271, 273, 280, 435 Yersinia pseudotuberculosis, 330 YFV. See Yellow fever virus YM-1, 318 ZAP-70, 32

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B

COLOR PLATE 1 • (chapter 1) Comparison between Drosophila Toll and mammalian TLR4 and IL-1R receptor signaling pathways. (A) Binding of the maturated Spaetzle ligand triggers Toll signaling in Drosophila. A signaling complex, including MyD88, Tube, and Pelle, is recruited to the intracytoplasmic tail of the Toll receptor. This process is followed by the phosphorylation of the IkB-homolog Cactus by a so far unidentified kinase. Phosphorylated Cactus is degraded by the proteasome, allowing the nuclear translocation of the NF-kB transcription factors Dorsal and/or DIF (in larvae and adult flies respectively), activating the expression of hundreds of genes, including those encoding some of the antimicrobial peptides (Drosomycin). (B) A simplified scheme of TLR4 and IL-1R signaling pathways in mammals. (For a more detailed description the readers are referred to recent reviews by Bhoj and Chen, 2009; and Kawai and Akira, 2009). Upon stimulation by their corresponding ligands, TLR4 and IL-1R recruit a signaling complex comprising MyD88, IRAK1 and 4, and TRAF6, which controls the activation of TAK1. TAK1 further activates the IkB kinase (IKK) complex, which includes two catalytic subunits, IKKa and IKKb, and a regulatory subunit IKKg. Activated IKK phosphorylates IkB, which is further degraded by the proteasome. Freed NF-kB translocates to the nucleus and controls the expression of genes encoding inflammatory cytokines. MyD88, myeloid differentiation factor 88; IRAK, IL-IR associated kinase; TAK1, transforming growth factor (TGF)-b activated kinase 1; TRAF, tumor necrosis factor receptor (TNFR) associated factor; DD, death domain; KD, kinase domain.

A

B

COLOR PLATE 2 • (chapter 1) Comparison between the IMD pathway in Drosophila and the TNF-a receptor pathway in mammals. (A) Upon binding of DAP-type PGN to the PGRP-LC receptor, the adaptor protein IMD is recruited to the signaling domain of the receptor with which it interacts via a so far unidentified third partner. This process leads to the activation and nuclear translocation of the NF-kB transcription factor, Relish, driving the expression of hundreds of genes including those encoding some of the antimicrobial peptides such as Diptericin. Relish activation implies two independent processes activated downstream of the adaptor protein IMD. The first process involves FADD, the caspase-8 homolog DREDD, and TAK1, and leads to the phosphorylation of Relish by the IkB kinase (IKK) signalosome complex. This complex includes a regulatory subunit, Kenny (IKKg), and a catalytic subunit, ird5 (IKKb). The mechanism leading to the activation of TAK1 and IKK is currently unknown but probably involves K63-linked polyubiquitination mediated by the ubiquitin conjugating enzyme complex (Uev1a and Ubc13), the RING-domain containing protein DIAP2 (which possibly functions as a ubiquitin ligase), and TAB2, which contains a zinc finger which potentially binds to K63linked polyubiquitin chains. The second process involves FADD and DREDD (with a recently demonstrated input from the IKK complex) and leads to the cleavage of Relish, separating the C-terminal ankyrin repeat inhibitory domain from the Rel-containing segment. Note that TAK1 also serves to activate the JNK signaling pathway. (B) Signaling via the tumor necrosis factor-1 receptor (TNF-1R). (For a more detailed description the readers are referred to a recent review by Bhoj and Chen, 2009). Binding of TNFa to the TNF-1R leads to the recruitment of the death domain (DD) adaptor protein TRADD. A signaling complex is further assembled around TRADD, including RIP1, TRAF2, TRAF5, cIAP1, and cIAP2. RIP1 is a DD-containing serine/threonine kinase (DD similar to that of Drosophila IMD) and the TRAF and IAP proteins are RING-domain ubiquitin ligases. RIP1 is polyubiquitilated within this complex, a process that requires Ubc13 and Uev1A. The TAK1 kinase complex binds to the K63-linked polyubiquitin chains of RIP1 via TAB2/3. TAK1 is thus activated and phosphorylates the IKK complex. This complex includes two catalytic subunits, IKKa and IKKb, and a regulatory subunit, IKKg/NEMO, which mediates the binding of the IKK signalosome to the polyubiquitin chains. Activated IKK, in turn, phosphorylates IkB, which is further degraded by the proteasome. As a consequence, NF-kB translocates to the nucleus and activates the expression of numerous genes, namely those encoding inflammatory cytokines. Following the activation of NF-kB signaling, TRADD and RIP1 dissociate from the membrane receptor complex and associate with FADD, which binds to the caspase-8, forming a cytoplasmic complex that is implicated in signaling to apoptosis. cIAP, cellular inhibitor of apoptosis protein; DED, death effector domain; DIAP2, Drosophila inhibitor of apoptosis protein 2; DREDD, death-related ced-3/ nedd2-like protein; FADD, fas-associated death domain; NEMO, NF-kB essential modulator; TAK1, transforming growth factor (TGF)-activated kinase 1; TAB, TAK1 binding protein; Ubc13, ubiquitin-conjugating enzyme 13; Uev1a, ubiquitin-conjugating enzyme E2 variant; RING, really interesting new gene; RIP1, receptor interacting protein 1; TRADD, TNF-1R-associated via death domain; TRAF, TNFR-associated factor.

COLOR PLATE 3 • (chapter 6) Structure of the classical and lectin pathway activation complexes. C1 contains a heterotetramer of C1r and C1s, in which C1r (orange) and C1s (purple) are linked N-terminal to N-terminal via their CUBI, EGF, and CUBII domains. Two such C1rs drimers combine through the CCP-serine protease domains of C1r to form the tetramer. Lectin pathway activation complexes contain homodimers of MASPs linked through the CUBI, EGF, and CUBII domains (dark green). C1r–C1s and MASP–MASP interactions are conserved, so that the C1rs tetramer is effectively equivalent to two MASP dimers.

COLOR PLATE 4 • (chapter 13) Phagocytosis of C. albicans and A. fumigatus by macrophages. (A) C. albicans (labeled in green) adheres to and is taken up by activated human macrophages. The nuclei of the human cells are shown in blue and the cellular shape is visible. C. albicans adheres to human macrophages as shown on top of the human cells. In addition, Candida hyphae invade or are ingested by the human cells as shown on the left side of the central cells (S. Luo & P. F. Zipfel, unpublished data). (B) A. fumigatus conidia expressing dsRed germinating within macrophages 6 h after phagocytosis by J774 macrophages (Behnsen et al., 2007).

A

B

C

D

E

F

COLOR PLATE 5 • (chapter 29) Giemsa-stained, thin-smear blood films showing circulating stages of P. falciparum and P. vivax in patients with malaria. In all photomicrographs, nuclear material appears magenta in color and the cytoplasm appears blue in color. (A) P. falciparum ring-stage parasites. It is not uncommon to find multiple-infected red blood cells (RBCs) in vivo. (B) P. falci parum sexual-stage gametocyte. The banana-shaped morphology is specific for P. falciparum and is not found in other Plasmodium spp. (C) P. vivax ring-stage parasites. Note that the cytoplasm of P. vivax ring-stage parasites is thicker than that of P. falciparum. (D) P. vivax trophozoites. Note the amoeboid appearance of trophozoites and the eosinophilic stippling of the parasitized RBC surface, which corresponds to the caveolae-vesicle complexes. (E) P. vivax schizont containing individual developing merozoites. (F) P. vivax sexual-stage gametocyte. All images were kindly provided by DPDx (the CDC’s website for parasitology identification) at www.dpd.cdc.gov/dpdx/.

COLOR PLATE 6 • (chapter 29) Cytoadherence interactions involving P. falciparum-infected red blood cells (RBCs). (A) Rosettes are formed by the binding of parasitized RBCs to multiple nonparasitized RBCs. The presence of parasites in the centrally located RBC is detected by the DNA-staining Hoechst dye (blue). Image kindly provided by Dr. Odile Mercereau-Puijalon, Institute Pasteur, Paris, France. (B) A cross section of a cerebral capillary showing adherence of parasitized RBCs. This autopsy image was taken from a Malawian child who died of severe malaria. Image kindly provided by Dr. Terrie Taylor, Michigan State University, East Lansing, MI. (C) Distribution and morphology of knobs on the surface of a P. falciparum-infected red blood cell (RBC). An atomic force micrograph of a trophozoite-infected RBC obtained from a naturally parasitized Malian child with malaria. The presence of a trophozoite within the RBC is detected by the DNA-staining YOYO-1 dye (green). Image kindly provided by Dr. Takayuki Arie, Osaka Prefecture University, Osaka, Japan.

A

B COLOR PLATE 7 • (chapter 29) Architecture of PfEMP-1 molecules encoded by the var genes of a single parasite clone, 3D7. (A) The var gene repertoire of 3D7 is predicted to encode 59 PfEMP-1 proteins, of which 51 are shown (the number of each architectural type present in the genome is shown at left). PfEMP-1 is a family of modular proteins composed of a series of domains— Duffy binding-like (DBL), cysteine interdomain region (CIDR) domain, and C2—in various orders, followed by a transmembrane (TM) domain and a cytoplasmic acidic terminal segment (ATS). With the exception of VAR2CSA, PfEMP-1 proteins begin with an N-terminal DBL domain followed by a CIDR domain. The number and order of DBL, CIDR, and C2 domains then vary considerably toward the carboxyl terminus of the protein. Most (38 out of 51) of the PfEMP-1 proteins shown are short variants containing only two DBL and two CIDR domains. In addition to variation in domain architecture, PfEMP-1 proteins show sequence variation between domains of the same classification (e.g., DBL1a; PfEMP-1 proteins can also be classified into three groups by their upstream promoter genotype: upsA, upsB, and upsC). The upsA group is associated with the presence of N-terminal DBL1a1 domains, some of which mediate rosetting and have been associated with severe malaria in some studies. PfEMP-1 variants belonging to the upsB and upsC groups, on the other hand, bind CD36 as they contain the tandem groups DBL1a and CIDR1a. (B) The binding properties of individual DBL and CIDR domains. Note that CIDR1a domains bind CD36, but only when next to an N-terminal DBL1a domain. Likewise, DBL2b binds to ICAM-1 when located next to a C2 domain. While other host receptors are known to bind additional DBL and CIDR domains (see panel A), many receptors remain unidentified. On the other hand, some host receptors (e.g., gC1qR) are known to mediate cytoadherence of P. falciparum-infected red blood cells but the putative PfEMP-1 domains they bind have yet to be identified. Figure adapted from Kraemer et al., 2006.

A

B

C

D COLOR PLATE 8 • (chapter 34) Development of uropathogenic Escherichia coli (UPEC) intracellular bacterial communities (IBCs) in the mouse urinary tract infection (UTI) model. Bladders from mice infected transurethrally with UPEC-expressing green fluorescent protein (GFP) were stretched, fixed, and stained with a cell surface marker (wheat germ agglutinin, red) and Hoeschst (blue) to visualize cell nuclei, and optical sections were taken on a Zeiss inverted epifluorescence microscope. (A) Early IBC characterized by cytoplasmic, loosely packed, rod-shaped UPEC bacteria. (B) Mature middle IBC with densely packed coccoid UPEC bacteria in a biofilm-like structure. (C) Three-dimensional reconstruction from optical sections of a late IBC, with a UPEC filament fluxing out of the IBC. In this case, DNA was stained with the dye TO-PRO3 (red). (D) Mature middle IBC under attack by a polymorphonuclear lymphocyte that fails to access UPEC bacteria in the IBC. Images graciously provided by Dr. Sheryl S. Justice.

COLOR PLATE 9 • (chapter 36) Structural features of the trypanosome variant surface glycoprotein molecule. Adapted from Chattopadhyay, 2005, with permission.

COLOR PLATE 10 • (chapter 36) VSG switch variants temporally express a mosaic surface coat (top). When trypanosomes are genetically altered to stably express a mosaic coat (bottom), these organisms fail to stimulate a T-independent B-cell response and thus escape early B-cell recognition during infection. Adapted from Dubois et al., 2005, and Mansfield et al., 2002, with permission.

COLOR PLATE 11 • (chapter 43) Predictive signatures for other vaccines. (a) A single archetypal signature that predicts T- and B-cell immunity and protective immunity for all vaccines. (b) Signatures might be highly vaccine specific, with each vaccine having a unique predictive signature. (c) The most likely scenario is that there will be “metasignatures,” that predict the immunogenicity or protective capacity of clusters of vaccines that work through similar mechanisms. For example, vaccines A, C, W, and N may mediate protection by stimulating high frequencies of polyfunctional CD81 T cells, and these may share a common signature. In contrast, vaccines L, R, O, and S may mediate protection by stimulating T cells that migrated to the lung, and these may have a common signature. By clustering all such signatures related to T-cell-mediated mechanisms of protection, one could have a metasignature that predicts the immunogenicity or protective capacity of virtually all T-cell-based vaccines. Similarly, one could have a metasignature for B-cell-based vaccines (e.g., vaccines that protect via neutralizing antibodies would have a common signature, whereas those that protect via opsonization antibodies would have a separate signature). Meta signatures may also exist for vaccines that mediate protection via other mechanisms.

COLOR PLATE 12 • (chapter 43) The vaccine chip. The identifying signatures that predict immunogenicity and/or protective capacity of many vaccines would enable the development of a vaccine chip. This chip would consist of perhaps 200 to 1,000 genes, organized in clusters. Each cluster of genes would predict a particular facet of the innate or adaptive immune response (e.g., magnitude of CD81 T-cell effector cell response, frequency of polyfunctional T cells, frequency of Th1/ Th2/Th17 balance, high affinity antibody titers, etc.). This would permit the rapid evaluation of vaccinees for the strength, type, duration, and quality of protective immune responses stimulated by the vaccine. Thus, the vaccine chip could be used to predict immunogenicity and protective capacity of virtually any vaccine in the future.

COLOR PLATE 13 • (chapter 47) Ex-vivo expression of IFN-g by TCRb1 cells at the bite site 6 h after feeding Phlebotomus papatasi on ears of naïve and pre-exposed mice (magnification 3 200) (S. Kamhawi and D. L. Sacks, unpublished data).

COLOR PLATE 14 • (chapter 49) Examination of Ziehl Neelsen-stained sputum smear samples by light microscopy to detect acid-fast bacteria has been the cornerstone for the diagnosis of tuberculosis for more than 100 years. (Photograph courtesy of Professor Juanita Bezuidenhout, Department of Pathology, Tygerberg Academic Hospital, South Africa.) Courtesy of Prof. J. Bezuidenhout, Division of Anatomical Pathology, Tygerberg Academic Hospital.

COLOR PLATE 15 • (chapter 49) Microscopic appearance of multiple tuberculous granulomas in the lung with central caseous necrosis. (Photograph courtesy of Professor Juanita Bezuidenhout, Department of Pathology, Tygerberg Academic Hospital, South Africa.) Courtesy of Prof. J. Bezuidenhout, Division of Anatomical Pathology, Tygerberg Academic Hospital.

COLOR PLATE 16 • (chapter 50) Map of the shrinking distribution of malaria from 1900 to 2007. Changes between 1946 and 1965 reflect the results of the first global malaria eradication campaign. Areas of stable and unstable malaria transmission are shown for 2007. Provided by the Malaria Atlas Project (www.map.ox.ac.uk) (Guerra et al., 2008; Hay et al., 2004).

COLOR PLATE 17 • (chapter 50) Map depicting the global distribution, by country, of drugresistant Plasmodium falciparum malaria in 1979, 1984, and 2009, based on literature searches and other publicly available data compiled by the WorldWide Antimalarial Resistance Network (WWARN, www .wwarn.org). (CQ, chloroquine-resistant P. falciparum; SP, sulfadoxine-pyrimethamine-resistant P. falciparum; ART, artemisinin-resistant P. falciparum.)

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