Nature Reviews Immunology 11, 71 (February 2011)

ReseaRch highlights Nature Reviews Immunology | AOP, published online 14 January 2011; doi:10.1038/nri2920

R E G U L AT O RY T C E L L S

Weight watchers There is a growing understanding of how host metabolism can affect the immune system. Now, a study by Procaccini et al. has described another important link between host energy status and immune function by showing that leptin, a hormone that is mainly produced by adipocytes and that controls food intake and energy expenditure, can activate mammalian target of rapamycin (mTOR) and regulate the proliferative capacity of regulatory T (TReg) cells. mTOR is a serine/threonine kinase that integrates signals from environmental nutrients and growth factors to control cell proliferation and differentiation. In initial experiments conducted in vitro, freshly isolated human TReg cells showed higher mTOR activity and an increased metabolic rate compared with purified effector T cells. Although TReg cells do not normally proliferate in response to in vitro T cell receptor (TCR) stimulation, transient inhibition of mTOR, through pretreatment with rapamycin, led to robust proliferation of TReg cells following culture with CD3- and CD28-specific antibodies. Extending these findings in vivo, the authors found that a single injection of rapamycin promoted TReg cell proliferation in mice, both in the steady state and after immunization with antigen. Additionally, in a model of experimental autoimmune encephalomyelitis (EAE), mice treated with rapamycin before EAE induction showed increased frequencies of TReg cells and decreased disease severity. Interestingly, although decreased mTOR activity seemed to be necessary for the initial phases of TReg cell proliferation, TReg cells that were actively proliferating in vivo

expressed high levels of phosphorylated mTOR. Furthermore, continuous treatment with rapamycin or silencing of mTOR expression with short hairpin RNA failed to reverse TReg cell anergy in vitro. Thus, although early, transient inhibition of mTOR activity could overcome TReg cell anergy, subsequent upregulation of mTOR activity seemed to be required to sustain TReg cell proliferation, indicating that the mTOR pathway has a dynamic role in TReg cell responsiveness. As previous work showed that leptin can be produced by, and inhibits the proliferation of, TReg cells, the authors predicted that this molecule might interact with the mTOR pathway. In support of this, addition of leptin to cultures of TCR-activated, rapamycintreated TReg cells led to increased activation of the mTOR pathway and prevented TReg cell proliferation. In addition, neutralization of leptin markedly reduced mTOR activity in cultured TReg cells, suggesting that autocrine production of leptin by TReg cells may promote their high mTOR activity in vitro. Finally, the authors examined the effects of acute starvation (which markedly reduces circulating levels of leptin and immune function) on the mTOR pathway and TReg cell function. Strikingly, starvation led to increased proportions of TReg cells in peripheral lymph nodes. Furthermore, TReg cells from starved mice showed markedly reduced mTOR activity and increased rates of proliferation in vitro compared with TReg cells from control animals. Taken together, this study describes the leptin–mTOR signalling pathway as an important link

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CORBIS

between host energy status and TReg cell activity. The authors conclude that oscillating mTOR activity is necessary for TReg cell activation and suggest that this may explain why TReg cells are unresponsive to TCR stimulation in vitro, where high levels of leptin and nutrients may sustain mTOR activation.

Yvonne Bordon

ORIGINAL RESEARCH PAPER Procaccini, C. et al. An oscillatory switch in mTOR kinase activity sets regulatory T cell responsiveness. Immunity 33, 929–941 (2010) FURTHER READING Finlay, D. & Cantrell, D. A. Metabolism, migration and memory in cytotoxic T cells. Nature Rev. Immunol. 14 Jan 2011 (doi:10.1038/nri2888)

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ReseaRch highlights Nature Reviews Immunology | AoP, published online 14 January 2011; doi:10.1038/nri2919

m AC R O P H AG E S

Preventing lipid overload Eaten too much over the holiday season? New research shows that macrophages in the mesenteric lymph nodes (MLNs) can offset the proinflammatory effects of overeating saturated fats by expressing angiopoietin-like protein 4 (ANGPTL4), which inhibits the generation of free fatty acids and subsequent lipid uptake by macrophages. After eating saturated fats, longchain fatty acids are incorporated as triglycerides into lipoprotein particles known as chylomicrons, which travel through the lymphatics before entering the blood. The triglycerides in chylomicrons are then hydrolysed by the enzyme lipoprotein lipase (LPL), which is highly expressed by endothelial cells and macrophages, to generate free fatty acids that fuel tissues such

as the heart and muscles. Based on the knowledge that saturated fatty acids have potent pro-inflammatory effects and that ANGPTL4 is an inhibitor of LPL activity, the authors examined the effects of ANGPTL4 on diet-induced obesity and its metabolic consequences. They observed that feeding mice that are deficient for ANGPTL4 (Angptl4–/– mice) a diet rich in saturated fats had lethal consequences, associated with fibrinopurulent peritonitis, intestinal inflammation, a wasting disease and fat-laden ascites fluid. Further analysis showed that these mice had an abundance of chylomicrons in ascites fluid and a marked leukocyte infiltration of the intestine and mesenteric adipose tissues. These abnormalities were preceded by a massive acutephase response, suggesting that fat-induced systemic inflammation was the cause. The authors also noted that the MLNs of Angptl4–/– mice on a highfat diet were dramatically enlarged compared with Angptl4–/– mice on a diet of medium-chain fatty acids, which are not incorporated into chylomicrons and do not flow through the lymphatics. Consistent with chylomicrons having a direct pro-inflammatory effect, the

MLNs of Angptl4–/– mice fed a high-fat diet contained a large number of lipidladen macrophages, known as foam cells, and incubation of peritoneal macrophages from Angptl4–/– mice with an emulsion of chylomicrons led to foam cell formation and the induction of inflammatory gene expression. These effects were shown to be the result of loss of ANGPTL4-mediated inhibition of LPL, as they could be reproduced by incubation with a synthetic inhibitor of LPL and prevented by treatment with recombinant ANGPTL4. Finally, the mechanism of inflammation induced by excess chylomicrons was found to involve activation of endoplasmic reticulum stress pathways, which have previously been linked to inflammation. Together, the findings suggest that ANGPTL4, the expression of which is upregulated in macrophages by chylomicron-derived fatty acids, is part of a feedback mechanism that protects MLN-resident macrophages from lipid overload and associated inflammation.

Lucy Bird

ORIGINAL RESEARCH PAPER Lichtenstein, L. et al. Angptl4 protects against severe proinflammatory effects of saturated fat by inhibiting fatty acid uptake into mesenteric lymph node macrophages. Cell Metab. 12, 580–592 (2010) fuRtHER REAdING Osborn, O., Sears, D. D. & Olefsky, J. M. Fat-induced inflammation unchecked. Cell Metab. 12, 553–554 (2010)

BRAND X

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in brief I M M U N O M E TA B O L I S M

The inflammasome-mediated caspase-1 activation controls adipocyte differentiation and insulin sensitivity Stienstra, R. et al. Cell Metab. 12, 593–605 (2010)

The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance Vandanmagsar, B. et al. Nature Med. 9 Jan 2011 (doi:10.1038/nm.2279)

Obesity is associated with chronic low-grade inflammation and is a known risk factor for a number of metabolic diseases, including type 2 diabetes. The link between obesity and inflammation has been unclear, but these studies show that obesity-induced activation of the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome is crucial for caspase 1-mediated activation of inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18, which promote insulin resistance. Stienstra et al. found that caspase 1 expression was upregulated during differentiation of adipocytes, and mice fed a high-fat diet showed markedly increased levels of caspase 1, IL-1β and IL-18 expression in adipose tissue. Elevated IL-1β production was shown to contribute to insulin resistance in adipose tissue, and adipocytes from NLRP3-deficient or caspase 1-deficient mice showed increased insulin sensitivity and were more metabolically active. Furthermore, treatment of obese mice with a caspase 1 inhibitor improved insulin sensitivity in these animals. In the second study, Vandanmagsar et al. showed a direct correlation between adiposity and the expression of NLRP3 and IL-1β in both humans and mice. Weight loss, resulting from calorie restriction or exercise, led to decreased NLRP3 expression and improved insulin sensitivity in adipose tissue. The authors found that ceramides (lipid molecules that are released into the circulation by adipocytes during progressive obesity) directly activate the NLRP3 inflammasome and induce caspase 1 activation in adipocytes and macrophages. NLRP3-deficient mice fed a high-fat diet showed increased insulin sensitivity and, interestingly, showed decreased expression of interferon-γ and reduced effector T cell numbers in adipose tissue. These results suggest that the NLRP3 inflammasome is also important for regulating adipose tissue T cell responses during obesity. I M M U N O M E TA B O L I S M

IL-17 regulates adipogenesis, glucose homeostasis, and obesity Zúñiga, L. A. et al. J. Immunol. 185, 6947–6959 (2010)

The pro-inflammatory cytokine interleukin-17 (IL-17) is upregulated in the blood of obese humans, but its role in metabolic disease remains unclear. This study shows that IL-17 is produced by γδ T cells in adipose tissue and acts as a negative regulator of adipogenesis and glucose metabolism. Comparison of mice fed a normal, low-fat or high-fat diet showed that increasing obesity promoted the accumulation of IL-17-producing γδ T cells in inguinal adipose tissue. Whereas most αβ T cells in adipose tissue produced interferon-γ (IFNγ) but little IL-17, adipose tissue γδ T cells produced high levels of IL-17 but low levels of IFNγ. Interestingly, IL-17 was shown to inhibit lipid uptake and insulin-induced glucose uptake by adipocytes, and to suppress adipogenesis. Young IL-17-deficient mice were more susceptible to diet-induced obesity than wild-type controls, but older IL-17-deficient mice were no longer protected from diet-induced obesity. The authors suggest that the positive metabolic effects of IL-17 may be overwhelmed by other mechanisms once obesity is established.

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vA C C I N E S

Foes of fungi are just 17 The incidence of systemic fungal infections has increased throughout the world, and this has prompted much interest in developing effective vaccines against these pathogens. T helper 1 (TH1) cells are believed to be crucial for protection from pathogenic fungi, but the exact role of TH17 cells has remained unclear and both protective and pathological functions for these cells have been reported. Now, a recent study suggests that TH17 cells, but not TH1 cells, are necessary for vaccine-induced protection of mice against Coccidioides posadasii, Histoplasma capsulatum and Blastomyces dermatitidis, which are the main causes of systemic mycoses in humans in North America. In initial experiments, the authors showed that a protective vaccine against B. dermatitidis induced both antigen-specific interferon-γ (IFNγ)-producing TH1 cells and interleukin-17 (IL-17)producing TH17 cells in mice. Following pulmonary infection with B. dermatitidis, TH1 and TH17 cells were rapidly recruited to the lungs of vaccinated, but not unvaccinated, mice. Most of the vaccinated animals cleared the infection within 6 days; however, unvaccinated mice could not clear B. dermatitidis and were moribund by day 15.

Interestingly, antibody-mediated blockade of IL-17 inhibited the ability of vaccinated mice to clear B. dermatitidis more profoundly than blockade of IFNγ. Furthermore, despite developing normal TH1-type immune responses, vaccinated mice that were deficient in IL-17 or the IL-17 receptor (IL-17R) were not protected from pulmonary B. dermatitidis infection. By contrast, mice that were deficient in molecules necessary for TH1-type immunity, including T-bet and IL-12R, were protected from subsequent B. dermatitidis infection following vaccination. Importantly, in each of the experiments, vaccine-induced TH17 cells promoted protective antifungal immunity without inducing immunopathology in the lungs. The authors extended these findings using T cell receptor-transgenic OT-I mice (which have few endogenous T cells and do not develop protective immunity following vaccination against B. dermatitidis). They showed that adoptive transfer of B. dermatitidis-specific transgenic T cells to OT-I mice prior to vaccination led to the development of IL-17-producing T cells, which protected against subsequent infection with B. dermatitidis. Transfer of B. dermatitidis-specific T cells that were deficient in T-bet or IL-12R also

NATuRE REVIEws | Immunology

promoted successful vaccination in OT-I mice, indicating that antigenspecific TH17 cells are necessary and sufficient for protective vaccination. Vaccine-induced TH17 cells were shown to protect mice from subsequent B. dermatitidis infection by recruiting and activating granulocytes and macrophages. Interestingly, although the pattern recognition receptor dectin 1 (also known as CLEC7A) was previously suggested to be necessary for TH17 cell responses to fungi, the authors found that vaccination-induced differentiation of TH17 cells did not require dectin 1, but instead depended on expression of the Toll-like receptor adaptor molecule myeloid differentiation primary response protein 88 (MYD88). Finally, they showed that TH17 cells are also required for vaccine-induced protection of mice from C. posadasii and H. capsulatum. Together, these results suggest that future vaccination strategies for human fungal infections should concentrate on priming effective TH17 rather than TH1 cell responses.

Yvonne Bordon

ORIGINAL RESEARCH PAPER Wüthrich, M. et al. Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice. J. Clin. Invest. 4 Jan 2011 (doi:10.1172/JCI43984)

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In the news One FLu Over? eschew the rest! Last year we had a vaccine surplus (Nature Rev. Immunol., Feb 2010), this year there isn’t enough (The Telegraph, 13 Jan 2011): is there ever any good news when it comes to our struggle with influenza virus? Amid all the media reports of death and despair, a group of scientists provided a glimmer of hope by showing that humans who recover from infection with swine flu (the 2009 H1N1 pandemic strain of influenza virus) may have increased resistance to all other strains of influenza virus (J. Exp. Med., 10 Jan 2010). The researchers, led by Patrick Wilson (University of Chicago) and Rafi Ahmed (Emory University, Atlanta), identified five antibodies from patients infected with swine flu in 2009 that could recognize all H1N1 strains from the last decade, as well as the 1918 ‘Spanish flu’ and deadly H5N1 ‘bird flu’ strains (BBC News, 11 Jan 2011). Strikingly, these antibodies protected mice from otherwise lethal doses of influenza virus. The study has important implications for vaccine development. “It says a universal influenza vaccine is really possible,” said Wilson (Reuters, 10 Jan 2011). The antibodies seem to protect against multiple strains of influenza virus because of their unusual tendency to bind the stalk region of viral haemagglutinin. Unlike other regions of haemagglutinin, the stalk region shows little variation between different influenza virus strains and is, according to Ahmed, “the Achilles’ heel” of the virus (Fox News, 10 Jan 2011). One word of warning: being overweight may reduce your chances of developing these ‘super’ antibodies. Researchers from the California Department of Public Health found that obese individuals have an up to 300% increased risk of death following infection with swine flu (Tehran Times, 13 Jan 2010). The mechanisms may be unclear, but these findings offer yet another fascinating link between host metabolism and immunity. Yvonne Bordon

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m AC R O P H AG E S

A transcription factor to call their own Not to be outdone by the everexpanding list of T cell subsets and their defining transcription factors, macrophages are let in on the act by new research showing that interferon regulatory factor 5 (IRF5) is a subsetdefining factor for M1 macrophages with both activating and repressive transcriptional functions. Monocyte-derived macrophages that are differentiated with granulocyte–macrophage colony stimulating factor (GM-CSF) adopt an M1 (classical) phenotype, which is associated with the production of pro-inflammatory cytokines such as interleukin-12 (IL-12). Conversely, differentiation with M-CSF leads to the polarization of M2 (alternative) macrophages that produce antiinflammatory cytokines such as IL-10. This study showed that treatment of human monocytes with GM-CSF but not M-CSF resulted in increased expression of IRF5.

In line with a role for IRF5 in macrophage plasticity, as well as in initial polarization, the conversion of M2 macrophages to M1 macrophages by culture with GM-CSF also resulted in increased IRF5 expression. Human M2 macrophages that were forced to express IRF5 had increased expression of IL-12p70 and IL-23 and decreased expression of IL-10, whereas inhibition of IRF5 expression in M1 macrophages resulted in the converse levels of cytokine production. These data show that IRF5 promotes the IL-12hiIL-23hiIL-10low cytokine profile that is associated with M1 macrophages, and further studies showed that this is the result of direct effects on transcriptional activity. The mRNA levels for these cytokines were modulated by IRF5 in a manner consistent with the protein data, and genome-wide expression analysis showed that IRF5 induces

the expression of M1-specific genes and represses the expression of M2-specific genes. Furthermore, IRF5 was shown to bind to the promoter regions of the genes encoding IL-12p40, IL-12p35, IL-23p19 and IL-10. Recruitment of RNA polymerase II to the IL10 promoter only occurred after the dissociation of IRF5 from this region. This is in keeping with a role for IRF5 in transcriptional inhibition of the IL10 gene, and is supported by the demonstration that IRF5 inhibits the expression of a reporter construct containing the IL10 promoter sequence. Mutations in IRF5 or the IL10 promoter region that disrupted IRF5–DNA binding prevented this inhibitory effect on reporter expression. These data indicate that the expression of GM-CSF at sites of inflammation can drive M1 macrophage polarization through increased IRF5 expression, which has both positive and negative effects on the transcription of macrophage subset-specific genes. In turn, M1 macrophages are known to drive pro-inflammatory T helper 1 (TH1) cell responses. Indeed, the forced expression of IRF5 by human M2 macrophages resulted in increased proliferation of MHC-mismatched T cells and the differentiation of both TH1 and TH17 cell populations.

Kirsty Minton

ORIGINAL RESEARCH PAPER Krausgruber, T. et al. IRF5 promotes inflammatory macrophage polarization and TH1–TH17 responses. Nature Immunol. 16 Jan 2011 (doi:10.1038/ni.1990)

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A u tO I m m u N I t y

Joint damage without antigen The development of various auto­ immune diseases, including rheuma­ toid arthritis, is thought to be due to a breakdown in CD4+ T cell tolerance for a tissue­specific antigen. However, several lines of evidence have suggested that cognate antigen recognition by CD4+ T cells may not always be necessary. Murakami et al. now show that initiation of CD4+ T cell­dependent arthritis in gp130F759/F759 mice involves the local accumulation of activated T helper 17 (TH17) cells in the absence of cognate antigen recognition. This triggers an interleukin­17A (IL­17A)­dependent IL­6 amplification loop, which the authors termed the ‘IL­6 amplifier’. gp130F759/F759 mice have enhanced IL­6 receptor­mediated signal­ ling and spontaneously develop a

rheumatoid arthritis­like disease as they age. In this study, the authors found that gp130F759/F759 mice engineered to express a single T cell receptor that recognizes a non­joint antigen also develop arthritis, indi­ cating that cognate antigen recogni­ tion was not involved. These mice had a higher number of TH17 cells in lymphoid tissues, and the concentra­ tions of IL­6 and IL­17A in the blood were increased. The authors hypothesized that local events in the joint (such as microbleeding) may contribute to joint inflammation by triggering the accu­ mulation of activated TH17 cells, so they transferred in vitro differentiated TH17 cells to gp130F759/F759 or control C57BL/6 mice that had undergone experimental microbleeding in one

nATuRe RevIewS | Immunology

leg. Arthritis developed in the leg in which microbleeding was induced (but not the other leg) in gp130F759/F759 mice following TH17 cell transfer, but did not occur in control mice, suggesting that the enhanced sensi­ tivity to IL­6 in gp130F759/F759 mice is required for disease. Microbleeding in the joint induced the localized expres­ sion of CC­chemokine ligand 20 (CCL20), which is a chemoattractant for CC­chemokine receptor 6 (CCR6)+ TH17 cells. In addition, IL­6­mediated signalling in type I collagen­expressing cells and local IL­17A production by TH17 cells were shown to be important for disease pathogenesis in this mouse model. So, putting these observations together, the following model emerges. A local event in the joint, such as microbleeding, induces the accumulation of TH17 cells through increased CCL20 expression, resulting in the activation of the IL­6 amplifier and disease develop­ ment. The authors propose that in humans, the availability of TH17 cells for such a model could be due to the known age­dependent increase in memory or activated phenotype T cells. Also, several factors, such as infection, may increase sensitivity to IL­6 in the tissue. Olive Leavy

ORIGINAL RESEARCH PAPER Murakami, M. et al. Local microbleeding facilitates IL‑6‑ and IL‑17‑dependent arthritis in the absence of tissue antigen recognition by activated T cells. J. Exp. Med. 10 Jan 2011 (doi:10.1084/jem.20100900)

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ReseaRch highlights Nature Reviews Immunology | AoP, published online 21 January 2011; doi:10.1038/nri2924

I m m u N E R E G u L At I O N

MicroRNAs keep microglia quiet MicroRNAs regulate gene expression in many biological processes, and now a new study shows that a brainspecific microRNA is a key regulator of microglial cell quiescence in the central nervous system (CNS), thereby helping to prevent CNS inflammation. Microglial cells are CNS-resident macrophages that, under normal conditions, have a resting phenotype that is characterized by low-level expression of CD45 and MHC class II molecules. During CNS inflammation (such as that which occurs in experimental autoimmune encephalomyelitis (EAE)), microglial cells become activated and are thought to contribute, together with peripheral macrophages that infiltrate the inflamed CNS, to the pathological processes. The mechanisms that maintain the unique resting phenotype of microglial cells are unknown, so Weiner and colleagues set out to investigate whether microRNAs might be involved. A comparison of the expression of 31 known microRNAs in macrophages isolated from different organs of healthy adult mice revealed that only CNS-resident microglial cells expressed the microRNA miR-124. During EAE,

however, miR-124 expression by microglial cells decreased, and CNS-infiltrating peripheral macrophages started to express low levels of miR-124 during the onset and recovery phases of the disease. This suggested that miR-124 expression correlates inversely with the activation state of microglial cells and macrophages in the CNS. Consistent with the idea that miR-124 regulates the activation state of macrophages, the authors showed that transfection of bone marrow-derived macrophages with miR-124 results in downregulation of activation markers, including CD45, MHC class II and CD86. miR-124 transfection also inhibited macrophage proliferation, promoted macrophage polarization to an M2 phenotype and altered cell morphology. Investigation of the mechanisms underlying these effects showed that miR-124 directly binds to the mRNA encoding the myeloid cell master transcription factor CCAAT/enhancer-binding protein-α (C/EBPα), causing downregulation of C/EBPα protein expression. Accordingly, mutation of the three predicted miR-124 binding sites in C/EBPα mRNA abolished the inhibitory effect. In turn, the reduced levels

NATURE REvIEWS | Immunology

of C/EBPα in miR-124-transfected cells caused decreased expression of the downstream transcription factor PU.1 and its target genes encoding CD45 and MHC class II. Returning to the in vivo setting, the authors showed that administration of miR-124 shortly after induction of EAE substantially ameliorated or prevented disease symptoms. Mice treated with miR-124 had less microglial cell activation and leukocyte infiltration in the CNS than control mice. These effects on disease were dependent on miR-124-mediated downregulation of C/EBPα, as they could be replicated by a small interfering RNA specific for C/EBPα but not by miR-124 lacking the C/EBPα binding sequence. Finally, evidence from co-culture experiments supports the hypothesis that signals from CNS stromal cells, such as astrocytes, and neurons cause macrophages to adopt a resting microglial cell phenotype through upregulation of miR-124. Lucy Bird ORIGINAL RESEARCH PAPER Ponomarev, E. D. et al. MicroRNA‑124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP‑α‑PU.1 pathway. Nature Med. 17, 64–70 (2011)

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Cy tO k I N E S

Regulating energy stores Stimulation of hepatocytes with IL‑4 shifted the metabolic reliance of these cells from fatty acid to glucose oxida‑ tion. STAT6, downstream of IL‑4, was shown to directly interact with peroxisome proliferator‑activated receptor‑α (PPARα) — an important regulator of the breakdown and oxidation of fatty acids — thereby inhibiting its transcriptional activity. This suggests that IL‑4–STAT6 signal‑ ling inhibits fatty acid breakdown in hepatocytes by inhibiting PPARα. The authors next determined whether IL‑4–STAT6 signalling affected metabolism during obesity. Although Stat6–/– mice were resistant to high‑fat diet‑induced obesity owing to increased energy

MACMILLAN NEW ZEALAND

Whether interleukin‑4 (IL‑4)‑ driven T helper 2 (TH2)‑type immune responses regulate nutrient metabolism and insulin sensitivity is unclear. In this study, Chawla and colleagues show that IL‑4, through activation of signal transducer and activator of transcription 6 (STAT6), enhances the anabolic actions of insulin to promote storage of glucose. This is mediated by inhibition of catabolic metabolism in the liver and attenuation of inflammation in white adipose tissue. IL‑4 was shown to induce STAT6 activation in the liver, but not in skeletal muscle or white adipose tissue, suggesting that this pathway might regulate hepatic metabolism.

expenditure, they exhibited decreased insulin sensitivity. This decrease in insulin action, which was primarily observed in the liver, resulted from a loss of STAT6‑mediated inhibition of PPARα, a transcription factor that has previously been shown to antagonize the anabolic actions of insulin and decrease glucose disposal. Conversely, administration of IL‑4 to wild‑type mice on a high‑fat diet improved glucose tolerance and insulin sensitivity. In this case, IL‑4 treatment inhibited PPARα trans‑ criptional activity in the liver, and attenuated the expression of nuclear factor‑κB (NF‑κB)‑regulated inflam‑ matory genes in white adipose tissue. Finally, using a model of allergic inflammation, the authors showed that polarization towards a TH2‑type immune response in high‑fat diet‑fed mice improves glucose tolerance and insulin sensitivity. Thus, this report highlights that IL‑4–STAT6 signalling, which is associated with TH2‑type immune responses, improves insulin sensi‑ tivity and may protect against the disease‑promoting effects of high‑fat feeding.

Olive Leavy

ORIGINAL RESEARCH PAPER Ricardo-Gonzalez, R. R. et al. IL-4/STAT6 immune axis regulates peripheral nutrient metabolism and insulin sensitivity. Proc. Natl Acad. Sci. USA 107, 22617–22622 (2010)

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Ly m P H O C y t E m I G R At I O N

Travel agents for two T helper 2 (TH2) cells are associated with protection from parasites but also drive immunopathological allergic responses. Dissecting the mechanisms that are involved in the migration of TH2 cells from lymph nodes to peripheral tissues has attracted much interest, as selectively inhibiting these pathways could be an effective new therapy for patients with allergies. Two recent papers in Nature Immunology further our understanding of TH2 cell trafficking, by showing that extracellular matrix protein 1 (ECM1) is necessary for TH2 cell egress from lymphoid tissues and CC-chemokine receptor 8 (CCR8) is needed for TH2 cell migration to allergic skin. Using microarrays, Li et al. had previously shown that ECM1, a glycoprotein with no known immune function, is highly expressed by TH2 but not TH1 cells. Confirming these findings, their new study demonstrated that cultured TH2 cells secrete higher

levels of ECM1 than other T cell subsets. Furthermore, the expression of ECM1 was found to be controlled by the TH2 cell-associated transcription factors signal transducer and activator of transcription 6 (STAT6) and GATA-binding protein 3 (GATA3). Exploring the significance of ECM1 expression, they found that TH2 cell differentiation was normal in ECM1-deficient mice; however, ECM1-deficient TH2 cells were unable to traffic to the lungs and could not promote disease in a mouse model of allergic airway inflammation. The inability of ECM1-deficient TH2 cells to promote allergic inflammation seemed to be due to the failure of these cells to exit lymphoid tissues. In keeping with this, the expression of sphingosine-1-phosphate receptor 1 (S1P1; also known as S1PR1), which has important roles in promoting lymphocyte egress, was decreased in ECM1-deficient TH2 cells. Retroviral transfection of ECM1-deficient TH2 cells with Ecm1 restored both the expression of S1P1 and S1P1dependent chemotaxis in these cells. Interestingly, ECM1-deficient TH2 cells also showed defective expression of CCR4, which has previously been linked to TH2 cell trafficking, but the authors did not further explore this finding. In the second study, Islam et al. found that mouse CC-chemokine ligand 8 (CCL8) is the only member of the monocyte chemoattractant protein (MCP) family to be constitutively expressed at high levels in the skin. Although other MCP family members promote chemotaxis through CCR2, mouse CCL8 was found to induce cell migration solely through CCR8 (which is also

nATURE REvIEwS | Immunology

a functional receptor for CCL1). Using a model of atopic dermatitis, the authors found that mice lacking CCL8 or CCR8 did not develop chronic TH2-type inflammation in the skin. By contrast, antibodymediated blockade of CCL1 could not protect mice from cutaneous disease. Expression of the TH2-type cytokine interleukin-5 (IL-5), but not that of IL-4 or IL-13, was significantly decreased in CCR8-deficient mice during induction of dermatitis, and this led to decreased eosinophilia and IgG1 class switching in these animals. The defective IL-5 response was a result of impaired recruitment of a subset of IL-5-producing TH2 cells; although these cells seemed to differentiate normally in CCR8-deficient mice, they accumulated in skindraining lymph nodes and could not enter the allergen-inflamed skin. Interestingly, in experiments with in vitro-derived TH2 cells, only cells that had undergone multiple rounds of differentiation under TH2-polarizing conditions upregulated CCR8 and IL-5, suggesting that CCR8 is important for recruiting highly differentiated TH2 cells to the skin. Finally, the authors extended their findings to humans, by showing ex vivo that CCR8+CD4+ T cells from healthy donors were enriched for IL-5 production, whereas CCR4+CD4+ T cells produced IL-4 but not IL-5.

Yvonne Bordon

ORIGINAL RESEARCH PAPERS Li, Z. et al. ECM1 controls TH2 cell egress from lymph nodes through re-expression of S1P1. Nature Immunol. 9 Jan 2011 (doi:10.1038/ni.1983) | Islam, S. A. et al. Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ TH2 cells. Nature Immunol. 9 Jan 2011 (doi:10.1038/ni.1984)

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ReseaRch highlights Nature Reviews Immunology | AOP, published online 21 January 2011; doi:10.1038/nri2923

H A E m At O P O I E S I S

Baby tolerance The human adaptive immune system starts to form at an early stage of fetal development (as early as gestation week 10), which contrasts with mice, in which the adaptive immune system only starts to develop around birth. Therefore, mechanisms must exist in humans to prevent a fetal immune response to maternal alloantigens. Reporting in Science, Mold et al. show that human T cells arise from different haematopoietic stem and progenitor cell (HSPC) populations during different stages of development and that fetal CD4+ T cells are biased towards immune tolerance. The authors isolated CD4+ T cells from fetal mesenteric lymph nodes after 18–22 weeks of gestation, and compared them with adult naive CD4+ T cells that were isolated from the blood. Fetal naive CD4+ T cells were more responsive than adult CD4+ T cells to allogeneic stimulation, and a higher proportion of the fetal CD4+ T cells developed into CD25+FOXP3+ regulatory T (TReg) cells after stimulation. Furthermore, fetal and adult CD4+ T cells (including fetal and adult TReg cells) had

distinct gene expression profiles, as determined by microarray analysis. These data suggest that fetal and adult CD4+ T cells are distinct populations and that fetal CD4+ T cells are biased towards immune tolerance. The authors next determined whether fetal and adult CD4+ T cells arise from different HSPC populations. During development, the HSPC pool first resides in the aorta–gonad–mesonephros region, then in the fetal liver and finally in the bone marrow, where most HSPCs are thought to reside throughout adulthood. The authors isolated human HSPCs from fetal liver, fetal bone marrow and adult bone marrow and transferred them to SCID-hu Thy/Liv mice (immunodeficient mice that are engrafted with human fetal thymus and liver tissue and are used to model human haematopoiesis), where they could develop into single positive (SP) thymocytes. A significantly higher number of forkhead box P3 (FOXP3)+ TReg cells arose from fetal

liver- and bone marrow-derived HSPCs than from adult bone marrow-derived HSPCs. Fetal HSPC-derived CD4+ SP thymocytes were highly responsive to allogeneic stimulation in vitro, and significantly more developed into TReg cells compared with adult HSPC-derived CD4+ SP thymocytes. In addition, the gene expression profiles of fetal liver- and fetal bone marrowderived CD4+ SP thymocytes were indistinguishable, but were distinct from the gene expression profile of adult bone marrow-derived CD4+ SP thymocytes. These observations suggest that haematopoiesis occurs in waves that generate distinct T cell populations at different times of development. Although it is still not clear whether fetal and adult HSPCs are distinct lineages or whether adult HSPCs arise from fetal HSPCs in the bone marrow, these data show that fetal and adult HSPCs give rise to distinct T cell populations, and that during development the initial waves of CD4+ T cells are biased towards tolerance.

Olive Leavy

ORIGINAL RESEARCH PAPER Mold, J. E. et al. Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans. Science 330, 1695–1699 (2010)

IS RB CO

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ReseaRch highlights

in brief MUCOSAL IMMUNOLOGY

Induction of colonic regulatory T cells by indigenous Clostridium species Atarashi, K. et al. Science 23 Dec 2010 (doi:10.1126/science.1198469)

There is currently much interest in the crosstalk between the gut microbiota and the immune system. This study shows that forkhead box P3 (FOXP3)+ regulatory T (TReg) cells are most abundant in the colon and, through the use of germ-free or antibiotic-treated specific pathogen-free (SPF) mice, that accumulation of these cells after weaning depends on the gut microbiota. Further analysis identified Clostridium spp. belonging to clusters IV and XIV as the specific component of the microbiota that induces colonic TReg cell accumulation. A defined mix of Clostridium spp. induced the production of transforming growth factor-β by intestinal epithelial cells (in a TLR-, NODand dectin 1-independent manner) and the accumulation of IL-10+CTLA4hi TReg cells in the colon. Finally, oral inoculation of neonatal SPF mice with Clostridium spp. suppressed the development of DSS-induced colitis and systemic IgE responses. dENdRITIC CELLS

Mucosal and systemic anti-HIV immunity controlled by A20 in mouse dendritic cells Hong, B. et al. J. Clin. Invest. 4 Jan 2011 (doi:10.1172/JCI42656)

The ubiquitin-modifying enzyme A20 (also known as TNFAIP3) is a negative feedback regulator of several important pro-inflammatory signalling pathways and controls the immunostimulatory function of antigen-presenting cells. Silencing of A20 mRNA may therefore affect the potency of dendritic cells (DCs) in the induction of HIV-specific immune responses. Injection of mice with A20-silenced, bone-marrow-derived DCs loaded with recombinant HIV envelope protein gp120 resulted in cellular and humoral gp120-specific immune responses, both in mucosal tissues and systemically. These DCs migrated more efficiently to the mesenteric lymph nodes than control DCs and induced the expression of gut-homing receptors on activated lymphocytes, partly through the production of retinoic acid. Furthermore, A20-silenced gp120-pulsed DCs enhanced cytotoxic T cell responses in the absence of CD4+ T cells. So, silencing of A20 may enhance the efficacy of DC-based vaccines against HIV. TUMOUR IMMUNOLOGY

CD169-positive macrophages dominate antitumor immunity by crosspresenting dead cell-associated antigens Asano, K. et al. Immunity 30 Dec 2010 (doi:10.1016/j.immuni.2010.12.011)

It is not known how antigen-presenting cells in the lymph node internalize and cross-present tumour-associated antigens to CD8+ T cells for the initiation of effective antitumour cytotoxic T lymphocyte (CTL) responses. In agreement with previous observations, subcutaneous injection of dead tumour cells activated tumour-specific CTLs. However, this did not induce the migration of CD11c+ DCs from the skin to the draining lymph node; instead, the dead cells travelled via lymphatic flow to the draining lymph node, where they were phagocytosed by CD169+ macrophages in a phosphatidylserine-dependent manner. Antitumour responses did not develop when dead tumour cells were administered to tumour-bearing mice that lacked CD169+ macrophages. Finally, CD11c+CD169+ macrophages (which mainly localized in the cortical and paracortical sinus) were shown to directly cross-present dead-cell-associated antigen to CD8+ T cells. So, CD169+ macrophages promote tumour immunity following tumour cell death. nature reviews | Immunology

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F O C U S O N m E TA b O l i S m A N d i m m U N O lO g y

Foreword FOCUS CONTENTS 85

98

Adipokines in inflammation and metabolic disease Noriyuki Ouchi, Jennifer L. Parker, Jesse J. Lugus and Kenneth Walsh Type 2 diabetes as an inflammatory disease Marc Y. Donath and Steven E. Shoelson

109 OPINION Metabolism, migration and memory in cytotoxic T cells David Finlay and Doreen A. Cantrell

Immunometabolism: an emerging frontier Abstract | Immunometabolism is an emerging field of investigation at the interface between the historically distinct disciplines of immunology and metabolism. Accelerating interest in this area is being fuelled by the obesity epidemic and the relatively recent realization that obesity affects the immune system and promotes inflammation, and that obesity-induced inflammation potentially promotes a variety of chronic conditions and diseases. The multilevel interactions between the metabolic and immune systems suggest pathogenic mechanisms that may underlie many of the downstream complications of obesity and offer substantial therapeutic promise. “To lengthen thy life, lessen thy meals.”

Benjamin Franklin, Poor Richards Almanac (1737).

Diane Mathis Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA. e‑mail: [email protected] Steven E. Shoelson Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA. e‑mail: steven.shoelson@ joslin.harvard.edu doi:10.1038/nri2922

It has long been recognized that effector cells of the immune system are required to ward off tumours and infectious agents. Likewise, it is well known that regulatory cells of the immune system rein in such responses, as well as guarding against immune dysregulation, such as that which occurs in allergy and autoimmunity. Even greater respect for this powerful homeostatic system has emerged over the past few years with the increasing appreciation that immune cells also affect important non-immune functions, including neurodegeneration, cardiovascular function and metabolism. This Focus issue of Nature Reviews Immunology, produced with support from sanofi-aventis, draws attention to an emerging frontier, immunometabolism — that is, the interplay between immunological and metabolic processes. On the one hand, it has emerged that certain supposedly non-immune pathologies result in mobilization of the innate and adaptive immune systems and, in the case of obesity, this promotes metabolic abnormalities, culminating in increased susceptibility to type 2 diabetes, cardiovascular diseases, cancer and neurodegeneration. On the other hand, it is now clear that the behaviours of lymphocytes and other leukocytes are controlled on many levels by internal metabolic properties. Dissection of the molecular underpinnings of the immunological–metabolic crosstalk has become a priority.

Obesity and chronic disease The obesity epidemic continues unabated in Western countries, and is rising even more dramatically throughout the rest of the world, paradoxically even

in the countries where poverty and malnutrition are most widespread. Coinciding with recent increases in obesity have been proportional increases in medical conditions with obvious metabolic connections, such as cardiovascular disease, type 2 diabetes, fatty liver disease and cirrhosis. Additional associations are being drawn between obesity and diseases that are less obviously linked to metabolic derangements, including asthma, Alzheimer’s disease and several forms of cancer. Inflammation has been aetiologically linked to the pathogenesis of each of these conditions, and as obesity is causally linked to a systemic low-grade subacute inflammatory state, as well as inflammation in adipose tissue, obesity-induced inflammation may be a common pathogenic denominator. Two articles in this Focus issue discuss the relationship between adipose tissue expansion and inflammation. The article by Ouchi et al.1 focuses on adipokines, which are bioactive proteins that are produced by adipose tissues and have hormonal or cytokine actions locally and in other tissues. The article by Donath and Shoelson2 discusses the immunological effects of expanding fat mass and inflammation in insulin resistance, and the effects of inflammation in pancreatic islets as these relate to the development and severity of type 2 diabetes.

Adipokines Some adipokines, such as leptin and adiponectin, are produced exclusively (or at least predominantly) by adipose tissues, whereas other so-called adipokines are more typical pro-inflammatory or anti-inflammatory cytokines that are best known for their roles in innate and adaptive immune responses. The list of proinflammatory adipokines that are produced by fat

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FOrEwOrd tissues includes tumour necrosis factor (TnF), interleukin-6 (IL-6), resistin, retinol-binding protein 4 (RBP4) and the closely related protein lipocalin 2, CC-chemokine ligand 2 (CCL2), IL-18, nicotinamide phosphoribosyltransferase (nAmPT; also known as visfatin) and CXC-chemokine ligand 5 (CXCL5). The production of each of these adipokines is increased with adipose tissue expansion, suggesting that this contributes to the pro-inflammatory state that is associated with obesity, and potentially to the deleterious consequences of obesity that are mediated by chronic inflammation. By contrast, anti-inflammatory adipokines and other cytokines produced in fat, including adiponectin, IL-10 (Ref. 3) and the WnT inhibitor secreted frizzled-related protein 5 (SFRP5)4, seem to decrease with fat mass expansion, and this could also contribute to the pro-inflammatory state associated with obesity and its deleterious consequences. Although all of the adipokines are found in adipose tissue, the relative amounts produced by adipocytes versus macrophages, endothelial cells, T cells and mast cells, for example, are in many cases unknown. Also unknown are the relative extents to which the adipokines act as primary inhibitors of insulin sensitivity and secretion as opposed to secondary mediators through their effects on leukocyte recruitment and activation.

obesity is causally linked to a systemic lowgrade subacute inflammatory state, as well as inflammation in adipose tissue

Inflammation in type 2 diabetes Attempts to target inflammation in type 2 diabetes have moved quickly for two main reasons. Foremost is the robustness of the clinical end point — that is, changes in measures of glycaemic control. Fasting blood glucose and glycated haemoglobin (HbA1c) levels are easily measured and highly accurate and reproducible. Fasting blood glucose changes within days to weeks of initiating a therapy, whereas HbA1c levels provide an 8–12 week integrated average blood glucose measure. It is therefore possible to test the efficacy of anti-inflammatory strategies within weeks for initial assessment and in months for highly predictive results. This is in contrast to assessments of drug efficacy and safety in other conditions such as cardiovascular disease, which require much larger trial sizes and duration — and hence much greater cost — to assess true clinical outcomes. Trials of drugs to prevent Alzheimer’s disease are even more challenging, as the common forms of the disease cannot be predicted before disease onset, and this makes trial size and duration, and costs, prohibitive. Clinical trials that assess effects on biomarkers that associate with cardiovascular disease or Alzheimer’s disease can be smaller and of shorter duration, but their ability to predict clinical outcomes is often weak or unknown. As trials in type 2 diabetes can be conducted using reasonable numbers of subjects and at a reasonable cost, they may be used as a screen for potential anti-inflammatory treatments for other obesity-induced chronic diseases that are more difficult to study. Completed and ongoing trials are testing this possibility. Three strategies discussed by Donath and Shoelson2 are the use of salicylates, such as salsalate,

and neutralization of either IL-1 or TnF. Small clinical trials report positive outcomes following selective blockade of IL-1 receptor type 1, either with specific antibodies or recombinant IL-1 receptor antagonist 5,6. Salsalate is a prodrug form of salicylate (an orally active, small-molecule anti-inflammatory drug) and has also been shown to lower blood glucose levels in patients with type 2 diabetes7–9. IL-1 antagonism and salsalate are both being tested further in larger clinical trials. Although small clinical trials using TnF blockade have not provided improvements in blood glucose levels in patients with type 2 diabetes, encouraging results in non-diabetic patients being treated with TnF blockers for other conditions suggest that this might be worth re-exploring.

Unique metabolic uses in immune cells A completely different perspective on the immunological–metabolic interface is the extent to which, and the precise mechanisms by which, typical cell-intrinsic metabolic processes influence the performance of immune cells. In most cases, immune cells use and respond to nutrients similarly to other cells, so it is the exceptions to the rules that may be most interesting. The serine/threonine kinases AKT1–3, AmPK (AmPactivated protein kinase), mTOR (mammalian target of rapamycin) and LKB1 (also known as STK11) are generally thought of as cellular nutrient sensors that help to maintain energy homeostasis by relaying signals that determine how cells respond to high or low levels of intracellular carbohydrates or amino acids. Finlay and Cantrell10 suggest that in addition to their more established roles in nutrient responses, AKT1–3, AmPK and LKB1 control a fate switch, from cytotoxic effector to memory CD8+ T cells. They argue that in CD8+ T cells the main role for the AKT proteins is to regulate repertoires of adhesion molecules and chemokine receptors and hence to control trafficking and migration, and that this is what determines the memory versus terminally differentiated effector decision in CD8+ T cells. A separate series of investigations looked at the effects of LKB1 in haematopoietic stem cells (HSCs)11–13. As noted above, in most cells LKB1 is a serine/threonine kinase that is upstream of AmPK (a master regulator of energy homeostasis) and mTOR complex 1 (mTORC1; a protein complex that controls protein synthesis and cell proliferation). These three reports showed that LKB1 regulates the function and dynamics of HSCs through pathways that are independent of AmPK and mTORC1. Deletion of Lkb1 in mice led to an initial expansion of HSCs and multipotent progenitors, but over time the cells were depleted and the mice became pancytopenic. moreover, an Lkb1–/– bone marrow transplant was unable to reconstitute the haematopoietic system in irradiated mice, again suggesting that the survival of HSCs depends on LKB1. Together, these recent studies showed that under certain conditions, immune cells may use metabolic pathways to control fate and function in ways that are different from other cells.

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F O C U S O N m E TA b O l i S m A N d i m m UrNEOwlO y FO Og rd in addition to their more established roles in nutrient responses, AKT1–3, AMPK and LKB1 control a fate switch, from cytotoxic effector to memory CD8+ T cells.

Thus, the emerging field of immunometabolism has already yielded some novel insights, which have theoretical and practical implications for future work. On the theoretical side, several important questions have been raised, notably: to what extent are obesity and inflammation triggered in parallel or in sequence? If they are mainly triggered in parallel, what is the common initiating signal? If in sequence, what signals link the two processes? Why does obesity-associated inflammation persist, as opposed to being held in check? By what pathway(s) does inflammation provoke type 2 diabetes, cardiovascular disease and other downstream pathologies? Can genetic and environmental factors reinforce or dissociate the link between metabolic and immunological abnormalities? On the practical side, the finding that inflammation mediates many of the pathological consequences of obesity raises the hope of exploiting the existing armamentarium of antiinflammatory drugs, or future ones, to treat patients with obesity-associated metabolic and cardiovascular disorders (and even perhaps some cancers and neurodegenerative diseases). underscoring this potential, the type 2 diabetes drug metformin has shown promise in cancer prevention14, and is being tested in trials for its ability to prevent various cancers. And even more to the point, a daily dose of aspirin (an anti-inflammatory salicylate) correlates with reduced death from several different cancers15.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Ouchi, N., Parker, J. L., Lugus, J. J. & Walsh, K. Adipokines in inflammation and metabolic disease. Nature Rev. Immunol. 11, 85–97 (2011). Donath, M. Y. & Shoelson, S. E. Type 2 diabetes as an inflammatory disease. Nature Rev. Immunol. 11, 98–107 (2011). Lumeng, C. N., Bodzin, J. L. & Saltiel, A. R. Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J. Clin. Invest. 117, 175–184 (2007). Ouchi, N. et al. Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity. Science 329, 454–457 (2010). Larsen, C. M. et al. Interleukin-1-receptor antagonist in type 2 diabetes mellitus. N. Engl. J. Med. 356, 1517–1526 (2007). Larsen, C. M. et al. Sustained effects of interleukin-1 receptor antagonist treatment in type 2 diabetes. Diabetes Care 32, 1663–1668 (2009). Goldfine, A. B. et al. Use of salsalate to target inflammation in the treatment of insulin resistance and type 2 diabetes. Clin. Transl. Sci. 1, 36–43 (2008). Fleischman, A., Shoelson, S. E., Bernier, R. & Goldfine, A. B. Salsalate improves glycemia and inflammatory parameters in obese young adults. Diabetes Care 31, 289–294 (2008). Goldfine, A. B. et al. The effects of salsalate on glycemic control in patients with type 2 diabetes: a randomized trial. Ann. Intern. Med. 152, 346–357 (2010). Finlay, D. & Cantrell, D. A. Metabolism, migration and memory in cytotoxic T cells. Nature Rev. Immunol. 11, 109–117 (2011). Nakada, D., Saunders, T. L. & Morrison, S. J. Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells. Nature 468, 653–658 (2010). Gurumurthy, S. et al. The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Nature 468, 659–663 (2010). Gan, B. et al. Lkb1 regulates quiescence and metabolic homeostasis of haematopoietic stem cells. Nature 468, 701–704 (2010). Gallagher, E. J. & LeRoith, D. Insulin, insulin resistance, obesity, and cancer. Curr. Diab. Rep. 10, 93–100 (2010). Rothwell, P. M. et al. Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials. Lancet 377, 31–41 (2010).

Competing interests statement

The authors declare competing financial interests: see Web version for details.

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REVIEWS Adipokines in inflammation and metabolic disease Noriyuki Ouchi*, Jennifer L. Parker‡, Jesse J. Lugus‡ and Kenneth Walsh‡

Abstract | The worldwide epidemic of obesity has brought considerable attention to research aimed at understanding the biology of adipocytes (fat cells) and the events occurring in adipose tissue (fat) and in the bodies of obese individuals. Accumulating evidence indicates that obesity causes chronic low-grade inflammation and that this contributes to systemic metabolic dysfunction that is associated with obesity-linked disorders. Adipose tissue functions as a key endocrine organ by releasing multiple bioactive substances, known as adipose-derived secreted factors or adipokines, that have pro-inflammatory or anti-inflammatory activities. Dysregulated production or secretion of these adipokines owing to adipose tissue dysfunction can contribute to the pathogenesis of obesity-linked complications. In this Review, we focus on the role of adipokines in inflammatory responses and discuss their potential as regulators of metabolic function. Insulin resistance A condition characterized by the inability of cells (in the muscle, liver and fat) to respond appropriately to endogenous insulin, resulting in increased blood glucose levels.

*Department of Molecular Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya, 466-8550 Japan. ‡ Molecular Cardiology/ Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany Street, W611, Boston, Massachusetts 02118, USA. Correspondence to K.W. e-mail: [email protected] doi:10.1038/nri2921 Published online 21 January 2011

Obesity has become a major worldwide health problem, not least because it is strongly associated with a number of diseases, including insulin resistance , type 2 diabetes, atherosclerosis and ischaemic heart disease, that reduce life expectancy and together have huge economic and societal consequences. Increasing evidence indicates that obesity is causally linked to a chronic low-grade inflammatory state 1,2, which contributes to the development of obesity-linked disorders, in particular to metabolic dysfunction. It is now well established that adipose tissue is not only involved in energy storage but also functions as an endocrine organ that secretes various bioactive substances 3,4. The dysregulated expression of these factors, caused by excess adiposity and adipocyte dysfunction, has been linked to the pathogenesis of various disease processes through altered immune responses. As such, much attention has been paid to developing a better understanding of the immunoregulatory functions of adipose tissue. New factors secreted by adipose tissue have been identified that either promote inflammatory responses and metabolic dysfunction or contribute to the resolution of inflammation and have beneficial effects on obesity-linked metabolic disorders. These findings lend additional support to the notion that an imbalance of pro- and anti-inflammatory adipokines secreted by adipose tissue contributes to metabolic dysfunction.

Obesity and inflammation Clinical observations. Obesity — in particular, excess visceral adiposity — is strongly associated with insulin resistance, hypertension and dyslipidaemia, which contribute to high rates of mortality and morbidity. Accumulating evidence indicates that a state of chronic inflammation has a crucial role in the pathogenesis of obesity-related metabolic dysfunction1,2. Indeed, clinical and epidemiological studies have described a clear connection between the development of low-grade inflammatory responses and metabolic diseases, particularly in the context of obesity and type 2 diabetes. Excess adipose mass (as occurs in obese individuals) is associated with increased levels of the pro-inflammatory marker C-reactive protein (CRP) in the blood5. Increased levels of CRP, and its inducer interleukin-6 (IL-6), are predictive of the development of type 2 diabetes in various populations5,6. In addition, interventions aimed at causing weight loss lead to reductions in the levels of pro-inflammatory proteins, including CRP and IL-6 (Ref. 7). The adipokine concept. Adipose tissue was traditionally considered to be a long-term energy storage organ, but it is now appreciated that it has a key role in the integration of systemic metabolism. This metabolic function is mediated, in part, by its ability to secrete numerous proteins. Factors that are secreted by adipose tissue are collectively

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ReVieWs

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Figure 1 | Adipose tissue depots. Adipose tissue is mainly found in subcutaneous and visceral depots. Under conditions of obesity, adipose tissue expands in these and other depots throughout the body. Common sites of adipose tissue accumulation 0CVWTG4GXKGYU^+OOWPQNQI[ include the heart, the kidneys and the adventitia of blood vessels. Differential adipokine secretion by various adipose tissue depots may selectively affect organ function and systemic metabolism.

referred to as adipokines3,4. Importantly, following the onset of obesity, the secretory status of an adipose tissue depot can be modified by changes in the cellular composition of the tissue, including alterations in the number, phenotype and localization of immune, vascular and structural cells. The expression of adipokines can also vary depending on the site of an adipose tissue depot (fIG. 1). The two most abundant depots are visceral and subcutaneous adipose tissues, which produce unique profiles of adipokines8,9. In addition, adipocyte depots occur throughout the body in association with multiple organs, including the heart and kidneys. Adipocytes are also found in the bone marrow, lungs and the adventitia of major blood vessels. In some instances, it has been shown that high-calorie diets can promote the development of a pro-inflammatory state in these depots in a similar manner to that observed in subcutaneous and visceral adipose tissue (for example, see Ref. 10). Although the functional importance of many of these individual adipose depots is generally not known, recent evidence suggests that diet-induced changes in their adipokine secretion can influence the function of the associated tissue11. brown adipose tissue, which is mainly found in infants and hibernating animals, is functionally distinct from white adipose tissue, and is not covered in this Review. Adipsin (also known as complement factor D) was identified as an adipokine in 1987 (Ref. 12). In 1993, tumour necrosis factor (TNF) was identified as a proinflammatory product of adipose tissue that is induced in models of diabetes and obesity, providing evidence for a functional link between obesity and inflammation13. subsequently, leptin was identified as an adipose tissuespecific secreted protein that regulates food intake and energy expenditure in an endocrine manner 14. similarly,

the identification of plasminogen activator inhibitor 1 (PAI1), an inhibitor of fibrinolysis, as an adipokine that is strongly upregulated in visceral adipose depots in obesity 15 suggested a mechanistic link between obesity and thrombotic disorders. At about the same time, adiponectin (also known as ACRP30 and ADIPOQ) was identified as an adipocyte-specific adipokine16–18. Adiponectin expression was found to be decreased in obesity, and studies in experimental organisms showed that adiponectin protects against several metabolic and cardiovascular disorders that are associated with obesity. These results were surprising as most adipokines stimulate inflammatory responses, are upregulated in obesity and promote obesity-induced metabolic and cardiovascular diseases. Collectively, these findings have led to the notion that metabolic dysfunction that is due to excess adipose tissue mass may partly result from an imbalance in the expression of pro- and anti-inflammatory adipokines, thereby contributing to the development of obesity-linked complications. Accordingly, the concept that adipokines function as regulators of body homeostasis has received widespread attention from the research community (TABLe 1). Infiltration of immune cells into adipose tissue. Adipose tissue is mainly comprised of adipocytes, although other cell types contribute to its growth and function, including pre-adipocytes, lymphocytes, macrophages, fibroblasts and vascular cells (fIG. 2a). Obesity can lead to changes in the cellular composition of the fat pad as well as to the modulation of individual cell phenotypes (BOX 1). Adipose tissues in obese individuals and in animal models of obesity are infiltrated by a large number of macrophages, and this recruitment is linked to systemic inflammation and insulin resistance19,20. moreover, the accumulation of adipose tissue macrophages is proportional to adiposity in both humans and mice19,20, and sustained weight loss results in a reduction in the number of adipose tissue macrophages that is accompanied by a decrease in the pro-inflammatory profiles of obese individuals21. macrophages are also more abundant in visceral than subcutaneous adipose tissue22, and this is in line with the belief that visceral adipose tissue has a more important role in the development of insulin resistance. However, it has been recently reported that macrophages accumulate in adipose tissues during the early phase of weight loss, presumably as a result of adipose tissue lipolysis23. Adipose tissue also contains fibroblasts, which produce extracellular matrix components. Recently, it has been shown that metabolically dysfunctional adipose tissue produces excess matrix components that may interfere with adipose mass expansion and contribute to metabolic dysregulation24. Thus, it is becoming increasingly evident that intercellular communication within adipose tissue is required for normal metabolic function. Examples of such intercellular communication include the counter-regulation between the adipocyte-derived anti-inflammatory factors adiponectin and secreted frizzled-related protein 5 (sFRP5) and the macrophage-derived pro-inflammatory factors TNF and wNT5a. under conditions of obesity, TNF and wNT5a are upregulated, whereas adiponectin and sFRP5 are downregulated3,4,25 (fIG. 2b).

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f o c u s o n m e ta b o l i s m a n d i m m uRneoVlo gy ieW s Table 1 | Sources and functions of key adipokines Adipokine

Primary source(s)

Binding partner or receptor

Function

Leptin

Adipocytes

Leptin receptor

Appetite control through the central nervous system

Resistin

Peripheral blood mononuclear cells (human), adipocytes (rodent)

Unknown

Promotes insulin resistance and inflammation through IL-6 and TNF secretion from macrophages

RBP4

Liver, adipocytes, macrophages

Retinol (vitamin A), transthyretin

Implicated in systemic insulin resistance

Lipocalin 2

Adipocytes, macrophages

Unknown

Promotes insulin resistance and inflammation through TNF secretion from adipocytes

ANGPTL2

Adipocytes, other cells

Unknown

Local and vascular inflammation

TNF

Stromal vascular fraction cells, adipocytes

TNF receptor

Inflammation, antagonism of insulin signalling

IL-6

Adipocytes, stromal vascular fraction cells, liver, muscle

IL-6 receptor

Changes with source and target tissue

IL-18

Stromal vascular fraction cells

IL-18 receptor, IL-18 binding protein

Broad-spectrum inflammation

CCL2

Adipocytes, stromal vascular fraction cells

CCR2

Monocyte recruitment

CXCL5

Stromal vascular fraction cells (macrophages)

CXCR2

Antagonism of insulin signalling through the JAK–STAT pathway

NAMPT

Adipocytes, macrophages, other cells

Unknown

Monocyte chemotactic activity

Adiponectin

Adipocytes

Adiponectin receptors 1 and 2, T-cadherin, calreticulin–CD91

Insulin sensitizer, anti-inflammatory

SFRP5

Adipocytes

WNT5a

Suppression of pro-inflammatory WNT signalling

ANGPTL2, angiopoietin-like protein 2; CCL2, CC-chemokine ligand 2; CXCL5, CXC-chemokine ligand 5; IL, interleukin; JAK, Janus kinase; NAMPT, nicotinamide phosphoribosyltransferase; RBP4, retinol-binding protein 4; SFRP5, secreted frizzled-related protein 5; STAT, signal transducer and activator of transcription; TNF, tumour necrosis factor.

Crown-like structure An aggregation of single or fused macrophages (also referred to as multinucleated giant cells) around a single adipocyte in adipose tissue. These structures are typically associated with obesity, adipose tissue dysfunction and chronic inflammation.

M1 or ‘classically activated’ macrophage A macrophage that is activated by Toll-like receptor ligands (such as lipopolysaccharide) and interferon-γ, and that expresses inducible nitric oxide synthase and nitric oxide, as well as other pro-inflammatory factors.

M2 or ‘alternatively activated’ macrophage A macrophage that is stimulated by interleukin-4 (IL-4) or IL-13, and that expresses arginase 1, the mannose receptor CD206 and the IL-4 receptor α-chain.

It has become evident that in addition to absolute fat quantity, qualitative aspects of adipose tissue function and cellular composition have an important effect on the systemic metabolic phenotype26. Indeed, body massmatched obese individuals can be divided into two categories: those that have fully dysfunctional metabolic control and those that have mildly dysfunctional metabolic control (fIG. 3). Obese individuals with the latter intermediate metabolic phenotype have lower levels of inflammatory marker expression and reduced cardiovascular risk compared with metabolically dysfunctional obese individuals27. In the same study, the classification of metabolically dysfunctional obese individuals correlated with the presence of crown-like structures, which are histological features that represent an accumulation of macrophages around dead adipocytes in inflamed adipose tissue28,29. because a key function of macrophages is to remove apoptotic cells in an immunologically silent manner to prevent the release of noxious substances, it is reasonable to speculate that the presence of crown-like structures in adipose tissue reflects a pro-inflammatory state that is due, in part, to an impairment of the macrophage-mediated phagocytic process. Consistent with this notion is the finding that the induction of adipocyte apoptosis in an inducible mouse model of lipoatrophy leads to macrophage accumulation in adipose tissues30.

However, the process may be more complex as a recent paper has reported that adipocyte death is not increased by obesity in humans31. Different subsets of macrophages are involved in obesity-induced adipose tissue inflammation. macrophages that accumulate in the adipose tissues of obese mice mainly express genes associated with an M1 or ‘classically activated’ macrophage phenotype, whereas adipose tissue macrophages from lean mice express genes associated with an M2 or ‘alternatively activated’ macrophage phenotype32. stimulation with T helper 1 (TH1)-type cytokines, including interferon-γ (IFNγ), or with bacterial products leads to the generation of m1 macrophages, which produce pro-inflammatory cytokines (including TNF and IL-6), express inducible nitric oxide synthase (iNOs) and produce reactive oxygen species (ROs) and nitrogen intermediates33. by contrast, macrophages are polarized to the m2 phenotype by TH2-type cytokines such as IL-4 and IL-13. m2 macrophages upregulate production of the antiinflammatory cytokine IL-10 and downregulate synthesis of pro-inflammatory cytokines. The transcription of several genes, including those encoding arginase 1, macrophage mannose receptor 1 and IL-1 receptor antagonist, is upregulated in m2 macrophages, through a programme that is reported to be regulated by the transcription factors peroxisome proliferator-activated receptor-γ (PPARγ)

NATuRE REvIEws | Immunology

vOLumE 11 | FEbRuARy 2011 | 87 © 2011 Macmillan Publishers Limited. All rights reserved

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Figure 2 | Components of adipose tissue. a | Adipocytes are the main cellular component of adipose tissue, and they are crucial for both energy storage and 0CVWTG4GXKGYU^+OOWPQNQI[ endocrine activity. The other cell types that are present are precursor cells (including pre-adipocytes), fibroblasts, vascular cells and immune cells, and these cells constitute the stromal vascular fraction of adipose tissue. Vascular cells include both endothelial cells and vascular smooth muscle cells, which are associated with the major blood vessels. The blood vessels in adipose tissue are required for the proper flow of nutrients and oxygen to adipocytes, and they are the conduits that allow for the distribution of adipokines. Vascular cells also secrete, and are responsive to, adipose tissue-secreted proteins. Other active adipose tissue components include macrophages and T cells, which have major roles in determining the immune status of adipose tissue. The fibroblast-derived extracellular matrix functions to provide mechanical support, and excess matrix can lead to adipose tissue dysfunction. Factors that are secreted by these different cellular components are critical for maintaining homeostasis in adipose tissue and throughout the body. b | Examples of intercellular communication between different adipose tissue cell types include the counter-regulation between adiponectin and tumour necrosis factor (TNF), and between secreted frizzled-related protein 5 (SFRP5) and WNT5a. Under conditions of obesity the pro-inflammatory factors (TNF and WNT5a) predominate.

and PPARδ34. Functionally, m2 macrophages are associated with the repair of injured tissues and the resolution of inflammation33. so, it has been suggested that m1 macrophages promote insulin resistance and m2 macrophages protect against obesity-induced insulin resistance35. Recent studies have described subsets of T cells that are present in adipose tissues and seem to be involved in the regulation of macrophage phenotype. CD4+ regulatory T cells are more abundant in the adipose tissues of lean mice and have a protective effect by inhibiting proinflammatory macrophages, leading to the suppression of insulin resistance36. CD8+ effector T cells and TH1 cell-associated factors can initiate the recruitment and activation of macrophages in adipose tissues and promote a pro-inflammatory cascade that is associated with

insulin resistance37,38. Thus, obesity-induced perturbations in the balance between TH1- and TH2-type signals may influence the recruitment and activation of macrophages in adipose tissues, thereby generating either a pathogenic and inflammatory environment or a noninflammatory and protective environment. However, the changes in the adipose tissue microenvironment that initiate T cell recruitment and macrophage activation are not fully understood. Nevertheless, it is important to bear in mind that obesity-associated changes in the cellular composition of adipose tissue complicates our understanding of whether a putative adipokine is expressed entirely by adipocytes or by recruited inflammatory cells (TABLe 1).

Pro-inflammatory adipokines The production of most adipokines is upregulated in the obese state, and these pro-inflammatory proteins typically function to promote obesity-linked metabolic diseases. In addition to leptin, TNF and IL-6, more recently identified adipokines that promote inflammation include resistin, retinol-binding protein 4 (RbP4), lipocalin 2, IL-18, angiopoietin-like protein 2 (ANGPTL2), CC-chemokine ligand 2 (CCL2), CXC-chemokine ligand 5 (CXCL5) and nicotinamide phosphoribosyltransferase (NAmPT) (TABLe 1), and this subset of factors is discussed in more detail below. It is the upregulation of these factors (as well as others) that leads to the development of a chronic inflammatory state and contributes to metabolic dysfunction. below, we briefly describe adipose tissue-derived proteins that generally have pro-inflammatory effects and discuss their metabolic regulatory properties. Leptin. The adipokine leptin is the product of the obese gene (ob; also known as Lep), which was identified in ob/ob mice by positional cloning 14. Leptin regulates feeding behaviour through the central nervous system. mice that lack leptin (ob/ob mice) show hyperphagia (abnormally increased feeding), obesity and insulin resistance, and the administration of leptin to ob/ob mice reverses these changes39. The administration of leptin to lipoatrophic mice (which lack subcutaneous adipose tissue and thus have low levels of leptin) also improves metabolic abnormalities, including insulin resistance and hyperlipidaemia40. Leptin has also been shown to be effective at improving metabolic dysfunction in patients with lipodystrophy or congenital leptin deficiency 41,42. However, leptin levels in the blood positively correlate with adipose mass, indicating the occurrence of leptin resistance, and obese individuals have high levels of leptin without the expected anorexic responses39. Leptin is structurally similar to the family of helical cytokines that includes IL-2 and growth hormone 1, and is thought to have pro-inflammatory activities. Indeed, leptin increases the production of TNF and IL-6 by monocytes and stimulates the production of CC-chemokine ligands (namely, CCL3, CCL4 and CCL5) by macrophages by activating the JAK2 (Janus kinase 2)–sTAT3 (signal transducer and activator of

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f o c u s o n m e ta b o l i s m a n d i m m uRneoVlo gy ieW s Box 1 | Vascular function in adipose tissue Recent interest has been paid to the status of the vasculature in adipose tissue. It has been shown that obesity can lead to capillary rarefaction in adipose tissue leading to localized hypoxia146,147. Theoretically, reductions in blood flow in obese adipose tissue could limit the delivery of nutrients, thereby contributing to insulin resistance. Perhaps more importantly, a restriction of blood flow in adipose tissue could contribute to an inflammatory state, possibly as a result of ischaemia-induced adipocyte necrosis and the subsequent recruitment of macrophages. Furthermore, obesity leads to the downregulation of anti-inflammatory factors, such as adiponectin, that pacify the vascular endothelium, and the upregulation of pro-inflammatory factors that activate endothelial cells and promote a dysfunctional phenotype11. In turn, the activated vascular endothelium expresses adhesion molecules and chemotactic factors that accelerate and localize inflammatory processes. Thus, the status of endothelial cell function may have an integral role both in mediating the effects of metabolic disease on the cardiovascular system and in controlling the metabolic state of the organism by influencing, either positively or negatively, the microenvironment in adipose tissue. Therefore, it can be posited that obesity favours a vicious cycle whereby endothelial cell dysfunction in the adipose tissue leads to metabolic dysfunction, reflected by adipokine dysregulation, adipocyte necrosis and inflammation120. In turn, metabolic dysfunction promotes endothelial cell dysfunction, both in the adipose tissue and systemic circulation, putting further stress on adipocytes. However, in contrast to these considerations, it has been reported that obese mice receiving anti-angiogenic reagents have a reduced body weight and adipose mass, and show increased metabolic rates148. Because there is a close interplay between adipogenesis and angiogenesis in obesity149, further analysis of the role of adipokines in controlling vascular growth and metabolic function during adipose tissue expansion should be of interest.

transcription 3) pathway 43,44. In monocytes, leptin also stimulates the production of ROs and promotes cell proliferation and migratory responses43,45. Leptin levels in the serum and adipose tissues are increased in response to pro-inflammatory stimuli, including TNF and lipopolysaccharide (LPs)46. Furthermore, leptin increases the production of the TH1-type cytokines IL-2 and IFNγ and suppresses the production of the TH2-type cytokine IL-4 by T cells or mononuclear cells47, thus polarizing T cells towards a TH1 cell phenotype. Consistent with these findings, leptin deficiency protects against liver damage in models of T cell-mediated hepatitis48. In addition, ob/ob mice are resistant to the induction of experimental autoimmune encephalomyelitis, owing to the polarization of T cells towards the TH2-type phenotype rather than the pathogenic TH1-type phenotype47,49. Thus, it is generally accepted that leptin acts as a pro-inflammatory adipokine. The metabolic syndrome A disorder characterized by the presence of at least three of the following features: large waist circumference (men: ≥40 inches; women: ≥35 inches), high levels of circulating triglycerides (≥150 mg dl–1), low levels of high-density lipoprotein (men: