Control of Innate and Adaptive Immune Responses During Infectious Diseases

Control of Innate and Adaptive Immune Responses during Infectious Diseases

Julio Aliberti Editor

Control of Innate and Adaptive Immune Responses during Infectious Diseases

Editor Julio Aliberti Associate Professor Divisions of Molecular Immunology and Pulmonary Medicine Cincinnati Children’s Hospital Medical Center and School of Medicine University of Cincinnati Cincinnati, OH, USA [email protected]

ISBN 978-1-4614-0483-5 e-ISBN 978-1-4614-0484-2 DOI 10.1007/978-1-4614-0484-2 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011936972 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Upon infection, pathogen and host perform a complex interaction that ultimately aims to achieve elimination of the invading microbe with the least amount of damage to host tissues and organs. Interestingly, both sides of this equation co-evolved several mechanisms that mediate pathogen recognition, initiation and expansion of immune responses, neutralization of toxic elements and elimination of replicating organisms and finally healing and remodeling of damaged tissues. On one side pathogens evolved mechanisms to evade recognition and killing, while on the other side, host express numerous (sometimes redundant) mechanisms of recognition and elimination of the pathogen. Nonetheless, it is clear that an absolute successful strategy on the pathogen side would be lethal to both host and pathogen. Therefore, several evasion mechanisms are seen among several microbes. The most successful ones are not necessarily the most abundantly found within the host, but those that can achieve transmission. On the other hand, hosts need a robust and extended immune response in order to expand memory cells. This critical balance is where the co-evolution between host and pathogens lies. This book covers several aspects of induction, control and evasion of host immune response during infectious diseases. Multiple aspects are covered and each chapter focuses on one prominent infectious agent. Cincinnati, OH

Julio Aliberti

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Contents

1

Resolution of Inflammation During Toxoplasma gondii Infection ........ Julio Aliberti

2

Mechanisms of Host Protection and Pathogen Evasion of Immune Response During Tuberculosis .............................. Andre Bafica and Julio Aliberti

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NKT Cell Activation During (Microbial) Infection ............................... Jochen Mattner

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Regulation of Innate Immunity During Trypanosoma cruzi Infection ....................................................... Fredy Roberto Salazar Gutierrez

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B Cell-Mediated Regulation of Immunity During Leishmania Infection ................................................................... Katherine N. Gibson-Corley, Christine A. Petersen, and Douglas E. Jones

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69

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Control of the Host Response to Histoplasma Capsulatum.................... George S. Deepe, Jr.

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Modulation of T-Cell Mediated Immunity by Cytomegalovirus .......... 121 Chris A. Benedict, Ramon Arens, Andrea Loewendorf, and Edith M. Janssen

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T Cell Responses During Human Immunodeficiency Virus (HIV)-1 Infection ............................................................................ 141 Claire A. Chougnet and Barbara L. Shacklett

Index ................................................................................................................. 171

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Contributors

Julio Aliberti, Ph.D. Associate Professor, Divisions of Molecular Immunology and Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center and School of Medicine, University of Cincinnati, Cincinnati, OH, USA [email protected] Ramon Arens Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Andre Bafica, M.D., Ph.D. Assistant Professor, Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianopolis, SC, Brazil andre.bafi[email protected] Chris A. Benedict Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA [email protected] Claire A. Chougnet Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation and Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA [email protected] George S. Deepe. Jr, M.D. Professor, Veterans Affairs Hospital, Cincinnati, OH, USA; Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH, USA [email protected] Katherine N. Gibson-Corley Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, USA Fredy Roberto Salazar Gutierrez, M.D., Ph.D. Assistant Professor, School of Medicine, Antonio Nariño University, Bogotá, Colombia [email protected]

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Contributors

Edith M. Janssen Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, University of Cincinnati College of Medicine, Cincinnati, OH, USA [email protected] Douglas E. Jones Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, USA [email protected] Andrea Loewendorf Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Jochen Mattner, M.D. Professor of Molecular Microbiology and Infection Immunology, University Hospital of Erlangen, Microbiology Institute – Clinical Microbiology, Immunology and Hygiene, Erlangen, Germany [email protected] Christine A. Petersen Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, USA Barbara L. Shacklett Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA, USA

Chapter 1

Resolution of Inflammation During Toxoplasma gondii Infection Julio Aliberti

Abstract Upon Toxoplasma gondii host infection, a powerful immune response takes place in order to contain dissemination of the parasite and prevent mortality. Once parasite proliferation is contained by IFN-J-dependent responses, nevertheless, parasite immune escape prevents complete clearance characterizing the onset of the chronic phase of infection, with a continuous (and powerful) cell-mediated immunity. Such potent responses are kept under tight control by several, non-redundant mechanisms that control pro-inflammatory mediators. Including cytokines, such as members of the IL-10 family, TGF-beta, the membrane receptors, ICOS, CTLA4 and a class of anti-inflammatory eicosanoids, the lipoxins. In this chapter we address the host strategies that keep pro-inflammatory responses under control during chronic disease. On the other hand, we approach the perspective of the pathogen, which pirates the host’s machinery to its own advantage as a part of the pathogen’s immuneescape mechanisms.

1.1

Introduction

Toxoplasmosis is caused by the protozoan parasite, Toxoplasma gondii. The pathogen can be found worldwide and is particularly prevalent in the United States, where it is estimated that more than 60 million people may be infected. Among those who are infected, few develop symptoms due to healthy immune system that usually prevents the parasite from causing illness. Nevertheless, within the high risk group are pregnant women and individuals with compromised immune systems.

J. Aliberti (*) Divisions of Molecular Immunology and Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center and School of Medicine, University of Cincinnati, Cincinnati, OH, USA e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_1, © Springer Science+Business Media, LLC 2012

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Ingestion of oocysts

predation

Feces Congenital transmission Ingestion of infected raw meat or water/food contaminated with oocysts

Fig. 1.1 Toxoplasma gondii life cycle. Cats become infected with T. gondii through predation of infected mice or rats. After cysts or oocysts are ingested the organisms are released and spread throughout the small intestine and then form oocysts, which are excreted and can potentially survive for long periods in the environment. Human acquire infection via in several routes: ingestion of infected food containing Toxoplasma cysts; ingestion of oocysts from contaminated hands or food; organ transplantation or blood transfusion from infected humans; transplacental transmission from an infected mother; and accidental inoculation of tachyzoites

Felines, including the house cat are definitive hosts in which it is observed the sexual stages of T. gondii and thus, are considered to be the main parasite reservoirs. Cats become infected with T. gondii by carnivorism (Fig. 1.1). After tissue cysts or oocysts are ingested, viable organisms are released and invade epithelial cells of the small intestine, where they undergo an asexual cycle followed by a sexual cycle and then form oocysts, which can be excreted. The unsporulated oocyst takes 1–5 days after excretion to sporulate (become infective). Although cats shed oocysts for only 1–2 weeks, large numbers may be shed. Oocysts can survive in the environment for several months and are remarkably resistant to disinfectants, freezing, and drying, but are killed by heating to 70°C for 10 min. The persistency of oocysts in the environment may enhance the infectious potential of the parasite. Humans may acquired T. gondii via different routes (Fig. 1.1): (a) Ingestion of: raw or undercooked and infected meat containing Toxoplasma cysts; oocysts from fecally contaminated hands or food; (b) Organ transplantation or blood transfusion from infected humans; (c) Transplacental transmission from an infected mother; and (d) Accidental inoculation of tachyzoites.

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Toxoplasma gondii, a protozoan apicomplexa parasite is highly virulent and can potentially invade and subsequently replicate within any nucleated host cell. Under natural conditions infection occurs by ingestion of parasite oocyst-contaminated food or water. Oocysts are complex structures formed in the digestive tract of the definitive host – felines which protect the parasites from heat and dehydration and can remain infective within the environment for long periods of time. Once ingested, oocyst rupture occurs within the host digestive system and the released parasites enter host cells through an active process mediated by the apical complex (Morisaki et al. 1995). Host cells include epithelial cells, resident macrophages and dendritic cells (Fig. 1.1, Life Cycle). Once intracellular, the parasites (tachyzoites) quickly replicate. Although definitive evidence is still required, it is proposed that circulating infected host cells (probably macrophages or DCs) might mediate spread of the parasite to several organs, including the liver. One current hypothesis proposes that the acute phase of infection resolves when the remaining fast-replicating parasites switch, probably as a response to immune attack, to a slow replicating form known as bradyzoites and seclude themselves in cysts in certain tissues, such as the central nervous system (CNS) and the retina (known as chronic or persistent infection) (Black and Boothroyd 2000). For a long time it was widely accepted that cysts containing bradyzoites were latent, biologically inactive structures that eventually died off or in some cases re-activated parasite replication. Today, however this concept has been challenged as it has been shown that cysts are dynamic structures, where parasites convert to tachyzoites. The conclusion is that this “dripping” effect in which tachyzoites are slowly released, continuously stimulating immune response. Therefore, when immune suppression caused by drugs or other infections, such as HIV, can lead to reversion from bradyzoites back to the fast replicating tachyzoites, which rupture cysts causing local tissue necrosis, thus characterizing the main pathology resulting from this infection. If reactivation occurs in the CNS, it is often lethal. During the early years of the AIDS epidemic, encephalitis due to reactivation of chronic T. gondii infection was one of the most relevant pathologies affecting immuno-depressed patients (Martinez et al. 1995). In nature, the main route for T. gondii transmission is through predation (i.e. felines preying on rodents), therefore an evolutionary advantage would be among pathogens that populate the host and simultaneously provide conditions to protect the host to carry as many parasites without killing it. In other words, this means to proliferate while promoting host survival. To achieve this, the parasite has evolved several mechanisms to induce a powerful immune response by the host, which prevents host death by controlling parasite growth. However, to avoid the potential collateral damage of such powerful pro-inflammatory reaction, the pathogen subverts the immune system allowing it to persist through the chronic phase of the disease, which can last for many years (Hay and Hutchison 1983). Herein, we discuss the immune response triggered by T. gondii and how hosts and pathogens make use of immune-regulatory pathways to promote host survival, which increases the probability of parasite transmission.

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1.2 1.2.1

J. Aliberti

Experimental T. gondii Infection Microbial Recognition and IL-12 Induction

A balanced interrelationship between host and parasite is highly dependent on the early induction of immune response after infection. Too much immune response and pathogen is swiftly cleared without causing disease. On the other hand, the absence of a proper timely host response may lead to uncontrolled pathogen replication and spread, often leading to the death of the host. Nevertheless, this is an over-simplification of the rather complex scenarios that take place during T. gondii infection. Although significant protection is achieved after infection, a relevant proportion of invading parasites evade immune effector mechanisms, i.e. tachyzoites turn infected cells incapable to secrete pro-inflammatory mediators (Walker et al. 2008), bradyzoites, hidden within tissue cysts populate immune privileged sites, such as the retina or the CNS. Therefore, T. gondii parasites can persist in the host even in the presence highly powerful immune response. To add further complexity to this interaction, several lines of evidence indicate that without innate immune responses, such as following NK-cell depletion, the initial IFN-J-dependent control of parasite replication is compromised and, in the case of NK-cell-depletion of T-cell-deficient mice, host resistance is lost resulting in host death, which indicates an important role for NK cells in the induction of a response (Sher et al. 1993; Hunter et al. 1994). IL-12 is a cytokine produced during pathogen recognition that is essential to trigger both NK cell as well as T cell-derived IFN-J production during T. gondii infection. The biological relevance of this cytokine was evidenced by the finding that IL-12-deficient animals are extremely susceptible to T. gondii infection (Gazzinelli et al. 1994). B cells, macrophages, neutrophils and DCs are known to produce IL-12 in vitro and in vivo (Denkers 2003). During T. gondii infection, macrophages, neutrophils and DCs can all produce detectable amounts of IL-12 after T. gondii infection (Denkers 2003). However, DCs – abundant producers of IL-12 in vivo – are the most relevant cell population for the development of a parasite-specific type 1 immune response. Reis e Sousa and colleagues observed that splenic mouse CD8D+ DCs produce IL-12 in response to T. gondii in the absence of co-stimulatory signals (Reis e Sousa et al. 1997). While macrophages require a cognate priming signal, i.e. IFN-J and neutrophil IL-12 production levels are relatively low when compared to DCs. In summary, DCs can either activate the immune system by recognition of parasitederived molecules or can harbor initial replication of the intracellular parasites. A cellular homogenate from culture-derived tachyzoites (STAg) was used in order to decipher which are the parasite components and their respective host receptors involved in DC IL-12 induction by T. gondii. Such approach seemed feasible since STAg was capable to induce markedly higher levels of IL-12 from in vitro stimulated splenic DCs than when the same cell populations are exposed to several other microbial products. Although the mechanisms underlying such responses are

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not completely understood, one potential interpretation came from studies showing that the chemokine receptor CCR5 plays an important role in the induction of IL-12 synthesis following stimulation with T. gondii STAg (Aliberti et al. 2000). The biological relevance of the unusual requirement for a chemokine receptor to participate in microbial recognition by DCs is supported by the fact that decreased IL-12 production is observed during acute infections in CCR5-deficient animals although defects in cell migration could also contribute to this susceptible phenotype. The phenotype of CCR5-deficient mice infected with T. gondii cannot be solely explained by the IL-12 production defects, other studies indicated that NK cells show defective migration patterns after oral infection, leading to a weaker initial NK-derived IFN-J secretion, resulting in susceptibility to infection (Khan et al. 2006). In summary, it seems clear that CCR5 plays several critical roles for the development of innate immunity after T. gondii infection. Both at the DC IL-12 induction level as well as inducing NK cell migration to infection foci. In the pursuit to identify the ligands with IL-12-inducing activity from T. gondii, a thorough analysis of this activity was performed in fractionated suspensions of secreted parasite proteins. Such analysis identified cyclophillin-18 (C-18) as one such component. T. gondii C-18 is a secreted prolyl-isomerase that can bind avidly to the immunosuppressant cyclosporine, which was therefore initially pursued as a potential therapeutic target for the treatment of toxoplasmosis (Aliberti et al. 2003b). C-18 was found to bind directly to human and mouse CCR5 with affinities comparable to its prototype ligand, the chemokine CCL4 (MIP-1E). Indeed, C-18 competed with the natural ligand CCL4 for binding to CCR5. It has been shown that endogenous CCR5 ligands can trigger IL-12 production. Nevertheless, the low levels of cytokine secretion observed under these conditions indicate that it may not have a critical influence on determining resistance to infection. Given that CCR5 is a coreceptor for HIV invasion, further studies showed that toxoplasma C-18 could inhibit the infection of monocytes by CCR5-tropic primary and laboratory HIV isolates (Golding et al. 2003, 2005). However, despite the evident stimulating activity of C-18 triggering IL-12 production by murine DCs, the resulting IL-12 levels observed after stimulation of DCs with C-18 are consistently lower than those seen after stimulation with whole parasite lysate or with a pool of tachyzoite-secreted proteins, indicating that pathways other than those initiated by CCR5/C-18 might also be important for IL-12 production by DCs (Aliberti et al. 2003b). Toll-like receptors (TLRs) have been investigated as a likely candidate for such cytokine induction. Mice deficient for the TLR adaptor protein MyD88 were found to have a pronounced defect in IL-12 production in response to STAg stimulation in vivo and in vitro. Moreover, upon T. gondii infection, MyD88-deficient hosts had high mortality due to a lack of protective IFN-J-mediated immunity (Scanga et al. 2002). Suggesting that the residual IL-12 produced in response to CCR5 was clearly not sufficient to provide any level of protection after infection. Obviating the predominant role of TLR’s in initiating innate IL-12 production in the presence of parasite derived molecules. TLR2 has been found to be involved in the development of resistance to infection with a large inoculum of T. gondii cysts (300 cysts/mouse) (Mun et al. 2003).

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Apparently, the defect seen in TLR2-deficient mice is related to inefficient activation of microbicidal functions, as a defect in nitric oxide production by macrophages was reported, whereas no defect in the production of IL-12 or any other pro-inflammatory cytokines, which are typical of innate microbial recognition, was seen (Mun et al. 2003). In an attempt to identify the TLR ligands present in STAg, a CCR5-independent IL-12-inducing activity was purified from parasite preparations, further analysis indicated the cytoplasmic protein profillin was the key molecule inducing CCR5independent, MyD88-sensitive DC IL-12 production in mice (Yarovinsky et al. 2005). In fact, a thorough evaluation of a panel of TLR-responsive elements in a cell reporter assay allowed for the identification of mouse TLR11 as the receptor of profillin (Yarovinsky et al. 2005). In the absence of TLR11, DC’s showed major reduction in IL-12 responses. Interestingly, no residual IL-12 was detected under these conditions, something seen previously with STAg-stimulated MyD88-deficient cells. Although the effects are dominant and TLR11-deficiency completely abolishes resistance to infection, some points remain unanswered, including the identity of the human TLR involved in T. gondii recognition – given that the human TLR11 homolog is a pseudogene. Moreover, profillin is not a secreted protein it is released only after tachyzoite rupture, suggesting that it may not necessarily be present at the initial stages where the killing mechanisms are still not active. Taken together, the recognition steps that lead to full IL-12 responses is a rather complex interaction that assures the host to produce vigorous DC-derived IL-12 when in the presence of parasite molecules. Such responses are essential for the development of protective adaptive immunity. The biochemical basis for the induction of the IL-12 genes has been studied extensively (for review see (Trinchieri 2003)). However, the transcription factors that are directly involved in IL-12 induction during in vivo infection with intracellular parasites, including T. gondii it is still not completely elucidated. IRF-8deficient mice cannot produce IL-12 during infection with T. gondii and fail to develop resistance to infection (Scharton-Kersten et al. 1997). This observation would directly implicate IRF-8, an interferon-inducible transcription factor that binds to interferon consensus sequences and promotes gene transcription, in the induction of IL-12 gene expression, but recent reports have pointed out that besides their IL-12-induction defect, IRF-8-deficient mice fail to develop the major DC subset involved in IL-12 production in response to STAg stimulation, the CD8D+ subset (Aliberti et al. 2003a; Tsujimura et al. 2003). Furthermore, the remaining DC subsets also had severe defects in response to microbial stimuli. Further studies are required to clarify whether the defect in IL-12 production in these mice precludes the DC developmental defect. NF-kB family members have been studied during T. gondii infection models, it is clear that NF-kB activation is a required step for the development of protective immune response to infection (Tato et al. 2006). However, the developmental abnormalities seen in animals genetically deficient of NF-kB make dendritic cell specific responses rather complex due to environmental and developmental deficiencies. It has been reported that p38 MAP kinases are required for macrophage IL-12 responses to STAg stimulation (Mason et al. 2004). On the other hand, JNK family

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of MAP kinases while have been associated with induction of IL-12 by some (Sukhumavasi et al. 2007), while other studies indicate that the play a negative, inhibitory role (Sukhumavasi et al. 2010). A more comprehensive analysis of this enzyme activity, targets and function remains to be reported. The unusual combination of receptors triggered by T. gondii molecules, i.e. CCR5 and TLR11 suggests that the transcriptional machinery might be unique, which could explain, at least in part, the unusually high IL-12 levels seen after exposure of DCs to STAg. An interesting paradox has been unveiled while studying cytokine responses in infected cells. In other words, both dendritic cells and macrophages failed to produce significant levels of IL-12 when exposed to live tachyzoites (Butcher et al. 2001). Such studies led to the hypothesis that dendritic cells may serve as a parasite shuttle during in vivo infection, providing protection while migrating throughout the host (Denkers and Butcher 2005). This set of reports support a scenario in which microbial recognition takes place through released proteins from live free-floating parasites or from infected cells. The mechanistic that leads to inhibition of infected cell responsiveness to microbial stimulation is still not clear, but it has been shown that intracellular parasites disrupt the cytoskeleton of the host cells, specially membrane proximal structures. It is possible that such disruption decouples the biochemical signaling apparatus that is associated with the recognition receptors. Another possibility is that infected cells produce an autocrine suppressive factor, although this possibility has not been fully investigated.

1.2.2

IFN-g, Th1 Cells and Microbicidal Activity

Once present in the infected host, IL-12 activates NK cells to produce IFN-J and drives the proliferation of type 1 CD4+ and CD8+ T cells, which produce even more IFN-J. IFN-J-producing cells is a central component to induce and maintain control of parasite proliferation and dissemination during both acute and chronic infection (Yap and Sher 1999). Several factors are driven by IFN-J activation that have been to shown to be involved in controlling intracellular parasite growth. Macrophages harboring intracellular parasites and activated with IFN-J can produce nitric oxide, which, in turn, is responsible for microbicidal/microbiostatic control of intracellular parasite growth. Intriguingly, even though IFN-J-induced microbicidal mechanisms are potent, the machinery is not 100% effective at eliminating parasites (Yap and Sher 1999). During the chronic phase, some parasites evade immunity and survive within the host for long periods, despite the continuing survey of the immune system in search of released parasites. Interestingly, among the genetic clusters upon which the strains of T. gondii are grouped, some present extremely high virulence, leading to rodent host death prior to opportunity for viable transmission. Such parasite strains have shown weaker innate immune activating properties. Suggesting that for the lack of some evolutionary pressure, those strains present little to no adaptation to mouse hosts.

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With the onset of the chronic phase parasites make use of two major mechanisms to evade immune responses: 1. Parasites become less susceptible to host microbicidal activity; and/or, 2. Parasites induce immunosuppressive factors that dampen immune effector activity, including the production of pro-inflammatory mediators. As an example of an evasion of immune response mechanism, it is well-known that a several microbes escape effector immunity through the actions of membrane receptors or cytoplasmic enzymes that inactivate or neutralize effector molecules, including a complement factors (Karp and Wills-Karp 2001), superoxide or nitric oxide. As an example, during T. gondii infection, complement receptors are activated inducing expression of peroxiredoxins (Son et al. 2001). It seems obvious to speculate that those evasion strategies may increase the frequency of persisting parasites within the host, despite ongoing potent immunity. As an alternative, modulatory or anti-inflammatory factors could be selectively enhanced by the pathogen leading to inhibition of the migration, proliferation or differentiation of effector cells at the infection foci, favoring the evasion of the pathogen from protective immunity and progression towards the development of chronic disease.

1.3

1.3.1

Pro-resolution Strategies as a Mechanism to Prevent Immunopathology Resolution Phase of the Inflammatory Response

In general, protective pro-inflammatory response ultimately clears the tissues of both the cause and consequences of tissue injury that can accompany host defense (Cotran and Pober 1990). If unresolved, acute inflammation may lead to chronic inflammation, scarring and eventual loss of function (Majno and Joris 1995). A growing list of reports indicated that, in addition to classic diseases associated with inflammation, for example psoriasis, periodontal disease and arthritis, uncontrolled inflammation governs the pathogenesis of many widely prevalent diseases including infectious, cardiovascular and cerebrovascular disease, cancer, obesity and Alzheimer’s disease (Libby 2002; Calder 2006) (Van Dyke and Serhan 2003). Prostaglandins and leukotrienes are initially produced locally at the inflammation site and are key in promoting the cardinal signs of inflammation. Of interest, another class of arachidonic acid-derived mediators, the lipoxins (LXs) and aspirin-triggered lipoxins (ATLs), are mediators recently recognized to perform both endogenous anti-inflammatory and pro-resolving actions (Serhan 2005, 2007). In the recent years, multiple previously unknown enzymatic pathways were identified to be present during the resolution phase. Those are derived from the precursors EPA and DHA. Both EPA and DHA are major n-3 fatty acids also widely known as the omega-3 PUFA or fish oils. The new mediators are biosynthesized during the

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evolution of locally contained inflammatory exudates. They possess potent actions in controlling the resolution (Serhan et al. 2000, 2002; Hong et al. 2003). Resolvins are endogenous, local-acting mediators that carry potent anti-inflammatory and immunoregulatory signals (Serhan et al. 2002). These include novel actions that are targeted to promote resolution, namely reducing neutrophil infiltration and regulating the cytokine-chemokine axis and reactive oxygen species and stimulating the uptake and clearance of apoptotic PMN as well as lowering the magnitude of the inflammatory response and associated pain (Serhan et al. 2000, 2002; Svensson et al. 2007). Protectins are biosynthesized in many organs and perform potent anti-inflammatory (Hong et al. 2003) as well as protective actions demonstrated for the novel and potent DHA-derived 10,17-docasatriene in animal models of stroke (Marcheselli et al. 2003) and Alzheimer’s disease (Lukiw et al. 2005). Both families, the resolvins and protectins, are potent local-acting agonists of endogenous anti-inflammation and promote resolution specific processes (Serhan 2007). IFN-J-dependent immune and its derived pro-inflammatory responses are potentially extremely toxic to the host. As an example, during chronic inflammatory diseases such as arthritis or Crohns’ disease, sustained or uncontrolled type 1 cytokine responses has been shown to cause serious damage to host tissues and organs. In order to prevent that potential damage, several host mediators and receptors have evolved to counter host-damaging responses. The homeostasis of the immune response is absolutely dependent on the presence of such counter regulatory pathways. This complex network of anti-inflammatory pathways, given its actions, has been seized by pathogens and used to their own benefit to prevent parasite eradication.

1.3.2

Interleukin-10

IL-10 is one of the most biologically active cytokine with anti-inflammatory properties besides TGF-E and IL-35. It can be produced by a growing list of activated immune cells, in particular monocytes/macrophages and T cell subsets including Tr1, Treg, and Th1 cells. It acts via activation of a transmembrane receptor complex, which is composed of IL-10R1 and IL-10R2, and controls the activities of several immune cells. In monocytes/macrophages, IL-10 inhibits the production of proinflammatory mediators and antigen processing and presentation. Furthermore, IL-10 plays a relevant role in the differentiation/proliferation of B and T cells. In general, its physiological relevance lies in the preventing over-whelming immune responses and, consequently, of tissue damage. Simultaneously, IL-10 enhances the “scavenger”-like activities. To highlight its relevance in inhibiting immune responses during infectious diseases, IL-10 is used by pathogens to evade the development of protective immunity. Viruses, such as EBV, encode a viral IL-10-homologue that can initiate the signaling cascade as the one triggered by the mammalian cytokine (Salek-Ardakani et al. 2002). Poxviruses carry genes that encode IL-10 receptor homologues; therefore, cells expressing such receptors when in the presence of IL-10 become refractory to

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IL-12 NK

tachyzoite

IFN-

DC 4

C-18

8

NO M

IFN-

TNF

Fig. 1.2 Induction of pro-inflammatory responses during T. gondii infection. Immediately after infection, host dendritic cells (DCs) produce IL-12 in response to products secreted by T. gondii, via the CCR5-binding cyclophillin-18 and the TLR11-activating profillin. IL-12 promotes the differentiation and proliferation of type 1, IFN-J-producing T cells (CD4+ and CD8+) and NK cells. In turn, IFN-J triggers host cells, including macrophages (MM) to exert microbicidal activity, such as the production of nitric oxide, expression IDO and tryptophan depletion or autophagy

pro-inflammatory signals (Haig 1998). Moreover, IL-10 gene transfer has been shown to have anti-inflammatory actions in various pathologies associated with increased IFN-J, IL-1 or TNF production (van de Loo and van den Berg 2002, Wille et al. 2001). Importantly, neutralization of IL-10 during chronic toxoplasmic encephalitis leads to increased leukocyte infiltration in the CNS, indicating a role for this cytokine in controlling CNS inflammation (Deckert-Schluter et al. 1997). IL-10-deficient mice show uncontrolled hyper-inflammatory reaction and fail to transition to chronic disease succumbing to T. gondii infection in the earlier to mid-acute phase. The animals show severe infiltration of leukocytes and hepatic necrotic lesions in the liver as well as focal necrosis in small intestines, concomitant to high levels of IFN-J and TNF production (Suzuki et al. 2000, Gazzinelli et al. 1996) (Fig. 1.2). IL-10 was hypothesized to mediate immune evasion during T. gondii infection, given its immune modulatory activities. Conversely, IL-10 has not been found to be directly related to the elements that potentially contribute to the mechanisms of T. gondii virulence. Furthermore, the over-production of IL-10 has been shown to perform no clear role in the pathways involved in driving persistence of T. gondii in the chronic stage (Wille et al. 2001). In summary, it is clear that, besides IL-10, several other mechanisms of immune modulation/evasion used by the parasite and by the host are relevant to allow mutual survival (host and pathogen) for enough time to permit successful transmission and, ultimately for parasite survival as a species (Fig. 1.3).

1.3.3

TGF-b

TGF-E, alongside with IL-10, is one of the most relevant cytokines that control effector function of the immune system. It is one of the main soluble factors released by regulatory T cells and plays an essential role in providing a suppressive environment that is essential for homeostasis of the mucosal surfaces, including the gut.

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11

M

IL-10

DC C-18

IL-12 4 8

IFN-

NO M TNF

Fig. 1.3 Central role of IL-10 in controlling pro-inflammatory responses during acute phase of T. gondii infection. The potential cytotoxic effects of such activity are controlled, during the midto-late acute phase, by IL-10 production at sites of high-level parasite replication, such as the liver and spleen. IL-10 down-regulates pro-inflammatory cytokine and chemokine expression, as well as the microbicidal activities of DCs, T cells, NK cells and macrophages

During the interaction with macrophages, T. gondii tachyzoites expose phosphatidyl serine leading to the release of active TGF-E by infected macrophages (Seabra et al. 2004). TGF-E is a well-known macrophage deactivator, including inhibition of NOS2 expression. Neutralization of TGF-E abolished the inhibition of NO production, thus reducing the persistence of intracellular T. gondii in activated macrophages. Furthermore, the up-regulation of Smad 2 and 3 in infected macrophages confirms that a TGF-E autocrine effect was caused by the T. gondii infection. It is clear that TGF-E is present during host/parasite interaction and that affects key aspects of host immune protective responses. The peroral route T. gondii infection – an experimental model for the mucosal host/pathogen interation that is characterized by ileitis mediated both by parasitemediated tissue destruction as well as by the host immune responses. Buzoni-Gatel et al. 2001 showed that intraepithelial lymphocytes present in the gut mucosa are the main producers of TGF-E (Butcher et al. 2001). CD8+ T cells differentiate into TGF-E-producing cells. The presence of this cytokine inhibits CD4+ T lymphocyte infiltration, macrophage activation and tissue destruction mediated by excessive IFN-J production (Mennechet et al. 2004). Consequently, the hallmarks of TGF-E exposure are indeed present in lamina propria-resident CD4+ T cells, the up-regulation of Smad2 and Smad3 (Fig. 1.4).

1.3.4

IL-22

IL-22 is a member of the IL-10 cytokine family and signals through a heterodimeric receptor composed of the common IL-10R2 subunit and the IL-22R subunit. IL-10 and IL-22 both activate the STAT3 signaling pathway. Unlike IL-10, which is produced by a variety of cell types, IL-22 is made by only a subset of activated immune cells, preferentially by Th17 T cells, but also by Th1 cells and conventional

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b

a

c IL-22

NO

M

8

TGF-

Gut Lumen

Th17

Gut Lumen

TGF4

Fig. 1.4 The role of TGF-E and IL-22 in controlling gut mucosal immune responses after oral infection with T. gondii. (a) During the interaction with macrophages, T. gondii tachyzoites expose phosphatidyl serine leading to the release of active TGF-E by infected macrophages. (b) Gut mucosa intraepithelial lymphocytes produce TGF-E that inhibits CD4+ T lymphocyte infiltration, macrophage activation and tissue destruction mediated by excessive IFN-J production. (c) IL-22 is made preferentially by Th17 T cells. The receptor for IL-22 is distinct and consists of IL-22R, which is expressed by epithelial cells and the ubiquitous IL-10R2. IL-22 mediates protection against mucosal damage during oral infection with T. gondii

NK cells, JG CD3+ T cells, noncytolytic NK cells, lymphoid tissue inducer cells, and skin-homing IL-13+ T cells. The receptor for IL-22 is distinct and consists of IL-22R, which most studies indicate is restricted to the surfaces of epithelial cells, keratinocytes, and some fibroblasts, and IL-10R2, which is ubiquitous. In fact, T. gondii oral infection triggers IL-22-mediated protection of the gut mucosal surfaces. Suggesting an additional mediator that provides an immune suppressive environment to control gut inflammation (Wilson et al. 2010).

1.3.5

IL-27

IL-27 is a heterodimeric cytokine composed of Epstein-Barr virus–induced gene 3 (EBI3) and p28. It is known to signal through a receptor complex composed of the IL-27 receptor and gp130. Expression of IL-27R is confined to immune cells, its partner gp130, a shared receptor component of several cytokines including IL-6, is widely expressed both in and out of the immune system. While IL-27 was initially known to promote T cell proliferation and the development of TH1 responses; it was subsequently indicated that it could suppress TH1 and TH2 responses during various parasitic infections. In agreement with this observation, IL-27R-deficient mice develop exaggerated T helper cell responses during the acute stages of toxoplasmosis, Chagas disease and leishmaniasis and after helminth challenge. Moreover, it was found that IL-27R-deficient mice develop exuberant CD4 + T cell responses in the CNS during chronic toxoplasmosis, in which the majority of the cells are expressing the pro-inflammatory cytokine IL-17 (Stumhofer et al. 2006). In fact, IL-27 could inhibit in vitro differentiation of naïve T cells into the Th17 subset. Thus, unveiling a new mechanism to control inflammation in a very sensitive environment, the central nervous system (Fig. 1.5).

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4

IL-27 4

IL-6 TGFTh17

8 Fig. 1.5 IL-27 and suppression of immune pathogenic cells in the central nervous system during chronic toxoplasmosis. Naive T cells suppressed the development Th17 cells mediated by IL-6 + TGF-E after exposure to IL-27

1.3.6

Lipoxin A4

“DC paralysis” was a phenomenon in which protection against T. gondii infection was conferred to IL-10-deficient mice by injection with STAg 24 h before T. gondii challenge, which downregulated IL-12 production by DCs (Reis e Sousa et al. 1999). The injection of STAg was found to trigger the endogenous release of an eicosanoid known as lipoxin A4 (LXA4). This mediator inhibited STAg-induced DC migration and IL-12 production in vivo and in vitro (Aliberti et al. 2002a). Lipoxins have been show to have potent anti-inflammatory properties in several disease models (Samuelsson 1991; Goh et al. 2003; Van Dyke and Serhan 2003; Kieran et al. 2004). Their actions include inhibition of leukotriene function, NK-cell function, leukocyte migration, TNF-induced chemokine production, NF-NB translocation, and chemokine receptor and adhesion molecule expression (Ramstedt et al. 1985; Clish et al. 1999; Hachicha et al. 1999; Bandeira-Melo et al. 2000; Ohira et al. 2004). Lipoxins are known to bind to two main receptors — a seven-transmembrane G-protein coupled receptor, ALX/FPRL-1 (Maddox et al. 1997), and a nuclear receptor, AhR (Schaldach et al. 1999). Evidence indicating that ALX is mediating some, if not all, of the anti-inflammatory actions of lipoxins in vivo came from observations reporting that mice over-expressing human ALX have shorter and less severe inflammatory responses (Devchand et al. 2003). Despite intense investigation, it is not yet clear which of the two receptors are most important for the triggering of lipoxin-derived anti-inflammatory responses. There is evidence of a role for suppressors of cytokine signaling (SOCS) molecules in the induction of the antiinflammatory effects seen after lipoxin exposure (Leonard et al. 2002). The SOCSfamily proteins, SOCS-1, -2 and -3, are thought to mediate their actions by binding to the intracellular domains of cytokine or hormone receptors, thereby blocking activation of downstream signaling pathways (Alexander and Hilton 2004). On the other hand, these proteins may act as part of a ubiquitin ligase molecular complex that lead to proteasome-dependent degradation of transcription factors via their poly-ubiquitinylation (Kile et al. 2002; Alexander and Hilton 2004). The molecular basis for lipoxin-induced SOCS expression and the control of pro-inflammatory responses is poorly understood (Fig. 1.6).

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M LXA4

DC C-18

IL-12

4

8

TNF M

IFN-

NO

Fig. 1.6 Lipoxins and control of pro-inflammatory cytokine production during chronic toxoplasmosis. With the onset of chronic disease, LXA4 is produced and controls pro-inflammatory cytokine responses, mostly at sites where parasite replication might be occurring, such as the CNS, without interfering with the microbicidal activity of macrophages

The biosynthetic cascades for the generation of lipoxin involve several complex trans-cellular pathways and therefore, it is unlikely to be only one cellular source for this mediator. Nevertheless, production of LXA4 seen upon STAg stimulation is completely dependent of 5-lipoxygenase, indicating that the biosynthetic pathways involving this enzyme were crucial in this experimental setting for the production of LXA4 (Aliberti et al. 2002b). 5-lipoxygenase is produced as a pro-peptide that is activated by cleavage, however, low levels of active 5-lipoxygenase are found in different cell types, including macrophages, platelets, DCs and neutrophils (Funk et al. 2002). The expression of a 5-lipoxygenase-activating protein (FLAP) seems to be the key signal for induction of 5-lipoxygenase activity. Although, at the moment, it is not completely clear which cells are the source of lipoxygenase activity in vivo during T. gondii infection, it is evident that 5-lipoxygenase is required for biosynthesis of LXA4. During T. gondii infection, serum levels of LXA4 were found to steadily increase over most of the acute phase, and plateau at high levels through chronic disease (Aliberti et al. 2002b). 5-lipoxygenase-deficient animals succumbed to T. gondii infection at the early onset of chronic disease. Immune responses against the parasite were found to be increased in the absence of 5-lipoxygenase, with significantly less brain cyst formation than in control animals. By contrast, excessive pro-inflammatory cytokine secretion and massive brain inflammatory cell infiltration was found. The excessive pro-inflammatory response in the brain ultimately caused the death of the 5-lipoxygenase-deficient hosts (Aliberti et al. 2002b).

1.3.7

Redundancy and Control of Inflammation

IL-10, IL-27, TGF-E and lipoxins share several biological functions in terms of controlling inflammation. Although it is tempting to suggest that they might play redundant roles, in vitro and in vivo evidence suggest otherwise. For example, the treatment of T. gondii-infected 5-lipoxygenase-deficient mice with IL-10 was

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able to control some of the inflammation, but concomitant reactivation of parasite proliferation resulted in failure to rescue animals from mortality (Aliberti et al. 2002b). In fact, it was also observed that IL-10 but not LXA4, effectively inhibited the microbicidal activity of macrophages (Aliberti et al. 2002b). Another interesting apparent discrepancy between IL-10 and LXA4 biological actions was associated with the pathological findings seen during T. gondii infections of IL-10- versus 5-lipoxygenase-deficient mice. While the former showed generalized lymphocytic infiltration and massive hepatic necrosis after infection, with little to no inflammation in the brain; both liver and CNS infiltration was observed during infection of 5-lipoxygenase-deficient mice, indicating that these two anti-inflammatory mediators are released in a time and organ controlled fashion and play differentiated intracellular inhibitory pathways. The biochemical pathways involved in inhibition of pro-inflammatory responses during infection also support the concept that regulatory mechanisms follow distinct intracellular targets. On one hand, while lipoxins trigger expression of the regulatory protein SOCS2, IL-10 mediate their inhibition via up-regulation of SOCS1/ SOCS3 and IL-27 do so via up-regulation of STAT3 (Machado et al. 2006). TGF-E, on the other hand, seems to mediate its functions via Smad proteins, including Smad2 and Smad3. It is likely that the particular aspects associated with each regulatory mediator, i.e. induction of tolerance, apoptosis, suppression of chemotaxis, can be traced back to their respective biochemical signature.

1.3.8

Induction of Endogenous LXA4 as an Pathogen Evasion Pathway

Several lines of evidence show that the anti-inflammatory actions of lipid mediators are used by pathogens, including fungi and helminths. The modulation of host immune responses is the desired effect. The 5-lipoxygenase activity after T. gondii infection was known to be associated with splenic macrophages (Aliberti et al. 2002b), the 15-lipoxygenase-expressing cell population was not known. In order to identify the cell populations involved in mediating 15-lipoxygenase activity after T. gondii infection, Bannenberg and colleagues isolated an enzymatic activity in tachyzoites exposed to calcium ionophore in the presence of arachidonic acid in vitro (Bannenberg et al. 2004). Moreover, proteomics analysis of tachyzoitederived lysates revealed the presence of peptides homologous to plant-derived type 1 lipoxygenases (Bannenberg et al. 2004). Therefore, it seems that the induction of lipoxin biosynthesis by T. gondii has been selected through the carrying of a plant-like lipoxygenase gene, which together with the actions of host-derived 5-lipoxygenase results in lipoxin production. High levels of lipoxin, subsequently, suppress immune responses providing hosts the ability to control parasite proliferation without suffering the damaging consequences of exuberant inflammation or tissue necrosis. The molecular basis for 5-lipoxygenase induction after parasite stimulation has not been clarified, it is known that this enzyme can be induced after leukocyte exposure to a

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General pathways for LXA4and Resolvins biosynthesis DHA AA

Pathogen-dependent LXA4 biosynthetic pathway

17S -Resolvins

HOOC COOH

COOH

(O)OH

5-LO

AA

OH

COOH

HO OH

5-LO COOH

COOH

12-LO

OOH OOH

COOH

O(O)H

15-LO

OH OH HO

COOH

LXA4

OOH

15-LO OOH

COOH

O(O)H

LXA4 OH OH

COOH

HO

Fig. 1.7 General and pathogen-dependent LXA4 biosynthetic pathways. (a) General pathways for LXA4 biosynthesis. Arachidonic acid (AA), which is released in response to inflammatory stimuli, is catalysed by 5-lipoxygenase (LO) to generate LTA4. This compound, secreted by leukocytes, is captured by neighboring platelets or endothelial cells, and, through the actions of 12- or 15-LO, respectively, is converted to LXA4. (b) Pathogen-dependent LXA4 biosynthetic pathway. After the generation of LTA4 in a 5-LO-dependent manner, it is catalyzed by pathogen-secreted 15-LO into LXA4, which is then secreted by the infected cell

variety of stimuli, including PGE2 (Levy et al. 2001). The interplay between these mediators, the induction of 5-lipoxygenase and the control of immune responses in vivo await further investigation. Another interesting observation that supports the argument for a role of T. gondii 15-lipoxygenase in immune evasion is the presence of such enzymatic activity in an organism that does not have lipids that could serve as substrates for lipoxygenases. Therefore, the substrate has to come from infected host cells. Pseudomonas aeruginosa 15-lipoxygenase-like enzyme is another example of a pathogen carrying an enzyme whose substrate is only present in host cells (Vance et al. 2004). P. aeruginosa is most commonly associated with chronic lung infections in patients with cystic fibrosis. It is possible that the bacteria may use the 15-lipoxygenase pathway leading to lipoxin biosynthesis to promote suppression of inflammation and persist throughout chronic disease. However, patients with cystic fibrosis fail to generate lipoxins in the lungs and the continuing proliferation of bacteria results in uncontrolled accumulation of activated neutrophils that ultimately lead to serious tissue damage with organ failure (Karp et al. 2004). This constitutes the major pathology for the lung form of cystic fibrosis. The relevance of pathogenderived 15-lipoxygenase given the lack of lipoxin generation in the lungs of patients with cystic fibrosis and the severity of disease still remains to be elucidated (Fig. 1.7). Mycobacterium tuberculosis is another example of a lung-invading bacterium that leads to a chronic disease, human tuberculosis is among the top infectious diseases worldwide with enormous public health relevance. M. tuberculosis infection is usually asymptomatic, granulomatous reaction in the lungs contain the bacilli and

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prevents its spreading, and a readily-detectable cell-mediated immunity is usually found in exposed patients (Chan and Flynn 2004). However, mycobacterial growth increases and transmission of viable bacilli occurs, along with granuloma disruption and organ function is compromised whenever the immunological status of the hosts is suppressed (Flynn and Chan 2003). Initial pulmonary colonization by M. tuberculosis is a latent process with very little reaction occurring in the organ. With the pathogen slowly establishing into the organ and with almost no intervention from the host innate immune system (Flynn and Chan 2003). Interestingly, it has been shown that in the absence of endogenously generated LXA4, mice become more resistant to infection, with longer survival rates, lower bacterial counts and higher type 1 cellmediated immunity against the bacilli (Bafica et al. 2005). The mechanisms involved in control of the immune response during tuberculosis is approached in detail in another chapter of this book. When comparing the outcomes of infections by T. gondii versus M. tuberculosis in 5-lipoxygenase-deficient animals a clear discrepancy becomes evident. This indicates a protective versus a host detrimental role for endogenously produced lipoxins, respectively. While T. gondii – a fast-replicating pathogen – that depends on keeping the host alive so that transmission can occur through predation, M. tuberculosis – slow growing, silent pathogen – requires high proliferation rates in lungs of infected hosts for transmission to occur. Nevertheless, both cases indicate that lipoxindependent inhibition of pro-inflammatory type 1 responses provides a favorable environment for pathogen transmission. Therefore, both the host and the pathogen rely on driving a well-balanced immune response.

1.4

Conclusions

In summary, mechanisms that contain the breadth, intensity and duration of proinflammatory functions of the immune system play apparent redundant roles. Nevertheless, animal knockout models indicate otherwise. A large body of evidence clearly establishes that TGF-E is a key player in modulating immune specific responses in the gut mucosal surfaces during oral infection with T. gondii. Another organ highly sensitive to the presence of inflammatory reactions is the brain, targeted by the parasite during chronic infection. In this case, evidence point to the protective effects of IL-27 by inhibiting the differentiation of pathogenic Th17 cells in the nervous tissue. While these mediators seem to play a localized role, several reports indicate that IL-10 plays a systemic role, with animals lacking IL-10 or its receptor showing widespread leukocyte infiltration, liver necrosis and mortality after T. gondii infection. Although the presence of lipoxins in the serum is preceded by IL-10, there is no evidence that the two mediators cross-regulate each other. Moreover, their anti-inflammatory actions are overlapping but not redundant. The emerging body of evidence including lipoxins as immune-regulatory mediators, and the potential use of their inhibitory effects for pathogen survival and replication, is still a poorly understood area of research. Relevant questions including the nature of

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the pathogen-derived signal (s) that induces lipoxin production, or whether the anti-inflammatory actions of lipoxins play a role in modulating the balance between Th1, Th2, Th17 and regulatory T cell responses await to be answered. And, in addition, the use of the lipoxygenase system and its mediators by pathogenic microbes as a general mechanism for evasion and manipulation of immune responses as well as pathogen persistence. The further investigation on the roles played by all of these mediators, their cellular sources, biochemical and cellular targets may provide the basis the development of novel therapeutic intervention strategies to enhance weak or inhibit undesirable pro-inflammatory immune responses in vivo. Acknowledgements Julio Aliberti is funded by grants from NIH (AI075038 and AI078969).

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Serhan, C. N. (2007). “Resolution phase of inflammation: novel endogenous anti-inflammatory and proresolving lipid mediators and pathways.” Annu Rev Immunol 25: 101–37. Serhan, C. N., C. B. Clish, et al. (2000). “Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.” J Exp Med 192(8): 1197–204. Serhan, C. N., S. Hong, et al. (2002). “Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals.” J Exp Med 196(8): 1025–37. Sher, A., I. P. Oswald, et al. (1993). “Toxoplasma gondii induces a T-independent IFN-gamma response in natural killer cells that requires both adherent accessory cells and tumor necrosis factor-alpha.” J Immunol 150(9): 3982–9. Son, E. S., K. J. Song, et al. (2001). “Molecular cloning and characterization of peroxiredoxin from Toxoplasma gondii.” Korean J Parasitol 39(2): 133–41. Stumhofer, J. S., A. Laurence, et al. (2006). “Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system.” Nat Immunol 7(9): 937–45. Sukhumavasi, W., C. E. Egan, et al. (2007). “Mouse neutrophils require JNK2 MAPK for Toxoplasma gondii-induced IL-12p40 and CCL2/MCP-1 release.” J Immunol 179(6): 3570–7. Sukhumavasi, W., A. L. Warren, et al. (2010) “Absence of mitogen-activated protein kinase family member c-Jun N-terminal kinase-2 enhances resistance to Toxoplasma gondii.” Exp Parasitol 126(3): 415–20 Suzuki, Y., A. Sher, et al. (2000). “IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii.” J Immunol 164(10): 5375–82. Svensson, C. I., M. Zattoni, et al. (2007). “Lipoxins and aspirin-triggered lipoxin inhibit inflammatory pain processing.” J Exp Med 204(2): 245–52. Tato, C. M., N. Mason, et al. (2006). “Opposing roles of NF-kappaB family members in the regulation of NK cell proliferation and production of IFN-gamma.” Int Immunol 18(4): 505–13. Trinchieri, G. (2003). “Interleukin-12 and the regulation of innate resistance and adaptive immunity.” Nat Rev Immunol 3(2): 133–46. Tsujimura, H., T. Tamura, et al. (2003). “ICSBP/IRF-8 retrovirus transduction rescues dendritic cell development in vitro.” Blood 101(3): 961–9. van de Loo, F. A. and W. B. van den Berg (2002). “Gene therapy for rheumatoid arthritis. Lessons from animal models, including studies on interleukin-4, interleukin-10, and interleukin-1 receptor antagonist as potential disease modulators.” Rheum Dis Clin North Am 28(1): 127–49. Van Dyke, T. E. and C. N. Serhan (2003). “Resolution of inflammation: a new paradigm for the pathogenesis of periodontal diseases.” J Dent Res 82(2): 82–90. Vance, R. E., S. Hong, et al. (2004). “The opportunistic pathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase.” Proc Natl Acad Sci USA 101(7): 2135–9. Walker, M. E., E. E. Hjort, et al. (2008). “Toxoplasma gondii actively remodels the microtubule network in host cells.” Microbes Infect 10(14-15): 1440–9. Wille, U., E. N. Villegas, et al. (2001). “Interleukin-10 does not contribute to the pathogenesis of a virulent strain of Toxoplasma gondii.” Parasite Immunol 23(6): 291–6. Wilson, M. S., C. G. Feng, et al. (2010) “Redundant and pathogenic roles for IL-22 in mycobacterial, protozoan, and helminth infections.” J Immunol 184(8): 4378–90. Yap, G. S. and A. Sher (1999). “Cell-mediated immunity to Toxoplasma gondii: initiation, regulation and effector function.” Immunobiology 201(2): 240–7. Yarovinsky, F., D. Zhang, et al. (2005). “TLR11 activation of dendritic cells by a protozoan profilinlike protein.” Science 308(5728): 1626–9.

Chapter 2

Mechanisms of Host Protection and Pathogen Evasion of Immune Response During Tuberculosis Andre Bafica and Julio Aliberti

Abstract An integrated response of the host is essential in health and disease. Upon microbial exposure, infected hosts strictly regulate immune responses to both contain pathogen dissemination and modulate immunopathology-associated effects, thus preventing mortality. In addition to a variety of molecules, such potent responses are kept under tight control by a class of anti-inflammatory eicosanoids, the lipoxins. Lipoxins are induced following exposure to several infectious agents and can function as immuno-modulatory molecules. A number of observations made in animal models of infection and human studies indicate that such lipid mediators play a critical role in controlling early as well as chronic immune responses. This chapter summarizes the role of cytokines and lipoxins in regulating innate immune responses to a major human pathogen, Mycobaterium tuberculosis.

2.1

Introduction

Despite more than 100 years of research, tuberculosis is still the most important bacterial infection worldwide. The discovery of Mycobacterium tuberculosis as the causative agent of TB was announced by Robert Koch in 1882 (Koch 1891). In his lecture Koch, who received the 1905 Nobel Prize for his discoveries, reminded his audience that one in seven human beings died of tuberculosis. Every year, it is estimated that tuberculosis causes around 1.5 million mortalities with 8 million new cases reported (Barber et al. 2009). In general, most infections are controlled by the host’s immune system, leading to latency with persistent/dormant bacteria. The World Health Organization estimates that one-third of the world population

A. Bafica (*) Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianopolis, SC, Brazil e-mail: andre.bafi[email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_2, © Springer Science+Business Media, LLC 2012

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carries the bacilli however only 10% of them will develop clinical disease. Acquired immunodeficiency syndrome (AIDS) and other immune-compromising conditions greatly increases the risk of developing active tuberculosis, supporting the observation that protective immunity suppress M. tuberculosis infection. The only vaccine against tuberculosis, M. bovis Bacillus Calmette Guerin (BCG), has proven to be of low efficacy against the most frequent outcome of tuberculosis, lung infection in adults (Fine 1995). Furthermore, with the current treatment requiring up to three drugs and a high degree of patient compliance, the number of multi drug resistant (MDR) isolates is on the rise in many areas of the world. The urgency to develop more effective vaccines or immunotherapies requires potent immune activating strategies at the interface of innate and adaptive immunity.

2.2 2.2.1

Infection and Innate Immunity Neutrophils

In the presence of pro-inflammatory stimulus, neutrophils are among the first innate immune cells to migrate from the blood to the foci. Neutrophils are professional phagocytes and express a range of receptors that can recognize opsonized and nonopsonized microbes, which are rapidly killed upon fusion of the phagosome with lysosomal compartments and specialized cytoplasmic granules that contain a vast arsenal of antimicrobial effector molecules including D-defensins, proteases as well as iron and siderophore-binding molecules, i.e. lactoferrin and lipocalin, respectively (Segal 2005; Appelberg 2007). In addition to direct microbicidal activity, neutrophils drive cell migration via production of chemokines as well as produce pro-inflammatory cytokines in response to microbial pattern recognition receptor stimulation. Neutrophillic infiltrates had been reported during acute pulmonary tuberculosis both in clinical studies and experimental infections (non-aerosol infection models) (Appelberg 1992; Condos et al. 1998; Schluger and Rom 1998; Lasco et al. 2004). However, the role of neutrophils during tuberculosis is still controversial. The lack of a selective neutrophil deficient model has stifled the studies in this area. However, investigators have used antibody-mediated depletion models to question the role of neutrophils during tuberculosis. Pedrosa and colleagues studied BALB/c infected with a high intravenous dose of M. tuberculosis Erdmann and the effect of early (days 0, 2 and 4 post infection) and late (days 16, 18 and 20 post infection) depletion of neutrophils (Pedrosa et al. 2000). While later neutrophil depletion did not show a quantifiable effect on M. tuberculosis proliferation in the lungs, earlier neutrophil depletions impaired development of protection against mycobacterial challenge. Further evidence in favor of an early protective role for neutrophils is provided by the observation that intra-tracheal injection of pre-activated neutrophils protected Fischer rats from infection with M. tuberculosis Kurono (Sugawara et al. 2004). On the other hand, other studies failed to show any protective effect when C57Bl/6

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mice were challenged via the aerosol route (Seiler et al. 2000), suggesting that neutrophils do not play an essential part in the early control of infection with M. tuberculosis. Yet in another report, the investigators compared the mouse strains genetically resistant (A/Sn) versus susceptible (I/St) to M. tuberculosis and concluded that upon aerosol infection with M. tuberculosis H37Rv, neutrophils contribute to pathology rather than protection (Eruslanov et al. 2005). This controversy may be partially explained by genetic differences among strains of experimental animals as well as different laboratory strains of M. tuberculosis. Another approach to the potential role of neutrophils during tuberculosis has been raised by a series of studies where it has been shown that apoptotic neutrophils can modulate the induction of acquired immunity by dendritic cells (Aleman et al. 2002, 2005, 2007; Tan et al. 2006), or provide “help” to M. tuberculosis-infected macrophages in vitro (Tan et al. 2006), via delivery of antimicrobial contents of neutrophil granules to M. tuberculosis-containing phagosomes shortly after uptake of apoptotic neutrophils, leading to improved killing of mycobacteria mediated by the neutrophil D-defensin HNP-1.

2.2.2

T Cells

The in vivo mouse model of low-dose aerosol M. tuberculosis exposure produces a very low cellular response, especially when comparing to those responses seen after some viral or other fast-replicating bacteria. These differences in robustness of responses may reflect an immune-modulatory activities triggered by the pathogen or may simply result from the inoculum route and dose. During natural as well as during experimental aerosol exposure, M. tuberculosis enters the lung in droplets of 3–5 Pm of diameter (typically generated by a cough of a diseased individual), only a few bacilli might be found within these droplets. Furthermore, those droplets will most likely settle within the alveolar space. It should also be noted that M. tuberculosis is relative slow grower, with estimated duplication time of approximately 28 h to double in vivo (Dunn and North 1995). Therefore, the optimal conditions for an invading bacilli to initiate the cascade that lead to powerful cellular immune response are extremely low and greatly dependent on the inoculum dose as well as the potential to reach professional antigen-presenting cells and secondary lymphoid tissues, such as the draining lymph nodes. Consistent with this observation is the fact that it is possible to detect early T-cell activation in the lymph nodes draining the lung but not within the lung itself (Chackerian et al. 2001). In order to investigate mycobacteria antigen-specific T cell activation, T-cell receptor transgenic (TCRTg) T cells specific for an IAb-restricted epitope of the early secreted antigenic target 6 kDa protein (ESAT-6) or for an IAb-restricted epitope of the essential mycolyl transferase of Mtb, Ag85The activation of naive T cells has been had been used. The published data show a significant up-regulation of an early marker of T cell activation (CD69) as well as proliferation of naïve T cell in the draining nodes of the lung, which is concomitant to the arrival of the bacilli in the node (Reiley et al. 2008;

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Wolf et al. 2008). The lag between bacterial proliferation in the lungs and the growth and differentiation of effector T cells constitute the basis for the apparent slow cellular immune response during M. tuberculosis.

2.2.3

Dendritic Cells

Fluorescent bacteria delivered by aerosol can be seen within dendritic cells (DCs) and cannot be detected in the lymph node (Wolf et al. 2007). More importantly, if the chemotactic signals responsible for DC migration towards the lymph node are absent, the appearance of M. tuberculosis in that organ is inhibited (Wolf et al. 2007). It has recently been shown that appearance of microbially activated (or infected) DCs in the lymph node is dependent on the presence of homodimers of IL-12p40 (Khader et al. 2006), previously supposed to act as an endogenous antagonist for the Th1promoting cytokine IL-12p70. This was supported by the findings that while mice that deficiency of IL-12p70 or IL-23 could induce accumulation of activated CD41 T cells in the lung 21 days after infection, mice that lacked IL-12p40 could not (Khader et al. 2006). The investigators found that this IL-12p40(2) was very rapid (under 3 h) and drove DC migration via up-regulation of responsiveness to CCL19/CCL21 (CCR7 ligands). If DC did not produce IL-12p40, then migration in response to CCL19 and CCL21 remained at the level of the non-activated DC, despite presence of M. tuberculosis (Khader et al. 2006). On the other hand, DCs lacking IL-12p35, IL-23p19, or both of these cytokine components respond to CCL19 and CCL21 when incubated with M. tuberculosis (Khader et al. 2006). The mechanisms by which IL-12p40(2) mediated up-regulation of CCR7 responsiveness is not yet understood. DC accumulation in the lymph node provides environmental cues to the immune system. Notably, bacterially activated DCs will drive the naïve T cell activation, growth and differentiation. In agreement with this observation, it was shown that M. tuberculosis-exposed DCs adoptively transferred to the lung could provide the signals to drive T-helper 1 cell development (Bhatt et al. 2004). These data also support the paradigm that the mechanisms that modulate the appearance of functional effector T cells depend directly upon the DC activation status and subset and that there are no redundancies that could compensate for by the intact host cells within the node.

2.2.4

Natural Killer Cells

Natural killer (NK) cells are innate granular lymphocytes (Lodoen and Lanier 2006; Newman and Riley 2007; Moretta et al. 2008) known to play a role in mediating in allograft rejection and killing of transformed and virus-infected cells. Additionally, NK cells secrete pro-inflammatory cytokines, most prominently IFN-J. NK cell activity is controlled by cytokines (IL-12, IL-18 and IFN-D in particular) and a complex repertoire of activating (e.g. NKG2D, natural cytotoxicity receptors) and inhibitory receptors (e.g. CD94-NKG2A).

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It has become evident that NK cells are capable of mounting a vigorous response to M. tuberculosis. Human NK cells express granulysin within their intra-cytoplasmic granules and it has been shown that this peptide could directly kill M. tuberculosis (Stenger et al. 1999). Moreover, human NK cells are known to directly lyse M. tuberculosis-infected monocytes and macrophages in vitro (Denis 1994; Vankayalapati et al. 2002). The activation occurs via triggering of NKG2D and NKp46 that bind to the stress-induced ligands UL16-binding protein 1 (ULPB1) and vimentin, respectively (Vankayalapati et al. 2002, 2005; Garg et al. 2006). Human NK cells can also actively inhibit mycobacterial growth via induction of apoptotic cell deaths (Brill et al. 2001; Millman et al. 2008). During experimental infections with M. tuberculosis it was found that NK cells accumulate start to accumulate in the lungs and secrete IFN-J after approximately 2 weeks after low dose aerosol infection with M. tuberculosis Erdmann (JunqueiraKipnis et al. 2003). NK cell depletion showed no effect on control of mycobacterial growth. Feng and colleagues provided evidence for this hypothesis when they demonstrated that NK cells are the principal source of IFN-J in T cell-deficient RAG2−/− animals after low-dose M. tuberculosis H37Rv infection. Most importantly, that NK cell-derived IFN-J was essential for controlling the pathology (Feng et al. 2006). In the absence of both NK and T cells (RAG2−/− Jc−/−), the control of mycobacterial growth was severely abolished, indicating that NK cells can drive cellular responses against mycobacteria.

2.2.5

Regulatory T Cells

Regulatory T cells (T regs) were found to accumulate in the lungs of mice during experimental M. tuberculosis infection and bacterial control is improved after their depletion (Scott-Browne et al. 2007). In humans, it was shown that mannose-capped lipoarabinomannan (ManLAM) drove expansion of CD4 + CD25 + FoxP3+ cells in samples from tuberculin responders but not from naïve individuals (Garg et al. 2008). ManLAM is thought to trigger macrophages to produce PGE2, which in turn allows for further Treg expansion (Garg et al. 2008). Interestingly, TB patients show higher frequency of Treg cells than in healthy tuberculin responders (Garg et al. 2008). During anti-viral therapy, AIDS/TB patients, show an increase in both effector T cells and T regs, however the regulatory activity of the T regs is defective (Seddiki et al. 2009).

2.3

Granuloma Formation and Containment of Bacilli

Granuloma is an organized collection of immune cells, including a large proportion of macrophages, within a tissue as a result of chronic unresolved/persistent inflammatory stimulus. The purpose of the granuloma for the host is two-fold.

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Firstly, through a fibrous capsule is thought to provide a physical containment of the infected area while preventing pathogen spread. Secondly, it aggregates a collection of immune effector cells including APCs, lymphocytes and granulocytesa crucial step during the development of immunity to infection. The tuberculous granuloma has been described by Ghon in 1912, however only in recent years an accurate depiction of primary human granulomas has allowed a better understanding of their role during tuberculosis. Frequently, an area of necrosis if found at the inner region of the tuberculosis granuloma. This is thought to be a consequence from previous extensive macrophage infection and killing. However, the granuloma also allows the chronic maintenance of M. tuberculosis in infected macrophages. Notably, while the core of the granuloma carries few antigen-presenting cells containing mycobacterial antigens, the periphery of the granuloma is enriched with organized aggregates of APC and proliferating lymphocytes and are thought to be a site of active immunity (Ulrichs and Kaufmann 2006). In summary, tuberculosis granuloma can be considered both an essential player in the protective immune response to the pathogen and a facilitator in the development of latency during chronic disease, which is hard for the immune system to tackle and is notoriously difficult to treat by conventional methods.

2.3.1

TNF

TNF has extensively studied during experimental mouse M. tuberculosis infections. It has been shown that it is central for the development of protective immune response both during acute and chronic disease, notably from the poor granuloma formation observed in vivo as well as defective macrophage activity (Flynn et al. 1995; Mohan et al. 2001; Algood et al. 2005). Other experimental M. tuberculosis infection models, such as zebrafish and nonhuman primate have shown that TNF is relevant for the mechanisms involved in overcoming acute infection and to prevent disease reactivation, however, granuloma formation is not affected by TNF depletion (Lin et al. 2010; Clay et al. 2008). More importantly, genetic polymorphism of the TNF receptor in humans has been associated with increased susceptibility to active TB in Africa (Moller et al.), which is strongly supported by the increased frequency of re-activated tuberculosis among patients treated with TNF antagonists (Keane 2004). Despite the majority of these cases were from tuberculosis reactivation, there is increased concern over the risk of fulminant acute tuberculosis within highly endemic areas. TNF is a pleiotropic cytokine that can affect several arms of the immune system. During early tuberculosis infection, it has been associated with the induction of adhesion molecules (Windish et al. 2009) and chemokines (Peters et al. 2001; Scott and Flynn 2002; Algood et al. 2004), which can perform potentially protective roles during the initiation and expansion of immune responses. Another possible aspect that can be affected by TNF is programmed cell death, as a mediator of apoptosis, TNF can be directly detrimental to the survival of mycobacteria within macrophages.

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Human TNF production by M. tuberculosis infected alveolar macrophages triggers apoptotic cell death, thereby reducing intracellular bacterial burden (Keane et al. 1997). Moreover, attenuated M. tuberculosis strains were found to increase apoptosis and, consequently induce stronger CD8 T cell protective responses, indicating that apoptosis is associated with a better outcome of infection (Hinchey et al. 2007).

2.3.2

IFN-g

IFN-J is a central cytokine in the initiation and effector function of cellular immune responses. During experimental M. tuberculosis infection, the absence of IFN-J leads to uncontrolled bacilli growth and mortality (Cooper et al. 1993). The downstream IFN-J-inducible enzymes required for generation of nitrogen and oxygen radicals, nitric oxide synthase 2 (NOS2) and p47phox, are also relevant for control of immunopathology (Cooper et al. 2002).

2.3.3

Lipoxins

We have reported evidence for the role of a pathway involving the 5-LO–dependent production of lipoxins that dampens M. tuberculosis–driven pro-inflammatory immune responses and regulates bacterial growth. Lung tissue from 5-LO-deficient animals display higher production of IL-12 and other pro-inflammatory cytokines, suggesting that 5-LO-dependent eicosanoids counter-balance such secreted proteins. Since 5-LO is required for both leukotriene and lipoxin biosynthesis, reconstitution experiments were performed to more directly assess the role of the latter group of eicosanoids in the regulation of mycobacterial growth in vivo. Importantly, administration of the stable lipoxin analog – ATLa2 to M. tuberculosis-infected 5-LO-deficient mice, restored both pulmonary mycobacterial loads and pro-inflammatory cytokine production. These observations demonstrated that deficiency in lipoxins is sufficient to explain the effects on bacterial growth and host response seen in the infected 5-LO–deficient animals. Interestingly, granuloma formation in 5-LO-deficient mice was found to be altered suggesting that lipoxins regulate chemotaxis of inflammatory monocytes to the site of infection and/or activation of macrophages in vivo (Bafica et al. 2005). Nevertheless, whether lipoxins directly control influx/efflux of lung antigen presenting cells is still unclear. A role for products derived from 5-LO, encoded by ALOX5 gene, in pulmonary tuberculosis was further supported in human association studies. In humans, ALOX5 gene comprises 14 exons and 13 introns approximately 82 kb on chromosome ten (10q11.2). ALOX5 promoter is GC-rich, and the region between 79 and 56 bp is essential for gene expression (Herb et al. 2008). In that region, a variable number of tandem repeats (VNTR) has been identified, consisting of [5c-GGGCGG-3c]2–8, which are targets for binding of the transcription factor Sp1. Insertions or deletions

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of the Sp1-binding motif have created variant alleles of two to eight repeats and depending on the number of repeats, Sp1 binding and subsequent gene expression is altered (Cohn et al. 2001; Wang et al. 2008). On the basis of the above mentioned results found in mice, Herb et al. hypothesized that naturally occurring ALOX5 variants might also be relevant to the phenotype of M. tuberculosis infection in humans (Herb et al. 2008). The authors found a strong association between low 5-LO activity genotypes and decreased TB risk, consistent with the observation that 5-LO deficiency in mice was associated with relative protection from TB. However, it remains to be demonstrated whether lipoxins directly regulate human immune responses during TB. While a number of questions need to be answered from these studies, such findings can be considered as an example of a gene regulating in vivo M. tuberculosis infection in both human and mice.

2.3.4

IL-10

IL-10 is a powerful inhibitor of the IL-12/IFN-J cellular immunity pathway. However, the role of this cytokine in TB is not well established. In humans, an increased macrophage IL-10 production upon TLR-stimulation such as LPS is associated with faster development of primary TB (Awomoyi et al. 2002). In mice, the genetic deletion of IL-10 causes an accelerated accumulation of lung DC migration in the draining lymph nodes after mycobacterial infection (Demangel et al. 2002). M. tuberculosis infection in a susceptible mouse strain – CBA – leads to increased lung macrophage IL-10 production, something that was not found in C57Bl/6 (resistant) mouse strain (Turner et al. 2002). Conversely, inhibition of IL-10 in CBA mice enhances control of bacterial burden during the chronic TB (Beamer et al. 2008).

2.3.5

TGF-b

It has been reported that an excess of TGF-E is produced during tuberculosis and its message is abundantly up-regulated during active M. tuberculosis infection. Consistent with that T-cell responses from active TB patients are significantly improved after TGF-E neutralization. TGF-E modulates multiple cells of the immune system, including T cells (activation and cytokine production), macrophage (migration, microbicidal activity, cytokine production). In agreement with this set of observations, the presence of TGF-E leads to increased growth of intracellular M. tuberculosis (Hirsch et al. 1994). On the other hand, its neutralization (Hirsch et al. 1994) as well as the addition of natural TGF-E inhibitors (Hirsch et al. 1997) caused a reduction in the growth of intracellular mycobacteria. Given its correlation with disease severity, it is likely that TGF-E-mediated macrophage deactivation is severe in patients carrying higher loads of bacilli.

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2.4 2.4.1

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Evasion of Immune Response Cell Wall Components

Mycobacteria are Gram positive and cannot be decolorized by acid alcohol and are therefore classified as acid-fast bacilli. Acid fastness is mostly due to higher amounts of mycolic acids (long chain cross-linked fatty acids) and other cell-wall lipids in the cell wall (Daffe and Draper 1998). Mycolic acid and other lipids are linked to underlying arabinogalactan and peptidoglycan (Daffe and Draper 1998). A variety of unique lipids, such as lipoarabinomannan (LAM), trehalose dimycolate and phthiocerol dimycocerate, anchor noncovalently with the cell membrane and have been shown to mediate the virulence of M. tuberculosis (Glickman and Jacobs 2001). Disruption of the gene involved in mycolic acid cyclopropanation caused a reduction in virulence (Glickman et al. 2000). The majority of exported proteins and protective antigens of M. tuberculosis are a cluster of related gene products termed the antigen 85 complex, each having fibronectin binding capacity and thus an important role in disease pathogenesis (Belisle et al. 1997). LAM is one of the major components of mycobacterial cell wall. It triggers TNF production from the macrophages (Chatterjee et al. 1992). Moreover, LAM can scavenge for potentially cytotoxic oxygen free radicals, inhibit protein kinase C activity and block the tran- scriptional activation of gamma interferon inducible genes in human macrophages. Thus, potentially contributing to the persistence of mycobacteria within mononuclear phagocytes (Chan et al. 1991).

2.4.2

Inhibition of Phagolysosome Fusion

Phagosome-lysosome fusion is a key event in killing intracellular pathogens (Moulder 1985). Upon engulfment by macrophages, most bacilli are directed to phagolysosomes (McDonough et al. 1993). Interestingly, however, individual M. tuberculosis bud out from the fused phagolysosomes into vacuoles refractory to fusion with the secondary lysosomes thus escape intracellular killing. M. tuberculosisderived sulfatides (anionic trehalose glycolipids) were shown to have such antagonistic fusion activity (Goren 1977). Virulent M. tuberculosis culture supernatants are enriched for ammonium, which were shown to inhibit phagolysosomal fusion by increasing the alkalinity within the intralysosomal environment (Gordon et al. 1980; Hart et al. 1983).

2.4.3

Mycobacterial Dormancy

M. tuberculosis can enter dormancy within host cells for extended times while remaining potentially active. During dormancy/latency of M. tuberculosis infection,

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the bacterium escapes the activity host immune cells. Experimental M. tuberculosis infection in mice is dependent on the glyoxylate shunt enzyme – isocitrate lyase, essential for fatty acid metabolism (McKinney et al. 2000). Its genetic disruption resulted in increased bacterial persistence as well as virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. Hypoxia was also found to be a major factor in the induction of non-replicating persistent mycobacterium (Wayne and Sohaskey 2001).

2.4.4

Modulation of Host Cell Signaling

Upon infection, host proteins are targeted by phosphatases and kinases of several pathogenic bacteria thereby modulating and potentially contributing to the establishment of the disease. The phagocytosis of M. tuberculosis by macrophages is associated with a several early signaling events, including the activation of Src protein tyrosine kinase family members. Such activation leads to the enhanced tyrosine phosphorylation of multiple target proteins and phospholipase D activation (Kusner et al. 1996). LAM derived from virulent M. tuberculosis strains can modulate intracellular signaling associated to bacterial survival via phosphorylation of proapoptotic protein downstream from the phosphatidylinositol 3-kinase-dependent pathway (Maiti et al. 2001). A major anti-phosphotyrosine reactive protein is present only in strains belonging to M. tuberculosis complex (Chow et al. 1994). Also, it has been shown that M. tuberculosis has two functional secreted tyrosine phosphatases that may interfere with the host cells signaling machinery (Koul et al. 2000).

2.5

Disease Reactivation

Presently, the drug regimen for the treatment of latent TB infection is of 9 months of isoniazid (2000), a drug for which the efficacy is dependent upon the synthesis of mycolic acid, which occurs only during active replication. Based on epidemiologic studies, the reactivation of latent TB may derive from: HIV, malnutrition, tobacco smoke, indoor air pollution, alcoholism, silicosis, insulin dependent diabetes, renal failure, malignancy, and immune-suppressive therapy (i.e., glucocorticoids) (Horsburgh 2004; Jick et al. 2006; Lonnroth and Raviglione 2008). AIDS and inhibitors of TNF are well-characterized mechanisms of disease reactivation. An increased incidence of TB was noted among patients upon treatment with TNF inhibitors (Keane 2004; Wallis 2009). Similar observations were made after TNF neutralization in non-human primates during latent tuberculosis (Lin et al. 2000). Importantly, although a high rate of reactivation (~65%) was noted in those animals, not all monkeys showed TB reactivation after short-term anti-TNF treatment (Keane 2004), indicating that TNF is an relevant but not the sole component in maintaining TB latency. AIDS and HIV infection is a central component in the resurgence of TB as a global health threat. HIV infection has been, by far, the most common risk for

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TB reactivation, almost tenfold higher among HIV patients rather than non-HIV patients (Horsburgh 2004). In contrast to the TB epidemiology from the pre-HIV era, an extremely higher rate of disseminated/extrapulmonary TB has been reported among AIDS patients (Small et al. 1994). Although CD4 T cell counts are directly associated with risk of TB reactivation, anti-retroviral therapy had no significant effect (Meintjes et al. 2008; Lawn et al. 2009). Moreover, optimal conditions CD4 T cell counts do not necessarily lower the risk of TB reactivation (Badri et al. 2002; Lawn et al. 2005), indicating that mere CD4 T cell counts may not necessarily reflect immune fitness to fight TB reactivation. In non-human primates, infection with SIV led to TB reactivation (Diedrich et al. 2010). The synergic effect between HIV and M. tuberculosis involve: HIV-induced loss of mycobacterial specific CD4 T cells, M. tuberculosis-induced increases in HIV load in serum and macrophages, shift from Th1 to Th2 response via alterations in IL-10, regulatory T cells, IL-12, IL-4, and TNF, loss of granuloma integrity, and alterations in apoptotic mechanisms (Djoba Siawaya et al. 2007).

2.6 Vaccines, Chemotherapy in the Interface with the Immune System BCG is currently the only vaccine available for TB, however it is well accepted that its efficacy, albeit low, is restricted to acute pulmonary disease. Several strains of BCG differ in immunogenicity (Aguirre-Blanco et al. 2007). It is known that BCG differs from M. tuberculosis due to the lack of RD-1 gene (Billeskov et al. 2007; Woodworth et al. 2008). Systemic delivery of BCG induce antigen-specific cells, thereby triggering protection, which is characterized by the appearance of IFN-J-producing T cells in the lung at day 14. Suggesting that timing for accumulation of antigen-specific cells in the lung that is key to protection (Mittrucker et al. 2007; Cooper 2009). Strategies aiming at improving protection after BCG vaccination focused on specific delivery of antigen(s) alongside the original vaccine. The route of this boost is important as BCG vaccinated mice boosted intranasally (but not intradermally) using a recombinant adenovirus expressing Ag85, show better levels of protection and infection control when compared to BCG alone. Moreover, the introduction of a boost to the BCG regimen with a vaccinia virus expressing M. tuberculosis Ag85A caused a significant expansion of long-lived memory T cells expressing IFN-J TNF, and IL-2 (Beveridge et al. 2007). Along these lines, BCG engineered to express the DC stimulating factor, GM-CSF was shown to improve cellular immunity after vaccination (Triccas et al. 2007). Furthermore, genetic deletion of the BCG SecA2 made host cells more susceptible to apoptosis induction and resulting in improved CD8 T cell induction (Hinchey et al. 2007). On the other hand, Flt3-ligand-expressing BCG had no effect in improving protection (Triccas et al. 2007). Interestingly, T reg depletion improved vaccine induced cellular responses, however, it failed to enhance protection levels (Quinn et al. 2008).

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In the mouse model, administration of defined antigens and adjuvants has been shown to result in the early T cell recruitment to the lungs upon challenge, resulting in better control of infection. Earlier T-cell responses are desirable effect in order to act to limit M. tuberculosis growth and positively affect the outcome of the infection, by limiting steady-state bacterial infection in the lungs in a timely manner.

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Glickman, M. S., J. S. Cox, et al. (2000). “A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis.” Mol Cell 5(4): 717–27. Glickman, M. S. and W. R. Jacobs, Jr. (2001). “Microbial pathogenesis of Mycobacterium tuberculosis: dawn of a discipline.” Cell 104(4): 477–85. Gordon, A. H., P. D. Hart, et al. (1980). “Ammonia inhibits phagosome-lysosome fusion in macrophages.” Nature 286(5768): 79–80. Goren, M. B. (1977). “Phagocyte lysosomes: interactions with infectious agents, phagosomes, and experimental perturbations in function.” Annu Rev Microbiol 31: 507–33. Hart, P. D., M. R. Young, et al. (1983). “Chemical inhibitors of phagosome-lysosome fusion in cultured macrophages also inhibit saltatory lysosomal movements. A combined microscopic and computer study.” J Exp Med 158(2): 477–92. Herb, F., T. Thye, et al. (2008). “ALOX5 variants associated with susceptibility to human pulmonary tuberculosis.” Hum Mol Genet 17(7): 1052–60. Hinchey, J., S. Lee, et al. (2007). “Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis.” J Clin Invest 117(8): 2279–88. Hirsch, C. S., J. J. Ellner, et al. (1997). “In vitro restoration of T cell responses in tuberculosis and augmentation of monocyte effector function against Mycobacterium tuberculosis by natural inhibitors of transforming growth factor beta.” Proc Natl Acad Sci USA 94(8): 3926–31. Hirsch, C. S., T. Yoneda, et al. (1994). “Enhancement of intracellular growth of Mycobacterium tuberculosis in human monocytes by transforming growth factor-beta 1.” J Infect Dis 170(5): 1229–37. Horsburgh, C. R., Jr. (2004). “Priorities for the treatment of latent tuberculosis infection in the United States.” N Engl J Med 350(20): 2060–7. Jick, S. S., E. S. Lieberman, et al. (2006). “Glucocorticoid use, other associated factors, and the risk of tuberculosis.” Arthritis Rheum 55(1): 19–26. Junqueira-Kipnis, A. P., A. Kipnis, et al. (2003). “NK cells respond to pulmonary infection with Mycobacterium tuberculosis, but play a minimal role in protection.” J Immunol 171(11): 6039–45. Keane, J. (2004). “Tumor necrosis factor blockers and reactivation of latent tuberculosis.” Clin Infect Dis 39(3): 300–2. Keane, J., M. K. Balcewicz-Sablinska, et al. (1997). “Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis.” Infect Immun 65(1): 298–304. Khader, S. A., S. Partida-Sanchez, et al. (2006). “Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection.” J Exp Med 203(7): 1805–15. Koch, R. (1891). “A Further Communication on a Remedy for Tuberculosis.” Br Med J 1(1568): 125–127. Koul, A., A. Choidas, et al. (2000). “Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.” J Bacteriol 182(19): 5425–32. Kusner, D. J., C. F. Hall, et al. (1996). “Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages.” J Exp Med 184(2): 585–95. Lasco, T. M., O. C. Turner, et al. (2004). “Rapid accumulation of eosinophils in lung lesions in guinea pigs infected with Mycobacterium tuberculosis.” Infect Immun 72(2): 1147–9. Lawn, S. D., L. G. Bekker, et al. (2005). “How effectively does HAART restore immune responses to Mycobacterium tuberculosis? Implications for tuberculosis control.” AIDS 19(11): 1113–24. Lawn, S. D., L. Myer, et al. (2009). “Short-term and long-term risk of tuberculosis associated with CD4 cell recovery during antiretroviral therapy in South Africa.” AIDS 23(13): 1717–25. Lin, P. L., A. Myers, et al. (2010). “Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model.” Arthritis Rheum 62(2): 340–50.

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Chapter 3

NKT Cell Activation During (Microbial) Infection Jochen Mattner

Abstract Invariant Natural Killer T (iNKT) cells constitute an innate-like lymphocyte population endowed with powerful immunomodulatory functions. Unlike conventional T cells, iNKT cells predominantly express a conserved semiinvariant T cell receptor (TCR), VD14-JD18/VE2, 7, 8 in mice and VD24-JD18/ VE11 in humans. These canonical TCRs in both species do not recognize peptides, but glycosphingolipid (GSL) patterns presented by CD1d on antigen presenting cells (APCs). The natural mechanisms for iNKT cell activation were unclear prior to the recent identification of their endogenous and exogenous GSL ligands. Microbes can employ two alternative strategies for iNKT cell activation as exemplarily shown here for Gram-negative bacteria: (a) recognition of endogenous GSLs – by-products of the complex mammalian GSL metabolic pathways – and the presence of interleukin-12 (IL-12), triggered by Toll-like receptor (TLR) signaling of infected APCs, are required for the early secretion of IFN-J by iNKT cells in response to Gram-negative, LPS-positive bacteria. Whereas iNKT cells are secondary to APC-mediated effects in infections with these bacteria, (b) iNKT cells accelerate the clearance of Gram-negative LPS-negative alphaproteobacteria due to the cognate recognition of GSLs in the cell wall of these alphaproteobacteria. Thus, the iNKT cell population represents a major innate recognition pathway for these LPSnegative, GSL-positive alphaproteobacteria that senses infection at sites where iNKT cells accumulate, such as the liver. In this context, iNKT cell activation upon microbial encounter may not only contribute to bacterial clearance, but may be even deleterious for the host, providing innate signals that break peripheral tolerance and unleash autoimmune effector cells.

J. Mattner (*) University Hospital of Erlangen, Microbiology Institute–Clinical Microbiology, Immunology and Hygiene, Erlangen, Germany e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_3, © Springer Science+Business Media, LLC 2012

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Introduction

T cells play a central role in the microbial immune defense. Conventional CD8+ and CD4+ T cells recognize peptide antigens presented by classical major histocompatibility complex (MHC) class I and II molecules. Upon antigen encounter, their T cell receptor (TCR) can re-arrange and shape the TCR repertoire due to the clonal expansion of the T cell population with the most suitable TCR for the presented antigen. In contrast to these conventional T cells, the specificity of invariant Natural Killer T (iNKT) cells is directed against glycosphingolipid (GSL) antigens presented by CD1d, a non-classical MHC class I molecule expressed on dendritic cells (DCs), macrophages and B cells. However, while classical MHC class I molecules are loaded within the endoplasmatic reticulum and present intracellular cytosolic peptides to cytotoxic CD8+ T lymphocytes, CD1d is loaded within the late endosome or the lysosome and presents endogenous or exogenous GSL antigens to iNKT cells. Like classical MHC class I, CD1d associates with E-2-microglobulin (E2m). Unlike classical MHC class I, CD1d molecules as all other CD1 molecules (see below) are nonpolymorphic and present GSLs rather than peptides to iNKT cells (summarized in (Bendelac et al. 2007)). Current models suggest that the hydrophobic tails of the GSL antigens bind into the groove of CD1d via hydrophobic interactions, while the polar head groups contact the TCR (Moody et al. 1997) and subsequently trigger iNKT cell activation. iNKT cells display a canonical VD14 TCR in mice and a VD24 TCR in humans in combination with a limited set of VE chains (VE8, VE7 and VE2 in mice and VE11 in humans) – that does not re-arrange upon antigen encounter – and release immediately copious amounts of cytokines and chemokines upon engagement of their TCR. Although iNKT cells are inherently autoreactive due to the recognition of endogenous GSL antigens, they react also to defined GSL structures in the cell wall of microbes (summarized in (Bendelac et al. 2007)). Thus, iNKT cells recognize distinct self and microbial GSLs and their TCR acts more like a pattern recognition receptor, identifying iNKT cells as an innate (−like) lymphocyte population. These characteristics establish the CD1/NKT system as another line of antimicrobial defense next to MHC class I and II as well as to the diverse pattern recognition receptors of the innate immune system that alternatively recognizes defined GSL antigens in addition to peptide antigens, lipoproteins, nucleic acids or polysaccharides (Brigl and Brenner 2004). These CD1d-restricted iNKT cells expressing the VD14 TCR can be distinguished from CD1a-, CD1b- and CD1c-restricted T cells as well as from CD1d-restricted T cells that express non-invariant TCRs and from a variety of other non-CD1drestricted T cells that express lineage receptors of NK cells (Eberl et al. 1999; Godfrey et al. 2004). Although non-VD14 CD1d-restricted T cells have been recently implicated in various diseases, this book chapter focuses mainly on canonical iNKT cells by providing an overview over GSL antigen presentation and iNKT cell function and summarizing the different GSLs recognized by iNKT cells during microbial infection as well as the functional consequences of this recognition for the host.

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NKT Cell Activation During (Microbial) Infection

3.2

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NKT Cells and the CD1 System

NKT cells form a heterogeneous cell population, which respond to (glyco-) lipid and/or GSL antigens presented by CD1. Although the CD1 heavy chain associates with E2 microglobulin similar as classical MHC class I, it contains in contrast to classical MHC class I several hydrophobic channels that bind the lipid portion of GSLs or other lipid-rich antigens (see also Sect. 3.4 – CD1d mediated presentation of glycolipids). The CD1 genes that diverged from the MHC gene complex several 100 million year ago (Kasahara 1997) can be divided into Group I (CD1a, b, c), Group II (CD1d), and Group III (CD1e) genes based on sequence similarities and their organization in the locus. While group I and II CD1 molecules are expressed on the cell surface and present GSL and/or (glyco-) lipid antigens, the fifth human CD1 molecule, CD1e, facilitates the loading of certain lipid antigens onto CD1b (de la Salle et al. 2005). While humans express all five CD1 genes, their expression is not conserved in all mammalian species: mice, for example, have been shown to express only CD1d (summarized in (Bendelac et al. 2007)). Defined by the recognition of distinct antigens presented by these CD1 groups, NKT cells can be distinguished into the following three categories:

3.2.1

Group I CD1 Molecules Recognizing NKT Cells (CD1a, b, c)

Because of its lipid-rich cell wall, Mycobacterium tuberculosis has been the prime candidate pathogen for studying (glyco-) lipid – reactive T cells in vitro. The first study published in this context described a T cell line specific for mycolic acids that are presented by CD1b (Beckman et al. 1994). Mycolic acids are not only found in Mycobacterium tuberculosis, but also are also constituents of the cell wall of Actinomyces, Corynebacteria, and Nocardia species (Brennan and Nikaido 1995; Moody et al. 2002). During subsequent studies, additional group I CD1 antigens in the cell wall of Mycobacterium tuberculosis have been identified: didehydroxymycobactin, presented by CD1a (Moody et al. 2004; Rosat et al. 1999), Ac2SGL, a sulfoglycolipid presented by CD1b (Gilleron et al. 2004), and hexosyl -1 – phosphoisoprenoids, presented by CD1c (Rosat et al. 1999; Beckman et al. 1996; Moody et al. 2000). Although Mycobacteria are a productive source for studying group I CD1-restricted T cell responses in vitro, the functional consequences of group I CD1 restricted T cell activation in vivo due to the lack of a suitable animal model is not understood. Interestingly, preferentially animals susceptible to infections with Mycobacteria like cattle express functional group I CD1 genes. Ongoing studies in cows may explain in the future the evolution of these group I CD1 – restricted T cell populations and their respective roles during mycobacterial infections (Kasmar et al. 2009).

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Group II CD1 Molecules Recognizing iNKT Cells (CD1d)

Group II CD1 genes have been preferentially studied in the mouse. CD1d is constitutively expressed on DCs, macrophages, and B cells (Roark et al. 1998; Brossay et al. 1997), particularly marginal zone B cells; CD1d is also expressed on cortical thymocytes, where its expression is essential for the development of iNKT cells (Bendelac 1995a, b, c), hepatocytes, hepatic Kupffer cells and endothelial cells lining liver sinusoids (Geissmann et al. 2005). Although CD1d is upregulated on microglial cells during inflammation (Busshoff et al. 2001), only relatively modest changes in CD1d expression occur upon TLR activation, infection or exposure to inflammatory cytokines on DCs, macrophages or B cells (Skold et al. 2005). Similar to the MHC class II system, most other solid tissue cells with the exception of the liver, where iNKT cells are abundantly found and non-antigen-presenting hematopoietic cells express low or undetectable levels of CD1d.

3.2.2.1

CD1d: Restricted Invariant NKT Cells (iNKT Cells, Type I NKT Cells)

iNKT cells constitute a population of T lymphocytes that predominantly use a conserved semi-invariant TCR with specificity for CD1d combined with glycosphingolipid lipid (GSL) ligands (Bendelac et al. 2007; Brigl and Brenner 2004; Kronenberg 2005). Reactivity towards alpha-GalactosylCeramide (D-GalCer), the prototypical iNKT cell ligand isolated from marine sponges distinguishes iNKT (or type I NKT) cells (Kawano et al. 1997) from non-classical (also summarized as type II) NKT cells (see Sect. 3.2.2.2). The iNKT cell TCR utilizes a canonical D-chain (VD14JD18 in mice and VD24-JD18 in humans) in combination with a limited set of VE chains (VE8, VE7 and VE2 in mice and VE11 in humans). Analysis of CD1d- and MHC-deficient mice demonstrated that iNKT cells (also called type I NKT cells) represent the majority of CD1d-restricted T cells (Kawano et al. 1997; Bendelac et al. 1994, 1995a, b, c). Their conserved TCR structure, their memory/effector differentiation – as indicated by the combined expression of CD44 and CD69 even in the absence of exogenously added GSL antigens (Bendelac et al. 1992) -, and their expression of a panoply of NK receptors suggest modalities of activation that are distinct from conventional T cells. The conserved semi-invariant structure of their TCR is remarkably similar to that of B-1 B cells and JG T cells (Bendelac et al. 2001) and transgenic expression of the antigen receptor of each of these cell-types was found to instruct the corresponding lineage, suggesting that interactions with self ligands dictate lineage commitment and differentiation. Developmental studies used D-GalCer loaded CD1d tetramers (Benlagha et al. 2000; Matsuda et al. 2000) to follow the iNKT precursors and demonstrated that their TCR is intrinsically autoreactive, inducing a phase of clonal expansion in the thymus prior to migration into peripheral organs and secondary acquisition of NK cell receptors (Bendelac et al. 2007; Benlagha et al. 2002).

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Using D-GalCer loaded CD1d tetramers iNKT cells have been identified in mice, rats, rhesus macaques, chimpanzees and humans (Matsuura et al. 2000; Pyz et al. 2006; Liu et al. 2006). The distribution of iNKT cells has been well characterized in mice and differs from the one of conventional MHC I- or MHC II- restricted T cells. iNKT cells represent only 0.2–0.5% of the T cell population in the blood, the thymus and the peripheral lymph nodes, 1–2% of lymphocytes in the spleen, mesenteric, and pancreatic lymph nodes, and up to 30% of T cells in the liver. Mechanisms causing that unique distribution have remained elusive. Whereas the precise distribution of iNKT cells within the lymphoid organs is still unknown, they have been shown to patrol along the liver sinusoids (Geissmann et al. 2005; Ohteki and MacDonald 1994) providing intravascular immune surveillance: most likely they crawl along a CXCL16 gradient as they express high levels of CXCR6. Upon engagement of their TCR by potent GSL antigens like alpha-GalCer, however, iNKT cells immediately arrest. Although the expression of CXCR6 on iNKT cells matches the expression of CXCL16 on endothelial cells lining the liver sinusoids, CXCR6 deficiency resulted in reduced survival rather than altered migration of iNKT cells. Like NK cells, iNKT cells constitutively express mRNA but not protein for IFN-J, a hallmark of their poised effector stage (Stetson et al. 2003). Unlike NK cells, however, iNKT cells also produce interleukin four and 13 (IL-4 and IL-13) and the contribution of both Th1 and Th2 cytokines has been demonstrated in vivo, in conditions where iNKT cells either improved or aggravated infectious, malignant or autoimmune diseases (Terabe et al. 2000). Like conventional memory T cells iNKT cells do not absolutely require classical costimulatory signals to secrete cytokines following TCR engagement (Uldrich et al. 2005). Indeed, using CD1d0 or Ja180 deficient mice that allow to distinguish iNKT cells from non-classical NKT cells, iNKT cells have been suggested to regulate various infectious, malignant and autoimmune diseases (Bendelac et al. 2007; Brigl and Brenner 2004; Kronenberg 2005; Terabe et al. 2000; Van Kaer 2005; Mars et al. 2004). The mechanisms of iNKT cell activation and the role of specific iNKT cell populations under these various conditions have remained unclear. Recent studies have, however, identified endogenous and microbial GlycoSphingoLipid (GSL) ligands that are critical for the activation of iNKT cells during infection with Gram-negative bacteria (see also Sect. 3.7) and begun to elucidate the functional consequences of this activation.

3.2.2.2

CD1d–Restricted Diverse NKT Cells (Type II NKT Cells, Non-classical NKT Cells)

Whereas iNKT (type I NKT) cells are defined by their reactivity to D-GalCer, there exist other populations of CD1d-restricted NKT cells that do not respond to D-GalCer. This heterogeneous group of cells are summarized as type II NKT cells; they supposedly recognize (glyco-) lipids as well and are characterized by a more diverse TCR repertoire than iNKT cells (Terabe and Berzofsky 2008; Behar et al. 1999b; Cardell et al. 1995). Although D-ManCer has been suggested as a potential ligand for a subpopulation of these type II NKT cells expressing a VD19-JD33 TCR

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(Shimamura et al. 2006, 2007, 2008), another study could not confirm this observation (Huang et al. 2008). As these non D-GalCer – reactive NKT cells form a very heterogeneous group of different cells and constitute at most organs sites for less than 1% of the T cells, the identification of single cell populations within these type II NKT cells is likely to be very problematic and may explain at least in part some of the discrepant results. Nonetheless, type II NKT cells have been described as critical for the promotion of liver pathology in a hepatitis B virus model (Vilarinho et al. 2007) and may play a role in the suppression of tumor immunity (Terabe and Berzofsky 2008). Although some progress has been made in characterizing and classifying these diverse NKT cell populations, their functional role and physiological ligands have remained unknown so far.

3.2.3

Group III CD1 Molecules (CD1e)

CD1e is present in many different animal species (De Libero and Mori 2006). CD1e protein is non-covalently associated with E2-microglobulin and shares an overall structure similar to that of the other CD1 molecules. However, rather than presenting antigens to TCRs, CD1e allows the processing and loading of microbial antigens with a large carbohydrate component onto CD1b as shown for phosphatidylinositol-mannosides (PIM6) (de la Salle et al. 2005). Thus, CD1e is indispensable for processing of glycolipids and lipoglycans to become optimally immunogenic and to ensure effective protective immunity to microbes expressing these antigens.

3.2.4

Species Distribution of CD1 Molecules

The CD1 system is highly conserved (Porcelli and Modlin 1999; Beckman and Brenner 1995) with CD1 analogues found in almost all mammalian species (Bendelac et al. 2007; Dascher and Brenner 2003) and in chicken (Miller et al. 2005; Salomonsen et al. 2005). The existence of CD1 in birds suggests that CD1 or its immediate ancestor was present in an ancient version of the MHC locus that arose several 100 million years ago as part of the evolving, early adaptive immune system. It is suspected that even jawed fish may contain CD1 (Dascher 2007). Different CD1 isotypes specialize in sampling partially overlapping sets of lipids in different cellular compartments, including the endosome and lysosome through tyrosine containing cytoplasmic motifs that bind adaptor proteins for clathrinmediated endocytosis. CD1 isoforms are also very diversely distributed in mammalians (Barral and Brenner 2007) and the functional consequences of this diverse distribution for the single species are not known; for example, humans contain one CD1a, b, c, d and e allele. Guinea pigs instead possess an extended family of CD1 genes (Dascher et al. 1999), where more than 11 potential CD1 genes including four CD1B and three CD1C orthologs were identified, whereas CD1d is the only

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representative in mice (Brigl and Brenner 2004). Rabbits show another pattern of distribution for CD1 expression: two CD1A genes and one CD1E gene (Hayes and Knight 2001) as well as orthologs for CD1B and CD1D have been reported (Calabi et al. 1989). Although iNKT cells, based on genomic and functional studies of CD1d, may be absent in cows (Van Rhijn et al. 2006), group I CD1 genes are detected predisposing this animal species for the analysis of Mycobacteria-infection and group I CD1 restricted T cells in vivo. In addition, the expression of the CD1 isoforms on different immune cells in humans is very diverse: CD1a is widely used as a key marker of Langerhans cells, although several DC subsets express CD1a as well (Crawford et al. 1989; Fithian et al. 1981; Sholl et al. 2007). Human Langerhans cells typically lack CD1d in situ, while dermal DCs and monocyte-derived DCs express CD1d (Caux et al. 1992; Gerlini et al. 2001; Nestle et al. 1993; Ochoa et al. 2008). The distribution of CD1 isoform expression and the accumulation of distinct immune cells at the site of infection/inflammation may therefore have important implications for the protection/susceptibility to certain pathogens, malignant diseases or inflammatory/ autoimmune disorders. As iNKT cells are the best characterized cell population and their biology can be studied in vivo in mice, we will focus in the following paragraphs exclusively on iNKT cells.

3.3

iNKT Cell Function

Our understanding of the function of iNKT cells is largely based on disease studies in iNKT cell-deficient CD1d0 or JD180 mice (Swann et al. 2004). iNKT cells play a role in the detection of bacteria and viruses as different as Pseudomonas aeruginosa, Streptococcus pneumoniae, Salmonella typhimurium, Mycobacterium tuberculosis, Listeria monocytogenes, Borrelia burgdorferi, Chlamydia spp., influenza, encephalomyocarditis, coxsackie B3 virus or cytomegalovirus (Nieuwenhuis et al. 2002; Kawakami et al. 2003; Brigl et al. 2003; Behar et al. 1999a; Szalay et al. 1999; Ranson et al. 2005; Kinjo et al. 2006; Kumar et al. 2000; Joyee et al. 2007, 2008; De Santo et al. 2008; Ilyinskii et al. 2006; Huber et al. 2003; Broxmeyer et al. 2007). iNKT cells were also shown to be key players in the natural rejection of chemicallyinduced primary sarcomas (Crowe et al. 2002), as well as transplanted tumors, either by enhancing or suppressing their immune rejection through the release of Th1 or Th2 cytokines, respectively. Again, neither the mechanism of iNKT cell activation nor the bias towards Th1 or Th2 cytokines in the respective mouse models has been elucidated so far. A role of iNKT cells in non-infectious inflammatory processes such as type I diabetes (T1D) (Gombert et al. 1996; Falcone et al. 1999; Godfrey et al. 1997; Hong et al. 2001; Sharif et al. 2001), allergic asthma (Akbari et al. 2003; Lisbonne et al. 2003; Meyer et al. 2007, 2008), systemic lupus erythematodes (SLE) (Zeng et al. 1998, 2000, 2003; Forestier et al. 2005) and ulcerative colitis (Fuss et al. 2004; van Dieren et al. 2007) has been reported as well.

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The involvement of iNKT cells in such diverse conditions is perplexing because it is unclear what common antigens could be induced and what the mechanisms of iNKT cell activation are. However, iNKT cells have been shown to be inherently autoreactive (Bendelac 1995a, b, c) and their autoreactivity is under the control of inhibitory NK receptors suggesting a model whereby induction of the self antigen(s) of iNKT cells combined with the loss of inhibitory MHC I ligands would result in their net activation. In addition, the fortuitous discovery of an agonist iNKT cell ligand, alpha-GalactosylCeramide (D-GalCer) (Kawano et al. 1997) and its use in vivo to characterize the consequences of iNKT cell activation, have shed some light on the cellular circuits and molecular mechanisms involved. D-GalCer was isolated from marine sponge extracts based on their ability to elicit potent rejection

a

Hydroxyl groups Anomericity

a-GalCer

HO

a-D-Galactose

OH O

HO

HN

OH

HO O

Linkage

Ceramide portion

Sphingomonas/Novosphingobium GSLs Glucuronosyl Series

HO

OH

HO HO

Sphingosine base

OH

Sugar moiety

b

Acyl chain

O

COOH O

O HO

HN

HO

HO O OH

c

OH

O HN

O OH

HO

iGb3

Galacturonosyl Series COOH O

HO

OH O HO

OH

O

OH O

O OH HO

O OH O

HN O

HO

iGb3

OH

Gal a1,3 Gal b1,4 Glc b1,1 Ceramide

Fig. 3.1 Glycosphingolipid (GSL) antigens of iNKT cells. (a) Marine sponge derived A-GalCer (KRN 7000), the prototypical iNKT cell ligand with a detailed description of its single components. (b) GSL-1 of Sphingomonas/Novosphingobium spp. has structural analogies with KRN 7000; GSL-1 containing either one glucose or one galactose head (alpha-glucuronylceramide or alphagalacturonylceramide) is a potent stimulator of cytokine production by iNKT cells compared to GSL-3 and GSL-4 that are not immunogenic. The rearrangement of their cell wall GSL composition may be used by Sphingomonas/Novosphingobium spp. to evade their recognition by the innate immune system. (c) Mammalian isoglobotrihexosylceramide (iGb3), or GalA1, 3GalB1, 4GlcB1, 1Cer, one of the endogenous physiological iNKT cell ligands. Note that the proximal glucose of iGb3 has a beta-anomeric linkage to ceramide, in contrast with the alpha-branched galactose or glucose of A-GalCer or Sphingomonas/Novosphingobium GSLs. However, the terminal sugar in the alpha-linkage appears to be predominantly responsible for the antigenicity of iGb3

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G IFN-

-G IFN

-G

MO

CD8+ N IF

NK 40 /CD L 40 CD

CD4+ DC

iNKT IL-12 ? Gr

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B IL-4

?

CD4+ CD25+

Fig. 3.2 Innate-like iNKT cell functions and the cellular and molecular network activated downstream of iNKT cell stimulation. Cellular and molecular networks activated by iNKT cells upon recognition of A-GalCer by the iNKT cell TCR. DCs and macrophages/Kupffer cells are the center of a cellular network of cross-activation, starting with the presentation of A-GalCer by CD1d to the iNKT cell TCR and the subsequent upregulation of CD40L on iNKT cells upon A-GalCer recognition, secretion of Th1 and Th2 cytokines and chemokines, and DC mediated priming of adaptive CD4 and CD8 T cell responses. iNKT cells provide also direct help to B cells for antibody production or indirect help via the activation of T helper cells; iNKT cells also rapidly trans-activate NK cells. Although interactions of iNKT cells with granulocytes and Tregs are suspected, no experimental evidence for the underlying mechanisms for these interactions has been provided to date

of B16 melanoma cells in mice (Kobayashi et al. 1995; Morita et al. 1995). D-GalCer consists of a sugar head group and a ceramide tail that can be divided in an acyl chain and a sphingosine base (Fig. 3.1). In vivo, it was shown using systemic administration of D-GalCer that iNKT cells participate in a prompt and widespread cellular and molecular activation cascade (Fig. 3.2). The sequence initially involves cognate interaction with, and cross-activation of CD1d expressing dendritic cells (DCs) and macrophages through CD40L/CD40 interactions, explosive release of cytokines and chemokines and powerful trans-activation of natural killer (NK) cells within 30–60 min (Ishikawa et al. 2005b; Fujii et al. 2003, 2004; Carnaud et al. 1999). Likewise, B cells, which also express CD1d, are activated to upregulate their costimulatory properties (Galli et al. 2003a, b) and antibody production referentially releasing IgG2a antibodies (Mattner et al. 2008). Besides these cognate interactions (Galli et al. 2003a, b; Mattner et al. 2008; Leadbetter et al. 2008), iNKT cells can help B cells also indirectly, via the enhancement of T helper (Th)-B-cell

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interactions (Galli et al. 2007; Tonti et al. 2009) and/or the release of cytokines or chemokines. iNKT cell ligands have been shown to constitute one of the most efficient adjuvant available (Fujii et al. 2003; Gonzalez-Aseguinolaza et al. 2002), particularly for antibody production and for priming cytotoxic T lymphocytes (CTLs) against non-replicating antigens, and are widely used in clinical trials (Giaccone et al. 2002; Nieda et al. 2004; Ishikawa et al. 2005a; Chang et al. 2005). In summary, the anti-microbial activity of iNKT cells could be due to direct elimination of CD1d expressing infected antigen presenting cells (APCs), cross-activation of NK cells, priming of MHC class I restricted anti-pathogen CTLs, and/or indirect cytokine/chemokine effects on the infected tissue or cell populations. The respective mechanisms involved in defending defined bacteria, viruses, parasites and fungi need to be elucidated in the respective infection models in the future.

Fig. 3.3 Loading of CD1d with GSLs within the late endosome/lysosome. Newly biosynthesized CD1d molecules reach the plasma membrane and are internalized through an AP-2/AP-3 clathrindependent pathway and trafficked to late endosomal/lysosomal compartments, where the exchange of GSLs is performed by lipid transfer proteins (LTPs). One important group of LTPs constitute the saposins that facilitate also the loading of CD1d with GSL antigens. CD1d extensively recycles between lysosome and plasma membrane, allowing further GSL exchange. Exogenous lipids bound to lipoproteins may enter the cell with VLDL (very low density lipoprotein) particles through the LDL receptor pathway, whereas microbial lipids can be released in the lysosome after fusion with the microbial phagosome and digestion of the microbe. The endogenous ligand iGb3 is produced through lysosomal degradation of iGb4 by B-hexosaminidase. Upon recognition if the sugar head of GSL antigens by the iNKT cell TCR, iNKT cells rapidly release copious amounts of Th1 and Th2 cytokines and chemokines

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3.4

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CD1d Mediated Presentation of GSLs

CD1d is a conserved family member of the MHC like CD1 molecules expressed on hematopoietic cells and APCs, whose general function is to capture cellular and microbial GSLs for presentation to the iNKT cell TCR (Bricard and Porcelli 2007). The CD1d lipid binding groove is composed of connected hydrophobic channels where the fatty acid (=ceramide) portion of the GSL is buried, whereas the polar sugar head is exposed for recognition by the iNKT cell TCR (Brigl and Brenner 2004; Zajonc et al. 2005; Koch et al. 2005; Borg et al. 2007). Mouse and human CD1d collect GSLs from the late endosome and lysosome, and their antigen presenting capacities are tightly dependent on lysosomal functions (Joyce and Van Kaer 2003). Endosomal trafficking is required for CD1d to acquire the natural ligands of iNKT cells in APCs (Chiu et al. 2002), and recently characterized lysosomal saposins and microsomal triglyceride transfer protein (MTP) as the lipid transfer proteins (LTPs) promote the editing of the GSL antigens (Zhou et al. 2004a; Winau et al. 2004; Kang and Cresswell 2004; Brozovic et al. 2004; Dougan et al. 2005, 2007) (Fig. 3.3). Saposins are also critical for the presentation of the natural endogenous iNKT cell ligand(s) by CD1d on the cell surface, as shown by the absence of iNKT cell activation in co-culture with saposin-deficient DCs in autoreactivity assays (Bendelac et al. 2007).

3.5

Self-GSL Antigens for CD1d Molecules

Reactivity against CD1d molecules in the absence of exogenous ligands like D-GalCer is a hallmark of peripheral iNKT cells and a requirement for their thymic selection and differentiation (Bendelac 1995a, b, c; Chen et al. 1997). Presentation of endogenous GSLs by CD1d underlies this autoreactivity of iNKT cells. As no experimental evidence has been reported to date that mammalian species can synthesize alphalinked GSLs, beta-linked GSLs residing within the late endosomes or lysosomes were considered as potential endogenous candidate antigens (Chiu et al. 1999, 2002; Bendelac et al. 1995; Roberts et al. 2002; Park et al. 1998; Spada et al. 1998; Stanic et al. 2003). Although being controversially discussed to date (Speak et al. 2007; Porubsky et al. 2007; Burrows et al. 2009), isoglobotrihexosylceramide (iGb3) has been identified as one of the physiological endogenous ligands underlying the autoreactivity and selection of iNKT cells (Zhou et al. 2004b; Li et al. 2009) (Fig. 3.1c). iGb3 stimulates human and murine iNKT cells, albeit its stimulatory capacity is lower than the one of D-GalCer (Zhou et al. 2004b; Xia et al. 2006). The activation of iNKT cells by iGb3 is blocked by the absence of CD1d in mice and by the application of anti-CD1d antibodies in humans and the autoreactivity of VD14 expressing iNKT cell hybridomas is lost when stimulated with thymocytes from littermates lacking the E-hexosaminidase B (=Hexb) enzyme. This E-hexosaminidase B enzyme is required for the generation of iGb3 by the mammalian GSL degradation pathways.

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However, iGb3 is also synthesized by the iGb3 synthase in the lysosome. Thus, the generation of GSLs is tightly controlled and the existence of two iGb3 generation pathways emphasizes the importance of the endogenous GSL supply for iNKT cells. These dual pathways may have more broad implications outside iNKT cell immunology and may be also important for the maintenance of some other likely evolutionary conserved functions as the GSL metabolism can be regulated at multiple levels, including through the control of enzyme expression, post-translational modifications and allosteric mechanisms (Bendelac et al. 2007). Both enzymes E-hexosaminidase B and iGb3 synthase are ubiquitously expressed in different tissues of mice (Speak et al. 2007). In the lysosome/late endosome of APCs, iGb3 can be loaded into CD1d and presented to the iNKT cell TCR as CD1d is constantly cycling between the late endosome/lysosome and the cell surface. Selflipid antigens like iGb3 may be also important in the folding and stable assembly of CD1d molecules. In some cases these endogenous antigens can be replaced by microbial antigens or other self-antigens while they traffic through the endocytic system. In general, the self-lipids reported to date are weaker agonists than the microbial antigens (see below) or D-GalCer (Bendelac et al. 2007; Zhou et al. 2004b; Gumperz et al. 2000). In addition, changes in the self-GSL repertoire in APCs upon microbial exposure have been found to skew towards more stimulatory self-lipids (De Libero et al. 2005; Paget et al. 2007) which may enhance autoreactivity of iNKT cells, although accessory signals are likely required for the full activation of iNKT cells (see below).

3.6

Activation of iNKT Cells During Bacterial Infection

Although iNKT cells are activated during infection with many different microbes including bacteria, viruses, parasites and fungi (summarized in (Bendelac et al. 2007; Brigl and Brenner 2004; Kronenberg 2005)), the mechanisms of iNKT cell activation have often not been elucidated. However, two alternative strategies for iNKT cell activation by Gram-negative bacteria have been described that induce functionally different iNKT cell responses. These two pathways that distinguish between the activation of iNKT cells by endogenous and exogenous GSL antigens likely have broader implications for iNKT activation by other pathogens:

3.6.1

Indirect, Bystander Activation of iNKT Cells by Gram-Negative, LPS-Positive Bacteria

Autoreactivity induced by endogenous GSL ligands like iGb3 and the presence of interleukin-12 (IL-12), triggered by Toll-like receptor (TLR) signaling of infected APCs, are required for the early secretion of IFN-J by iNKT cells during

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immune responses against Gram-negative, LPS-positive bacteria (Brigl et al. 2003; Mattner et al. 2005) (Fig. 3.4a). TLR signaling on APCs may be thereby not only important for the induction of inflammatory cytokines, but also for the accumulation of endogenous GSL antigens that may enhance the autoreactivity of iNKT cells (De Libero et al. 2005; Paget et al. 2007). In this context, it has been reported that Toll-like receptor (TLR) signaling inhibited alpha-Gal-A activity in APCs triggering subsequent accumulation of GSL antigens including iGb3 in the lysosome and enhanced iNKT cell activation (Darmoise et al. 2010). However, microbial invasion is detected by TLRs expressed on APCs, and iNKT cells function downstream of this primary event. iNKT cells are also just one of several IFN-J-producing cell-types recruited during infection with Gram-negative, LPS-positive bacteria like Salmonella (Brigl et al. 2003). This may explain why iNKT-deficient mice do not appear to be particularly susceptible to Salmonella.

Fig. 3.4 Dual recognition of self and microbial GSLs during microbial infections. (a) Infection by Gram-negative, LPS-positive bacteria like Salmonella activates TLR4 through LPS and induces IL-12, augmenting iNKT cell autoreactivity induced by endogenous GSL ligands like iGb3 (indirect microbial recognition). (b) Infection by Gram-negative, LPS-negative Sphingomonas/ Novosphingobium spp. induces the direct activation of iNKT cells through recognition of microbial cell wall alpha-glucuronylceramides or alpha-galacturonylceramides. Subsequent DC activation through CD40/CD40L interactions and IL-12 release augments the activation of iNKT cells

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3.6.2

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Direct, Cognate Recognition of GSL Antigens in the Cell Wall of Gram-Negative LPS-Negative Alphaproteobacteria

Another pathway of iNKT cell activation involves the direct recognition of microbial GSLs in the cell wall of Gram-negative alphaproteobacteria (Mattner et al. 2005; Kinjo et al. 2005; Sriram et al. 2005) that substitute for lipopolysaccharide (LPS) (Kawahara et al. 1999; Kawahara et al. 2001). Specifically, alphagalacturonylceramides or alphaglucuronylceramides in the cell wall of Sphingomonas/ Novosphingobium spp. have been identified as strong iNKT cell antigens (Mattner et al. 2005, 2008; Kinjo et al. 2005) (Fig. 3.1b). Thereby, only the alphagalacturonylceramides or alphaglucuronylceramides (summarized as GSL-1) that express one sugar were stimulatory (Long et al. 2007). Alphagalacturonyl- or -glucuronylceramides containing three or four sugars (summarized as GSL-3 and GSL-4) did not stimulate iNKT cells in co-culture with DCs and are likely not truncated into GSL-1 as the glycosidase necessary to cleave glucosamine from GSL-3 and 4 might be absent or inactive in the lysosom. Although most Sphingomonas/Novosphingobium spp. express preferentially GSL-1, changes in the ratio of GSL-1 to GSL-3 and - 4 might be exploited by bacteria to avoid detection under certain circumstances. Nonetheless, iNKT cell-deficient CD1d−/− and JD18−/− mice clear an infection with Sphingomonas/Novosphingobium and other LPS-negative alphaproteobacteria like Ehrlichia (Lin and Rikihisa 2003) slower than wild type mice (Mattner et al. 2005; Kinjo et al. 2005; Sriram et al. 2005; Stevenson et al. 2008). Thus, iNKT cells and CD1d represent a major innate recognition pathway for this class of bacteria. As both bacterial species are ubiquitously found in the environment (Barbeau et al. 1996; Brodie et al. 2007; Cavicchioli et al. 1999; Selmi et al. 2003; Shi et al. 2001) and as TLRs appear to be partially (Mattner et al. 2005, 2008) or completely (von Loewenich et al. 2004) dispensable for their detection, it is tempting to speculate that iNKT cells may have evolved due to microbial pressure to specifically target alphaproteobacteria lacking cell-wall ligands for TLRs. Intravenous injection of high doses of Sphingomonas causes also septic shock in wild type, but not iNKT cell-deficient mice highlighting the importance of iNKT cells in promoting pathological cytokine storms (Mattner et al. 2005). Although some of the remaining TLR ligands in Sphingomonas may induce DC activation, its cell wall GSLs subsequently induce DC activation, probably through CD40L/CD40 interaction with iNKT cells (Fig. 3.3) and subsequent IL-A2 production, as reported for D-GalCer (Fujii et al. 2003, 2004) suggesting that preferentially the iNKT cell signal is critical for the activation of the innate immune response. These distinct innate detection pathways for the two groups of bacteria may not only affect early immune responses, but also influence the adaptive immune reaction. This discovery of GSL antigens in LPS-negative alphaproteobacteria may even have broader implications: it is known that extracts from the marine sponge Agelas mauritianus have different immunological properties depending on the season and their location. As this marine sponge was the original source where D-GalCer was identified from and as these sponges are often colonized by D-proteobacterial

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symbionts, particularly by Sphingomonas spp. (Dieckmann et al. 2005) which have been also described as coral pathogens (Cavicchioli et al. 1999; Rosenberg and Ben-Haim 2002; Miller et al. 2003), the marine sponge D-GalCer may in fact have originated from bacterial symbionts. The presence of iNKT cell antigens within the marine environment supports the suspicion that CD1 may be even present in jawed fish (Dascher 2007). Alpha-galactosyldiacylglycerols expressed by Borrelia burgdorferi (Kinjo et al. 2006), the Gram-negative LPS-negative agent of Lyme disease, as well as purified phosphatidylinositolmannoside (PIM4) of Mycobacterium tuberculosis (Fischer et al. 2004) have been also described as microbial iNKT cell ligands.

3.7 3.7.1

Activation of iNKT Cells During Non-bacterial Infection Viral Infections

Several reports have also addressed the role of iNKT cells during viral infections: for example, iNKT cells accelerate the clearance of influenza A (De Santo et al. 2008), encephalomyocarditis (Ilyinskii et al. 2006) or coxsackie B3 virus (Huber et al. 2003), whereas the viral burden and the subsequent course of infection remains unaffected in the absence of iNKT cells during infections with lymphocytic choriomeningitis virus (Roberts et al. 2004), mouse cytomegalovirus (Broxmeyer et al. 2007), and vaccinia virus (Renukaradhya et al. 2005) despite iNKT cell activation. Although these studies implicate a role of iNKT cells in the detection of these viral pathogens, other viruses have developed mechanisms to evade the immune recognition by iNKT cells: HSV-1 specifically inhibits the recycling of CD1d from the lysosome to the cell surface (Yuan et al. 2006), an essential pathway for GSL antigen presentation to iNKT cells. The HIV proteins Nef and gp120 downregulate the expression of CD1d on the cell surface as well (Chen et al. 2006; Cho et al. 2005; Hage et al. 2005), and the infection of T cells expressing CD4 may contribute to reduced iNKT cell numbers in HIV patients, although no studies have investigated that question and its functional consequences to date. However, absence of iNKT cells may contribute to the lethal outcome of infections with Epstein-Barr virus in patients with X-linked lymphoproliferative (XLP) immunodeficiency syndrome due to SAP and XIAP mutations (Pasquier et al. 2005; Rigaud et al. 2006; Nichols et al. 2005). Although SAP-Fyn signaling is important for the development of iNKT cells (Griewank et al. 2007) and although SAP deficient mice do not as efficiently clear gammaherpesvirus 68, the equivalent of EBV as wild type mice (Yin et al. 2003), patients with XLP suffer from additional immune defects affecting preferentially CD8+ T and NK cell responses that may contribute to the susceptibility to EBV. Those defects need to be evaluated in the context of iNKT cell deficiency. The abundance of iNKT cells in the liver may on the other hand contribute to the antiviral effects of D-GalCer in a transgenic Hepatitis B virus (HBV) model (Kakimi et al. 2000);

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however, the application of D-GalCer as monotherapy for patients with chronic hepatitis B infection resulted in a strong decrease of iNKT cells and did not clearly affect HBV DNA and alanine aminotransferase levels (Woltman et al. 2009). The nature of the iNKT cell ligand(s) and the mechanism(s) of iNKT cell activation involved in these infectious conditions remain to be determined. It also needs to be elucidated which of these effects reflect a specific viral evasion or an immune defense strategy (Bendelac et al. 2007).

3.7.2

Parasitic, Helminth and Fungal Infections

Glycosylphosphatidylinositol (GPI)-anchored surface antigens of Plasmodium have been reported as iNKT cell ligands (Schofield et al. 1999). GPI-activated iNKT cells promote the production of IgG antibodies to the circumsporozoite antigens of malaria, one of the key components of protective immune responses in humans. However, additional studies could not confirm the role of GPIs in iNKT cell activation and the CD1d dependency of antibody responses (Molano et al. 2000; Romero et al. 2001). iNKT cells are also activated during infections with other parasites including different Trypanosoma, Leishmania and Schistosoma spp.; however, the functional relevance of this activation appears to be species specific and the anti-parasite responses may be controlled by different iNKT cell subsets (Duthie and Kahn 2005, 2006; Duthie et al. 2005a, b; Procopio et al. 2002; Stanley et al. 2008; Wiethe et al. 2008; Campos-Martin et al. 2006; Mattner et al. 2006; Ishikawa et al. 2000; Mallevaey et al. 2006, 2007). It is believed that TLR-induced IL-12 or other inflammatory cytokines augment the CD1d-mediated iNKT cell autoreactivity during most of these infections (indirect, by-stander iNKT cell activation – see also Sect. 3.6.1). However, a subset of liver iNKT cells recognizes lipophosphoglycan of Leishmania donovani (Amprey et al. 2004) (direct, cognate recognition of iNKT cell antigens – see also Sect. 3.6.2) and the seize of the pool for this iNKT cell subset may control parasitic replication. iNKT cells also contribute to the clearance of Leishmania spp. in visceral organs (Mattner et al. 2006); however, parasite elimination at the peripheral sites during cutaneous Leishmaniasis may be mainly mediated by iNKT cell-independent mechanisms (Mattner et al. 2006) considering the very low iNKT cell numbers in peripheral lymph nodes (7 days). These results indicate that innate immunity remains intact despite the absence of IL-10. Although the lack of IL-10 promotes more rapid fungal clearance, it does not afford protection from the deleterious effects of neutralization of TNF-D or IFN-J (Deepe and Gibbons 2003). In IL-10−/− mice, memory T cells are increased in number and the potency of their protective activity is enhanced.

6.5.3

Chemokines and Chemokine Receptors

Forty members comprise the chemokine superfamily. They are divided into four (D to G) families based on the spacing of the first two conserved cysteines at the NH2 terminus. Another categorization is the inducible (i.e., inflammatory) or the constitutive chemokines. The former are produced in response to pathogens and mediate a number of effector properties including cell trafficking and T cell differentiation (Rossi and Zlotnik 2000; Sallusto et al. 2000; Mellado et al. 2001; Rot and von Andrian 2004). Chemokine receptors and chemokines manifest promiscuity which creates redundancy in chemokine actions (Sallusto et al. 2000).

6.5.3.1

CCR2 and Its Ligands

Mice lacking the chemokine receptor CCR2 are more susceptible to infection with H. capsulatum. These mice manifest several immunological defects including a decrement in the number of inflammatory cells recruited to the lungs. The principal reduction is in inflammatory 0M and DC. Progression of infection in CCR2−/− mice is caused by elevation of IL-4 that is produced by several cell populations including 0M and DC (Szymczak and Deepe 2009). This increase in IL-4 is also associated with an increase in signatures of alternatively activated MM including arginase 1, FIZZ1, Ym1, and transferrin receptor but not a decrement in NO, which is a typical consequence of an increase in arginase. A role for arginase in impairing immunity has been suggested since administration of L-arginine improves fungal elimination (Fig. 6.2). As previously mentioned, IL-4 alone may lead to death of animals that are otherwise intact. Since the lungs of CCR2−/− mice contain far fewer cells, especially phagocytes, it is likely that MM will encounter excessive IL-4 and thus become alternatively activated (Fig. 6.3). Consequently, their anti-Histoplasma activity is impaired. CCR2 binds four chemokines, and CCL2 exclusively binds CCR2. The murine CCL2-CCR2 axis is exceedingly important in shaping the development of type 1 or 2 immunity by altering generation of IL-12 or by promoting the production of IL-4 by activated T cells (Luther and Cyster 2001). The lack of CCL2 does not mimic the

Fig. 6.2 Treatment with L-arginine but not D-arginine modifies the fungal burden of CC2−/− mice infected intranasally with H. capsulatum. Wild-type (WT) or CCR2−/− mice were infected with 2 × 106 yeast cells intranasally and on day 6 they were given 7.5 mg of L- or D-arginine intraperitoneally. They were sacrificed on day 7, and the number of colony-forming units (CFU) from lungs was quantified. The results represent mean ± Standard error the mean (SEM) of six mice. * = p < 0.05

Fig. 6.3 The network operative in CCR2−/− mice. The inability of CCL2 and CCL7 to signal leads to increased production of IL-4 by a number of cell populations including CD4+, macrophages, and dendritic cells. IL-4 can act as a paracrine or in a autocrine fashion. One of the major effects of this excessive IL-4 is to alternatively activate macrophages. Thus, these cells upregulate several genes including arginase 1, FIZZ1, Ym1, and transferrin receptor. The increase in arginase 1 likely impairs killing, and elevated numbers of transferrin receptors promotes uptake of iron that facilitates growth. Arg 1 = arginase 1; IL-4 = interleukin 4, and TR = transferrin receptor

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absence of CCR2 in murine histoplasmosis since the CCL2−/− mice efficiently eradicate infection. Only the absence of CCL2 and CCL7 mirrors what is found in CCR2−/− mice (Szymczak and Deepe 2009). Hence, CCL2 and CCL7 cooperate to constrain IL-4 generation. The increase in IL-4 in CCR2−/− mice is in part caused by the paucity of DC migrating into lungs. Supplementation of lungs with antigenloaded DC suppresses IL-4 but not the fungal burden. The effect of DC is dependent on the presence of MHC class II and expression of CD40.The increased IL-4 levels and fungal burden in CCR2−/− mice are restored to that of wild-type when DC are transferred and CD4+ cell are eliminated prior to infection (Szymczak and Deepe 2010). This finding strongly suggests that a regulatory T cell population exists in Histoplasma-infected CCR2−/− mice.

6.5.3.2

CCR5 and Its Ligands

CCR5 is best known as a co-receptor for the human immunodeficiency virus (Oppermann 2004), but it is a mediator that is crucial for attracting a number of cell populations including regulatory T cells. Unlike the progressive infection in CCR2−/− mice, CCR5−/− manifest an accelerated elimination of fungus during the time when T cell-mediated immunity is activated. By contrast the infection in knockouts is slightly worse between days 3 and 7. The heightened severity during the innate response is probably a result of fewer inflammatory cells in lungs thus creating a suboptimal protective response. Accelerated elimination of Histoplasma at day 14 and beyond is caused by a shift in the regulatory T cell/Th17 balance. The numbers of the former are sharply depressed in lungs while there is a concomitant increase in Th17 cells (Fig. 6.4). Examination of the cytokine profile of the lungs provides an insight of why Th17 cells are increased. There is higher amounts of IL-6 and IL-23 and lower quantities of transforming growth factor (TGF)-E, three factors critical in shaping the emergence of the Th17 cells (Bettelli et al. 2007). Neutralization of IL-17A does increase fungal burden but only to the level of that observed in wild-type and does not cause a progressive infection (Kroetz and Deepe 2010). This effect of IL-17A neutralization is strikingly distinct from the modest effect observed in wild-type mice. Taken together these findings imply in murine histoplasmosis that a decrement of regulatory T cells promotes IL-17 but the converse is not true. Thus, regulatory T cells appear to be the dominant driving force in dictating that balance.

6.5.4

Leukotrienes

Leukotrienes are important to H. capsulatum elimination (Medeiros et al. 1999). Inhibition of leukotriene synthesis is associated with a decrease in IFN-J which may explain in part the impaired immunity. The absence of leukotriene synthesis hampers

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Fig. 6.4 CCR5 dictates the balance between regulatory T cells and Th17 cells. In wild-type mice there is a balance between these two populations. When IL-17A neutralized, there is not an expansion of regulatory T cells, but the balance is shifted. However, there is only a slight change in the fungal burden in these animals. In the absence of CCR5, the balanced is more dramatically tipped and favors Th17. The associated decrease in regulatory T cells is marked by increases in IL-6, IL-23, and IL-17A and a concomitant decrease in TGF-E. These changes are not observed in wildtype mice given anti-IL-17A. IL-6 = interleukin 6; IL-23 = interleukin 23; IL-17A = interleukin 17A; TGF-E = transforming growth factor-E, and Treg = regulatory T cells

the recruitment of memory T cells into lungs of mice. Moreover, the lack of leukotrienes is accompanied by an upregulation of IL-10, which is known to depress immunity, and TGF-E (Medeiros et al. 2008). It is tempting to speculate that the increase in IL-10 and TGF-E means that regulatory T cells may be activated in mice unable to synthesize leukotrienes.

6.6

Conclusions

This chapter summarizes the current findings regarding host control of the protective immune response to the pathogenic fungus, H. capsulatum. It highlights the many mediators and the cells that must cooperate to effect optimal clearance. Although much is known, there are still major gaps in our knowledge regarding molecular and cellular control of infection. Answers to the many unsolved questions will likely have a clinical impact as patients receive newer immunotherapies that may be beneficial but also may induce serious infectious complications. The knowledge gained from pursuit of these studies may facilitate the creation of biologicals that are more precise in modulating immunity.

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Chapter 7

Modulation of T-Cell Mediated Immunity by Cytomegalovirus Chris A. Benedict, Ramon Arens, Andrea Loewendorf, and Edith M. Janssen

7.1

Introduction

The herpesviruses have coevolved with their vertebrate hosts for over one hundred million years (McGeoch et al. 2000), resulting in a finely tuned equilibrium with the immune system. Consequently, all herpesviruses employ a multitude of strategies to modulate the host immune response, facilitating the establishment of lifelong latency and/or persistence in the face of a robust innate and adaptive immune response. Cytomegalovirus (CMV, a E-herpesvirus) is the largest of the herpesviruses, with a genome of ~230 kB in size encoding >200 open reading frames (orfs). Approximately ~60% of the encoded genes are not essential for replication of virus in tissue culture where there is no selective pressure from the host immune system and are predicted to perform immunomodulatory functions and facilitate establishment of latency (Murphy et al. 2003; Brocchieri et al. 2005). CMV directly targets dendritic cells (DC) and exploits the DC’s crucial role in the regulation of innate and adaptive anti-viral immune responses to promote replication and establish latency while preventing host pathology.

C.A. Benedict (*) Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA e-mail: [email protected] E.M. Janssen (*) Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, University of Cincinnati College of Medicine, Cincinnati, OH, USA e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_7, © Springer Science+Business Media, LLC 2012

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CMV Epidemiology and Pathology

Human (H)CMV is highly prevalent throughout all geographic locations and socioeconomic groups and infects –depending on the country- between 50% and 90% of a population. HCMV is usually acquired early in life as an asymptomatic, subclinical infection (Zanghellini et al. 1999). However, if primary HCMV infection occurs in the developing fetus or neonate (before full immune system development) consequences can be severe, with HCMV being the most common infectious cause of congenital birth defects (Gaytant et al. 2002). HCMV establishes a latent infection in many cell lineages, including monocyte precursors and stromal cells, and persists for life in its host. In healthy, persistently infected individuals huge numbers of CMV-specific T cells accumulate over time (i.e. “memory inflation”), a process recently associated with the immune risk profile and immunosenescence in the elderly (Pawelec et al. 2005). Reactivation of HCMV and subsequent systemic HCMV viremia can occur upon immunosuppression (e.g. organ transplant recipients, patients receiving immunosuppressive drugs, and HIV-infected patients) (Rubin 2001; Steininger et al. 2006), and these cases represent significant causes of morbidity and mortality if not controlled by antiviral drug therapy. Importantly, studies identify CMV infection as risk factor for increased arterial blood pressure, and a co-factor in aortic atherosclerosis (Streblow et al. 2008; Cheng et al. 2009). Recently, CMV infection in humans has been suggested to be associated with several human malignancies like glioblastoma. It has been proposed that CMV may directly infect tumor cells, or tumor stem cells, potentially increasing their malignancy through the process of oncomodulation (Michaelis et al. 2009).

7.3

Immune Response to CMV

Over the last decades, many cellular components, including DCs, NK cells, macrophages, B cells and CD8 and CD4 T cells have been identified to participate in the anti-CMV immune response. Although CMV replication is largely species restricted all CMVs show significant genomic homology and exhibit similar tissue tropism, pathogenesis and temporal regulation of gene expression (Rawlinson et al. 1996; Vink et al. 2000; Davison et al. 2003; McGregor et al. 2004; Powers and Fruh 2008a, b). As a consequence, research of CMV infection in animal models has accelerated the dissection of the molecular mechanisms that carefully balance CMV infection and host immune responses (Brocchieri et al. 2005).

7.3.1

DCs in CMV Infection

DCs have multiple key regulatory roles role in CMV infection. DCs play a crucial role in the initiation and regulation of the immune response, both through their

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direct production of key cytokines (e.g. Type I IFND/E and IL12) as well as through their interaction with NK cells. In addition, DCs are critical regulators of the adaptive T cell response to CMV. DCs not only activate naïve CD4 and CD8 T cells, they also dictate the acquisition of T cell effector functions and confer the capacity for T cell survival, homeostasis, and memory formation. Importantly, CMV also directly infects DCs and myeloid lineage cells, and these cells have been shown to be reservoirs of latent CMV infection (Sinclair 2008a, b; Rolle and Olweus 2009). Perhaps not surprisingly given their central role in promoting anti-viral immune responses, many viruses have evolved strategies to specifically modulate DC phenotype and/or function. CMV directly infects DCs in vivo, and encodes several gene products that specifically interfere with various aspects of DC function (Raftery et al. 2001) (Table 7.1).

Table 1 CMV immune modulatory gene products Gene

Primate CMV homologues

Antigen presentation MCMV m04 (gp34) MCMV m06 (gp48) MCMV m152 HCMV US2 (gpUS2)

RhCMV 182

HCMV US3 (gpUS3)

HCMV US6 (gpUS6) HCMV US10 (gpUS10) HCMV US11 (gpUS11) HCMV UL82 (pp71) HCMV UL83 (pp65) HCMV UL40 HCMV UL142 (gpUL142) RhCMV Rh178 Co-stimulation MCMV m138 (fcr-1) MCMV m147.5/modB7-2 HCMV UL144 (HVEM) homolog

RhCMV 184 RhCMV 189 RhCMV 112 (pp65-2)

Cellular target/interacts with Binding of MHC I, Complex with H2-Dk is recognized by Ly49P Reduces surface expression of MHC I Reduces surface expression of MHC I MHC I heavy chain degradation. HLA-DRA, HLA-DMA degradation. Assembly of MHC I. Reduces association of MHC II A/B complexes with the invariant chain. Inhibition of TAP MHC I MHC I heavy chain degradation MHC I Binding and inactivation of NKp30, Accumulation of MHC II Increases HLA-E, recognized by HLA-E restricted TCRAB+ CD8+ CTLs (MHC)–class-I–chain–related A (MICA), a ligand for NKG2D MHC I heavy chain translation Fc-portion of mIgG, B7-1, MULT-1, H60, Rae-1varepsilon B7-2 Binds BTLA; Activates NFKB (continued)

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Table 1 (continued) Gene Cytokine homologues HCMV UL111a (cmvIL-10); LAcmvIL-10 (alt. splicing)

Primate CMV homologues

Cellular target/interacts with

RhCMV IL-10, BaCMV IL-10, AGCMV IL-10

Binds hIL-10R. Inhibits IFN-G, TNF-A, MHC I and II and NFKB. Stimulates STAT 3. Induces IDO.

NK cell recognition MCMV m157 LY49h RhCMV: Rhesus CMV; BaCMV: Baboon CMV; AGCMV: African Green Monkey CMV

7.3.2

NK and NKT Cell Response to CMV, the “Innate-to-Adaptive Transition”

NK cells play a critical role in the innate or “early” immune control of CMV infection. Humans lacking NK cells cannot control HCMV infection (Biron et al. 1989). Similarly, mice lacking NK cells or the Ly49H NK cell receptor that recognizes the MCMV m157 protein show enhanced virus production in the spleen, lung and liver during the peak of infection and are more susceptible to CMV-induced death at highdose infection (Bukowski et al. 1984; Andrews et al. 2005; Andoniou et al. 2006). Recent studies show that optimal activation of NK cells requires interaction between DC and NK cells via NKG2D activating receptor and cytokine production (IL-12, IL-18, type I IFN) by DCs. In turn, activated NK cells accelerate the induction of anti-viral CD8 T cell responses through early control of the virus and prevention of overproduction of anti-viral type I IFN that would otherwise ablate DC populations and thereby delay the priming of CD8 T cells (Andoniou et al. 2005; Walzer et al. 2005; Robbins et al. 2007). In addition, NKT cells are also activated early during MCMV infection in an IL-12 and TLR9 dependent fashion, independently of antigen presentation by CD1d (Tyznik et al. 2008). As NKT cells promote both NK cell and T cell responses in various settings, this suggests a potential role for NKT cells in promoting the adaptive immune response to CMV infection (van Dommelen et al. 2003; Wesley et al. 2008).

7.3.3

CMV Specific CD8 and CD4 T Cell Responses

Although NK cells are important cellular players for early innate defense against MCMV, both CD8 and CD4 T cells play a critical role and are absolutely required for the eventual control and clearance of MCMV infection (Holtappels et al. 2008). The contribution of CD8 T cells was originally suggested by the inability of Balb/c SCID mice to control MCMV infection (Welsh et al. 1991), and that adoptively transferred CD8 T cells restrict systemic MCMV replication in irradiated

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Balb/c mice MCMV (Reddehase et al. 1985). Interestingly, B6 RAG−/− mice (lacking T and B lymphocytes) control MCMV replication effectively for the first ~25 days of infection, but eventually succumb to infection shortly thereafter due to in vivo selection of viral variants with mutations in m157 that circumvent NK-mediated control (French et al. 2004). Studies using a mouse model mimicking CMVreactivation and viral-induced pneumonia following bone marrow transplantation (a serious clinical complication associated with HCMV infection) indicate that CD8 T cells are strictly required for resolution of this disease, with CD4 T cells not being able to substitute in this particular model (Podlech et al. 1998). In humans, the presence of HCMV-specific CD8 T cells is associated with protection from virusinduced disease, and forms the basis for prophylactic cellular immunotherapy procedures for prevention of HCMV disease (Riddell and Greenberg 1997). HCMV-specific CD4 T cells correlate strongly with disease protection in patients. (Jonjic et al. 1989, 1990, 1994). Convincing data for a role of CD4 T cells in immune control of HCMV has come from the ubiquitous cases of HCMV-induced disease in AIDS patients, especially before the advent of highly active antiretroviral therapy (HAART) (Steininger et al. 2006). In mice, CD4 T cells can contribute to control of primary, systemic CMV infection, and are necessary to restrict persistent replication in select tissues and promote antibody responses (Walton et al. 2008). CD4 T cells are absolutely required for the control of MCMV replication in the acinar epithelial cells of the salivary gland, and can functionally substitute for immune defense to MCMV in mice depleted of CD8 T cells (Jonjic et al. 1989, 1990). Recent work has revealed that the apparent inability of CD8 T cells to control persistent MCMV replication at select anatomical sites (e.g. the salivary gland) is due, at least in part, to the efficient inhibition of MHCI-mediated antigen presentation by three separate immune modulatory genes (m04, m06 and m152) (Lu et al. 2006). There is an intertwined relationship for CMV-specific CD4 and CD8 T cell responses; Priming of CD8 T cell responses in the presence of CD4 T cell “help” promotes the generation and/or maintenance of specific subsets of MCMV-specific CD8 memory T cells (Janssen et al. 2003; Sun and Bevan 2003; Snyder et al. 2009). In line with this observation in the mouse, the long term maintenance of HCMVspecific CD8 T-cells in cellular immunotherapy procedures is greatly enhanced by the co-infusion of HCMV-specific CD4 T-cells (Greenberg and Riddell 1999).

7.3.3.1

Subsets of CMV-Specific T Cells

In HCMV infected individuals, gradual increase (“memory inflation”) a large number of oligoclonal HCMV-specific of distinctive memory CD4 and CD8 T cell subsets exists with diverse specificities for many viral antigens, and these T cell populations display an almost exclusive characterized by the surface marker phenotype of CCR7-CD27-CD28 (van Lier et al. 2003; van Leeuwen et al. 2004; Vescovini et al. 2007).Memory T cells of this specific phenotype are not observed nearly to this extent for other chronic virus-specific T-cell populations including HIV, EpsteinBarr virus and hepatitis C virus (van Lier et al. 2003). Both mouse and rhesus CMV

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also induce characteristic CD8 and CD4 T cell memory subsets that undergo memory inflation and exhibit a similar “effector memory” phenotype. (Pitcher et al. 2002; Karrer et al. 2003; Munks et al. 2006; Arens et al. 2008). During MCMV infection, distinct antigen-specific CD8 T cell responses develop that do not or minimally undergo contraction (T cell contraction is typically observed during viral infections). These unique CMV-specific responses increase to significant numbers over the course of the ensuing months and are maintained at relatively high numbers during the lifespan of the host (Sierro et al. 2005; Munks et al. 2006). Thus far it is unknown what determines the development of these phenotypically unique T cell responses during CMV infections. The immunomodulatory effects of CMV on DCs (as discussed later), the balance of CMV latency and reactivation and CMV’s cellular tropism might all directly or indirectly contribute. Importantly, the phenotype of these CMV-specific inflationary T cells does not correlate with that of “exhausted” memory T cells seen in models of chronic LCMV or HCV infection (Barber et al. 2006; Boni et al. 2007). It is quite likely that the “smoldering” persistent phase of CMV replication primes/restimulates T cells at a much lower level than does these other chronic viral infections, perhaps due to both viral immune modulation and a more modest antigen load.

7.4

Induction of Adaptive T Cell Responses

Adequate activation of T cells that results in proliferation and acquisition of effector functions has been shown to require multiple positive signals from the DC to the T cell. The first signal, which is antigen-specific, is provided through the TCR which interacts with peptide-MHC molecules on the DCs. The additional signals, the costimulatory signals, are antigen nonspecific and are provided by the interaction between co-stimulatory molecules expressed by the DC and the T cell.

7.4.1

Requirement for MHC-TCR Interactions

During infection DCs present viral antigens via their MHC class I molecules to CD8 T cells and via their MHC class II molecules to CD4 T cells. There are two basic pathways by which DCs can process and present viral antigens: the exogenous pathway where viral antigens originate from the uptake of dying infected cells, and the endogenous pathway, that requires direct infection of the DC (Mellman 2005; Trombetta and Mellman 2005; Vyas et al. 2008). Thus, CMV-infected DCs fall victim to the immune-evasive machinations of the virus, while uninfected DCs are able to present viral antigens solely from exogenous sources. In the classic exogenous antigen presentation pathway, the infected material will be shuttled through endosomal and lysosomal compartments where it is cleaved/proteolyzed by various enzymes. MHC class II molecules are synthesized

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in the endoplasmic reticulum (ER) and transported from the ER via the Golgi compartments to endosomal vesicles. These vesicles fuse with the vesicles in which antigen degradation occurs to form a specialized compartment where peptide loading onto MHCII molecules takes place. Subsequently, the peptide-MHC class II complexes are transported to the cell surface for presentation to T cells. In the non-classical exogenous pathway, peptides derived from extracellular antigens are presented by MHC class I molecules in a process called cross-presentation or cross-priming; the viral proteins are cleaved into peptide fragments by the multicatalytic proteasome complexes in the cytosol. The fragments are transported to the TAP transporter and passed to the endoplasmatic reticulum (ER) where peptide loading onto class I molecules occurs. The MHC class I – peptide complex is then transported to the cell surface via the Golgi apparatus and secretory pathway (Cresswell et al. 2005; Guermonprez and Amigorena 2005). The classical endogenous antigen presentation pathway predominantly involves MHC class I molecules and normally presents peptides derived from intracellular (self) antigens. Upon infection, CMV utilizes the protein synthesis machinery of the infected cell to replicate and as a consequence viral antigens have the capacity to be targeted for MHC class I presentation and to a lesser degree for MHC class II presentation.

7.4.2

Costimulation in T Cell Priming

Although MHC-peptide recognition by the T cell receptor (TCR) on the T cell is crucial for initial activation, it will lead to anergy or non-responsiveness without appropriate additional costimulation. Costimulation can be provided by: (i) B7.1 (CD80) and B7.2 (CD86) molecules on the DC that ligate CD28 on the T cell (Greenwald et al. 2005); (ii) TNF (receptor) family members (CD40-CD40L, CD27-CD70, CD134 (OX40)CD134L and CD137 (4-1BB)-CD137L, CD30-CD30L) (Greenwald et al. 2005); and (iii) soluble mediators such as cytokines and chemokines that support the survival of the T cells and influence the phenotype of the T cells. Importantly, DCs also express molecules that provide negative signaling and can prevent T cell activation or inhibit ongoing T cell responses which is crucial to maintain peripheral tolerance and prevent pathology associated with lymphoproliferative aberrations. Binding of B7 family members B7.1 and B7.2 to cytotoxic T lymphocyte antigen (CTLA)-4 on activated T cells has been shown to be critical in the inhibition of T cell responses. In addition, binding of the B7 family members programmed death ligand (PD-L1, CD274; B7-H1) and PD-L2 (CD273/B7-DC) to PD-1 on T and B cells, and TNF receptor family member herpesvirus entry mediator (HVEM), to B and T lymphocyte attenuator (BTLA) have been shown to negatively regulate T cell activation. Besides cell-associated inhibitory molecules, DC express various soluble molecules like interleukin (IL)-10, Tumor Growth Factor (TGF)-E, and indoleamine 2,3-dioxygenase (IDO) that have been shown to negatively affect T cell activation, and inhibit ongoing T cell responses (Greenwald et al. 2005).

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The current models view the activation of naive T cells as a sum of positive and negative signals dictated by the relative expression of ligands and receptors on T cells and DC. T cells receiving a “positive net signal” from DC will undergo proliferation and acquire effector functions. Based on the type of signals, CD4 T cells will develop into T-helper (Th) 1 cells, producing IFN-J, TNF-D, and IL-2, Th2 cells that produce IL-4, -5, and −13, regulatory T cells that have been shown to secrete IL-10 and TGFE, Th17 cells secreting IL-17, or follicular helper cells (Tfh) that secrete IL-21. CD8 T cells predominantly develop into IFN-J, TNF-D, and IL-2 producing T-cytotoxic (Tc)1 cells (Woodland and Dutton 2003),and to a lesser degree into Tc2 cells (IL-4, IL-5), IL17 producing Tc cells, or Tc with regulatory capacity. In contrast, T cells receiving a “negative net signal” from the DC will be deleted or become anergic/nonresponsive.

7.5

CMV Modification of T Cell Activating Molecules

Evidence is emerging that CMV alters the DC cosignaling rheostat, shifting the balance of positive and negative signals delivered to the T cells (Benedict et al. 2008; Sinclair 2008a, b; Rolle and Olweus 2009). Infection of DC by MCMV leads to the induction of a so-called “paralyzed” phenotype, characterized by the down-regulation of MHC class I and II, costimulatory molecules (e.g. B7 molecules) and proinflammatory cytokines (Andrews et al. 2001). As a consequence these infected DC are unable to promote mixed lymphocyte reactions (MLR) or activate T cells (Andrews et al. 2001; Mathys et al. 2003). Similar negative effects on T cell activation have been reported for HCMV infection of DCs or monocytes (Odeberg and Soderberg-Naucler 2001; Moutaftsi et al. 2002; Hertel et al. 2003). Importantly, recent studies show that infected DCs not only passively prevent T cell activation by restricting positive cosignals, but also actively suppress T cells by enhancing negative cosignals, potentially leading to very different biological consequences and subsequent fates for virus-specific T cells upon interaction with these virally manipulated DCs (Fig. 7.1).

7.5.1

Viral Modification of MHC Class I Expression

As MHC-peptide expression is an absolute requirement for the activation of CD4 and CD8 T cells, it is not unexpected that all CMVs have been found to encode multiple gene products that target MHC antigen presentation (Yewdell and Hill 2002). MCMV encodes three known genes that interfere with antigen-presentation through the MHC class I pathway: m04, m06, and m152 (Pinto, Munks et al. 2006). m152 retains MHC class I molecules in the ER-Golgi intermediate compartment, while m06 redirects MHC class I molecules to the lysosmes (Gold et al. 2002). Expression of either one or both of these gene products results in

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CMV infection DC

T cell TCR CD8

MHC B7.1 B7.2

+

CD28

+

+

BTLA

UL144 MHC

TCR CD8

B7.1 B7.2

CD28

CD40

CD40L

CD40

CD40L

PD-L1

PD-1

-

PD-L1

PD-1

+ IFN-  IL-12

cytokine receptor

positive net signal

proliferation effector function memory

-

cytokine receptor

+

-

-

IL-10 IDO

negative net signal

anergy tolerance deletion

Fig. 7.1 CMV mediated modulation of MHC class I/II and positive and negative cosignaling molecules on infected DC

downregulation of MHC class I expression on infected cells (Pinto et al. 2006). The precise mechanism by which m04 functions to restrict T cell recognition of MHCI-peptide remains unknown, as m04-MHCI complexes are cotransported to the surface of infected cells, but recent evidence indicates a key role for m04 in inhibiting NK cell-mediated recognition of infected cells through its interaction with H-2DK (Kleijnen et al. 1997; Kavanagh et al. 2001; Kielczewska et al. 2009). Comparably, HCMV encodes 4 glycoprotein products, US2, US3, US6, and US11 that interfere with MHC class I expression (van der Wal et al. 2002). US2 and US11 relocate the heavy chain of the MHC class I the cytosol where it is targeted for proteasomal degradation (Jones and Sun 1997; Lee et al. 2005). US3 has been shown to interfere with the MHC class I assembly pathway, while US6 inhibits peptide transport through the TAP pore preventing the loading of peptide into the MHC groove (Jones et al. 1996; Ahn et al. 1997). Recently Rhesus CMV has been shown to encode a novel inhibitor of MHC class I expression not encoded by HCMV or MCMV, Rh178. Unlike the MHC class I modulating genes m06, m152, US2, US3, US6 and US11 that affect existing MHC molecules, Rh178 targets the MHC class I heavy chain signal peptide and interferes with heavy chain translation (Powers and Fruh 2008a, b). Interestingly, infection of C57Bl/6 mice with an MCMV mutant virus lacking three “immunoevasion” genes that regulate MHC class I expression (m04/m06/ m152) did not alter the immunodominance hierarchy and magnitude of the CD8 T-cell response, and it has been hypothesized that this may be because CD8 T cell responses to CMV are largely generated/primed by DC via cross-presentation/

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exogenous pathways (Gold et al. 2004; Munks et al. 2007). It may also be that although CMV effectively reduces the expression of MHC class I on the surface of infected DC, this reduction is not complete, and is likely sufficient to lower the strength-of-signal that CMV-specific T cells receive as opposed to completely inhibiting it. In support of this, studies have indicated that sufficient MHC/peptide complexes remain expressed on the surface of CMV-infected DC to allow for the initiation of proximal events leading to T cell priming/activation, but restrict the full differentiation of these T cells to a full-blown effector phenotype (Benedict et al. 2008).

7.5.2

Viral Regulation of MHC Class II Expression

Like MHC class I, CMV infection has also been shown to reduce MHC class II expression. However, the molecular mechanisms that confer this phenomenon are not well understood. HCMV infected DC show delayed and decreased MHC class II biosynthesis (Lee et al. 2006; Kessler et al. 2008). When overexpressed US2 can promote the degradation of human leukocyte antigen (HLA)-DR- and -DM- (Rehm et al. 2002). Similarly, overexpression of US3 results in its binding to class II /ß complexes in the endoplasmic reticulum (ER), reducing their association with the invariant chain, preventing efficient trafficking of to the class II loading compartment and resulting in significantly reduced peptide-loaded class II complexes (Hegde et al. 2003). However, more recent experiments in both monocyte derived DC and Langerhans DC suggest that US2/US3 may not function similarly to inhibit MHCII in HCMV infected cells (Kavanagh et al. 2001; Lee et al. 2006). CMV has also been shown to target molecules involved in the proteolytic degradation process. HCMV was reported to decrease expression of MHC II-associated proteases cathepsins S, Z, B, H and L and asparagine-specific endopeptidase (AEP) which correlated with lessefficient proteolytic degradation of peptide substrates by HCMV-infected DCs in vitro (Kessler et al. 2008). Besides these direct mechanisms affecting antigen presentation, CMV has also been described to exploit indirect pathways to affect MHC expression. Various studies have indicated that virally encoded IL-10 reduces both MHC class I and class II expression and may impede anti-viral T cell responses both during acute and latent infection (Cheung et al. 2009; Slobedman et al. 2009).

7.5.3

Regulation of B7 Costimulation

T cells require costimulatory signals for optimal activation and acquisition of effector functions (Wang and Chen 2004; Greenwald et al. 2005). The B7-CD28 costimulatory pathway consists of B7.1 and B7.2 that both bind to the activating

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receptor CD28 and the inhibiting receptor CTLA-4. High levels of B7.1 and B7.2 expression are restricted to APCs. B7.2 is constitutively expressed at low levels and rapidly upregulated, whereas B7.1 is inducibly expressed. CD28 is constitutively expressed on the surface of T cells while CTLA-4 expression is rapidly upregulated following T cell activation. Engagement of CD28 on naïve T cells by either B7.1 or B7.2 ligands concomitant with TCR signaling provides a potent positive costimulatory signal to T cells, resulting in the induction of IL-2 transcription, expression of CD25 and entry into the cell cycle (Sharpe 2009). Importantly, TCR interaction in the absence of CD28 engagement leads to T cell tolerance, anergy or deletion (Wang and Chen 2004; Greenwald et al. 2005). The MCMV m138 gene product, originally identified as an Fc receptor homologue, facilitates the down-regulation of B7.1 (Mintern et al. 2006), while m147.5 inhibits expression of B7.2 (Loewendorf et al. 2004). Both of these viral proteins target the B7 proteins at a post-transcriptional level, similar to CMV targeting of MHC expression. Human CMV also down-regulates B7/1/B7.2 from the surface of infected DC -although the responsible gene product(s) are yet to be identified. The relevance of B7-CD28 interactions in promoting CMV-specific T cell responses was recently shown by our group. Mice lacking B7.1/B7.2 or CD28 showed marked reductions in the initial expansion of MCMV-specific CD8 and CD4 T cells, and never controlled persistent MCMV replication in the salivary gland (Arens et al. 2011a, b). Interestingly, MCMV-specific inflationary memory CD8T cell responses did reach levels equivalent to those seen in wild-type mice by ~ 100 days after infection, suggesting the regulation of these populations is different from the “stable” memory pool of MCMV-specific CD8 T cells (Snyder et al. 2008), but even these populations eventually crashed in mice lacking B7-CD28 signaling by day ~ 200 after infection. Importantly, using a mutant of MCMV incapable of restricting B7.1 and B7.2 expression resulted in selective increases in MCMV-specific CD4 and CD8 T cell responses when compared to wild-type virus, proving that CMV modulation of this costimulatory pathway does dampen virus-specific T cell responses in vivo. It is becoming apparent that other positive cosignaling pathways can enhance, alter or substitute for the requirement for B7-CD28 signaling. Interestingly, CMV has also been shown to impinge upon some of these pathways by downregulating CD40 and ICAM-1 (Moutaftsi et al. 2002; Benedict et al. 2008). In addition, HCMV infection has been reported to induce the shedding of CD83 from infected DC, also resulting in the inhibition of T cell proliferation (Senechal et al. 2004).

7.5.4

Modulation of Soluble Positive Cosignals

CMV has been shown to affect the production of DC-derived cytokines that play roles in the maturation of DC, activation of NK cells and the priming, survival and effector function acquisition of T cells. Although the molecular mechanisms of the inhibition of cytokine production has not been identified, various studies have shown that HCMV and/or MCMV inhibit production of IL-2, IL-6, IL-8, IL-12, and TNFD in infected

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DCs (Moutaftsi et al. 2002; Beck et al. 2003). Importantly, this reduced cytokine production is maintained when infected DC are exposed to activated T cells or inflammatory stimuli like LPS. Most reports indicate that only infected DCs show decreased capacity for cytokine production while other DC that encounter virus through other pathways show increased cytokine production (Andrews et al. 2001; Tyznik et al. 2008). However, various studies, using total DCs from individuals suffering from HCMV induced hepatitis or mononucleosis, indicate that disregulation of cytokine production can be observed in both infected and bystander DC (Varani et al. 2009).

7.6

Viral Modification of T Cell-Inhibitory Signals

To further tip the balance from a “positive net signal”, to a “negative net signal”, CMV has evolved many strategies to modulate molecules that provide negative cosignals in the priming of T cells.

7.6.1

Regulation Via the PD-1/PDL-1 Pathway

Recently the B7 family has been expanded with PD-L1 (CD274/B7-H1) and PD-L2 (CD273/B7-DC) that have been show to provide negative signals that limit, terminate and/or attenuate T cell responses through its receptor PD-1 (CD279) (Wang and Chen 2004; Greenwald et al. 2005). PD-L1 is constitutively expressed on many cells types of hematopoietic and non-hematopoietic origin, whereas PD-L2 is transiently induced on DCs and macrophages upon activation. PD-1 is inducibly expressed upon activation on T, B and NK-T cells and confers a negative signal when engaged simultaneously with the TCR or BCR (Sharpe 2009). Early studies reported that PD-1 signaling resulted in cell cycle arrest, whereas later studies indicate that PD-1 signaling promotes death, either through the direct engagement of death pathways or indirectly by down-regulating survival signals and growth factors (Latchman et al. 2001; Dong et al. 2002; Petrovas et al. 2006; Keir et al. 2008). Our recent study showed that MCMV-infected DCs actively upregulate PD-L1 while down regulating MHC class I/II and positive costimulatory molecules (Benedict et al. 2008). T cells activated by the infected DC had a stunted phenotype characterized by poor survival, poor proliferation and lack of acquisition of effector functions. Blocking PD-L1 on the DC or PD-1 on the T cells using antibodies significantly improved survival, proliferation and effector functions, illustrating the importance of “negative cosignaling” in the induction CMV-specific T cell responses (Benedict et al. 2008). Interestingly, in B7.1/B7.2 double deficient mice which show a significantly compromised initial expansion of MCMV-specific T cell responses, CD8 T cell responses can be restored to wild-type levels at day 8 by blocking PD-1 signaling, while PD-1/PD-L1 blocking studies in WT mice resulted in very modest or no increases in CD8 T cell responses (Arens et al. in press). In total, these results

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strongly suggest that the B7-CD28 pathway is closely intertwined and counterbalanced by the PD-L1/2-PD-1 pathway, both in the case of cross-presented CMV antigen and in DC directly infected by CMV. Besides the direct effect on the T cells, it has been suggested that reverse signaling through PD-L1 on DC by PD-1 reduces the expression of CD40, CD80 and CD86 and increases IL-10 production, thereby reinforcing the immune-suppressive or “paralyzed” DC phenotype (Blocki et al. 2006; Kuipers et al. 2006; Van Keulen et al. 2006).

7.6.2

Regulation of HVEM/BTLA Interactions

CMV also exploits additional negative cosignaling pathways by targeting the recently identified B and T cell attenuator (BTLA), another Ig superfamily member with homology to CTLA-4 and PD-1 (Ware 2008). The ligand for BTLA was recently found to be a member of the TNF receptor superfamily, the herpes virus entry mediator (HVEM/HveA), establishing a new paradigm in cross-family ligand receptor interactions (Ware 2008). All primate CMVs encode an orthologue of HVEM in their genome, the canonical member being the ul144 orf in HCMV (Lurain et al. 1999), which binds to BTLA and is a potent inhibitor of CD4 T cell proliferation. Interestingly, UL144 has lost its ability to bind to LIGHT, the TNFfamily cytokine that binds HVEM and functions as a positive costimulatory system for T cells. This highlights the coevolution of CMV with its host, usurping the negative cosignaling functions of HVEM-BTLA interaction, while “losing” the positive aspects of HVEM-LIGHT signaling in the UL144 protein (Benedict et al. 1999; Ware 2008).

7.6.3

Immune Regulation via Cytokines

Besides membrane-associated molecules, CMV has been shown to affect the induction, production and sensitivity to various cytokines and/or chemokines that interfere with the induction of adaptive responses. A prime example of this is the CMV vIL-10, which has been shown to suppress the induction of the immune responses. HCMV encodes a biological active IL-10 (UL111a) that has low sequence similarity to other vIL-10 molecules, and similar IL-10 orthologues are contained within all the primate CMV genomes (Lockridge et al. 2000). CmvIL-10 has been shown to decrease production of proinflammatory cytokines and chemokines involved in anti-viral responses. A recent study demonstrated that cmvIL-10, like IL-10, can directly suppress synthesis of type I interferons (IFN) in plasmacytoid dendritic cells (pDC). In addition, cmvIL-10 has been shown to prevent the upregulation of costimulatory molecules and MCH class I and II molecules (Muller et al. 1998; Chang et al. 2004). Besides the prevention of the induction of activating molecules,

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cmvIL-10 has been suggested to actively downregulate MHC class I and class II proteins while upregulating indoleamine 2,3-dioxygenase (IDO), a molecule with potent immunoregulatory activity (Raftery et al. 2004). Both IDO and IL-10 have been described to inhibit T cell activation, proliferation and acquisition of effector functions, while promoting the induction of regulatory T cells. Although all of these pathways would be excellent means of viral sabotage, the in vivo evidence that CMV usurps these pathways to shape the antiviral immune response is still sparse. Importantly, cmvIL-10 is transcribed during latency in myeloid cells, and is currently the only bonified HCMV latency-associated gene where a functional consequence has been demonstrated. A HCMV mutant with a UL111a deletion was compromised in its ability to establish, maintain and reactivate from experimental latency. Additionally, MHC class II levels increased significantly on the surfaces of cells infected with the deletion virus, indicating a functional role for cmvIL-10 in regulating antigen presentation in infected cells. Finally, HCMV-specific CD4 T cells responded more vigorously to myeloid progenitors infected with the UL111a deletion virus, demonstrating that viral IL-10 expression during latency plays an important role in the regulation of T cell responses. (Cheung et al. 2009; Slobedman et al. 2009).

7.7

Biological Consequences

The direct infection of DCs by CMV demonstrates the ingenious approach the virus has evolved to manipulate the anti-viral immune response in the host. Naïve virus-specific T cells encountering infected DC receive suboptimal MHC and positive costimulation, while receiving amplified levels of negative signals. These virus-specific T cells will undergo apoptosis, become anergic or non-responsive, or may develop a regulatory or suppressive phenotype. As a result, the virus has eliminated or modified a significant proportion of the virus-specific T cell population that could have been adequately activated by DC that present viral antigens from exogenous sources. Moreover, the production of immune-suppressive factors by infected DC may instill a less than optimal antigen-presenting capacity in noninfected DC, providing an additional mechanism for the fine-tuning of the quality, quantity and kinetics of the anti-viral response. CMV has shown to interfere with apoptosis pathways in the infected DC (Miller-Kittrell and Sparer 2009), allowing for extended persistence of the infected DC thereby maximizing the transient immune suppression during acute infection in the host. However, the immunesuppressive machinations of CMV may not only affect the induction of anti-CMV response; the possibility exists that these strategies may impose a more profound and/or generalized inhibition of immune responses. Although there is no direct evidence that individuals infected with HCMV show compromised immunity, there has been much recent speculation regarding the potential role that lifelong, persistent infection with CMV may have on promoting immune senescence in the aging population (Pawelec et al. 2005).

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Tyznik, A. J., E. Tupin, et al. (2008). “Cutting edge: the mechanism of invariant NKT cell responses to viral danger signals.” J Immunol 181(7): 4452–6. van der Wal, F. J., M. Kikkert, et al. (2002). “The HCMV gene products US2 and US11 target MHC class I molecules for degradation in the cytosol.” Curr Top Microbiol Immunol 269: 37–55. van Dommelen, S. L., H. A. Tabarias, et al. (2003). “Activation of natural killer (NK) T cells during murine cytomegalovirus infection enhances the antiviral response mediated by NK cells.” J Virol 77(3): 1877–84. Van Keulen, V. P., B. Ciric, et al. (2006). “Immunomodulation using the recombinant monoclonal human B7-DC cross-linking antibody rHIgM12.” Clin Exp Immunol 143(2): 314–21. van Leeuwen, E. M., E. B. Remmerswaal, et al. (2004). “Emergence of a CD4 + CD28- granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus infection.” J Immunol 173(3): 1834–41. van Lier, R. A., I. J. ten Berge, et al. (2003). “Human CD8(+) T-cell differentiation in response to viruses.” Nat Rev Immunol 3(12): 931–9. Varani, S., G. Frascaroli, et al. (2009). “Human cytomegalovirus targets different subsets of antigen-presenting cells with pathological consequences for host immunity: implications for immunosuppression, chronic inflammation and autoimmunity.” Rev Med Virol 19(3): 131–45. Vescovini, R., C. Biasini, et al. (2007). “Massive load of functional effector CD4+ and CD8+ T cells against cytomegalovirus in very old subjects.” J Immunol 179(6): 4283–91. Vink, C., E. Beuken, et al. (2000). “Complete DNA sequence of the rat cytomegalovirus genome.” J Virol 74(16): 7656–65. Vyas, J. M., A. G. Van der Veen, et al. (2008). “The known unknowns of antigen processing and presentation.” Nat Rev Immunol 8(8): 607–18. Walton, S. M., P. Wyrsch, et al. (2008). “The dynamics of mouse cytomegalovirus-specific CD4 T cell responses during acute and latent infection.” J Immunol 181(2): 1128–34. Walzer, T., M. Dalod, et al. (2005). “Natural killer cell-dendritic cell crosstalk in the initiation of immune responses.” Expert Opin Biol Ther 5 Suppl 1: S49-59. Wang, S. and L. Chen (2004). “Co-signaling molecules of the B7-CD28 family in positive and negative regulation of T lymphocyte responses.” Microbes Infect 6(8): 759–66. Ware, C. F. (2008). “Targeting lymphocyte activation through the lymphotoxin and LIGHT pathways.” Immunol Rev 223: 186–201. Welsh, R. M., J. O. Brubaker, et al. (1991). “Natural killer (NK) cell response to virus infections in mice with severe combined immunodeficiency. The stimulation of NK cells and the NK celldependent control of virus infections occur independently of T and B cell function.” J Exp Med 173(5): 1053–63. Wesley, J. D., M. S. Tessmer, et al. (2008). “NK cell-like behavior of Valpha14i NK T cells during MCMV infection.” PLoS Pathog 4(7): e1000106. Woodland, D. L. and R. W. Dutton (2003). “Heterogeneity of CD4(+) and CD8(+) T cells.” Curr Opin Immunol 15(3): 336–42. Yewdell, J. W. and A. B. Hill (2002). “Viral interference with antigen presentation.” Nat Immunol 3(11): 1019–25. Zanghellini, F., S. B. Boppana, et al. (1999). “Asymptomatic primary cytomegalovirus infection: virologic and immunologic features.” J Infect Dis 180(3): 702–7.

Chapter 8

T Cell Responses During Human Immunodeficiency Virus (HIV)-1 Infection Claire A. Chougnet and Barbara L. Shacklett

Abstract The defining features of the acquired immunodeficiency are the “persistent and profound selective decrease in the function as well as number of T lymphocytes of the helper/inducer subset and a possible activation of the suppressor/cytotoxic subset”, as described in 1982 (Mildvan, D., U. Mathur, et al. (1982). “Opportunistic infections and immune deficiency in homosexual men.” Ann Intern Med 96(6 Pt 1): 700–4). Nowadays, although depletion of CD4+ T-cells remains a hallmark of Human Immunodeficiency Virus (HIV) infection, the multifactorial nature of the disease provoked by infection by HIV-1 or Simian Immunodeficiency Virus (SIV) in “non-natural” hosts is generally acknowledged, in that no unique immune alteration has been identified that can fully explain the plethora of dysregulation associated with the development of pathogenic HIV and SIV infection. This review will focus on what we know (or do not know) about T cell responses during HIV infection. This choice reflects the main expertise of the authors, and the major theme of this book. However, other aspects of adaptive and innate immunity should not be overlooked. Notably, HIV-specific antibodies, including neutralizing antibodies, are an important defense of the adaptive immune system, although HIV appears to quickly evade the effect of these antibodies. Recent studies have also highlighted the role of innate immunity in protection against HIV/SIV. In addition, several cellular antiretroviral restriction factors, either constitutively expressed or induced by interferons, have been identified, which provide considerable resistance to retroviral infection. For more on these topics, we refer readers to recent reviews summarizing these crucial aspects of the virus/host interaction

C.A. Chougnet (*) Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation and Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA e-mail: [email protected] J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2_8, © Springer Science+Business Media, LLC 2012

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(Kuritzkes, D. R. and B. D. Walker (2007). HIV-1 Pathogenesis, Clinical Manifestations and Treatment. Fields Virology. D. Knipe and P. M. Howley. Philadelphia, PA, Lippincott Williams & Wilkins. 2: 2187–2214; Levy, J. A. (2007). HIV and the Pathogenesis of AIDS. Washington, D.C., ASM Press; Zwick, M. B. and D. R. Burton (2007). “HIV-1 neutralization: mechanisms and relevance to vaccine design.” Curr HIV Res 5(6): 608–24; Alter, G. and M. Altfeld (2009). “NK cells in HIV-1 infection: evidence for their role in the control of HIV-1 infection.” J Intern Med 265(1): 29–42; Neil, S. and P. Bieniasz (2009). “Human immunodeficiency virus, restriction factors, and interferon.” J Interferon Cytokine Res 29(9): 569–80; Stamatatos, L., L. Morris, et al. (2009). “Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine?” Nat Med 15(8): 866–70; Strebel, K., J. Luban, et al. (2009). “Human cellular restriction factors that target HIV-1 replication.” BMC Med 7: 48; Altfeld, M., L. Fadda, et al. (2011). “DCs and NK cells: critical effectors in the immune response to HIV-1.” Nat Rev Immunol 11(3): 176–86).

8.1 8.1.1

Background HIV Life Cycle

HIV-1 is a primate lentivirus that infects cells of the human immune system, predominantly CD4-expressing cells, i.e. CD4+ T cells, macrophages and dendritic cells, although in vitro infection of non-classical CD4− cells, such as hepatocytes or astrocytes, has also been reported (Lopez-Herrera et al. 2005; Xiao et al. 2008). From the 5c- to 3c-ends, the HIV-1 genome includes three structural genes: the gag (group-specific antigen), pol (polymerase), and env (envelope) genes (reviewed in (Freed and Martin 2007)). The Gag and Pol proteins are produced as a Gag-Pol precursor protein whose synthesis requires a (−1) ribosomal frameshift at the junction between the two overlapping translational reading frames of Gag and Pol (Jacks et al. 1988). This frameshift is directed by a highly conserved RNA secondary structure. The Gag-Pol precursor protein is then cleaved autocatalytically by its own Protease (PR) domain (Kramer et al. 1986). The Gag polyprotein is cleaved into the MA (matrix, p17), CA (capsid, p24), NC (nucleocapsid, p7) and several smaller proteins (Freed and Martin 2007). The Pol polyprotein is cleaved into the viral enzymes PR (protease, p10), RT (reverse transcriptase and RNAse-H, p51/66) and IN (integrase, p32) (di Marzo Veronese et al. 1986). The envelope (Env) glycoproteins are also synthesized as a polyprotein precursor, gp160, which is processed by a cellular protease during Env trafficking to the cell surface (Decroly et al. 1994). Gp160 processing results in the generation of the surface Env glycoprotein (SU, gp120), which interacts with the cellular receptor and coreceptor for HIV (Sattentau and Weiss 1988) (Lifson et al. 1986), and the transmembrane glycoprotein (TM, gp41) (Veronese et al. 1985). In addition to these structural genes, HIV-1 also encodes a number of regulatory and accessory proteins. These include Tat

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(transactivator of transcription) (Arya et al. 1985), which is critical for transcription from the HIV-1 LTR (Selby et al. 1989)(Starcich et al. 1985); Rev (regulator of virion) (Sodroski et al. 1986), which plays a role in the transport of viral RNAs from the nucleus to the cytoplasm (Malim et al. 1989); Nef (negative factor) (Franchini et al. 1986; Terwilliger et al. 1986); Vif (virion infectivity factor) (Strebel et al. 1987); Vpr (viral protein R); and Vpu (viral protein U) (Strebel et al. 1988) (Cohen et al. 1988). Very briefly, HIV replication proceeds in a series of stages: following interaction with its receptor (CD4) and co-receptor (a chemokine receptor, mainly CCR5 or CXCR4) (Bleul et al. 1996; Dragic et al. 1996; Oberlin et al. 1996), and after fusion with the host cell membranes, the virion capsid is released into the cytoplasm. From there, it undergoes a series of still-controversial steps leading to uncoating and nuclear import of the viral nucleic acid (reviewed in (Arhel 2010)). Once inside the nucleus, the viral RNA is reverse transcribed into DNA, which is integrated into the host cell chromosome. Messenger RNAs encoding viral accessory and structural proteins are transcribed from integrated proviral DNA. “Early” regulatory proteins Tat and Rev are the first viral proteins synthesized. Tat positively regulates transcription from the viral promoter, and Rev facilitates transport of mRNAs encoding the structural proteins (Gag, Pol, Env) from the nucleus to the cytoplasm. “Late” structural proteins are then synthesized and assembled along with the viral genomic RNA into infectious progeny virions that bud from the cell membrane (reviewed in (Cullen 1991)(Freed and Martin 2007)). All steps of the viral life cycle have become targets for therapy. Inhibitors of viral reverse transcriptase and protease are usually combined to form the regimen called “Highly Active Anti-Retroviral Therapy” (HAART). Inhibitors of virus entry (chemokine receptor inhibitors or Enfuvirtide, a fusion inhibitor) and viral integrase are added to HAART if it is failing (Eggink et al. 2010; Zolopa et al. 2010 ; Gilliam et al. 2011).

8.1.2

Experimental Models to Study HIV Infection

Immune cells from rodents are generally resistant to HIV infection, because of barriers to infection at the level of entry (i.e., differences between human and rodent CD4 and CCR5) and viral gene expression (i.e., differences in factors required for HIV gene expression) (Goffinet et al. 2007; Tervo et al. 2008; Michel et al. 2009). Therefore, most of the knowledge we possess on HIV infection comes from ex vivo studies of cells from HIV-infected subjects, as well as from the experimental model of progressive infection that Asian macaques developed after infection by SIV. SIV infection of Rhesus macaques (RM) exhibits many features similar to that of progressive HIV infection in humans (Chakrabarti et al. 1987; Franchini et al. 2002; Pandrea et al. 2009), and this model remains the best choice for pathogenesis and vaccination studies. SIV naturally infects African nonhuman primates (referred to as SIV “natural hosts”), and is generally non-pathogenic in these species

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(Hahn et al. 2000). These SIV natural hosts however can develop AIDS, and when they do, the spectrum and pathogenesis of the disease are very similar to those encountered in humans or rhesus macaques with AIDS (reviewed in (Pandrea et al. 2009)). Obviously, the usefulness of SIV/RM model is limited by its use of heterologous viruses, particularly for vaccine studies. Many groups have thus invested considerable effort in developing humanized mice as models of infection (for review, see (Denton and Garcia 2009; Legrand et al. 2009; Van Duyne et al. 2009)). An increasing number of studies are performed with these systems; however, whether they will constitute a suitable alternative to the SIV/RM model to conduct pathogenesis studies is very much debated. We have therefore focused this review on what we have learned from studies of HIV-infected individuals and SIV-infected hosts, RM or natural hosts.

8.1.3

A Brief Summary of HIV Transmission

Despite our advances in dissecting the HIV life cycle, the identity of the “founder” cell following mucosal HIV infection is not yet clearly ascertained. Following mucosal exposure of RM to high doses of SIV, the virus crosses the mucosal epithelial barrier within hours and establishes a small founder population of infected cells, mainly “resting” CD4+ T cells (reviewed in (Haase 2010)). This founder population undergoes local expansion during the first week of infection, before propagating systemically, throughout the secondary lymphoid organs. Analysis of HIV envelope sequences in people with acute infection with clade B virus shows that the great majority of these subjects had evidence of productive infection by a single virus (Keele et al. 2008). Phenotypic analysis of transmitted or early founder envelopes revealed a consistent pattern of CCR5 dependence, masking of coreceptor binding regions, and equivalent or modestly enhanced resistance to fusion inhibitors and broadly neutralizing antibodies compared with those from chronically infected subjects (Keele et al. 2008). On the other hand, dendritic cells with detectable viral antigens reach the draining lymph nodes much earlier – 18–24 h after exposure (Hu et al. 2000). Many researchers have thus suggested that dendritic cells are the first cells interacting with HIV. These cells could then transmit the virus to CD4+ T cells, through cis- or trans-infection (Teleshova et al. 2003; Wu and KewalRamani 2006; Cavrois et al. 2008). However, arguing against the latter model, the draining lymph nodes are not the site where productive infection is first detected (Zhang et al. 1999). Although models of mucosal infection in RM have been informative, they have some important weaknesses that need to be remembered because they contribute to the lack of precise knowledge about the founder cells. First, the dose of virus used to obtain a reliable infection of RM is very high compared to the doses that humans are usually exposed to, although many investigators have started using repeated low doses of viruses to infect macaques, a model that better mimics human transmission

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(Alpert et al. 2010; Liu et al. 2010; Reynolds et al. 2010). Second, free viruses, not cell-associated viruses, are used in RM infection, another major difference with the human situation. Third, results are likely to differ depending on the route of transmission (vaginal, rectal, penile). Fourth, it is difficult to reproduce in RM the complexity of human transmission, particularly the changes due to concomitant mucosal infections.

8.2 8.2.1

CD4+ T Cells and HIV Is HIV/AIDS a Disease of Depletion?

CD4 depletion is a hallmark of progressive HIV/SIV infection (Fig. 8.1). Loss of CD4+ T cells from peripheral blood during the chronic phase of infection is quite slow in HIV-infected humans or SIV-infected RM (Mellors et al. 1997), although this loss is closely associated with HIV morbidity and mortality. Opportunistic infections generally start developing when CD4 blood counts plummet under 200/ ul (Crowe et al. 1991). However, seminal studies performed by R. Veazey and colleagues more than 10 years ago showed that CD4 depletion in the gastrointestinal (GI) tract was both more severe and earlier than that observed in the blood (Veazey et al. 1998). These findings in SIV-infected RM were later confirmed in HIVinfected patients (Brenchley et al. 2004; Mehandru et al. 2004). As most transmitted viruses use CCR5 for entry, activated/memory CD4+ T cells, which express CCR5, are preferential targets of HIV/SIV infection compared to naïve (CCR5− ) CD4+ T cells. Accordingly, studies of SIV-infected RM have shown a high level of infection in GI tract CCR5+ memory CD4+ T cells, which are subsequently depleted in less than 3 weeks after infection (Mattapallil et al. 2005). GI devastation leads to microbial translocation, although additional failure of liver macrophages appears to be necessary to lead to elevated plasma LPS levels (Hofer et al. 2010), which are associated with immune activation and poor outcome (Brenchley et al. 2006). However, the contribution of the CD4 depletion occurring in the GI tract to HIV pathogenesis must be interpreted with caution. Indeed, acute loss of GI CD4+ T cells also occurs during nonpathogenic SIV infection, although it is less dramatic than it is in progressive HIV/SIV infection, and mucosal CD4+ T cells somewhat recover in these natural hosts during the chronic phase of infection (Gordon et al. 2007; Milush et al. 2007; Pandrea et al. 2007). Similarly, although CD4 depletion in the GI tract occurs very early after HIV infection, the majority of HIV-infected individuals do not develop AIDS for years. Taken together, these studies suggest that devastation of the GI tract is an important, but not unique, contributor to HIV pathogenesis. Moreover, a different dynamic between virus and immune system is likely at play in the “natural” versus “non-natural” hosts (reviewed in (Pandrea et al. 2009; Sodora et al. 2009; Brenchley et al. 2010)). Reasons for decreased CD4 numbers caused by HIV infection remain under intense scrutiny. Direct cytopathic effects of the virus have been implicated

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Fig. 8.1 CD4+ T cells are the primary target cells for HIV-1 (macrophages and dendritic cells, not shown, may also be infected). HIV-specific CD4+ T-cells may be preferentially targeted (Douek et al. 2002). CD4+ T-cells are rapidly depleted from the gut, either through direct infection (Mattapallil et al. 2005), or bystander apoptosis (Li et al. 2005). CD4+ T-cell dysfunction is apparent in early infection (Shearer et al. 1991; Rosenberg et al. 2000), with important implications for the ‘help’ normally provided to B-cells and CTL. HIV Nef mediates downregulation of MHC class I molecules, protecting infected cells from CTL surveillance (Collins et al. 1998). Meanwhile, cytotoxic T-cells (CTL) progressively exhibit the characteristics of exhaustion or functional senescence (Wherry et al. 2007); in addition, high-avidity CTL responses drive viral sequence ‘escape’ (Borrow et al. 1997). B-cell polyclonal activation is a hallmark of HIV disease, also beginning in early infection (Levesque et al. 2009). Neutralization escape is another important mechanism by which HIV circumvents immune control (Frost et al. 2005; Frost et al. 2008). Finally, systemic effects of HIV infection, not reviewed in detail here, include gut epithelial impairment, thymic dysfunction, generalized immune activation, and lymphoid tissue fibrosis. For additional detail on these topics, see (McCune et al. 2000), (Brenchley and Douek 2008)(Hunt 2007)

(Mattapallil et al. 2005), although they do not appear to account for all HIV-induced cell death. Instead, increased susceptibility of “bystander” CD4+ T cells (i.e. cells that do not exhibit markers of active HIV replication) to apoptosis is well described (Li et al. 2005). Apoptosis is always difficult to visualize in vivo, but expression of molecules associated with T cell death is increased in lymphoid tissues of HIVinfected and SIV-infected progressors (Badley et al. 1998; Herbeuval et al. 2005), strongly supporting the involvement of apoptosis in HIV pathogenesis. Increased apoptotic death of CD4+ T cells follows cross-linking of CD4 by HIV gp120 (Banda et al. 1992), which appears to be mediated by ligand-receptor systems of the tumor necrosis factor superfamily (principally FasL/Fas and TRAIL/DR).

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CD4 cross-linking induces FasL expression not only on T cells but also on monocytes/macrophages, which could indirectly contribute to the apoptotic death of uninfected CD4+ T cells (Oyaizu et al. 1997). Furthermore, CCR5+ CD4+ T cells preferentially express the receptor for IFN-D/E, and may therefore be particularly susceptible to the pro-apoptotic effect of type I IFNs (Boasso et al. 2008). Other HIV proteins have also been implicated in cell death, including Nef through membrane permeabilization (Laforge et al. 2007) and Tat, through alteration of the Egr1-PTEN-Akt and p53 pathways (Dabrowska et al. 2008). Importantly, a recent study reported that the death of “bystander” CD4+ T cells also involves abortive HIV infection, as inhibitors of HIV entry or fusion, but not of reverse transcription, prevented their death. Incomplete viral reverse transcripts were shown to accumulate in the cytoplasm of the cells, and to kill them through activation of a caspasedependent pyroptotic response (Doitsh et al. 2010).

8.2.2

Is HIV/AIDS a Disease of Immune Dysregulation?

CD4+ T cells are functionally abnormal in the majority of HIV-infected individuals and SIV-infected RM. These defects start occurring early in the course of HIV infection, prior to the decline of circulating CD4+ T cell numbers (Shearer et al. 1991, 1998; Rosenberg et al. 2000). Moreover, loss of in vitro CD4+ T cell responses and of in vivo delayed-type hypersensitivity were predictive of disease progression and time to death in HIV-1-infected individuals before the advent of HAART (Miedema et al. 1994; Dolan et al. 1995). Many CD4+ T-cell abnormalities have been defined ex vivo, and can be summarized as follows: (1) decreased polyfunctionality, particularly of HIV-specific T cells, in that activated CD4+ T cells exhibit decreased proliferation and production of IL-2, but not IFN-J and TNF-D (Palmer et al. 2004; Sun et al. 2005), and defective upregulation of some activation markers, such as CD40 ligand, but not CD69 or OX40 (Zhang et al. 2004); (2) upregulation of the inhibitory molecules CTLA-4 (Kaufmann et al. 2007), PD-1 (Day et al. 2006; D’Souza et al. 2007) and Tim-3 (Kassu et al. 2010). Many HIV-specific CD4+ T cells from untreated subjects coexpress PD-1, CTLA-4, and Tim-3, in contrast to cytomegalovirus- or varicella-zoster virus-specific CD4+ T cells in these patients. Coexpression of all three inhibitory receptors on HIV-specific CD4+ T cells is more strongly correlated with viral load than with the expression of each receptor individually (Kassu et al. 2010); (3) increased expression of the apoptosis markers TRAIL DR5 and increased percentage of CD4+ T cells entering apoptosis (Herbeuval et al. 2005); (4) blunted T cell signaling induced by TCR cross-linking, IL-2 or PMA/ionomycin (Cayota et al. 1994; Bostik et al. 2001; Schweneker et al. 2008; Nyakeriga et al. 2009). Interestingly, there may be a genetic basis underlying maintenance of strong polyfunctional CD4+ T-cell responses, as HIV-infected controllers with the strongest HIV-specific CD4 responses in the gut mucosa also possessed HLA-DRB1*13 and/or HLA-DQB1*06, two class II HLA alleles previously associated with non-progression (Ferre et al. 2010a).

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Notably, there is a discrepancy between the ex vivo picture, in which CD4+ T cells from HIV-infected patients exhibit increased expression of many activation markers, including increased basal level of kinase phosphorylation and CD40L, and the in vitro picture, in which CD4+ T cells from patients respond poorly to stimulation (Zhang et al. 2004; Schweneker et al. 2008). These findings suggest a model of chronic “tickling” of the adaptive immune system, which precludes its full response to pathogens. Of interest, binding and cross-linking of CD4, besides their induction of apoptosis, also affect the function of conventional CD4+ T cells from normal uninfected individuals, inhibiting the up-regulation of activation markers (Chirmule et al. 1995; Tamma et al. 1997; Zhang et al. 2004) and the formation of the immunological synapse (Nyakeriga et al. 2009). These data provide a potential mechanism for the decreased response to TCR stimulation of CD4+ T cells in HIVinfected patients. HIV-CD4+ T cell interaction also involves an activated form of the heterodimeric integrin D4E7, normally associated with mucosal localization of lymphocytes, inducing increased LFA-1 expression on CD4+ T cells, which favors the formation of the virus-cell synapse (Arthos et al. 2008). However, whether the D4E7-gp120 interaction and subsequent signaling play a role in the dysfunction of CD4+ T cells or of other immune cells is still unknown. Finally, interactions with other HIV proteins have been implicated in CD4 dysfunction: for example, HIV Nef can directly inhibit CD4+ T cell function by disturbing tyrosine phosphorylation at the immunological synapse (Thoulouze et al. 2006).

8.2.3

Does HIV Particularly Affects Certain CD4+ T Cell Subsets?

Th1/Th2 cells: Based on the fact that production of IL-2 and T cell proliferation, but not IL-10 responses, were lost during chronic infection, Mario Clerici and Gene Shearer proposed in 1993 that the imbalance in the “type-1” versus “type-2” responses was a key factor in the immune dysregulation associated with HIV infection (Clerici and Shearer 1993), and this viewpoint was vigorously debated at the time. As mentioned above, the HIV field has since moved on to the concept of decreased polyfunctionality, particularly of HIV-specific T cells, in that activated CD4+ T cells exhibit decreased proliferation and production of IL-2, but somewhat preserved IFN-J and TNF-D production, data that do not encompass the traditional Th1/Th0/Th2 concepts. In terms of susceptibility to HIV infection, early work showed that HIV-1 spreads better through cultures of Th2 cells than Th1 cells (Maggi et al. 1994; Tanaka et al. 1997), although these results were not confirmed by all studies (Mikovits et al. 1998). Although cell surface density of CCR5 molecules was higher in Th1 versus Th2 subsets, preferential infection and entry of Th1 cells by R5 HIV-1 was not associated with preferential replication, as eventually the R5-virus replicated to a higher level in Th2 cells (Moonis et al. 2001). Th2 subsets

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expressed higher levels of CXCR4 than Th1 subsets and were more susceptible to HIV infection by X4-viruses (Moonis et al. 2001). Interestingly, new studies reported higher levels of two cellular antiretroviral restriction factors, APOBEC3G and APOBEC3F, in Th1 cells than in Th2 cells, and HIV-1 produced from Th1 cells had decreased infectivity, compared to virions produced from Th2 cells (Vetter et al. 2009), data that provide a mechanistic insight on why Th2 are more susceptible to in vitro infection than Th1 cells. Th17 cells produce IL-17, as well as other cytokines such as IL-21 and IL-22, in response to stimulation through the TCR and play an important role in antibacterial responses, promoting recruitment of neutrophils, as well as inducing proliferation of enterocytes and production of antibacterial defensins (Korn et al. 2009). Remarkably, IL-17 production induced by Salmonella typhimurium infection in the ileum was inhibited in SIV-infected macaques, leading to a much-exacerbated bacterial disease (Raffatellu et al. 2008). Early depletion of Th17 cells is observed in the gut mucosa of SIV-infected RM that progress to disease, but not in nonpathogenic SIV-infection of sooty mangabeys nor, importantly, in elite controller SIV-infected RM (Brenchley et al. 2008; Cecchinato et al. 2008). In highly viremic RM, IFN-J-producing cells predominated over IL-17-producing cells and the frequency of Th17 cells at mucosal sites was negatively correlated with plasma virus level (Cecchinato et al. 2008). During chronic infection, the frequency of IL-17-producing cells was severely decreased in all lymphoid tissues in pathogenic as compared to non-pathogenic SIV infection (Favre et al. 2009). These findings were recently confirmed in humans, as progressive HIV disease was associated with the loss of Th17 cells both in peripheral blood and rectosigmoid biopsies (Favre et al. 2010). Systemic immune activation (as evidenced by increased Ki67 expression) also correlated with the loss of IL-17–producing cells from the intestine (Gordon et al. 2010). Such alteration likely contributes to the disruption of the mucosal barrier during pathogenic SIV/HIV-1 infection, leading to increased microbial translocation (Estes et al. 2010), but the underlying mechanisms of this loss remain unclear. Although it is clear that Th17 cells are susceptible to SIV infection both in vitro and in vivo, whether they are preferentially infected in vivo compared to Th1 cells is debated, with discrepant data coming from different studies (Cecchinato et al. 2008; Gosselin et al. 2010). Of note, a recent study implicated a tryptophan catabolite in the Th17 loss (Favre et al. 2010). Regulatory T cells (“Treg cells”, defined here as CD3+CD4+FOXP3+ cells) are essential for maintaining host homeostasis, as evidenced by the catastrophic autoimmunity developed by mice or humans in which FoxP3 is not functional. However, Treg cells also dampen effector responses to pathogens, and may thus hamper the capacity of the hosts to control chronic infections (Li et al. 2008; Feuerer et al. 2009; Josefowicz and Rudensky 2009). Comparison of progressor and nonprogressor HIV-infected patients and SIV-infected RM showed an association between increased frequency of Treg cells in lymphoid tissues and high viral loads or reduced anti-viral cytotoxic T cell activity (Andersson et al. 2005; Epple et al. 2006;

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Estes et al. 2006; Nilsson et al. 2006; Boasso et al. 2007). The Th17/Treg cell balance in peripheral blood and in rectosigmoid biopsies is severely altered in progressors (Favre et al. 2010). However, it should be noted that highly pathogenic SIV infection (in nemestrina or fascicularis macaques) leads to decreased Treg cell frequency in tissues, particularly in the gut (Chase et al. 2007; Qin et al. 2008), suggesting either inter-species variations or altered Treg cell-virus interactions during rapid progression. Inhibitory functions of Treg cells appear to be preserved in HIV-infected patients and SIV-infected RM (Hryniewicz et al. 2006; Kinter et al. 2007; Thorborn et al. 2010). However, Treg cells likely play a complex balancing role during HIV-1 infection. They also limit non-specific immune activation (Eggena et al. 2005; Chase et al. 2007; Ndhlovu et al. 2008), and likely limit HIV infection/replication in susceptible target cells (Liu et al. 2009) (Moreno Fernandez et al. 2011), which could play a beneficial role during early HIV infection when the effector immune cells are not yet activated. In support of this hypothesis, first, Treg cells are activated very early following SIV infection in African green monkeys, which could limit harmful generalized activation and allow for this infection to remain non-pathogenic (Kornfeld et al. 2005); second, in utero activation of Treg cells in HIV-exposed uninfected children has been postulated to contribute to the lack of vertical transmission by reducing T cell activation (Legrand et al. 2006); and third, CTLA-4 blockade during acute primary SIV infection in rhesus macaques reduced Treg cell numbers and increased viral replication at mucosal sites (Cecchinato et al. 2008). Treg cells could also maintain some protective role during chronic infection by limiting HIV infection/replication, although their dampening effect on HIV/SIV specific responses (Aandahl et al. 2004; Weiss et al. 2004; Hryniewicz et al. 2006; Kinter et al. 2007), as well as their production of fibrosis-inducing TGF-E1 (Estes et al. 2007), may tilt the balance towards a detrimental role during chronic infection. Like many other immune processes, Treg cells thus behave as a double-edged sword during HIV/SIV infection. Mechanisms underlying increased Treg cell frequency in tissues during HIV/SIV infection are not well understood, and are likely multiple. HIV and SIV can infect Treg cells both in vivo and in vitro, but Treg cells appear to be relatively less frequently infected than non-Treg memory T cells in vivo (Estes et al. 2006; Allers et al. 2010), which is in agreement with our data showing that Treg cells were less susceptible than non-Treg to in vitro infection by R5 viruses (Moreno-Fernandez et al. 2009). Binding and cross-linking of CD4 may have different effect on Treg cells than on other subsets: although CD4+ T cells generally show increased susceptibility to apoptosis upon CD4 cross-linking (see above), the same pathway promotes in vitro survival of the Treg cell subset (Nilsson et al. 2006). Increased proliferation of Treg cells may also promote their relative accumulation, as Treg cells express high levels of the cycling marker Ki67, compared to other non-Treg CD4+ T cells, and this difference is exacerbated in HIV-infected individuals (Chougnet et al., in preparation). Finally, the role of peripheral conversion in Treg cell accumulation has not yet been thoroughly examined, but HIV-exposed plasmacytoid dendritic cells (PDC) induced a higher rate of in vitro FOXP3 induction in naïve T cells than unexposed PDC (Manches et al. 2008).

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8.3 The CD8+ T-cell Response to HIV 8.3.1

CD8+ T Cells Contribute Significantly to Immune Control of HIV

One of the body’s major defense mechanisms against viral infection is the CD8+ T-cell. These cells may act through a combination of mechanisms including direct cytolysis, mediated by cytolytic granules that specifically target and eliminate virally infected host cells (Bangham 2009), and through non-cytolytic mechanisms termed “viral suppression” (Freel et al. 2010)(Killian et al. 2011). Several findings are frequently cited as strong evidence that CD8+ T-cells contribute significantly to immune control of HIV: first, during acute HIV infection, the emergence of HIVspecific CD8+ T-cells coincides temporally with the decline in peak viremia (Koup et al. 1994); second, viral escape mutations have been documented in response to certain HIV/SIV-specific CD8+ T-cell responses (Borrow et al. 1997; Goulder and Watkins 2004; Leslie et al. 2004); third, experimental removal of circulating CD8+ T-cells in SIV-infected rhesus macaques leads to a rapid surge in viremia (Jin et al. 1999; Schmitz et al. 1999); fourth, it is clear that infected individuals differ widely in their ability to control HIV, and recent findings support a role for MHC class I-restricted, CD8+ T-cell responses in the establishment and maintenance of control in the elite controllers (discussed below) (Fellay et al. 2007; Pereyra et al. 2010). Nevertheless, although HIV-specific CD8 responses may be quite robust, they ultimately fail to contain the infection in the vast majority of infected individuals. Cytolytic CD8+ T cells (CTL) have traditionally been defined by their ability to induce death in MHC class I-compatible target cells expressing foreign peptides, an outcome quantified in vitro by measuring 51Cr released by dying target cells into a culture supernatant. This approach has numerous shortcomings, and has been largely replaced by surrogate assays, such as Elispot and intracellular flow cytometry, that measure the production of various cytokines, chemokines and cytolytic granule constituents by the CD8+ T-cells upon TCR stimulation (Doherty 1998; McMichael and O’Callaghan 1998; Maecker 2009). However, in the absence of well-defined correlates of protection from HIV infection and/or disease progression, it remains unclear which T-cell functions are most desirable or most strongly associated with a positive outcome (reviewed in (Appay et al. 2008; Makedonas and Betts 2011)). Candidates for “most valuable correlate of CD8+ T-cell immunity”, based upon studies of chronic HIV infection, long-term nonprogression, and in some cases vaccine trials, have included: “polyfunctionality”, i.e., the capacity to respond to TCR stimulation by producing multiple effector molecules (Betts et al. 2006), proliferative capacity (Migueles et al. 2002), production of perforin, granule loading and/or degranulation (Migueles et al. 2008; Hersperger et al. 2010, 2011), non-cytolytic suppression of HIV replication (Freel et al. 2010, 2011), T-cell avidity (Almeida et al. 2007, 2009), localization of HIV-specific T-cells to mucosal tissues (Critchfield et al. 2007, 2008; Ferre et al. 2009, 2010a, b), and the predominance of specific memory subsets (Letvin et al. 2006; Hansen et al. 2009; Chattopadhyay and Roederer 2010).

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Lessons from HIV Controllers

In the absence of clear-cut correlates of protection revealed by vaccine studies, many clinical studies have attempted to identify correlates of disease non-progression in the small subset of infected persons known as “HIV Controllers”. These rare individuals, likely representing less than 1–2% of all infected persons, are able to control viral replication to levels undetectable by standard assays, without antiretroviral therapy. Over the past 5 years, several genome-wide association studies (GWAS) of HIV controller cohorts have attempted to identify genetic correlates of HIV non-progression (Fellay et al. 2007, 2009; Dalmasso et al. 2008; Pelak et al. 2010; Pereyra et al. 2010). To date, the consensus from these studies is that polymorphisms localized to the MHC class I region of chromosome 6, notably the alleles HLA-B57 and B27, are most strongly associated with HIV control (Pereyra et al. 2010). Protection associated with certain MHC class II alleles (i.e., DRB1*01, DRB1*0701, DRB1*1303, and the haplotype DRB1*1301/2-DQB1*06), acting either independently or in combination with protective MHC class I alleles, was reported earlier (Malhotra et al. 2001; Ferre et al. 2010a; Vingert et al. 2010; Julg et al. 2011), but these associations have not been confirmed in larger scale GWAS. Interestingly, synergy of certain combinations of MHC class I and II alleles was suggested by studies in rhesus macaques (Giraldo-Vela et al. 2008), and further investigation of this issue is warranted (Bashirova et al. 2011). What properties conferred by “protective” MHC class I alleles might be responsible for their unique association with HIV control? First, B57 and B27 alleles may recognize highly conserved HIV epitopes that cannot be mutated without significant loss of viral fitness (Schneidewind et al. 2007, 2008; Miura et al. 2009a, b). Several reports suggest that Gag-specific CD8+ T-cell responses are associated with relative immune control, while Env-specific responses are not (Kiepiela et al. 2004; Ferre et al. 2010b). In the case of HLA-B27, a single immunodominant epitope (KK10) is located in a structurally constrained region involved in interactions between the virion capsid protein (p24) and the host protein cyclophilin A (Schneidewind et al. 2007, 2008). Viral escape from KK10-specific CTL frequently occurs late in infection and is associated with loss of immune control. In the case of HLA-B57, four immunodominant epitopes are located within p24; of these, the TW10 epitope is targeted by CTL early in infection. TW10 escape mutations occur rapidly and have a negative impact on viral fitness during the early phase of infection; however, many escape variants also elicit strong CTL responses (Brockman et al. 2007). Thus, the combination of strong CTL responses and reduced viral fitness likely contributes to immune control in this case. Intriguingly, GWAS have implicated another MHC class I region in control of HIV viral load, as indicated by a single nucleotide polymorphism (SNP) located 35 kb upstream of the HLA-C gene (reviewed in (Bashirova et al. 2011)). HLA-C proteins present antigenic peptides to CTL, but can also serve as ligands for killer

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immunoglobulin-like receptors (KIR) that regulate natural killer (NK) cell function. Thus, protective effects of HLA-C polymorphisms could be mediated via CTL and/ or NK cells, and this topic is an important area for future study. In contrast, certain MHC class I polymorphisms, notably HLA-B*35 alleles, have been associated with rapid progression to AIDS (Gao et al. 2001; Bashirova et al. 2011). The HLA-B*35 family of alleles has been subdivided into two groups according to peptide-binding specificity and disease association. B*35-PY allotypes, including B*3501, bind peptides with the amino acids proline and tyrosine at positions 2 and 9, respectively, and are not associated with a rapid disease course. In contrast, B*35-Px allotypes, which include B*3502, B*3503, B*3504, and B*5301, prefer residues other than tyrosine at position 9, and are associated with accelerated progression to AIDS. There remains some debate as to the mechanistic basis for these associations, as in vitro binding studies have failed to find strong differences in peptide-binding affinity between the two variants (Huang et al. 2009). At least part of the HLA effect on HIV control is thought to be due to interactions between killer immunoglobulin-like receptors (KIR) and MHC class I molecules (reviewed in (Carrington et al. 2008)). Most studies to date agree that there is an association of KIR3DL1/S1 and certain HLA-B alleles with disease outcome. KIR3DL1, an inhibitory KIR, binds HLA-B allotypes containing the Bw4 epitope, particularly those with an isoleucine rather than a threonine at position 80 (i.e., Bw480I), such as HLA-B*57 (Martin et al. 2007). The stimulatory receptor KIR3DS1, which is highly similar to KIR3DL1, may not interact directly with HLA-Bw4 (Gillespie et al. 2007); however, the combination of KIR3DS1 and HLA-Bw4-80I has been associated with slow disease progression in some (Qi et al. 2006; Martin et al. 2007) but not all cohorts (Barbour et al. 2007). Thus, HIV control may be influenced by interactions between MHC class I molecules and NK activating/inhibitory receptors, although the fine details of these interactions remain to be elucidated. A recent study made use of a novel in silico model for thymic selection to predict the outcome of thymic selection when the repertoire of self-reactive peptides was varied in diversity (Kosmrlj et al. 2010). The model was used to calculate the number of peptides from the human proteome (i.e., “self” peptides) that could be bound by the different HLA molecules. Strikingly, two MHC class I molecules associated with protection, human HLA-B*5701 and rhesus macaque Mamu-B*17, were predicted to recognize far fewer self-peptides than alleles not associated with protection, such as human HLA-B*0701 or macaque Mamu-A*02. Previous studies in mice have demonstrated that T cells that develop with exposure to a limited selfpeptide repertoire are significantly more cross-reactive than T-cells that develop with exposure to the full range of self-peptides (Huseby et al. 2005). Accordingly, the work of Kosmrlj et al. suggests that, by encountering fewer self-peptides during thymic selection, T-cells restricted by HLA-B*5701 or Mamu-B*17 should be capable of recognizing a broader range of variant peptides once in the periphery. This model also provides a potential explanation for the puzzling link between HLA-B*57 and a predisposition to autoimmune psoriasis and hypersensitivity reactions (Bhalerao and Bowcock 1998; Chessman et al. 2008).

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CD8+ T-cell Responses During Acute/Early HIV Infection

Recent studies have shed additional light on the breadth and specificity of the acute phase T-cell response and its relationship to the establishment of the viral load set point (Altfeld et al. 2006; Streeck et al. 2007, 2008, 2009). The viral load set point is a strong prognostic indicator (Mellors et al. 1996). In a study involving more than 400 patients with acute/early infection, CD8+ T-cell responses were mapped to the epitope level (Streeck et al. 2009). In this study, those patients whose acute/earlyphase CD8+ T-cell responses were strongly focused towards immunodominant epitopes had lower viral load set points than those whose responses were distributed across a variety of epitopes, without a clear immunodominance hierarchy. This was true whether or not patients with protective MHC class I alleles (i.e., HLA-B27 and B57) were included in the analysis. Notably, the association between immunodominance hierarchy and plasma viral load was lost during chronic infection (Streeck et al. 2009). The ability of HIV/SIV-specific CD8+ T-cells to localize to tissue sites of viral replication during acute/early infection may also be critical in establishing the host-pathogen balance. In RM infected intravaginally, SIV-specific CD8+ T-cell responses in cervicovaginal and GI mucosal tissues remained weak for at least 1–2 weeks following initial infection (Reynolds et al. 2005). This pre-immune “window” allowed viral replication and mucosal CD4+ T-cell depletion to occur. More recently, a comparative study of acute lymphocytic choriomeningitis virus (LCMV) infection of mice and SIV infection of RM, visualizing both viral mRNA and MHC class I tetramer staining at multiple time points post-infection (Li et al. 2009), showed that control of viral replication is directly related to the ratio of virusspecific CD8+ T-cells to virus-infected cells in tissues. Taken together, these studies suggest that in addition to response specificity and magnitude, the localization of HIV/SIV-specific T-cells proximal to tissue foci of viral replication is critical in determining outcome.

8.3.4

CD8+ T-cell Function, and Dysfunction, During Chronic HIV Infection

Much work has been devoted to characterizing the cytolytic functions of CD8+ T-cells, as well as their ability to release cytokines, chemokines, and induce non-cytolytic suppression of viral replication. As with CD4+ T-cells, CD8+ T-cell dysfunction and ‘exhaustion’ during chronic infection appear to be major contributors to the failure to control HIV. The phenomenon of immune exhaustion has been well documented in mice infected with LCMV (Wherry and Ahmed 2004). In this model, T-cell functions are progressively lost, beginning with proliferative capacity and IL-2 production, followed by loss of TNF-D production, whereas IFN-J production persisted until the final stages of exhaustion (Wherry et al. 2003, 2007).

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The contribution of CD4+ T-cell loss/dysfunction to CD8+ T-cell dysfunction is not well understood, but several recent studies started tackling the issue. Notably, help of CD4+ T cells appears critical to HIV-specific CD8 proliferation and control of autologous virus replication (Lichterfeld et al. 2004; Chevalier et al. 2011), and this effect may be mainly linked to IL-21-producing CD4+ T cells (Chevalier et al. 2011). Other CD4 factors likely contribute to CD8 function, including Treg cell control of CD8 effector cells (see Treg cell section above). Premature “ageing” of the immune system has been invoked as a significant contributor to HIV-specific CD8+ T-cell dysfunction (Appay and Rowland-Jones 2002). High levels of antigenic stimulation appear to drive the induction of a “senescent” phenotype, characterized by expression of cell surface antigens such as PD-1, lymphocyte activation gene-3 (LAG-3), Tim-3 and CD57, as well as telomere shortening and decreased responsiveness to TCR stimulation (Day et al. 2006; Petrovas et al. 2006; Trautmann et al. 2006; Jones et al. 2008). Several inhibitory receptors play a role in the contraction of the immune response following clearance of infection (Blackburn et al. 2009). Blocking these pathways in vivo can lead to increased CD8+ T-cell function, with potential implications for HIV immunotherapy (Barber et al. 2006; Freeman et al. 2006; Velu et al. 2009). However, expression of a single inhibitory receptor such as PD-1 does not necessarily indicate a senescent phenotype, as recent studies indicate that up to seven different receptors participate in complex pathways of negative regulation during chronic viral infection (Blackburn et al. 2009).

8.4

8.4.1

Effect of Anti-retroviral Therapy on T Cell Responses: Successes and Limitations HAART-Mediated Reconstitution of the Immune System

HAART has transformed a diagnosis of HIV infection from a likely death sentence to a prospect of a life-long, serious, but mostly treatable, disease. Current US and European guidelines recommend starting therapy in individuals with CD4 counts less than 350/PL (http://www.aidsinfo.nih.gov/ ContentFiles) and (Clumeck et al. 2008). A meta-analysis of more than 20,000 patients starting HAART showed that deferring therapy until CD4 counts were lower than 350/PL was associated with higher rates of development of AIDS and death than starting therapy in the 351–450/PL range (Sterne et al. 2009). In terms of CD4 reconstitution post HAART, a recent study of the AIDS Clinical Trials Group showed an initial phase (~8 weeks) characterized by expansion and redistribution of memory CD4+ T cells, followed by a second phase with reconstitution of both naïve and memory CD4+ T cells, as well as reduction of CD4 and CD8 activation (Robbins et al. 2009), data that are in agreement with the results of previous small-scale studies (reviewed in (Lederman 2001)). If delayed, HAART never restores CD4 counts to levels found in healthy volunteers

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(Robbins et al. 2009). In addition to these quantitative differences, qualitative differences were also noted. In particular, patients starting with lower baseline CD4 counts exhibit modest increase in CD4+ naive cells and greater increase in CD4+ memory cells. Functional amelioration of CD4+ T cells occurs post-HAART, but remains limited. First, HAART generally reconstitutes immune responses against prevalent microbial antigens, but HIV-1-specific responses remain largely dysfunctional (Rinaldo et al. 1999; Chougnet et al. 2001), which explains why most people with chronic HIV-1 infection cannot control viremia after HAART interruption (reviewed in (Lederman 2001)). Second, although expression of TRAIL and FasL is reduced in circulating and tonsil CD4+ T-cells from HAART-treated patients, tissue expression of DR5 and Fas remains higher in treated patients than in HIV-uninfected subjects (Herbeuval et al. 2009). Third, T-cell activation (defined as a high percentage of CD4+CD38+HLA-DR+) persists among treated patients, whatever their baseline CD4 counts. Fourth, we found that recruitment of lck and actin to the immunological synapse was ameliorated in the CD4+ T cells of HAART-treated patients, but responses remained lower than those measured in uninfected subjects (Nyakeriga et al. 2009). Fifth, suppressive HAART was associated with low Treg cell frequency in lymphoid tissues compared to untreated patients (Andersson et al. 2005), as well as decreased Treg cell frequency in the blood (Montes et al. 2011), but this frequency remained higher than that measured in uninfected subjects (Lim et al. 2007; Kolte et al. 2009). Intestinal biopsies from HIV-infected patients who were on long-term HAART showed that when effective CD4+ T cell restoration (>50% compared to uninfected controls) was achieved, enhanced Th17 responses and, in general increased polyfunctionality of anti-HIV cellular responses, were also found, but these functional ameliorations did not occur if reconstitution was more limited (Macal et al. 2008). Incomplete CD4+ T-cell repopulation of lymphoid tissue, including the gut, has been linked to collagen deposition and fibrosis mediated by TGF-E secretion (Schacker et al. 2005; Estes et al. 2008). Although HAART can be very effective at limiting viral replication, HIV-specific CD8+ T-cell responses generally wane in patients on HAART (Kalams et al. 1999; Ogg et al. 1999; Spiegel et al. 1999), suggesting that maintenance of these responses is to some extent antigen-driven. In patients interrupting HAART, viral load rebound is accompanied by increased CD8+ T-cell responses (Ortiz et al. 2001). In crosssectional studies, CD8+ T-cells from blood and mucosal tissues of patients on HAART secreted fewer cytokines in response to HIV peptide stimulation as compared to T-cells from HAART-naïve patients, suggesting that HAART leads to a decline in “polyfunctionality” as well as response magnitude (Critchfield et al. 2008). The reasons for persistent immunological defects and incomplete CD4+ T-cell repopulation despite prolonged suppressive treatment are still unclear despite multiple studies, but potential mechanisms include ongoing low-level HIV replication, perhaps in specific tissue reservoir sites, irreversible structural changes in the lymphoid compartments, and incomplete reconstitution of the gut mucosal integrity leading to persistent low levels of bacterial translocation.

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In Search of Efficient Immune Based Therapies

Searching for interventions that could ameliorate immune functional reconstitution, alone or combined with HAART, is still of high priority in HIV research. The goal of such interventions is to transform progressors into non-progressors, and thus delay or prevent starting HAART (if given alone), or to enhance control of HIV infection in patients undergoing HAART, and thus lower the viral set-point after treatment removal, or prevent failure of HAART. Several strategies have been tested or are currently tested. Broadly, they include cytokine supplementation, antibodies against negative regulators (CTLA-4, PD-1/PD-L1), and therapeutic vaccinations. In terms of cytokines, IL-2 was first evaluated. Despite a substantial and sustained increase in CD4 cell counts, addition of IL-2 to HAART has yielded no clinical benefit in either of two large international studies. On the contrary, IL-2 therapy increased the incidence of life-threatening clinical events in patients with the highest baseline CD4 counts (Abrams et al. 2009). The mechanisms underlying these deleterious effects are unclear but could be related to the induction of CD4+ T cells with characteristics of Treg cells (Velilla et al. 2008; Weiss et al. 2010) and/or that IL-2 had pronounced pro-inflammatory effects in patients with higher numbers of CD4+ T cells at baseline. IL-7 therapy appears more promising (Levy et al. 2009), and is currently being tested in Phase I/II randomized placebo clinical trials. Blocking anti-CTLA-4 combined with ART was not efficient in chronically SIVinfected RM. Despite a modest improvement in CD4 and CD8 anti-viral responses and a slight decrease in viral RNA in tissues of animals receiving anti-CTLA-4 antibody plus HAART compared to HAART alone-animals, anti-CTLA-4 antibody did not bring significant functional improvement (Hryniewicz et al. 2006). Moreover, treatment of RM with the same blocking anti-CTLA-4 antibody during the acute phase of infection was detrimental, inducing higher viral loads and accelerated mucosal CD4+ depletion (Cecchinato et al. 2008). Blockade of the PD-1/PD-L1 pathway is generating considerable interest. Velu and colleagues presented the first in vivo study to show enhancement of SIV-specific immune response using a blocking PD-1 Ab (Velu et al. 2009). In vivo blockade of the PD1/PDL1 pathway appeared safe and led to prolonged survival of SIV-infected RM. However, viral loads returned to pre-blockade levels within a few weeks. These studies highlight the problems associated with immune-based therapies in HIV/SIV infection: blockade of one inhibitory pathway will likely be insufficient to provide sustained benefits, but combined blockade of several regulatory pathways may provoke severe auto-immune reactions in treated patients. In terms of therapeutic vaccines, there are currently a number of clinical trials using dendritic cells, with the goal of enhancing control of HIV infection in patients undergoing HAART. We refer the reader to a recent review written by C. Rinaldo, which summarizes the strategies currently developed, and the difficulties researchers face when designing such treatments (Rinaldo 2009). Another tested approach was the combination of several types of HIV vaccines to early HAART (the QUEST trial). Results were very disappointing, as vaccinated subjects did not control HIV-1

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after treatment discontinuation better than those receiving the placebo, despite displaying significantly increased IFN-J expression by HIV-specific CD4+ and CD8+ T-cells (Kinloch-de Loes et al. 2005).

8.5

Concluding Remarks

Since the discovery of HIV more than 25 years ago, the field has come a long way despite many setbacks. We now clearly understand that HIV is a master at evading and impairing all arms of the immune system, while using basic mechanisms of T cell activation to its advantage. However, we also understand better its “Achilles’ heel”, revealed by the study of elite controllers, both humans and RM. This knowledge is currently being applied to design better vaccine strategies, better immunebased therapies, and better use of HAART, although most HIV researchers have stopped believing that we will ever find a “silver bullet”. Beyond this progress, HIV researchers also have been forced to develop better ways to probe the immune systems of humans and nonhuman primates, as appropriate rodent models were not available. The past and current studies of CD4/CD8 function, lymphocyte homeostasis and cell-cell interactions conducted in HIV-infected populations are providing significant insights regarding the human immune system, insights that will continue to be useful to many other research fields. Acknowledgments The authors thank the members of their laboratories and their colleagues for useful discussions, as well as their mentors without whom they would not have joined the HIV research field. CAC wants to particularly thank Dr Gene Shearer. CAC is supported by the National Institutes of Health (R01 AI068524, R01 AG033057 and U01 HL101800). BLS is supported by the National Institutes of Health (R01 AI057020, R01 DE021273, P01 AI083050, and R21 NS069219) and the American Foundation for AIDS Research (AmFAR 107854-RGRL).

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Index

A Aliberti, J., 1, 23–34 Anti-retroviral therapy and T cell responses HAART AIDS and death, 155 CD4 count levels and healthy volunteers, 155–156 CD4+ T cells amelioration, 156 diagnosis, HIV infection, 155 immunological defects and interruption, 156 limiting viral replication, 156 Treg cell activation and frequency, 156 immune based therapies blocking anti-CTLA–4, 157 goal and IL–2 effects, 157 PD–1/PD-L1, 157 vaccines, 157–158 Arens, R., 121

B Bacillus Calmette Guerin (BCG), 24 Bacterial and non-bacterial infection, iNKt cells bystander indirect activation description, 50–51 microbial infections, dual recognition, 51 Salmonella and TCR signaling, 51 direct recognition, microbial GSLs Agelas mauritianus, 52–53 alphaglucuronylceramides, 52 description, 52 lyme disease, 53 Sphingomonas/Novosphingobium, 52

gram-negative bacteria, 50 parasitic, helminth and fungal infections Candida albicans and Aspergillus fumigatus, 54 control parasitic replication, 54 Plasmodium, 54 role and anti-parasite responses, 54 viral infections detection, viral pathogens, 53 Hepatitis B virus (HBV) model, 53–54 immune defense strategy, 54 iNKT role, 53 lethal outcome, Epstein-Barr virus, 53 role and SAP-Fyn signaling, 53 Bafica, A., 23–34 B and T lymphocyte attenuator (BTLA), 133 B cell-mediated regulation, Leishmania antibodies, 86 arm, immune system, 85–86 CMI, 85 humoral immunity, 86 IL–1 and IL–6, 88 intercellular pathogen clinical infection, 90–91 description, 89 murine models, 92–93 Th1 vs. Th2, 89–90 intracellular pathogen antibodies role, 86–87 CMI effects, 86 immune control, Ehrlichia chaffeensis, 87 proinflammator vs. anti-inflammatory, 87–88 Th1 response, FcJR, 86 macropages activation and antibodies

J. Aliberti (ed.), Control of Innate and Adaptive Immune Responses during Infectious Diseases, DOI 10.1007/978-1-4614-0484-2, © Springer Science+Business Media, LLC 2012

171

172 B cell-mediated regulation, Leishmania (cont.) functions, 88–89 NADPH oxidase, 89 nitric oxide (NO) production, 88 superoxide production, 89 Benedict, C.A., 121 BTLA. See B and T lymphocyte attenuator Buzoni-Gatel, D., 11

C CD1 system and NKT cells description, 41 group II (CD1d) description, 42 in mouse, 42 restricted diverse type II iNKT cells, 43–44 restricted type I iNKT cells, 42–43 group III (CD1e), 44 group I (CD1a, b, c), mycobacterium tuberculosis, 41 mediated presentation, GSLs, 49 MHC gene complex, 41 self-GSL antigens endogenous presentation, 49 E-hexosaminidase B, 50 isoglobotrihexosylceramide (iGb3), 49–50 regulation, 50 self-lipid antigens, 50 species distribution birds, 44 existence, 44 human and guinea pigs, 44–45 isoforms, 44 mycobacteria-infection, 45 CD4+ T cells and HIV affecting subsets regulatory T cells, 149–150 Th17 cells, 149 Th1/Th2 cells, 148–149 AIDS and depletion disease cell loss, morbidity and mortality, 145 cross-linking, 146–147 cytopathic effects, virus, 145–146 GI loss, 145 IFNs effects, 147 increases “bystander” cells, 146 “natural” vs. “non-natural” hosts, 145 primary target cells, 146 immune dysregulation abnormalities, 147

Index chronic “tickling”, 148 defects, 147heterodimeric integrin D4E7, 148 in vitro CD4+ T cells, 147 CD8+ T cells responses, HIV acute/early infection, 154 function and dysfunction, chronic infection ageing, immune system, 155 characterization, 154 contribution, 155 immune exhaustion and LCMV, 154 immune control, 151 lessons and HIV controllers antiretroviral therapy, 152 GWAS, 152 HLA-B*35 alleles and AIDS, 153 human HLA-B*5701, Mamu-B*17, 153 in silico model, 153 MHC class I and II alleles, 152 natural killer (NK) cell function, 152–153 Cell mediated immunity (CMI), 85–87 Chemokine receptors CCR2 binds CCL2, 111, 113 inability, CCL2 and CCL7, 112 L-arginine, fungal elimination, 111, 112 CCR5 IL–17A neutralization, 113 role, 5 T and Th17 cells, 113, 114 Chougnet, C.A., 141 CMI. See Cell mediated immunity (CMI) CMV. See Cytomegalovirus Culture-derived tachyzoites (STag), 4, 13 Cytomegalovirus (CMV) adaptive T cell responses costimulation, 127–128 MHC-TCR interactions, 126–127 apoptosis pathways, 134 CD8+ and CD4+ T cells HCMV, 125 immunomodulatory effects, DCs, 126 MCMV replication, 124–125 memory inflation, 125–126 DCs, 122–124 epidemiology and pathology HCMV infection, 122 risk factor, 122 herpesviruses, 121 immunosuppressive machinations, 134 modification, T cells B7 costimulation, 130–131 cytokines, 133–134 HVEM/BTLA interactions, 133

Index MHC expressions, 128–130 PD–1/PDL–1 pathway, 132–133 soluble positive cosignals, 131–132 NK and NKT cells, 124

D Debbabi, H., 11 Deepe, G.S. Jr., 99 Dendritic cells (DCs) CMV cytokines, 122–123, 131–132 HCMV infection, 126 immune modulatory gene products, 123–124 MCMV, 128, 132 MHC molecules, 126 negative net signal, 128, 129 NK cells, 124 PD-L1, 132 positive net signal, 128, 129 cross-presentation pathway, DC, 104 cytomegalovirus (CMV), 122–124 Histoplasma capsulatum bind and ingest, 103, 104 cross-presentation pathway, 104 population, 101 yeast cells, 103, 104 immunomodulatory effects, 126 intracellular lifestyle, 104 microbial recognition, 4 neutrophils, 104 paralysis, 13 production, DC, 131–132

G Gibson-Corley, K.N., 85–93 Glycosphingolipid (GSL) bacterial and non-bacterial infection Agelas mauritianus, 52–53 alphaglucuronylceramides, 52 description, 52 lyme disease, 53 Sphingomonas/Novosphingobium, 52 CD1 system and NKT cells endogenous presentation, 49 E-hexosaminidase B, 50 isoglobotrihexosylceramide (iGb3), 49–50 mediated presentation, GSLs, 49 regulation, 50 self-lipids reportoire, 50 iNKT cell activation GSL antigens and structures, 40

173 in humans, 56 self-GSL antigens, 49–50 Gram-negative, LPS-positive bacteria, 50–53 Granuloma formation and bacilli tuberculosis chronic maintenance, 28 description, 27–28 fibrous capsule, 28 IFN-J, 29 IL–10, 30 lipoxins, 29–30 TGF-E, 30 TNF, 28–29 Gutoerrez, F.R.S., 69–81

H HAART. See Highly Active Anti-Retroviral Therapy (HAART) HCMV. See Human cytomegalovirus Hepatitis B virus (HBV) model, 53–54 Herpesvirus entry mediator (HVEM), 133 Highly Active Anti-Retroviral Therapy (HAART), 143, 155–156 Histoplasma capsulatum characterization, inflammatory response MI, 101 neutrophils, 100–101 T and B cells, 101 chemokines and chemokine receptors CCR2, 111–113 CCR5, 113 cytokines GM-CSF, 108 IL–1, 108 IL–4, 110 IL–10, 111 IL–17 and Th17, 109 IL–12/IFN-J axis, 107–108 TNF-D, 109–110 description, 99–100 granuloma intracellular pathogens, 101 T cells, 102 intracellular lifestyle DC, 104 MI, 102–103 neutrophils, 103–104 leukotrienes, 113–114 lymphoid cell, infection control B cells, 107 T cells, 104–107 TCR, 105–106

174 Human cytomegalovirus (HCMV) CD4 and CD8 T cells, 125 glycoprotein products, 129 IL–10, 133–134 NK cells, 124 Human immunodeficiency virus (HIV)–1 anti-retroviral therapy effect HAART, 155–156 search immune based therapies, 157–158 CD4+ T cells depletion disease, 145–147 immune dysregulation, 147–148 regulatory T cells, 149–150 Th17 cells, 149 Th1/Th2 cells, 148–149 CD8+ T-cells function and dysfunction, chronic infection, 154–155 immune control, 151 lessons and controllers, 152–153 responses, acute/early infection, 154 cell-cell interaction, 158 discovery, 158 experimental models limitation, heterologous viruses, 144 rodents, 143 SIV and Rhesus macaques (RM), 143–144 immune system, humans and honhumans, 158 life cycle CD4 T cells, 142 gag, pol and env genes, 142 HAART, 143 mRNA encoding, 143 replication, 143 Tat and Rev, transport RNAs, 142–143 T cell activation, 158 transmission cis-/trans-infection, 144 clade B virus, 144 draining lumph nodes, 144 mucosal exposure, 144 phenotypic analysis, 144 route and mucosal infections, 145 virus dose and human transmission, 144–145 HVEM. See Herpesvirus entry mediator

I IL. See Interleukin Immunopathology mechanism, Toxoplasma gondii

Index IL–27 and suppression, 12–13 IL–22 role, 11–12 inflammation redundancy and control, 14–15 interleukin–10 central role, 11 mediators and antigen processing, 9 modulatory activities, 10 neutralization, 10 pathogens and poxviruses, 9–10 pro-inflammatory responses, 10 TGFE and IL–35, 9 lipoxin A4 control, 14 “DC paralysis”, 13 injection, STAg, 13 Mycobacterium tuberculosis, 16–17 pathogen evasion, 15–16 production, 14 receptors and evidence role, 13 resolution phase cardinal signs, 8 controlling and promoting, 9 homeostasis, 9 omega–3 PUFA/fish oils, 8–9 tissue injury, cause and consequences, 8 TGF-E description, 10 macrophage deactivator, 11 mucosal host/pathogen interaction, 11 role, 12 iNKT cells, humans CD1d-restricted, 55 infections, Novosphingobium/ Sphingomonas spp.. osocomial and septic shock, 56 xenobiotic-metabolizing properties, 55–56 PBC association GSL recognition role, 56 pathogenesis, 56–57 PDC-E2 homologues, 57 primary biliary cirrhosis (PBC), 55 VD24 iNKT cells, 55 Innate immunity regulation, Trypanosoma cruzi cardiomyopathy, 70–71 cell migration, 79 Chagas’ disease, 69 characterization, 70 cytokines, 78 description, 69 development, cardiac damages, 71 diagnosis, 70

Index GIPL and Tc52 activation, 73 intracellular parasite, 73 production, TGF-E and IL–10 and IL.12p70, 73 TLR2 activation, 72 TLR2-/-and MyD88-/-mice, 73 GIPL recognition, 73–74 glycoinositolphospholipids (GIPL), 71 in human beings, 70 interaction and evolution, 71 life cycle, 70 mechanisms, innate immune system, 72 MMP, 80 nitric oxide (NO) APC and T cells and induce apoptosis, 75 deficiency impact, 76 donors and control, 75 production and role, 75 response mechanisms, 75 versatile immune mediators, 75 NLR participation, 74–75 phagocytic cells intracellular replication, 78 intracellular signaling, 77–78 macrophages activation, 76 mechanisms, 78 parasite killing, 77 TLR, 71 TLR9 involvement, 74 Interleukin (IL) cmvIL–10, 133–134 HCMV, IL–10, 133–134 Histoplasma capsulatum IL–1, 108 IL–4, 110 IL–10, 111 IL–17 and Th17, 109 IL–12/IFN-J axis, 107–108 IL–1 receptor, 108 IL–17A neutralization, 113 IL–12 induction, inflammation resolution (see Toxoplasma gondii infection) immune based therapies, IL–2 effects, 157 Leishmania, IL 1 and 6, 88 prevent immunopathology mechanism IL–22, 11–12 IL–27, 12–13 Toxoplasma gondii IL–10, 9 IL–27 and suppression, 12–13 IL–12 induction (see Toxoplasma gondii and inflammation resolution) IL–22 role, 11–12

175 Trypanosoma cruzi IL–10, 73 IL.12p70, 73 tuberculosis, IL–10, 30 Invariant Natural Killer T (iNKT) cell activation, microbial infection bacterial infection bystander indirect activation, 50–51 cognate recognition, GSL antigens, 52–53 CD1 presentation, 49 CD1 system division, 41 formation, 41 group I (CD1a, b, c), 41 group II (CD1d), 42–44 group III, (CD1e), 44 species distribution, 44–45 function antibody production, 48 anti-microbial activity, 48 autoreactivity, 46 bacteria and virus detection, 45 circuits and molecular mechanisms, 46 deficient CD1d0/JD180 mice, 45 interactions, 47–48 mechanisms, 48 natural and immune rejection, 45 systemic administration, 46–47 Th1 or Th2 cytokines, 45 trans-activation, 47 type I diabetes, 45–46 GSL antigens and structures, 40 in human correlation, 55 Novovosphingobium/Sphingomonas spp., 55–56 primary biliary cirrhosis, 56–57 MHC class I, 40 mouse model infection, 57–58 non-bacterial infection parasitic, helminth and fungal infections, 54 viral infections, 53–54 role, 40 self-GSL antigens, 49–50 TCR, cell population, 40 V124 TCR, human, 40 V114 TCR, mice, 40

J Janssen, E.M., 121 Jones, D.E., 85–93

176 K Koch, R., 23

L Lipoxins M. tuberculosis in humans, ALOX5, 29–30 5-LO-dependent, 29 risk, 30 role, 29 Toxoplasma gondii control, 14 “DC paralysis”, 13 injection, STAg, 13 mycobacterium tuberculosis, 16–17 vs. Mycobacterium tuberculosis, 17 pathogen evasion, 15–16 production, 14 receptors and evidence role, 13 Loewendorf, A., 121

M Macrophages (MM), Histoplasma capsulatum CD11/CD18 adhesin receptors, 102, 103 CD8+ cytotoxic T cells, 106 cross-presentation pathway, DC, 104 granuloma, 101 growth inhibition, 103 IL–4, 110 neutrophils, 103 yeast cells, 102, 103 Major histocompatibility complex (MHC) CD1 system and NKT cells, 41 CD8+ T cells responses, HIV, 152 class I expression antigen-presentation, 128–129 glycoprotein products, 129 “immunoevasion” genes, 129–130 class II expression antigen presentation, 130 proteolytic degradation process, 130 CmvIL–10, 133–134 cytomegalovirus (CMV), 126–130 dendritic cells, 126 iNKT cell activation, 40 MCMV, 128–129 TCR interactions cross-presentation, 127 exogenous and endogenous pathway, 126–127 Matrix Metalloproteinases (MMP), 80

Index Mattner, J., 31–58 MCMV. See Murine cytomegalovirus MHC. See Major histocompatibility complex Mixed lymphocyte reactions (MLR), 128 MM. See Macrophages MLR. See Mixed lymphocyte reactions Mouse model infection, 57–58 Murine cytomegalovirus (MCMV) CD8 and CD4 T cells, 124–125 m138 gene, 131 MHC class I pathway, 128–129 m157 protein, 124

N NADPH oxidase, 87, 89 Natural killer (NK) cells absence, 27 accumulation, 27 CMV DCs, 124 MCMV m157 protein, 124 depletion, 27 role, 26 Natural vs. non-natural hosts, 145 Neutorphils Histoplasma capsulatum DC, 104 human defensins, 103–104 IL–4, 110 yeast cells, MM, 103 tuberculosis acute pulmonary tuberculosis, 24 definition, 24 depletion, 24 mouse strains, 25 potential role, 25 protection, 24–25 Nitric oxide (NO) Leishmania, 88 Trypanosoma cruzi deficiency impact, 76 donors and control, 75 production and role, 75 response mechanisms, 75 versatile immune mediators, 75 NK cells. See Natural killer cells

P PDL–1. See Programmed death ligand Petersen, C.A., 85–93 Programmed death ligand (PDL–1), 132–133 Proinflammator vs. anti-inflammatory, 87–88

Index S Shacklett, B.L., 141 Superoxide production, 89

T T cell receptor (TCR) cell population V124 TCR, human, 40 V114 TCR, mice, 40 Histoplasma capsulatum CD3+, 105 protective immunity, 105–106 pulmonary infection, 105 reactivation histoplasmosis, 106 interactions cross-presentation, 127 exogenous and endogenous pathway, 126–127 lymphoid cell, infection control, 105–106 T cells B7 costimulation CD28 and CTLA–4, 130–131 MCMV m138 gene, 131 positive cosignaling pathways, 131 costimulation B7 family, 127 positive and negative net signals, 128, 129 TNF receptor and soluble mediators, 127 cytokines cmvIL–10 and IDO, 133–134 HCMV, 134 production, DC, 131–132 Histoplasma capsulatum CCR5, 113, 114 CD4+ and CD8+ cells, 104–105 granuloma, 102 IL–1 receptor, 108 PD ligands, 106 promote immunity, 106 receptors, CD3+, 105 regulatory, 107 TNF-D production, 105, 109 HVEM/BTLA interactions, 133 MHC expressions, 128–130 MHC-TCR interactions cross-presentation, 127 exogenous and endogenous pathway, 126–127 MLR, 128 PD–1/PDL–1 pathway B7-CD28 pathway, 132–133

177 cell cycle arrest, 132 negative cosignaling, 132 TCR. See T cell receptor Th1 vs. Th2, 89–90 TNF-D. See Tumor necrosis factor-alpha Toll-like receptors (TLRs) microbial recognition, 5–6 signaling, 51 Trypanosoma cruzi TLR, 71 TLR2 activation, 72 TLR2-/-and MyD88-/-mice, 73 TLR9 involvement, 74 Toxoplasma gondii and inflammation resolution cysts and bradyzoites, 3 description, 1 felines, cat, 2 in human, 2 IFN-J, Th1 cells and microbicidal activity activation factors and parasite strains, 7 components, 7 immune responses mechanisms, 8 investigation, 18 life cycle, 2 microbial recognition and IL–12 induction biochemical signaling, 7 CCR5 role, 5 complexity and protection, 4 cyclophillin–18, 5 cytoplasmic protein profillin, 6 hypothesis, dendritic cells, 7 IFN-J, 4 immune response and pathogen, 4 IRF–8, mice, 6 macrophages, neutrophils and DCs, 4 p38 MAP kinases, 6–7 TLRs, 5–6 transcription factors, 6 use STAg, 4–5 natural conditions infection, 3 parasite replication, 3 prevent immunopathology mechanism endogenous LXA4, 15–17 IL–22, 11–12 IL–27, 12–13 inflammation redundancy and control, 14–15 interleukin–10, 9–10 lipoxin A4, 13–14 resolution phase, 8–9 TGF-B, 10–11 protozoan apicomplexa parasite, 3 survival, oocysts, 2, 3

178 Toxoplasma gondii and inflammation resolution (cont.) symptoms development and risk, 1 tachyzoites replication and “dripping” effect, 3 transmission, 3 Toxoplasma gondii vs. Mycobacterium tuberculosis, 17 Tuberculosis, host protection and pathogen evasion BCG and treatment, 24 BCG vaccine, 33–34 disease reactivation AIDS and TNF, 32 drug and treatment, 32 effects, 33 epidemiology, 32 HIV, 32–33 granuloma formation and bacilli (see Granuloma formation and bacilli) history, 23 immune response cell wall components, 31 host cell signalling, 32 mycobacterial dormancy, 31–32 phagosome-lysosome fusion, 31

Index infection and innate immunity dendritic cells, 26 natural killer cells, 26–27 neutorphils, 24–25 regulatory T cells, 27 T cells, 25–26 risk, 24 WHO, 23–24 Tumor necrosis factor-alpha (TNF-D), Histoplasma capsulatum primary and secondary infection, 109 T cells, 106 TNF receptors, 109–110

V Vaccines BCG, 33 HIV, 33 Viral infections detection, viral pathogens, 53 Hepatitis B virus (HBV) model, 53–54 immune defense strategy, 54 iNKT role, 53 lethal outcome, Epstein-Barr virus, 53 role and SAP-Fyn signaling, 53