Adipocytokines: mediators linking adipose tissue, inflammation and immunity

Adipocytokines: mediators linking adipose tissue, inflammation and immunity Herbert Tilg and Alexander R. Moschen Abstract There has been much effort recently to define the role of adipocytokines, which are soluble mediators derived mainly from adipocytes (fat cells), in the interaction between adipose tissue, inflammation and immunity. The adipocytokines and leptin have emerged as the most abundant adipocyte products, thereby redefining adipose tissue as a key component not only of the endocrine system, but also of the immune system. Indeed, as we discuss here, several adipocytokines have a central role in the regulation of insulin resistance, as well as many aspects of inflammation and immunity. Other adipocytokines, such as visfatin, have only recently been identified. Understanding this rapidly growing family of mainly adipocyte-derived mediators might be of importance in the development of new therapies for obesity-associated diseases. Atherosclerosis A chronic disorder of the arterial wall characterized by endothelial damage that gradually induces deposits of cholesterol, cellular debris, calcium and other substances. These deposits finally lead to plaque formation and arterial stiffness. Christian Doppler Research Laboratory for Gut Inflammation and Department of Medicine, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria. Correspondence to H.T. e-mail: herbert.tilg@uibk.ac.at doi:10.1038/nri1937 Published online 22 September 2006 The incidence of obesity and its associated disorders is increasing markedly worldwide. Obesity predisposes individuals to an increased risk of developing many diseases, including atherosclerosis, diabetes, nonalcoholic fatty liver disease, certain cancers and some immune-mediated disorders, such as asthma 1 3. In addition to these associations between obesity and disease, research in the past few years has identified important pathways that link metabolism with the immune system and vice versa. Many of these interactions between the metabolic and immune systems seem to be orchestrated by a complex network of soluble mediators derived from immune cells and adipocytes (fat cells) 1. In mammals, adipose tissue occurs in two forms: white adipose tissue and brown adipose tissue. Most adipose tissue in mammals is white adipose tissue and this is thought to be the site of energy storage. By contrast, brown adipose tissue is found mainly in human neonates and is important for the regulation of body temperature through non-shivering thermogenesis. In addition to adipocytes, which are the most abundant cell type in white adipose tissue, adipose tissue also contains pre-adipocytes (which are adipocytes that have not yet been loaded with lipids), endothelial cells, fibroblasts, leukocytes and, most importantly, macrophages (FIG. 1). These macrophages are bone-marrow derived and the number of these cells present in white adipose tissue correlates directly with obesity. Adipose tissue is no longer considered to be an inert tissue functioning solely as an energy store, but is emerging as an important factor in the regulation of many pathological processes. Various products of adipose tissue have been characterized, and some of the soluble factors produced by this tissue are known as adipocytokines. The term adipocytokine is used to describe certain cytokines that are mainly produced by adipose tissue, although it is important to note that they are not all exclusively derived from this organ. Adiponectin, leptin, resistin and visfatin are adipocytokines and are thought to provide an important link between obesity, insulin resistance and related inflammatory disorders 1 6. Adiponectin and leptin are the most abundant adipocytokines produced by adipocytes. Various other products of adipose tissue that have been characterized include: certain cytokines, such as tumour-necrosis factor (TNF), interleukin-6 (IL-6), IL-1 and CC-chemokine ligand 2 (CCL2; also known as MCP1); mediators of the clotting process, such as plasminogen-activator inhibitor type 1; and certain complement factors 1,2 (FIG. 1). These products have well-known roles in the immune system, and although some of them are also produced by adipocytes, they are not normally considered to be adipocytokines; nonetheless, they have important roles at the interface between the immune and metabolic systems. Obesity is associated with a chronic inflammatory response, which is characterized by abnormal cytokine 772 OCTOBER 2006 VOLUME 6 www.nature.com/reviews/immunol

Lean adipose tissue Complement factors Complement factors are components of the complement system. Activation of these factors, which involves proteolytic cleavage of serum and cell-surface glycoproteins, leads to the formation of a terminal cell-lytic complex inside the cell membrane of a target cell. Complement fragments such as C3a and C5a have important proinflammatory properties, such as vasodilation, chemotaxis and opsonization. Adipocyte Blood vessel Macrophage Adipocytokines Adiponectin Leptin Resistin Weight gain Obese adipose tissue Apoptotic adipocyte Crosstalk Pro-inflammatory cytokines and chemokines TNF IL-6 CCL2 Macrophagederived factors Resistin (human) IL-1β Figure 1 Adipose tissue: cellular components and molecules synthesized. Expansion of the adipose tissue during weight gain leads to the recruitment of macrophages through various signals, which might include chemokines synthesized by adipocytes, such as CC-chemokine ligand 2 (CCL2). These macrophages are found mainly around apoptotic adipocytes. Various mediators synthesized by adipocytes and resident macrophages might contribute to local and systemic inflammation. The overall adipocytokine cytokine cocktail might favour a pro-inflammatory milieu. IL, interleukin; TNF, tumour-necrosis factor. production, increased synthesis of acute-phase reactants, such as C-reactive protein (CRP), and the activation of pro-inflammatory signalling pathways 1. Although there is no doubt that pro-inflammatory pathways are activated in the adipose tissue itself in cases of obesity, the relative contribution of adipocytes as a source of the circulating and systemically active cytokines, adipocytokines and chemokines remains unclear. The adipose tissue of obese individuals also contains a large number of macrophages, which are an additional source of soluble mediators in the adipose tissue 7,8 (FIG. 1). However, although macrophages in adipose tissue seem to be the main source of TNF, adipocytes contribute almost one third of the IL-6 concentration in the circulation of patients who are obese 9. CCL2, produced by adipocytes, has recently been identified as a potential factor contributing to macrophage infiltration into adipose tissue 10. Once macrophages are present and active in the adipose tissue, they, together with adipocytes and other cell types present in the adipose tissue, might perpetuate a vicious cycle of macrophage recruitment and production of pro-inflammatory cytokines. In humans, adipocytokines function as hormones to influence energy homeostasis and to regulate neuroendocrine function. As cytokines, they affect immune functions and inflammatory processes throughout the body. The field of adipocytokines has attracted tremendous interest recently and the knowledge that has accumulated might lead to the development of new therapeutics. Here, we provide an overview of recent advances in our view of the role of adipocytokines in inflammation and immunity. Insulin resistance: an inflammatory disease Systemic chronic inflammation has been proposed to have an important role in the pathogenesis of obesityrelated insulin resistance 1,11. Biomarkers of inflammation, such as TNF, IL-6 and CRP, are present at increased concentrations in individuals who are insulin resistant and obese, and these biomarkers predict the development of type 2 diabetes mellitus and cardiovascular diseases (BOX 1). The first link between obesity, an increase in expression of the pro-inflammatory cytokine TNF, and insulin action was reported by Hotamisligil and colleagues 11. Their findings in rodents showed that adipocytes directly express TNF and led to the concept of a role for inflammation in obesity. These observations were paralleled by human studies showing increased TNF expression in the adipose tissue of individuals who were obese, and decreased TNF expression after weight loss 12. Evidence supporting a key role for TNF in obesityrelated insulin resistance came from studies showing that ob/ob mice (leptin-deficient mice with evidence of insulin resistance) that were also deficient for TNF or TNF receptors (TNFRs) had improved insulin sensitivity in diet-induced obesity compared with TNF- and TNFR-sufficient ob/ob mice 13. In searching for the mechanisms involved in inflamma tion-induced insulin resistance, Yuan and co-workers identified the inhibitor of nuclear factor-κb (NF-κB) kinase-β (IKKβ) pathway of NF-κB activation as a mediator of TNF-induced insulin resistance 14. They showed that overexpression of IKKβ in a human embryonic kidney cell line (the HEK293 cell line) attenuated insulin signalling, and that ob/ob mice expressing only one copy of the gene encoding IKKβ (Ikbkb) were protected against the development of insulin resistance 14. The JUN N-terminal kinase (JNK) family of serine/ threonine protein kinases, which are activated by many inflammatory stimuli including TNF and ligation of Toll-like receptors (TLRs) are also important regulators of insulin resistance in mouse models of obesity 15. In both genetic and dietary animal models of obesity, JNK activity is increased in the liver, muscle and adipose tissue, and loss of JNK1 prevents insulin resistance 15. Another important mechanism involved in insulin resistance is endoplasmic-reticulum (ER) stress 16. ER stress leads to suppression of signalling through the insulin receptor in a rat hepatocyte cell line through activation of JNKs and the subsequent serine phosphorylation of insulin receptor substrate 1 (IRS1), which is one of the main mediators of insulin signalling and thereby controls NATURE REVIEWS IMMUNOLOGY VOLUME 6 OCTOBER 2006 773

Box 1 Insulin resistance, obesity and inflammation Insulin regulates the uptake, oxidation and storage of fuel in insulin-sensitive tissues, such as the liver, skeletal muscle and adipose tissue, and also macrophages. Obesity, in particular visceral obesity, which is the accumulation of adipose tissue inside the abdominal cavity, is associated with resistance to the effects of insulin (insulin resistance) on peripheral glucose and fatty-acid utilization, often leading to type 2 diabetes mellitus. With the recent trend for individuals to be more obese, a large increase in the prevalence of insulin resistance in westernized countries is expected. Insulin resistance, together with the associated hyperinsulinaemia and hyperglycaemia, and the presence of pro-inflammatory mediators might lead to a state of vascular endothelial dysfunction, an abnormal lipid profile, hypertension and vascular inflammation, all of which promote the development of atherosclerotic cardiovascular disease. Subclinical, low-grade inflammation might have an important role in the pathogenesis of insulin resistance and type 2 diabetes mellitus. Population studies show a strong correlation between the levels of pro-inflammatory biomarkers, such as C-reactive protein, interleukin-6 and tumour-necrosis factor, and perturbations in glucose homeostasis, obesity and atherosclerosis. However, although epidemiological correlations have been established, the exact cellular and molecular mechanisms that link obesity and insulin resistance are unknown. Insulin resistance might be partly precipitated or accelerated by an acute-phase reaction as part of the innate immune response, in which large amounts of pro-inflammatory mediators and insufficient amounts of anti-inflammatory mediators, such as, are released from adipose tissue. Type 2 diabetes mellitus A disorder of glucose homeostasis that is characterized by inappropriately increased blood-glucose levels and resistance of tissues to the action of insulin. Recent studies indicate that inflammation in adipose tissue, liver and muscle contributes to the insulin-resistant state that is characteristic of type 2 diabetes mellitus, and that the anti-diabetic actions of peroxisome-proliferatoractivated receptor-γ (PPARγ) agonists result, in part, from their anti-inflammatory effects in these tissues. ob/ob mice Mice with a spontaneous mutation in the gene encoding leptin (chromosome 6) that leads to decreased leptin production. These mice are severely obese and develop noninsulin-dependent diabetes mellitus. Endoplasmic-reticulum stress (ER stress). A response by the ER that results in the disruption of protein folding and in the accumulation of unfolded proteins in the ER. sensitivity to insulin. Furthermore, mice expressing only a single copy of the gene encoding X-box-binding protein 1 (XBP1), which is a transcription factor that regulates a large number of genes in the ER-stress response, develop insulin resistance 16. Other factors involved in the regulation of insulin resistance are IL-6 and various suppressor of cytokine signalling (SOCS) proteins 17,18. Therefore, several pro-inflammatory cytokines, SOCS proteins, ER stress, the IKKβ pathway of NF-κB activation and JNK signalling pathways are all associated with the development of insulin resistance, indicating that various pro-inflammatory mediators released by adipocytes, in addition to the initially described proinflammatory cytokine TNF, link the immune system with obesity-related insulin resistance. By studying mice with conditional deletion of Ikbkb in either hepatocytes or myeloid cells, Michael Karin s laboratory showed that mice lacking IKKβ in hepato cytes retain insulin responsiveness in the liver but develop insulin resistance in muscle and fat in response to a high-fat diet, obesity or aging 19. By contrast, mice lacking IKKβ in myeloid cells retain global insulin sensitivity and are protected from insulin resistance 19. Therefore, this study indicates that myeloid cells, probably macrophages, regulate systemic insulin sensitivity and are involved in inflammation-associated insulin resistance, whereas hepatic IKKβ expression is required for insulin resistance in the liver. Selective activation of NF-κB, causing continuous low-level expression of IKKβ, in hepatocytes from a transgenic mouse model leads to moderate systemic insulin resistance 20. In this study, insulin resistance was decreased by systemic neutralization of IL-6, which also resulted in decreased expression of SOCS1, SOCS2 and SOCS3 in the liver. Therefore, not only is a complex network of mediators involved in the regulation of insulin resistance, but various cell types in addition to adipocytes are also involved, including hepatocytes, macrophages and muscle cells. Adiponectin Although is synthesized mainly by adipocytes, it is also expressed by skeletal muscle cells, cardiac myocytes and endothelial cells 21 23 (FIG. 2a). It has sequence homology with a family of proteins that are characterized by an amino-terminal collagen-like region and a carboxy-terminal, complement factor C1q-like globular domain 24 26. Adiponectin exists as a full-length protein, as well as a proteolytic cleavage fragment, consisting of the globular C-terminal domain (which is known as globular ). It is thought that a leukocyte elastase, secreted by activated monocytes and/or neutrophils, mediates this cleavage process and generates the globular fragment of. Globular adiponection can trimerize after cleavage, but cannot oligomerize further 27. Full-length can exist as: a trimer (known as low-molecular-weight adipo nectin); a hexamer, which consists of two trimers linked by a disulphide bond (known as middle-molecularweight ); and a high-molecular-weight 12- to 18-mer (FIG. 2a). Adiponectin circulates at high concentrations in human serum (5 10 mg per ml, compared with leptin, which circulates at a concentration of a few ng per ml) and it has a wide range of biological activities 9. Serum levels of are markedly decreased in individuals with visceral obesity and states of insulin resistance, such as non-alcoholic fatty liver disease, atherosclerosis and type 2 diabetes mellitus, and levels correlate inversely with insulin resistance 28. Both trimers and other oligomers of are present in the circulation, whereas the presence of the globular fragment in the serum in humans has been questioned 29,30. It has been suggested recently that the ratio, and not the absolute amounts, of high-molecular-weight and low-molecular-weight in the serum might be crucial in determining insulin sensitivity 31. In support of the importance of circulating high-molecular-weight in protecting against insulin resistance, moderate weight loss leads to a relative increase in the ratio of high-molecular-weight to middle-molecular-weight and a decrease in the amount of low-molecular-weight in the serum 32. Two receptors for have been identified recently (ADIPOR1 and ADIPOR2). ADIPOR1 is expressed widely in mice, whereas ADIPOR2 is expressed mainly in the liver 33. Whereas globular seems to activate mainly ADIPOR1, ADIPOR2 engages mainly with the full-length variant of 33. In addition, T-cadherin, which is expressed by many cells including endothelial cells and smooth muscle cells, seems to function as a receptor for middle-molecular-weight and highmolecular-weight, but not for the trimeric (low-molecular-weight) and globular forms 34. TNF suppresses the transcription of in an adipocyte cell line, which might explain the lower levels of serum in individuals who are 774 OCTOBER 2006 VOLUME 6 www.nature.com/reviews/immunol

a b Endothelial cell Low-molecularweight IKKγ IKKα IKKβ TNFR1 Globular Globular IκB p50 p65 NF-κB Immunological function of TNF IFNγ IL-10 IL-1RA ADIPOR1 Adipocyte Middle-molecularweight Low-molecularweight AMPK SREBP1C PPAR PPRE p50 p65 NF-κB-binding motif Skeletal and cardiac myocytes High-molecularweight ADIPOR2 Metabolic function of AMPK β-oxidation GLUT4 translocation ACC (Malonyl-CoA) Figure 2 Adiponectin: sources, structure and effects on pro- and antiinflammatory cytokines. a Adiponectin is produced mainly by adipocytes, but other cell types, such as skeletal and cardiac myocytes and endothelial cells, can also produce this adipocytokine. Adiponectin exists as a full-length trimer (low-molecular-weight form), as well as a proteolytic cleavage fragment (globular ). The full-length trimer can dimerize to form a hexamer (middle-molecular-weight form), which can then oligomerize to form a polymer (high-molecular-weight form). It has been proposed that cleavage of full-length by a leukocyte elastase secreted by activated monocytes and/or neutrophils generates the globular fragment. b Adiponectin interacts with at least two known cellular receptors (ADIPOR1 and ADIPOR2). Activation of ADIPOR1 and/or ADIPOR2 by stimulates the activation of peroxisomeproliferator-activated receptor-α (PPARα), AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase 33. Adiponectin regulates the expression of several pro- and anti-inflammatory cytokines. Its main anti-inflammatory function might be related to its capacity to suppress the synthesis of tumour-necrosis factor (TNF) and interferon-γ (IFNγ) and to induce the production of anti-inflammatory cytokines such as interleukin-10 (IL-10) and IL-1 receptor antagonist (IL-1RA). Activation of PPARs exerts anti-inflammatory effects through inhibition of the transcriptional activation of proinflammatory response genes. ACC, acetyl-coa carboxylase; GLUT4, glucose transporter type 4; IKK, inhibitor of nuclear factor-κb (IκB) kinase; NF-κB, nuclear factor-κb; PPRE, peroxisome-proliferator response element; SREBP1C, sterol-regulatoryelement-binding protein 1C; TNFR, TNF receptor. obese 35. Expression of is also regulated by other pro-inflammatory mediators such as IL-6, which suppresses transcription and translation in an adipocyte cell line 36. Weight loss is a potent inducer of synthesis 37, as is activation of peroxisomeproliferator-activated receptor-γ (PPARγ) by its ligands thiazolidinediones, which are important for the treatment of type 2 diabetes mellitus 38,39. Circulating levels of, however, are affected by many other factors including gender, age and lifestyle. Role in innate and adaptive immunity. Early studies indicated that had an anti-inflammatory effect on endothelial cells through the inhibition of TNF-induced adhesion-molecule expression 40. In addition, -deficient mice have higher levels of expression of mrna encoding TNF in adipose tissue and higher TNF concentrations in plasma compared with -sufficient mice 35. Adiponectin inhibits NF-κB activation in endothelial cells and interferes with the function of macrophages 40,41 (FIGS 2b,3a); treatment of cultured macrophages with markedly inhibited their phagocytic activity and production of TNF in response to stimulation with lipopolysaccharide (LPS) 41. Adiponectin also induces the production of important anti-inflammatory cytokines, such as IL-10 and IL-1 receptor antagonist (IL-1RA), by human monocytes, macrophages (FIG. 3a) and dendritic cells (DCs), and suppresses the production of interferon-γ (IFNγ) by LPS-stimulated human macrophages 42. Through ADIPOR1, globular suppresses TLR-induced NF-κB activation 43, indicating that negatively regulates macrophage responses to TLR ligands, which is probably of relevance in innate immune responses. The presence of in T-cell proliferation assays resulted in a decreased ability to evoke an allogeneic T-cell response 42 (TABLE 1), and also markedly reduced the phagocytic capacity of macrophages. However, stimulation of DCs with did not result in any changes in cell-surface marker expression, phagocytic capacity or ability to stimulate allogeneic T-cell proliferation, which might indicate that mainly affects the function of macrophages and not DCs 42. There might, however, be certain situations in which has pro-inflammatory effects. In the presence of LPS, high-molecular-weight was shown to augment the translation of CXC-chemokine ligand 8 (CXCL8; also known as IL-8) by human macrophages 44. Low- and high-molecular-weight share some biological effects on monocytes, such as the induction of apoptosis, the activation of AMP-activated protein kinase (AMPK) and the suppression of scavengerreceptor expression by macrophages 45. However, in this study 45, high-molecular-weight also induced the secretion of IL-6 by human monocytes, whereas only the low-molecular-weight form had antiinflammatory effects by decreasing IL-6 production in response to LPS and inducing IL-10 synthesis. The exact NATURE REVIEWS IMMUNOLOGY VOLUME 6 OCTOBER 2006 775

a Adiponectin b Leptin PPAR PPRE Globular ADIPOR1 p38 IL-10 IL-1RA CD14 Leptin OBR b LPS IκB p50 p65 NF-κB TLR4 ERK TNF IFNγ Microorganism NOS2 ROS Apoptosis Monocyte Phagocytosis Macrophage Microorganism Monocyte Activation Proliferation Migration Phagocytosis Macrophage roles of the different full-length and globular forms of in inflammation and immunity remain to be defined. Role in insulin resistance and inflammation. In obese animals, treatment with decreases hyperglycaemia and levels of free fatty acids in the plasma, and improves insulin sensitivity 9,46. Furthermore, -deficient mice develop diet-induced insulin resistance on a high-fat, high-sucrose diet 35. Specific PPARγ agonists, such as thiazolidinediones, improve insulin sensitivity by mechanisms that are largely unknown. Circulating levels of are significantly upregulated in vivo after activation of PPARγ 38,39. Mice lacking not only have decreased hepatic insulin sensitivity but also have reduced responsiveness to PPARγ agonists, which indicates that is an important contributor to PPARγ-mediated improvements in insulin sensitivity 47. Adiponectin stimulates β-oxidation in rat hepatocytes and downregulates expression of sterol-regulatory-element-binding protein 1C (SREBP1C), which is the main transcription factor regulating expression of genes encoding mediators of lipid synthesis (FIG. 2b). Sustained peripheral, ectopic expression of decreases the development of diet-induced obesity and improves insulin sensitivity 48. Together, these studies strongly support a major role for in regulating insulin sensitivity. c Resistin Unknown receptor p38 STAT3 PI3K P P ERK CD14 LPS TNF IL-6 IL-12 TLR4 TNF IL-1β p50 p65 IL-6 IL-12 NF-κB-binding motif Resistin Monocyte?? Phagocytosis Macrophage Microorganism Figure 3 Effects of various adipocytokines on the monocyte macrophage system. Adipocytokines exert different effects on the innate immune system and either suppress or activate the monocyte macrophage system. a Adiponectin, through interaction with its receptor ADIPOR1 (and ADIPOR2), suppresses the nuclear factor-κb (NF-κB)-dependent synthesis of tumour-necrosis factor (TNF) and interferon-γ (IFNγ), and induces the production of interleukin-10 (IL-10) and IL-1 receptor antagonist (IL-1RA). Adiponectin also induces apoptosis of monocytes and inhibits phagocytosis by macrophages. b Leptin signals through its receptor OBR b to induce activation of the mitogen-activated protein kinases (MAPKs) p38 and extracellular-signal-regulated kinase (ERK) and of signal transducer and activator of transcription 3 (STAT3). This results in production of the pro-inflammatory cytokines TNF, IL-6 and IL-12. Leptin also induces the production of nitric-oxide synthase 2 (NOS2) and, thereby, reactive oxygen species (ROS), enhances macrophage phagocytosis, and induces the activation, proliferation and migration of monocytes. c The receptor for resistin is unknown, but this adipocytokine induces the activation of p38, ERK and phosphatidylinositol 3-kinase (PI3K). Resistin increases the production of TNF, IL-1β, IL-6 and IL-12. Its effect on monocyte and macrophage functions is not known. Whereas can be considered an anti-inflammatory strategy of the adipose organ, leptin and resistin have dominant pro-inflammatory features. IκB, inhibitor of NF-κB; LPS, lipopolysaccharide; PPAR, peroxisome-proliferator-activated receptor; PPRE, peroxisome-proliferator response element; TLR4, Toll-like receptor 4. 776 OCTOBER 2006 VOLUME 6 www.nature.com/reviews/immunol

Table 1 Effects of adipocytokines on the immune system and linked diseases Adipocytokine Inflammatory effect Effects on immunity Associated diseases Innate Adaptive Adiponectin Anti-inflammatory Endothelial adhesion molecules 40 NF-κB 40,41,43 TNF 35 IL-6(REF. 42) IFNγ 42 IL-10 (REF. 42) IL-1RA 42 Phagocytosis 42 Pro-inflammatory CXCL8 in presence of LPS 44 Leptin Pro-inflammatory TNF 68,71 IL-6 (REF. 68) IL-12 (REF. 68) Neutrophil activation (CD11b) 70 ROS 70 Chemotaxis 70 NK-cell function 72 Resistin Pro-inflammatory TNF 86,87 IL-1β 86 IL-6 (REF. 86) IL-12 (REF. 86) NF-κB 87 Endothelial adhesion molecules (VCAM1 and ICAM1) 88 Visfatin ND IL-6 (REF. 119) IL-8 (REF. 119) Apoptosis of neutrophils 98 B-cell lymphopoiesis 117 T-cell responses 42 ND Lymphopoiesis 73 Thymocyte survival 73 T-cell proliferation 64 T H 1 response (IL-2 and IFNγ) 64 T H 2 response (IL-4) 64 Insulin resistance and type 2 diabetes mellitus 33,35 Atherosclerosis 57,58,60 Experimentally induced liver disease: nonalcoholic and alcoholic fatty liver disease 49 ; CCl 4 liver fibrosis (REF. 50); LPS-treated KK-A y mice (REF. 51); and experimentally induced hepatitis (ConA) 52 Cardiac injury 55,56 Cancer 102,103 Inflammatory bowel disease 112 Rheumatoid arthritis 115 Insulin resistance 9 Experimentally induced hepatitis (ConA) 75,76 EAE and antigen-induced arthritis 4,77 Experimentally induced colitis: CD4 + CD45RB hi T-cell transfer 78 ; and IL-10- deficient mice 113 Asthma 110 Cancer 104 ND Insulin resistance (mice) 5 Type 2 diabetes mellitus (mice) 80 Rheumatoid arthritis 86 Atherosclerosis 92 Non-alcoholic fatty liver disease 118 Chronic kidney disease 94 ND Insulin resistance and type 2 diabetes mellitus 95 Acute lung injury 97 Sepsis 98 X CCl 4, carbon tetrachloride; ConA, concanavalin A; CXCL, CXC-chemokine ligand; EAE, experimental autoimmune encephalomyelitis; ICAM, intercellular adhesion molecule; IFNγ, interferon-γ; IL, interleukin; IL-1RA, IL-1 receptor antagonist; LPS, lipopolysaccharide; ND, not determined; NF-κB, nuclear factor-κb; NK, natural killer; ROS, reactive oxygen species; T H, T helper; TNF, tumour-necrosis factor; VCAM, vascular cell-adhesion molecule. Collagen-like region The amino-terminal domain of contains a signal sequence that is followed by a stretch of 22 collagen-like repeats, consisting of 7 perfect Gly-X-Pro repeats and 15 imperfect Gly-X-Y repeats (where X and Y are different amino acids), which similar to procollagen allows the assembly of three full-length molecules to an trimer. C1q-like globular domain The carboxy-terminal globular domain of, which has marked homology to several other proteins, including subunits of the complement factor C1q. The liver is one of the main insulin-sensitive tissues and insulin resistance has an important role in the development of non-alcoholic fatty liver disease. Therefore, several studies have assessed the roles of fat, inflamma tion and in experimental liver disease. Adiponectin has anti-inflammatory effects in various animal models of liver inflammation. Administration of has beneficial effects in both alcoholic and non-alcoholic fatty liver disease in mice, by suppressing the expression of TNF in the liver. Adiponectin also decreases hepatomegaly, steatosis and the levels of liver enzymes 49. In addition, attenuates liver fibrosis in the carbon-tetrachloride liver-fibrosis model 50 and protects against endotoxininduced liver injury in another model of fatty-liver disease, the KK-A y obese mouse model 51. Sennello and colleagues studied concanavalin A (ConA)-induced hepatotoxicity in lipodystrophic transgenic mice (which constitutively express a truncated form of SREBP1c and lack virtually all white adipose tissue) and lean, wildtype control mice 52. Serum levels were low in lipodystrophic mice compared with controls, and the administration of protected lipo dystrophic mice from hepatotoxicity and protected primary hepatocytes from TNF-induced cell death. AMPK is an evolutionarily conserved sensor of the energy status of a cell, and it has a crucial role in controlling the systemic energy balance by regulating food intake, body weight, and glucose and lipid homeostasis 53. AMPK increases the sensitivity of a cell to both insulin and thiazolidinediones. Full-length stimulates AMPK phosphorylation and activation in the liver, whereas globular has this effect in both skeletal muscle and liver tissue 54. Each of these effects of could be inhibited by the use of a dominant-negative AMPK mutant, further supporting the model that glucose utilization occurs through activation of AMPK 54. Adiponectin protects the myocardium from injury by protecting cardiac muscle cells from apoptosis through activation of AMPK signalling 55,56. Ischaemia reperfusion in -deficient mice resulted in increased myocardial-infarct size, myocardial apoptosis rate and TNF expression compared with -sufficient mice. All of these effects were NATURE REVIEWS IMMUNOLOGY VOLUME 6 OCTOBER 2006 777

Visceral obesity Accumulation of adipose tissue inside the abdominal cavity, in particular at omental and mesenteric regions, which are drained by the portal vein and therefore have direct access to the liver. T-cadherin A member of the cadherin family of transmembrane glycoproteins that mediate cell-adhesive interactions. Peroxisome-proliferatoractivated receptor-γ (PPARγ). A nuclear receptor that is a master transcriptional regulator of metabolism and fat-cell formation. The activity of PPARγ can be modulated by the direct binding of small molecules thiazolidinediones. PPARγ has anti-inflammatory properties by limiting the availability of limited cofactors or blocking promoters of pro-inflammatory genes. IL-1 receptor antagonist (IL-1RA). A secreted protein that binds to IL-1R, thereby blocking IL-1R downstream signalling. IL-1RA inhibits the pro-inflammatory properties of IL-1α/β. Carbon-tetrachloride liver-fibrosis model Intraperitoneal or oral administration of hepatotoxic carbon tetrachloride (CCl 4 ) to mice is a commonly used model of both acute and chronic liver injury. CCl 4 causes hepatocyte injury that is characterized by centrilobular necrosis followed by hepatic fibrosis. KK-A y obese mice The spontaneous A y mutation (agouti signal protein; yellow) was introduced onto the KK strain background. KK-A y heterozygous mice have yellow hair pigment and black eyes and develop hyperglycaemia, hyperinsulinaemia, glucose intolerance and obesity by 8 weeks of age. decreased by the administration of in both -deficient mice and wild-type mice 56. These studies indicate that several important induced effects are mediated through the activation of AMPK (FIG. 2b). Adiponectin might have an important role in the pathophysiology of atherosclerosis. Adiponectin-deficient mice had a two-fold greater neointimal (that is, inner vessel surface) formation in response to an external vascular injury than did wild-type mice 57. Furthermore, protected apolipoprotein E (APOE)-deficient mice (mice lacking a key component in cholesterol metabolism) from atherosclerosis 58. This experimental evidence is paralleled by several clinical reports that support the observation that hypoaemia is associated with the development of atherosclerosis 59 61. So, is an important mediator in the regulation of insulin resistance and can suppress inflammation in various animal models. This adipocytokine also has a crucial role in suppressing macrophage activity, not only in adipose tissue but also in other tissues such as the liver. Decreased synthesis of, as is observed in individuals who are obese, might lead to dysregulation of the controls that inhibit the production of pro-inflammatory cytokines, thereby leading to the production of increased amounts of pro-inflammatory mediators. One of the main challenges in understanding the physiology of this adipocytokine will be to understand why circulating levels decrease with the onset of obesity. Also of great interest is how this decrease might affect the cytokine adipocytokine milieu, resulting in an overwhelmingly pro-inflammatory state. Leptin The role of leptin in modulating the immune response and inflammation has become increasingly evident and has been reviewed recently 4. In addition to regulating neuroendocrine function, energy homeostasis, haematopoiesis and angiogenesis, this adipocytokine is an important mediator of immune-mediated diseases and inflammatory processes 4. Similar to, leptin is produced mainly by adipocytes. However, unlike, leptin is considered to be a pro-inflammatory cytokine and it has structural similarity to other pro-inflammatory cytokines such as IL-6, IL-12 and granulocyte colonystimulating factor. The main function of leptin is control of appetite 4. Indeed, mice with a mutation in the gene encoding leptin (ob/ob mice) or the gene encoding the leptin receptor (db/db mice) have obese phenotypes and are used in many studies as mouse models of obesity. However, these mice also have various defects in cell-mediated and humoral immunity 62 64. Serum levels of leptin reflect the amount of energy stored in the adipose tissue and are proportional to overall adipose mass in both mice and humans 4,65. Serum levels are 2 3 times higher in women than in men, even when adjusted for age and body-mass index (BMI). In animal models, expression of leptin is increased in conditions that are associated with the release of pro-inflammatory cytokines, as induced during acute inflammatory conditions such as sepsis 66,67. An increase in leptin levels and a decrease in expression of mrna encoding the full-length isoform b receptor (OBR b, which is one of at least six alternatively spliced isoforms, each of which has a cytoplasmic domain of a different length) has been observed in diet-induced obese rats 4. In addition to adipose tissue, leptin is produced by several other tissues, including placenta, bone marrow, stomach, muscle and perhaps the brain 4. Therefore, pro-inflammatory mediators and obesity seem to be the main factors responsible for increased leptin synthesis. Role in innate and adaptive immunity. In monocytes and macrophages, leptin increases the production of pro-inflammatory cytokines such as TNF, IL-6 and IL-12 (REF. 68) (FIG. 3b). It also upregulates the expression of pro-inflammatory and pro-angiogenic factors, such as CCL2 and vascular endothelial growth factor, respectively, in human hepatic stellate cells 69. This effect in human stellate cells is mediated through activation of NF-κB, as well as other signalling intermediates, including the serine/threonine protein kinase AKT, which is the main downstream target of phosphatidylinositol 3-kinase 69. Leptin also activates neutrophils, as assessed by increased expression of CD11b, and stimulates the proliferation of human circulating monocytes in vitro and upregulates the expression of activation markers such as CD25 (also known as IL-2Rα) and CD71 (the transferrin receptor) on these cells 9,70. Leptin-induced TNF production by murine peritoneal macrophages is inhibited by globular through suppression of the phosphorylation of extracellular-signal-regulated kinase 1 (ERK1), ERK2 and p38 (REF. 71). Furthermore, leptin stimulates neutrophil chemotaxis and the production of reactive oxygen species (ROS) by these cells, and regulates natural killer (NK)-cell differentiation, proliferation, activation and cytotoxicity 72. Most of these pro-inflammatory effects are mediated through the long isoform of the leptin receptor (OBR b ), which is expressed mainly by endothelial cells and various leukocytes. The effects of leptin on adaptive immunity have been well studied 4 (FIG. 4). Leptin induces the proliferation of naive CD4 + CD45RA + T cells, but inhibits the proliferation of memory CD4 + CD45RO + T cells in a mixed lymphocyte reaction (MLR) 64. In T-cell proliferation assays with mouse cells, leptin increased production of the T helper 1 (T H 1) cytokines IL-2 and IFNγ, and suppressed production of the T H 2 cytokine IL-4 (REF. 64). Administration of leptin reversed the immunosuppressive effects of acute starvation in mice 64 and provided a survival signal for thymocytes, thereby protecting the mice from starvationinduced lymphoid atrophy. Leptin has also been shown to increase thymic cellularity in ob/ob mice 73. Despite this evidence of a role for leptin in immune responses in vitro and in mouse models, it is currently unclear whether leptin influences immune responses in humans. Role in inflammation. The role of leptin in inflammation is incompletely understood. Endogenous leptin protects against TNF-mediated toxicity. Ob/ob mice and 778 OCTOBER 2006 VOLUME 6 www.nature.com/reviews/immunol

Lipodystrophic transgenic mice Transgenic mice that express a truncated, constitutively active form of the sterol-regulatoryelement-binding protein 1C (SREBP1C) transcription factor under the control of the adipose-specific ap2 promoter. Lipodystrophic mice have low plasma leptin levels, hyperphagia, hyperglycaemia and hyperinsulinaemia. Body-mass index (BMI). This is the most frequently used method to gauge an individual s deviation from normal body weight. The BMI is the quotient of body weight (in kg) through the square of height (m²). Underweight: <20; ideal: 20 25; overweight: >25; obese: >30. Mixed lymphocyte reaction A tissue-culture technique that is used for the in vitro testing of the proliferative response of T cells from one individual to lymphocytes from another individual. TNF-mediated toxicity The injection of tumournecrosis factor (TNF) into animals, which results in acute anorexia, weight loss, shock and even death. Experimental autoimmune encephalomyelitis (EAE). An experimental model of multiple sclerosis that is induced by immunization of susceptible animals with myelin-derived antigens, such as myelin basic protein, proteolipid protein or myelin oligodendrocyte glycoprotein. Adiponectin B-cell lymphopoiesis B-cell number COX1/COX2 and PGE2 in stromal cells Thymic homeostasis Apoptosis Thymocyte number T-cell proliferation T H 1 cytokines TNF IFNγ IL-10 CD4 + CD8 + T cells CD4 + CD8 T cells T H 2 cytokines IL-4 Leptin IgG2a switch Figure 4 Effects of adipocytokines on adaptive immunity. Only limited data are available to describe the potential effects of (and resistin) on adaptive immunity. In terms of T-cell proliferation, suppresses the ability to evoke allogeneic T-cell responses. Adiponectin affects T helper 1 (T H 1)-cell immunity by suppressing the production of interferon-γ (IFNγ) and tumour-necrosis factor (TNF), and by inducing production of the anti-inflammatory cytokine interleukin-10 (IL-10). In terms of lymphopoiesis, suppresses B-cell development through the induction of prostaglandin synthesis. Leptin is the best studied adipocytokine and has many influences on adaptive immunity, such as inducing a switch towards T H 1-cell immune responses by increasing IFNγ and TNF secretion, the suppression of T H 2-cell responses, and inducing the production of IgG2a by B cells. Leptin promotes the generation, maturation and survival of thymic T cells, and it increases the proliferation of and IL-2 secretion by naive T cells. COX, cyclooxygenase; PGE2, prostaglandin E2. db/db mice, as well as mice treated with a leptin-receptor antagonist, had increased sensitivity to the lethal effects of TNF 74. The addition of exogenous leptin protected against TNF-mediated toxicity in ob/ob mice, but did not increase the protective effect of endogenous leptin in wild-type mice. Also, in various mouse models of inflammation, such as ConA-induced hepatitis, leptin deficiency has a protective effect by decreasing the production of pro-inflammatory T H 1 cytokines and shifting the immune response towards a T H 2-type response 75. Several studies have investigated the susceptibility of ob/ob and db/db mice to experimentally induced auto immune diseases 4,9,76. Ob/ob mice are resistant to experimental autoimmune encephalomyelitis, but become susceptible to disease after leptin administration 77. Leptin administration also exacerbates disease in wild-type mice through increased synthesis of T H 1 cytokines 77. Similar data were obtained from models of intestinal inflammation, where CD4 + CD45RB hi T cells from db/db mice had a decreased capacity to cause colitis when transferred to severe combined immunodeficient (SCID) mice, compared with cells from wild-type mice 78. Decreased IFNγ production was observed early on in SCID mice that received db/db T cells compared with wild-type T cells, indicating that leptin affects the immune response, probably through its interaction with the long isoform of its receptor (OBR b ), which is expressed by CD4 + CD45RB hi T cells 78. Leptin administration also increases disease severity and accelerates autoimmune diabetes in female non-obese diabetic (NOD) mice. Recent clinical reports in patients with autoimmune diseases have shown that high serum levels of leptin might be either a contributing factor to or a marker of disease activity 4,70. Taken together, it seems clear that leptin has proinflammatory effects. This could be detrimental in many animal models of inflammatory and autoimmune disease, but it might be protective in several infectiousdisease settings, such as during the early immune response to pulmonary tuberculosis, following infection with Gram-negative pneumococci and during viral myocarditis 4,9. However, the direct interaction of the two main adipocytokines and leptin is not well understood and this might have important implications in understanding the role of these adipocytokines in obesity-associated disorders. Resistin The adipocytokine resistin was discovered by three independent groups 79 81. Resistin (also known as FIZZ3), which is a 114-amino-acid polypeptide, was originally shown to induce insulin resistance in mice 80. It belongs to a family of cysteine-rich proteins, also known as resistin-like molecules (RELMs), that have been implicated in the regulation of inflammatory processes 79. Resistin was shown to circulate in two distinct forms: a more prevalent high-molecular-weight hexamer and a substantially more bioactive, but less prevalent, low-molecular-weight complex 82. mrna encoding resistin can be found in mice and humans in various tissues, including adipose tissue, the hypothalamus, adrenal gland, spleen, skeletal muscle, pancreas and gastrointestinal tract 5. Although resistin protein synthesis in mice seems to be restricted to adipocytes, in humans, adipocytes, muscle, pancreatic cells and mononuclear cells such as macrophages can synthesize this protein. Expression levels of the gene encoding resistin have been shown to be higher in human peripheral-blood mononuclear cells (PBMCs) than in adipocytes; however, comparative protein data are not available 5. So, it still remains to be shown which cell type in humans is mainly responsible for systemic production and for the high circulating levels of resistin. Remarkably, at the protein level, human resistin is only 55% identical to its mouse counterpart, which indicates that it might not be evolutionarily well conserved across species. In human PBMCs, expression of resistin mrna is markedly increased by the pro-inflammatory cytokines IL-1, IL-6 and TNF, and by LPS, whereas IFNγ and leptin had no effect 83. Similarly, stimulation of human macrophages with LPS led to increased resistin mrna expression, and administration of LPS to humans resulted in a marked increase in the level of resistin in the serum 84. The induction of resistin synthesis can be NATURE REVIEWS IMMUNOLOGY VOLUME 6 OCTOBER 2006 779

Atherosclerotic aneurysm A localized dilation of a blood vessel by more than 50% of its diameter owing to atherosclerotic structural damage of the vessel wall. attenuated by PPARγ agonists 5,84. Accordingly, treatment of patients with type 2 diabetes mellitus with the PPARγ agonist pioglitazone decreased serum levels of resistin 85. In addition, several factors such as pituitary, steroid and thyroid hormones, adrenaline, β3-adrenoreceptor activation, endothelin-1 and insulin modulate resistin expression 5,84. Role in immunity. Resistin strongly upregulates the expression of TNF and IL-6 by human PBMCs and induces arthritis after injection into the joints of healthy mice 86. These pro-inflammatory properties of resistin were abrogated by an NF-κB inhibitor, showing the important role of NF-κB in resistincontrolled inflammatory reactions. Resistin has also been shown to accumulate in the inflamed joints of patients with rheumatoid arthritis and its levels correlate with markers of inflammation 5. Human resistin stimulates synthesis of the pro-inflammatory cytokines TNF, IL-1, IL-6 and IL-12 by various cell types through an NF-κBdependent pathway 83,87 (FIG. 3c). In further support of its pro-inflammatory profile, resistin also upregulates the expression of vascular cell-adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1) and CCL2 by human endothelial cells and induces these cells to release endothelin-1 (REF. 88). Role in inflammation and insulin resistance. Resistin has been implicated in the pathogenesis of obesityassociated insulin resistance and type 2 diabetes mellitus in mouse models 80, whereas such a role in humans is still debated 89 91. Although a clear function for resistin in humans is still lacking, its pro-inflammatory properties indicate that it has a role in inflammatory processes 5. Macrophages infiltrating human atherosclerotic aneurysms secrete resistin 92. Resistin and have reciprocal effects on vascular endothelial cells: resistin induces the expression of VCAM1, ICAM1 and pentraxin-3, whereas downregulates the expression of these molecules 93. Increased levels of resistin in chronic kidney disease are associated with impaired renal function and inflammation, but not with insulin resistance 94. Therefore, this adipocytokine, at least in humans, has many features of a pro-inflammatory cytokine and could have a role in inflammatory diseases with or without associated insulin resistance. These pro-inflammatory effects, however, are based on a small number of studies and much more information is required to characterize resistin more fully in both mice and humans. Visfatin and other new adipocytokines Visfatin (also known as PBEF) has recently been identified as an adipocytokine that is secreted by adipocytes in visceral fat and that decreases insulin resistance 95. This molecule binds to and activates the insulin receptor but does not compete with insulin, which indicates that the two proteins bind different sites on the insulin receptor. Visfatin was originally identified as PBEF (pre-b-cell colony-enhancing factor) more than 10 years ago and since then, it has been linked to several inflammatory disease states such as acute lung injury 96,97. Furthermore, expression of visfatin has been shown to be upregulated in activated neutrophils and to inhibit the apoptosis of neutrophils 98. Future studies of the cell biology of this natural insulin mimetic and potential inflammation-regulating adipocytokine should help to define its role in insulin resistance and associated inflammatory disorders. Hida and colleagues recently identified a new adipocytokine termed VASPIN (visceral adipose-tissuederived serine protease inhibitor), which has similarities to in that it improves insulin sensitivity. Preliminary studies indicate that VASPIN might also have anti-inflammatory effects, as it suppresses the production of TNF, leptin and resistin 99. Serum retinol-binding protein 4 (RBP4) is another recently characterized adipocytokine 100. Until recently, the sole function of RBP4 was thought to be the delivery of retinol to tissues. However, in patients with type 2 diabetes mellitus, serum levels of RBP4 are increased. Expression of RBP4 is also increased in adipose tissue of mice lacking the glucose transporter GLUT4, and consistent with this, expression of GLUT4 is selectively decreased in adipocytes from obese individuals or individuals with type 2 diabetes. Treatment with fenretinide, a synthetic retinoid that increases urinary excretion of RBP4, normalizes serum levels of RBP4 and decreases insulin resistance in mice with obesity induced by a high-fat diet. Transgenic overexpression of human RBP4 or injection of recombinant RBP4 in normal mice causes insulin resistance. Therefore, decreasing the concentration of RBP4 could be an interesting strategy for the treatment of individuals with type 2 diabetes mellitus. Role in cancer and immune-mediated diseases In humans, the prevalence of cancer and some immunemediated diseases (such as asthma) is increased or disease activity is more severe in individuals who are obese 2,3. Although there is currently only limited evidence for this model, mainly derived from correlative studies, it is proposed that adipocytokines could link obesity with these diseases. Adiponectin has anti-angiogenic effects, through the inhibition of endothelial-cell proliferation and migration 101. In a mouse tumour model, markedly inhibited primary tumour growth in a caspase-dependent manner and it resulted in endothelial-cell apoptosis 102. Prostate cancer is also associated with obesity, and fulllength inhibits the growth of prostate-cancer cells at physiological concentrations 103. Many studies have investigated the effect of leptin on different cancer types in experimental cellular and animal models 104. Most of the studies indicate that leptin can potentiate the growth of cancer cells (breast, oesophageal, gastric, pancreatic, colorectal, prostate, ovarian and lung carcinoma cell lines), whereas seems to decrease cell proliferation. Epidemiological studies indicate that obesity is a significant risk factor for the development of cancer, although the exact mechanisms of this have not yet been identified 2. Recently, it has been shown that patients with various types of cancer, including gastric, endometrial, prostate and breast cancer, have low 780 OCTOBER 2006 VOLUME 6 www.nature.com/reviews/immunol