Cytokines in inflammatory bowel disease

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1 Nature Reviews Immunology AOP, published online 22 April 2014; doi: /nri3661 REVIEWS Cytokines in inflammatory bowel disease Markus F. Neurath Abstract Cytokines have a crucial role in the pathogenesis of inflammatory bowel diseases (IBDs), such as Crohn s disease and ulcerative colitis, where they control multiple aspects of the inflammatory response. In particular, the imbalance between pro-inflammatory and anti-inflammatory cytokines that occurs in IBD impedes the resolution of inflammation and instead leads to disease perpetuation and tissue destruction. Recent studies suggest the existence of a network of regulatory cytokines that has important implications for disease progression. In this Review, we discuss the role of cytokines produced by innate and adaptive immune cells, as well as their relevance to the future therapy of IBD. Innate lymphoid cells (ILCs). Cells that develop from a common lymphoid progenitor but that do not express lineage markers associated with other lymphocytes. These cells rapidly secrete effector cytokines in response to activation and have been subdivided into three main groups on the basis of whether they produce T helper 1 ( 1)-, 2- or 17 type cytokines. Acute-phase proteins A group of proteins including C reactive protein, serum amyloid A and fibrinogen secreted by hepatocytes into the blood in increased or decreased quantities in response to trauma, inflammation or disease. These proteins can be inhibitors or mediators of inflammatory processes. Department of Medicine 1, University of Erlangen- Nürnberg, Kussmaul Campus for Medical Research, Erlangen, Germany. e mail: markus.neurath@ uk erlangen.de doi: /nri3661 Published online 22 April 2014 Inflammatory bowel diseases (IBDs), such as Crohn s disease and ulcerative colitis, are chronic relapsing disorders of the gastrointestinal tract that are characterized pathologically by intestinal inflammation and epithelial injury 1,2. However, several important clinical and pathological features differ between Crohn s disease and ulcerative colitis (BOX 1), suggesting that they represent independent clinical entities. Both disorders are associated with marked morbidity and can have a major impact on an individual s quality of life and their ability to work, which highlights the need for optimized anti-inflammatory therapy. Cytokines have been directly implicated in the pathogenesis of IBD in recent genetic and immunological studies, and they seem to have a crucial role in controlling intestinal inflammation and the associated clinical symptoms of IBD 3. Studies in mouse models of IBD have shown that the modulation of cytokine function can be used for therapy and have identified new cytokines as potential therapeutic targets for chronic intestinal inflammation 4,5. The key role of cytokines is also highlighted by the fact that blockade of tumour necrosis factor (TNF) is now commonly used as a standard therapy for IBD in the clinic 1. Studies in recent years have identified a major role of both genetic and environmental factors in the pathogenesis of IBD 1,2 (FIG. 1). A combination of these IBD risk factors seems to initiate alterations in epithelial barrier function thereby allowing the translocation of luminal antigens (for example, bacterial antigens from the commensal microbiota) into the bowel wall. Subsequently, aberrant and excessive cytokine responses to such environmental triggers cause subclinical or acute mucosal inflammation in a genetically susceptible host 3. In patients that fail to resolve acute intestinal inflammation, chronic intestinal inflammation develops that is induced by the uncontrolled activation of the mucosal immune system. In particular, mucosal immune cells such as macrophages, T cells and the recently discovered subsets of innate lymphoid cells (ILCs) seem to respond to microbial products or antigens from the commensal microbiota by producing cytokines that can promote chronic inflammation of the gastrointestinal tract. Cytokines not only drive intestinal inflammation and associated symptoms, such as diarrhoea, but may also regulate extra-intestinal disease manifestations (for example, arthralgia or arthritis) and systemic effects in IBD 3,6. For instance, interleukin 6 (IL 6) induces the release of acute-phase proteins by the liver, whereas TNF has been implicated in the development of arthritis and cachexia. Furthermore, cytokines seem to have a crucial role in the pathogenesis of progressive and destructive forms of IBD that are associated with complications such as intestinal stenosis, rectal bleeding, abscess and fistula formation, and the development of colitis-associated neoplasias 2,7,8. In this Review, we discuss the pleiotropic roles of the cytokines that are produced by innate and adaptive immune cells in IBD, and discuss their relevance for future therapeutic approaches. Cytokines and IBD pathogenesis Altered patterns of cytokine production by immune cells from the periphery and the lamina propria of patients with IBD were initially described in the mid to late 1980s and early 1990s 9,10. However, the functional relevance of the observed changes in terms of the clinical activity NATURE REVIEWS IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1

2 Box 1 Key facts about inflammatory bowel diseases Crohn s disease Incidence: 3.1 to 20.2 cases per 100,000 individuals per year Risk factors: genotype and environment (for example, smoking as a risk factor for aggressive forms of disease) Onset of disease: usually between 15 and 40 years Location: inflammation frequently affects distal ileum and colon Pathology: discontinuous, patchy gut inflammation with skip lesions Histology: transmural inflammation (all layers of the bowel wall) Symptoms: diarrhoea, abdominal cramping, fever, anaemia, weight loss and fatigue Extra-intestinal inflammatory manifestations: various organs and systems are affected, for example, joints, skin, liver, eye, mouth and blood (coagulation) Complications: stenosis, abscess formation, fistulas and colon cancer Ulcerative colitis Incidence: 2.2 to 19.2 cases per 100,000 individuals per year Risk factors: genotype and environment (smoking and appendectomy are protective factors) Onset of disease: usually between 15 and 40 years Location: inflammation affects the colon only (distal colitis or proctitis (55%), left-sided colitis (25%) and pancolitis (20%)) Pathology: continuous inflammation from the rectum to proximal parts of the colon Histology: superficial inflammation (mucosa and submucosa) Symptoms: diarrhoea (bloody), abdominal cramping, anaemia, weight loss and fatigue Extra-intestinal inflammatory manifestations: various organs and systems are affected, for example, joints, skin, liver, eye, mouth and blood (coagulation) Complications: severe bleeding, toxic megacolon, rupture of the bowel and colon cancer Cachexia A condition of severe weight loss, muscle wasting and debility that is caused by prolonged disease and is thought to be mediated by neuroimmunoendocrine interactions. of IBD remained unclear. Subsequently, gene-knockout mice deficient for regulatory cytokines (such as IL 2 and IL 10) were found to develop spontaneous colitis, which highlighted a crucial role of cytokines in intestinal inflammation 3. Moreover, studies in experimental mouse models of IBD indicated that the administration of recombinant anti-inflammatory cytokines or the neutralization of pro-inflammatory cytokines could be used for both the prevention and the therapy of chronic intestinal inflammation 3,4,11. Additional evidence from genetic studies has suggested a crucial role for cytokines and cytokine-producing immune cells in IBD patho genesis. In fact, genome-wide association studies (GWASs) have identified several IBD susceptibility loci that contain genes that encode proteins involved in cytokine and chemokine receptor signalling, and T helper ( ) cell responses for example, signal transducer and activator of transcription 1 (STAT1), STAT3, STAT4, CC chemokine receptor 6 (CCR6), CC chemokine ligand 2 (CCL2), CCL13, IL 12 receptor (IL 12R), IL 23R and Janus kinase 2 (JAK2). Further studies have identified IBD risk loci that contain genes that encode cytokines (for example, IL 2, IL 21, interferon γ (IFNγ), IL 10 and IL 27) thus highlighting a potentially major role for these cytokines in disease pathogenesis 12. In particular, recent work has found that loss of function mutations in the genes encoding IL 10 and IL 10R are associated with a very early-onset form of IBD that is characterized by severe intractable enterocolitis in infants, and that disease in IL 10R deficient patients can be alleviated by haematopoietic stem cell transplantation 13. Collectively, these observations were consistent with the idea that cytokines have a fundamental role in controlling mucosal inflammation in IBD. On the basis of the above findings, several attempts were made to treat patients with IBD using recombinant anti-inflammatory cytokines or antibodies specific for pro-inflammatory cytokines. The effects of treating patients with anti-inflammatory cytokines (such as IFNβ, IL 10 and IL 11) were disappointing 14 16, but a major breakthrough came with the first clinical use of a neutralizing antibody specific for TNF (infliximab) in patients with Crohn s disease 17. Anti-TNF therapy resulted in marked clinical improvement and macroscopic healing of the inflamed mucosa on endoscopy (mucosal healing) in Crohn s disease. As there were similar findings in patients with ulcerative colitis, anti-tnf therapy with several chimeric, humanized or fully human antibodies (such as adalimumab, certolizumab pegol, golimumab and infliximab) is now considered a crucial backbone of biological therapy in IBD 1,2,18,19. The clinical efficacy of this approach has led to a new era of anti-cytokine therapies for the treatment of IBD. However, neutralization of other cytokines, such as IFNγ (with fontolizumab), did not improve the clinical symptoms of Crohn s disease 20, and the IL 17A specific antibody secukinumab even led to an aggravation of Crohn s disease 21. This suggests the existence of a specific network of cytokines that regulates mucosal inflammation. In this Review, we discuss the role of pro-inflammatory and anti-inflammatory cytokines in the pathogenesis of IBD with a special focus on the mucosal immune cells that produce these cytokines, as well as on new approaches for cytokine-based therapy. Antigen-presenting cell-derived cytokines in IBD The IL 1 family and IBD. Lamina propria dendritic cells (DCs) and macrophages are key antigen-presenting cells (APCs) that are found in the inflamed mucosa in IBD. Following activation, which occurs in response to components of the commensal microbiota and Tolllike receptor (TLR) signalling, these cells produce large amounts of pro-inflammatory cytokines, such as IL 1β, IL 6, IL 18 and TNF 22. A significant decrease in the ratio of IL 1 receptor antagonist to IL 1 was found in the intestinal mucosa of patients with Crohn s disease and patients with ulcerative colitis when compared with control subjects, which indicates increased activation of the IL 1 system in IBD 23. Subsequent studies in mouse models of colitis have suggested that IL 1 family members possess pleiotropic functions that are dependent on the phase of the disease. IL 1β promoted innate immune pathology in Helicobacter hepaticustriggered intestinal inflammation by augmenting the recruitment of granulocytes and the activation of ILCs 24. Furthermore, in the T cell transfer model of colitis, IL 1R signalling in T cells controlled the early accumulation and survival of pathogenic CD4 + T cells in the colon. Although treatment with an IL 1R antagonist suppressed acute immune complex-induced colitis 2 ADVANCE ONLINE PUBLICATION

3 Phase I: pre-disease stage Phase II: acute intestinal inflammation Phase III: chronicity or resolution Phase IV: tissue destruction and complications Necroptosis A programmed form of necrotic cell death that is regulated by receptor-interacting protein kinase 1 (RIPK1) and RIPK3. Genetic factors Antimicrobial peptides Autophagy Handling of bacteria Chemokines Cytokines Microbial product Initiating triggers Impaired barrier function Environmental factors Microorganisms Diet Infections Stress NSAIDs Appendectomy Smoking Antibiotics Translocation of microbial products Immune cell activation Bacteria Failure of regulatory mechanisms T Reg cell Macrophage Effector T cell Activation of Chronic inflammation effector cells Fibrosis, stenosis, abscess, fistula, cancer and extra-intestinal manifestations Production of pro-inflammatory mediators Figure 1 Conceptual framework for the pathogenesis of IBD. Genetic and environmental factors induce impaired barrier function in the intestinal mucosa. Initiating triggers may involve infections in some patients. Altered barrier function subsequently induces the translocation of commensal bacteria and microbial products from the gut lumen into the bowel wall, which leads to immune cell activation and cytokine production. If acute mucosal inflammation cannot be resolved by anti-inflammatory mechanisms and the suppression of pro-inflammatory immune responses, chronic intestinal inflammation develops. In turn, chronic inflammation may cause complications of the disease and also tissue destruction, which are both driven by mucosal cytokine responses. DC, dendritic cell; IBD, inflammatory bowel disease; NSAIDs, non-steroidal anti-inflammatory drugs; T Reg cell, regulatory T cell. in rabbits 25, TNF blockade (but not IL 1 blockade) was effective in treating chronic dextran sodium sulphate (DSS)-induced colitis in mice 26. Collectively, these findings suggested that IL 1 has a prominent role in the initiation, rather than in the perpetuation, of colonic inflammation. However, blockade of the IL 1 family member IL 18 a cytokine that is mainly induced in macrophages and epithelial cells in patients with Crohn s disease 27 had beneficial effects in several murine models of acute and chronic colitis 28. Furthermore, deficiency of the IL 1β converting enzyme (ICE; also known as caspase 1) an enzyme that cleaves IL 1β and IL 18 into active cytokines protected mice from DSS-induced colitis, which suggests that blockade of IL 1 family members may be relevant for the therapy of chronic intestinal inflammation 29. IL 6 and IBD. IL 6 production by lamina propria macrophages and CD4 + T cells is increased in experimental colitis and in patients with IBD 30,31 (FIG. 2). In particular, CD14 + CD33 + CD68 + CD163 low myeloid cells that express some macrophage-associated and DC associated markers were found to produce high amounts of IL 6 and IL 23 (REFS 32,33). IL 6 binds to the soluble IL 6R (sil 6R), and the IL 6 sil 6R complex then activates intestinal target cells by binding to the gp130 surface molecule (also known as IL 6R subunit-β). Therefore, IL 6 can exert pro-inflammatory functions by activating multiple target cells, including APCs and T cells. In particular, the IL 6 sil 6R complex prevents programmed cell death (apoptosis) of mucosal T cells and activates pro-inflammatory cytokine production by these cells 30. However, IL 6 may also have important homeostatic functions by stimulating the proliferation and expansion of intestinal epithelial cells (IECs). Interestingly, blockade of IL 6 signalling with monoclonal antibodies was effective in suppressing chronic intestinal inflammation in mouse models, which suggests IL 6 as a potential therapeutic target in IBD 30,34. This effect was associated with the induction of T cell apoptosis and the reduced production of pro-inflammatory cytokines, such as IFNγ, TNF and IL 1β. On the basis of these promising results, initial studies were carried out using an IL 6R specific antibody (tocilizumab; also known as MRA) to block IL 6 signalling in patients with Crohn s disease. Antibody-mediated blockade of IL 6 signalling led to clinical responses in subgroups of patients with Crohn s disease 35. However, further studies are warranted to determine the therapeutic potential of this approach in IBD. Membrane-bound TNF and soluble TNF in IBD. TNF is produced as a transmembrane protein from which soluble TNF is released by proteolytic cleavage via the metalloproteinase TNF-converting enzyme (TACE; also known as ADAM17). The production of both membrane-bound and soluble TNF by lamina propria mononuclear cells is markedly augmented in patients with IBD. In particular, CD14 + macrophages, adipocytes, fibroblasts and T cells from patients with IBD have been shown to produce large amounts of TNF 3,33,36. TNF may exert various pro-inflammatory functions in colitis by binding to its receptors TNFR1 and TNFR2 followed by the intracellular activation of the transcription factor nuclear factor κb (NF κb). Furthermore, TNFR1 signalling may cause cell death via the activation of receptor-interacting protein kinase 1 (RIPK1) and caspase 3 proteins. TNF signalling in colitis drives pleiotropic pro-inflammatory effects, including augmented angiogenesis, the induction of Paneth cell death via necroptosis, the production of matrix metalloproteinases by myofibroblasts, the activation of macrophages and effector T cells, and the direct damage of IECs via myosin light chain kinase (MLCK) activation (FIG. 3). Recent clinical and experimental studies have shown that membrane-bound TNF, rather than soluble TNF, has a major role in driving intestinal inflammation. Consistent with this, neutralization of membrane-bound TNF has NATURE REVIEWS IMMUNOLOGY ADVANCE ONLINE PUBLICATION 3

4 Lumen Lamina propria IEC Goblet cell Bacteria ILC1 IFNγ ILC3 IL-18 NK cell ILC1 IFNγ IL-17A IL-17F IL-22 Homeostasis Inflammation Antimicrobial peptides, such as defensins Paneth cell Neutrophil Retinoic acid, TGFβ IL-12 Macrophage CD103 + DC IL-6 IL-23 TNF 1 cell 2 cell 17 cell T Reg cell T-bet GATA3 RORγt FOXP3 IFNγ IL-6 TNF IL-5 IL-6 IL-13 TNF IL-17A IL-17F IL-21 IL-22 IL-10 TGFβ Crohn s disease Ulcerative colitis Homeostasis Ulcerative colitis Crohn s disease Resolution Figure 2 Cytokines in the pathogenesis of IBD. In patients with inflammatory bowel disease (IBD) and in experimental mouse models of colitis, pro-inflammatory and anti-inflammatory cytokines have been shown to be produced by various cells of the mucosal immune system in response to environmental triggers, such as commensal microorganisms. In particular, dendritic cells (DCs), neutrophils, macrophages, natural killer (NK) cells, intestinal epithelial cells (IECs), innate lymphoid cells (ILCs), mucosal effector T cells (T helper 1 ( 1), 2 and 17) and regulatory T (T Reg ) cells produce cytokines in the inflamed mucosa. The key transcription factors and cytokines produced by T helper cell subsets in IBD-affected mucosa are shown. The balance between pro-inflammatory and anti-inflammatory cytokines in the mucosa regulates the development and potential perpetuation of mucosal inflammation in patients with IBD. The dashed arrow indicates that ILCs, which produce cytokines that are involved in intestinal inflammation, may respond to IL-18. GATA3, GATA-binding protein 3; IL, interleukin; RORγt, retinoic acid receptor-related orphan receptor-γt; TGFβ, transforming growth factor-β; TNF tumour necrosis factor.? been shown to induce T cell apoptosis and was effective in suppressing experimental colitis in mice, whereas activation of TNFR2 (which is induced by membranebound but not soluble TNF) on T cells was found to aggravate colitis activity 41,42. Clinically, treatment of IBD with antibodies that neutralize both soluble TNF and membrane-bound TNF (such as infliximab and adalimumab) was highly effective and was shown to induce T cell apoptosis in vivo, whereas agents that preferentially block soluble TNF (for example, etanercept) had no therapeutic effect 1,2,36,43,44. Thus, the development of strategies that more specifically target the membranebound TNF TNFR2 interaction is of potential interest for future therapy of IBD. IL 12 family members and IBD. Members of the IL 12 family of heterodimeric cytokines (such as IL 12, IL 23, IL 27 and IL 35) are produced by APCs during intestinal inflammation. For instance, both DCs and macrophages showed augmented production of IL 12 (which is composed of p35 and p40 subunits; also known as IL 12 subunit-α and IL 12 subunit-β, respectively) in Crohn s disease but not in ulcerative colitis, which suggests that activated APCs may favour 1 cell differentiation and activation in Crohn s disease 22,45. Similarly, these cells produce large amounts of IL 23 (which is composed of the IL 12p40 subunit and a p19 subunit; the p19 sub unit is also known as IL 23 subunit-α) in patients with Crohn s disease 46 ; this cytokine perpetuates local 17 cell responses and suppresses regulatory T (T Reg ) cell activity. The therapeutic potential of the above cytokines has been demonstrated in experimental models of colitis and in clinical trials in IBD using neutralizing antibodies 11, For instance, antibodies specific for the common p40 subunit of IL 12 and IL 23 (briakinumab (also known as ABT874) and ustekinumab) were tested in patients with active Crohn s disease 51,52. These studies indicated an increased rate of clinical response to antibody therapy as compared with placebo, particularly in patients who were resistant to TNF antagonists, thereby suggesting new avenues for therapy in patients with Crohn s disease who do not respond to anti-tnf therapy. It is currently unknown whether antibodies that target the IL 23p19 subunit, rather than the shared p40 subunit, might be more suitable for Crohn s disease therapy, but studies in several models of experimental colitis have suggested that IL 23 rather than IL 12 drives chronic intestinal inflammation 47,48. 4 ADVANCE ONLINE PUBLICATION

5 Source Target cell Effector mechanism Consequence Macrophage DC T cell Adipocyte Fibroblast TNF Endothelial cell Macrophage Paneth cell Myofibroblast Effector T cell IEC IL-1, IL-6 and TNF IEC: MLCK Paneth cell: RIPK1 and RIPK3 Figure 3 Central role of tumour necrosis factor in the pathogenesis of IBD. In inflammatory bowel disease (IBD), increased amounts of soluble and membrane-bound tumour necrosis factor (TNF) are produced by various immune and stromal cell populations, such as macrophages, dendritic cells (DCs), effector T cells, adipocytes and fibroblasts. TNF has been shown to exert various pro-inflammatory functions in the inflamed mucosa in IBD. In particular, TNF induces hypervascularization and angiogenesis, augments pro-inflammatory cytokine production by macrophages and T cells, causes barrier alterations and promotes cell death of intestinal epithelial cells (IECs) and Paneth cells. TNF also promotes tissue destruction by increasing the production of matrix metalloproteinases (MMPs) by myofibroblasts and drives T cell resistance to apoptosis via the induction of TNF receptor-associated factor 2 (TRAF2) and the activation of nuclear factor κb (NF κb). TNF-specific antibodies may alleviate disease by simultaneously suppressing several pro-inflammatory pathways in patients with IBD. IL, interleukin; MLCK, myosin light chain kinase; RIPK, receptor-interacting protein kinase; TIMP1, tissue inhibitor of matrix metalloproteinases 1. TIMP1 TRAF2 and NF-κB Angiogenesis and hypervascularization Activation, pro-inflammatory cytokine production and suppression of regulatory macrophages IEC: cell death and impaired barrier function Paneth cell: necroptosis MMP-induced tissue destruction and impaired migration Apoptosis resistance, cell survival and IL-6 synthesis Several studies have suggested that IL 27 (which is composed of p28 (also known as the IL 27 subunit-α) and Epstein Barr virus induced gene 3 (EBI3; also known as IL 27 subunit-β)) exerts pro-inflammatory effects in the context of chronic intestinal inflammation. For instance, in IL 10 deficient animals with spontaneous colitis, IL 27R deficiency in T cells reduced colitis activity 53. Furthermore, IL 27R deficient T cells failed to induce disease in a T cell transfer model of colitis due to impaired 1 type cytokine production and the expansion of T Reg cell populations, and p28 deficient mice did not develop colitis upon transfer of T cells due to the reduced production of IL 1 and IL 6 by APCs 54,55. However, other investigators have found that IL 27 has an anti-inflammatory effect or no effect in models of colitis For instance, IL 27 was not required for the development of spontaneous colitis in mice with a myeloid-specific deletion of STAT3 (REF. 57), which suggests that the functions of this cytokine are dependent on the model that is used. Instead, IL 35 (which is composed of EBI3 and IL 12p35) was found to control colitis activity in this model and the administration of recombinant IL 35 reduced colitis activity by suppressing the pro-inflammatory cytokine responses of T cells. IFN production by APCs in IBD. In addition to secreting IL 12 family members, APCs are also capable of producing various cytokines of the IFN family (including IFNα and IFNβ) 59. In colitis, intestinal bacteria that enter the mucosa after epithelial damage or following the exogenous administration of CpG oligodeoxy nucleotides have been shown to activate TLR9 and induce the production of IFNα and IFNβ by mucosal plasmacytoid DCs 60. These cytokines can promote epithelial regeneration or the induction of IL 10 producing T Reg cell subsets. Mice that are deficient in the type I IFN receptor exhibited more severe experimental colitis than wild-type mice 60. The administration of TLR9 agonists or recombinant IFNβ suppressed the severity of experimental colitis in recombination-activating gene 1 (RAG1)-deficient mice 60. However, treatment with recombinant IFNβ1a was safe but had no therapeutic benefit in patients with steroid-refractory ulcerative colitis 16, which suggests that an IFNβ-based approach is not ideally suited for IBD therapy. By contrast, a CpG-containing oligonucleotide was recently used to successfully treat several patients with steroid-resistant ulcerative colitis 61, which indicates that immunostimulatory approaches to induce IFN production might be effective for IBD therapy. Taken together, the above findings suggest that the targeting of distinct cytokines that are produced by APCs is of key relevance for IBD therapy. The targeting of TNF has already been shown to be an effective method for suppressing chronic intestinal inflammation in certain patients. In addition, several alternative approaches to the cytokine-based therapy of IBD have been developed, but these still require further evaluation in controlled clinical trials. NATURE REVIEWS IMMUNOLOGY ADVANCE ONLINE PUBLICATION 5

6 Cytokines and innate lymphoid cells in IBD ILCs are a recently discovered group of cells that control innate immunity at mucosal surfaces (FIG. 2). These cells are now recognized as an important source of IFNγ and of IL 23 inducible pro-inflammatory cytokines, such as IL 17A and IL 17F, which mediate experimental innate immune-mediated colitis 62. In human IBD, an expansion of IL 17 producing ILCs that express CD127 (also known as IL 7Rα) and CD56 (also known as neural cell adhesion molecule 1) was noted in the inflamed mucosa of patients with Crohn s disease but not in patients with ulcerative colitis 63. Further studies also identified the expansion of a human intraepithelial ILC1 subset that produces IFNγ in response to stimulation with IL 12 and IL 15 in patients with Crohn s disease 64. In addition, an IL 15 dependent subset of intestinal ILCs that expresses NKp46 (also known as natural cytotoxicity triggering receptor 1) was identified as an important source of CC chemokine ligand 3 (CCL3), which may amplify intestinal inflammation via the recruitment of CCR1 + inflammatory monocytes in Crohn s disease 65. Finally, functional studies have revealed that T betexpressing ILC1s contribute to pathology in a model of innate colitis that is induced by CD40 specific antibodies, and thus these ILCs might be a new therapeutic target 66. In addition to IFNγ and IL 17, IL 22 is produced by mucosal ILCs via signalling events that involve the tyrosine-protein kinase LYN 67. Moreover, IL 22 is produced by neutrophils, DCs, γδ T cells and effector αβ T cells in experimental colitis IL 22 induces the production of antimicrobial peptides, such as defensins and regenerating islet-derived (REG) proteins, by IECs and thus influences the colitogenic potential of the microbiota and also affects intestinal barrier function. The functional relevance of IL 22 was shown by the finding that the administration of recombinant IL 22 protected mice from DSS-induced or trinitrobenzene sulphonic acid (TNBS)-induced colitis 68,71,72. However, the pro-inflammatory effects of IL 22 were recently noted in innate immune-mediated colitis 73, which suggests that IL 22 may have multifaceted roles in mucosal inflammation. Cytokines and effector T cells in IBD T cells are implicated in the pathogenesis of IBD by virtue of the detection of high numbers of T cells in the inflamed bowel wall, the secretion of large amounts of T cell-derived pro-inflammatory cytokines, and the requirement for T cells in various animal models of chronic intestinal inflammation 1,2,4,74. Interestingly, lamina propria T cells in IBD are hyporesponsive to T cell receptor stimulation and thus are critically dependent on co-stimulatory factors, such as IL 6 and TNF signalling, to prevent apoptosis 30,36. These cytokines are produced by cells in the local microenvironment or by the T cells themselves upon activation of transcription factors such as nuclear factor of activated T cells cytoplasmic 2 (NFATC2) and IFN-regulatory factor 4 (IRF4) 36,75,76. 1 cell-associated cytokines. With regard to their cytokine profiles, studies have shown that the production of IFNγ and IL 2 by lamina propria and lymph node T cells is increased in patients with Crohn s disease compared with patients with ulcerative colitis and healthy controls 74,77 79 (FIG. 2). Furthermore, 1 cellassociated surface receptors (such as IL 12Rβ2) and transcription factors (such as STAT4 and T bet) were shown to be expressed by lamina propria T cells in patients with Crohn s disease 80,81. Taken together, these observations indicate that 1 cells are present in the intestinal lamina propria of patients with Crohn s disease, as T bet and STAT4 are key regulators of 1 cell differentiation STAT4 deficiency in T cells protected mice from experimentally induced colitis, whereas the overexpression of STAT4 exacerbated colitis, which suggests that 1 cells promote pathology in this setting 81,85,86. However, although antibodies specific for IFNγ were therapeutically effective in a T cell transfer model of colitis in mice 4, treatment with the IFNγ-specific antibody fontolizumab had no such effect in patients with Crohn s disease 20, suggesting that the targeting of a single 1 type cytokine might not be effective clinically. 2 cell-associated cytokines. In contrast to the lamina propria T cells in Crohn s disease, lamina propria T cells from patients with ulcerative colitis have been shown to produce the 2 type cytokines IL 5 and IL 13 (REFS 74,87,88) and to express the 2 cellassociated transcription factor GATA-binding protein 3 (GATA3) 81 (FIG. 2). However, these cells only show low levels of IL 4 production, which suggests that they do not display all of the features of classical 2 cells 74. Instead, it was shown that ulcerative colitis is associated with the presence of CD1d restricted non-classical natural killer T (NKT) cells that have an atypical cytokine response that is characterized by 2 cell-associated cytokines such as IL 13 (REF. 87). Functionally, IL 13 has been shown to promote fibrosis and to cause altered tight junction function in, and apoptosis of, IECs thereby driving mucosal ulceration 88. The potential therapeutic relevance of this effect was shown in mouse studies in which antibodies specific for IL 13 suppressed disease in an oxazoloneinduced model of colitis 89. Similarly, antibodies specific for IL 25 (a cytokine that is produced by IECs) or its receptor (IL 17RB) suppressed IL 13 production by NKT cells and alleviated oxazolone-induced colitis 90. On the basis of these findings, clinical trials with IL 13 specific antibodies (namely, anrukinzumab and tralokinumab) are ongoing in patients with ulcerative colitis. However, recent studies did not find elevated IL-13 levels in patients with ulcerative colitis 91. Thus, clinical trials of anti IL 13 therapy will be required to clarify the functional relevance of IL 13 in patients with ulcerative colitis. Interestingly, the first results from a recent Phase II study of the IL 13 specific antibody tralokinumab have not shown a significant reduction of clinical activity scores in patients with ulcerative colitis ADVANCE ONLINE PUBLICATION

7 17 cell-associated cytokines. 17 cells have been recognized as an important new T cell subset in recent years. These cells are abundant in the intestine, most notably in the terminal ileum where they are induced by cytokines (such as IL 6 and IL 23) that are upregulated in response to components of the normal microbiota (for example, segmented filamentous bacteria (SFB) in mice) 93,94. On the basis of the identification of polymorphisms in genes that encode proteins of the IL cell pathway in patients with IBD (for example, IL 23R, IL 12p40, JAK2, STAT3 and CCR6), various studies have analysed the expression of 17 type cytokines in the mucosa of patients with IBD 95,96. These studies have shown that there is increased production of 17 cell-associated cytokines, such as IL 17A and IL 17F, by lamina propria T cells in both Crohn s disease and ulcerative colitis (FIG. 2). 17 cells also produced IL 26 and some IFNγ 97,98. 1 derived IL 21, APC-derived IL 23 and TNF-like protein 1A (TL1A; also known as TNFSF15) were shown to induce or perpetuate 17 type cytokine production in IBD 34,35,99. Furthermore, mucosal T cells from patients with IBD expressed the 17 cellassociated surface markers CD161 and IL 23R, and the 17 cell-associated transcription factors retinoic acid receptor-related orphan receptor γt (RORγt; which is encoded by RORC), STAT3 and IRF4 (REFS 30,75,95,100). Functionally, 17 type cytokines, such as IL 17 and IL 21, were found to mediate pro-inflammatory functions including the upregulation of TNF, IL 1β, IL 6 and IL 8, the recruitment of neutrophils and the secretion of matrix metalloproteinases by intestinal fibroblasts These findings suggested that 17 type cytokines may induce tissue destruction in IBD. Consistent with this, the increased expression of the 17 cell-associated cytokine IL 26 has been noted in patients with Crohn s disease and this cytokine augmented pro-inflammatory cytokine production cells may also produce anti-inflammatory cytokines, such as IL 22, that control epithelial cell proliferation, wound healing and the production of antimicrobial proteins such as defensins, mucins, and REG3β and REG3γ proteins via STAT3 activation 72. However, other studies have shown that another T cell subset (namely, the 22 cell subset) is an important source of IL 22 in the intestine and have found a marked reduction of these cells in patients with ulcerative colitis, but not in patients with Crohn s disease 104. Studies in mouse models of experimental colitis have shown that the absence or neutralization of IL 17A or IL 17F alone had no effect, or even aggravated disease activity, in a T cell transfer model of colitis 105. By contrast, deficiency of IL 21, IRF4 or RORγ led to a marked suppression of colitis activity 75,105,106. Furthermore, IL 22 treatment was found to protect mice from T celldependent colitis 107. To date, clinical targeting of 17 cells in patients with Crohn s disease has been restricted to the use of secukinumab, an IL 17A specific neutralizing antibody. However, secukinumab treatment has been reported to be ineffective in treating Crohn s disease and is associated with higher rates of adverse events than placebo therapy 21. Taken together, the production of both pro-inflammatory and anti-inflammatory cytokines by cell subsets has a crucial role in shaping disease in mouse models of IBD 49,89,105,108. However, given the marked plasticity shown by colitogenic effector T cell subsets 109, their multifaceted pattern of cytokine production and the disappointing results from anti-ifnγ and anti IL 17A clinical studies 21,110, the targeting of T cell subsets themselves or the simultaneous targeting of multiple cytokines (rather than targeting a single effector cytokine) may hold promise for future therapy of IBD. Cytokines and regulatory T cells in IBD T Reg cells suppress effector T cell responses and have been found to have a major protective role in experimental colitis by producing anti-inflammatory cytokines such as IL 10 and transforming growth factor-β (TGFβ) (FIG. 2). The functional relevance of these findings was highlighted by the observation that mice with T cellspecific inactivation of the genes encoding IL 10 or TGFβ lack functionally active T Reg cells, fail to suppress pro-inflammatory cytokine production by APCs and effector T cells, and spontaneously develop chronic intestinal inflammation T Reg cells are themselves important targets of IL 10, and IL 10R signalling in T Reg cells leads to the activation of the transcription factor STAT3 (REF. 117). Subsequent studies have revealed that the overwhelming majority of mucosal T Reg cells express the key transcription factor forkhead box P3 (FOXP3), however mucosal T Reg cells that lack FOXP3 have been identified in the small intestine 118. Free fatty acids derived from the normal microbiota regulate the size and function of the inducible intestinal T Reg cell pool and protect against experimental colitis in a free fatty acid receptor 2 (FFAR2)-dependent manner 119. T Reg cells suppress the pro-inflammatory functions of mucosal macrophages and effector T cells by producing TGFβ, which leads to the intracellular activation of SMAD3 and SMAD4 proteins However, effector T cells from patients with IBD have been shown to overexpress SMAD7, which inhibits TGFβ signalling, and these cells may therefore become resistant to TGFβ-mediated suppression 120,121. The functional relevance of this finding was shown by studies of mice in which transgenic overexpression of Smad7 resulted in the resistance of colitogenic T cells to T Reg cell-mediated suppression. Based on this concept, SMAD7 antisense oligonucleotides have been tested as a new therapeutic option in patients with Crohn s disease and have shown remarkable beneficial effects in a recent Phase I clinical study 123. Although single nucleotide polymorphisms (SNPs) in IL10 have been associated with IBD in a GWAS 12, treatment with recombinant IL 10 was not an effective therapy in patients with Crohn s disease 11. Additionally, no defect in regulatory cells was found in the majority of patients with IBD. In fact, it was found that T Reg cells in the mucosa of patients with Crohn s disease express FOXP3 and suppress effector T cell activity 124. However, although the marked accumulation of effector T cells was seen in intestinal lesions of patients with Crohn s disease, there was only NATURE REVIEWS IMMUNOLOGY ADVANCE ONLINE PUBLICATION 7

8 a moderate expansion of T Reg cell populations, which suggests that their anti-inflammatory functions may be insufficient to suppress the overwhelming activity of effector T cells. The therapeutic relevance of these findings was highlighted by a recent study, which showed that the systemic administration of ovalbumin-specific T Reg cells to patients with Crohn s disease was well tolerated and had dose-related anti-inflammatory clinical effects 125. However, further controlled studies are required to fully explore the therapeutic potential of T Reg cells and T Reg cell-derived cytokines in patients with IBD. Cytokines and stromal cells In addition to APCs and lymphocytes, it is now recognized that various non-immune cells such as epithelial cells, sub-epithelial myofibroblasts, adipo cytes and stromal fibroblasts can also produce pro-inflammatory cytokines in IBD 37,40. For example, myofibroblasts have been identified as an important source of TNF and can also produce other cytokines such as IL 6 (FIG. 3). Accordingly, cytokines derived from these non-immune cells may activate mucosal immune cells such as APCs and T cells and may thereby contribute to mucosal inflammation in IBD. Moreover, these cells may respond to cytokines that are produced by immune cells and thereby contribute to tissue destruction in IBD that can be targeted by anti-cytokine agents. For instance, in patients with Crohn s disease, anti-tnf agents have been shown to target myofibroblasts by suppressing their production of collagen, enhancing their migration and inducing the production of tissue inhibitor of matrix metalloproteinases 1 (TIMP1; also known as metallo proteinase inhibitor 1) 38. Thereby, anti-tnf agents can specifically block matrix remodelling and tissue destruction in IBD. Cytokines and intestinal epithelial cells The proliferation and expansion of IECs is of crucial importance for the closure of erosions and ulcers, improvement of intestinal barrier function and healing of the inflamed mucosa in IBD 19. Recent studies have found that such mucosal healing is critically dependent on cytokines that are produced by cells in the local microenvironment and by the IECs themselves (FIG. 4). These cytokines have been shown to control IEC activation, survival, migration, differentiation and their secretion of antimicrobial peptides. IBD-associated ulcer IBD-associated cancer IEC Bacteria Tumour 1 cell 2 cell 17 cell IL-6 TNF IFNγ IL-6 TNF IL-22 DC IL-6 TNF IFNγ IL-6 IL-27 TNF Macrophage ILC1 DC IL-22 IL-6 TNF Macrophage IL-1 IL-6 TNF IL-6 IL-6 TNF IL-21 IL-22 Fibroblast 1 cell 2 cell 17 cell Neutrophil Figure 4 The crucial role of cytokines and epithelial cells on the battlefield: mucosal healing and cancer in IBD. Intestinal epithelial cells (IECs) are exposed to numerous pro-inflammatory and anti-inflammatory cytokines during chronic intestinal inflammation in inflammatory bowel disease (IBD). These cytokines are produced by cells in the local microenvironment and by IECs themselves. Local cytokine responses have major effects on mucosal healing and cancer development in patients with IBD. The cellular sources of key cytokines and their signalling cascades that regulate IEC survival, cell death and proliferation are shown. In the context of mucosal healing in ulcers (left), green boxes indicate beneficial effects of cytokines, whereas red boxes highlight pathogenic effects of cytokines. In colitis-associated cancer (right), blue boxes indicate pro-tumour effects of cytokines. DC, dendritic cell; IFN, interferon; IL, interleukin; ILC, innate lymphoid cell; cell, T helper cell; TNF, tumour necrosis factor. 8 ADVANCE ONLINE PUBLICATION

9 IEC production of IL 1 family members. IECs can produce various cytokines of the IL 1 family including IL 18, IL 33 and IL 37. The increased production of IL 33 by IECs and sub-epithelial myofibroblasts has been described in patients with ulcerative colitis, but not in patients with Crohn s disease 126. The administration of IL 33 in acute experimental DSS-induced colitis led to a slight aggravation of inflammation via ST2 (also known as IL 1RL1) signalling, whereas IL 33 treatment ameliorated disease activity in chronic DSSinduced colitis. In the chronic DSS-induced colitis model, IL 33 was shown to suppress 1 type cytokine responses and to induce 2 like immune reactions. Furthermore, IL 33 induced neutrophil influx during chronic inflammation was shown to reduce the translocation of pathogenic bacteria across the damaged epithelium 127. As IL 33 deficiency suppressed DSSinduced colitis activity 128, these findings suggest that IL 33 has mainly pro-inflammatory functions during chronic colitis. Accordingly, blockade of the IL 33 ST2 signalling pathway suppressed DSS-induced colitis, which suggests that targeting of IL 33 might be of interest for future therapy of IBD 129. IEC production of IL 37. IL 37 is a recently discovered cytokine that potently suppresses innate immune responses. Epithelial cell expression of IL 37 was found to be increased in patients with ulcerative colitis compared with healthy controls 130. The functional relevance of IL 37 was suggested by studies using transgenic expression of human IL37 in haematopoietic cells in mice 131. Transgene-induced IL 37 production protected mice from experimentally induced colitis and was associated with reduced IL 1β and TNF production by lamina propria cells. Production of cytokines by IECs regulates barrier function. The exposure of IECs to various pro-inflammatory and anti-inflammatory cytokines at the interface with commensal microbiota has a fundamental role in mucosal healing (FIG. 4). Several pro-inflammatory cytokines, such as IFNγ and TNF, have been shown to alter tight junction activity and to induce apoptosis of IECs 37,132. This leads to the loss of barrier function and aggravates the epithelial erosions and ulcers that are present in colonic inflammation. By contrast, other cytokines such as IL 22 (REF. 72), induce IEC activation and survival via Table 1 Selected key cytokine activities implicated in the pathogenesis of IBD Cytokine Source in the mucosa Potential function in the pathogenesis of chronic intestinal inflammation IFNα and IFNβ DCs Promote epithelial regeneration and induce IL 10 producing cells IFNγ T cells and ILCs Activates macrophages, augments antigen processing and induces death of epithelial cells TNF Macrophages, DCs and T cells Activates fibroblasts, stimulates pro-inflammatory cytokine production and angiogenesis, induces death of epithelial cells, mediates T cell resistance against apoptosis and induces cachexia IL 1 Neutrophils and macrophages Augments recruitment of neutrophils, stimulates IL 6 production by macrophages, activates ILCs and promotes tumour development IL 6 Macrophages, fibroblasts and T cells Activates T cells and prevents apoptosis, induces macrophage activation, recruits immune cells, activates acute-phase proteins, induces proliferation of epithelial cells and favours tumour growth IL 10 T cells Suppresses pro-inflammatory cytokine production by antigen-presenting cells and T cells and induces STAT3 signalling in regulatory T cells IL 12 Macrophages and DCs Induces 1 cell differentiation via STAT4 activation in T cells, stimulates 1-type cytokine production and activates ILCs IL 13 T cells and inkt cells Induces alterations of intestinal epithelial cells and barrier function IL 17A and IL 17F T cells and ILCs Induce pro-inflammatory and anti-inflammatory effects in the mucosa and IL 17A exerts pro-fibrotic functions IL 18 Macrophages, DC and epithelial cells Augments production of pro-inflammatory cytokines IL 21 1 cells Induces production of TNF, IL 1, IL 6 and IL 8 in the mucosa, recruits neutrophils, induces secretion of matrix metalloproteinases by fibroblasts and favours tumour development IL 22 γδ and αβ T cells, ILCs, neutrophils and DCs Activates production of antimicrobial peptides by epithelial cells, induces proliferation of epithelial cells and favours tumour development via STAT3 activation IL 23 Macrophages and DCs Activates mucosal immune cells such as T cells and macrophages, augments TNF production and stabilizes the phenotype of effector 17 cells IL 27 Macrophages Exerts pro-inflammatory effects by inducing T cell activation and 1-type cytokine production and exerts anti-inflammatory effects by blocking T cell expansion and inhibiting cytokine production by neutrophils IL 33 Epithelial cells and myofibroblasts Suppresses 1-type cytokine production and induces neutrophil influx IL 35 DCs Blocks the production of pro-inflammatory cytokines by mucosal immune cells IL 37 Epithelial cells Suppresses innate mucosal immune responses and reduces IL 1β and TNF production DC, dendritic cell; IBD, inflammatory bowel disease; IFN, interferon; IL, interleukin; ILC, innate lymphoid cell; inkt cell, invariant natural killer T cell; STAT, signal transducer and activator of transcription; cell, T helper cell; TNF, tumour necrosis factor. NATURE REVIEWS IMMUNOLOGY ADVANCE ONLINE PUBLICATION 9

10 Bispecific antibody Tocilizumab Soluble IL-6 receptor Ustekinumab Infliximab, adalimumab, certolizumab and golimumab γ c subunit IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, IL-21R Target more than one cytokine gp130 IL-6R, IL-11R, LIFR, OSMR IL-6 β c subunit IL-3R, IL-5R, GM-CSFR IL-12 IL-12Rβ2 IL-12R, IL-23R IL-23 TNFR TNFR1, TNFR2 Soluble TNF Membranebound TNF Tofacitinib JAK1, JAK3 JAK1, JAK2, TYK2 JAK2 JAK2, TYK2 TRAF2 Activation STAT P Dimerization STAT P P STAT Translocation STAT P P Pro-inflammatory gene expression T cells in Crohn s disease: STAT1, STAT3 and STAT4 T cells in ulcerative colitis: STAT3 and STAT6 Figure 5 Cytokine signalling in IBD. Cytokine signalling pathways and intracellular Janus kinase (JAK) signal transducer and activator of transcription (STAT) signalling cascades in mucosal immune cells are shown. In IBD, the activation of certain STATs in mucosal T cells results in augmented cytokine production. Several pro-inflammatory cytokines have been implicated in IBD pathogenesis and are potential targets for therapy. Antibodies targeting soluble tumour necrosis factor (TNF) and membrane-bound TNF (such as infliximab, adalimumab, certolizumab pegol and golimumab) are routinely used in the clinic. In addition, cytokine blockers (for example, tocilizumab, which targets interleukin 6 (IL 6) and ustekinumab, which targets the p40 subunit of IL 12 and IL 23) have been recently tested in clinical studies. In addition, inhibitors of JAK and STAT signalling (for example, the JAK3 and JAK1 inhibitor tofacitinib, which blocks IL 2, IL 4, IL 7, IL 9, IL 15 and IL 21 signalling) have yielded promising results in clinical trials. Future therapy of IBD may also use bispecific tetravalent dual variable domain IgG (DVD Ig) antibodies. Finally, the identification of specific cytokines and cytokine expression patterns that are unique to certain subsets of patients with IBD may open new avenues for future personalized medicine for these disorders. β c, cytokine receptor common subunit-β; γ c, cytokine receptor common subunit-γ; gp130, IL 6R subunit-β; LIFR, leukaemia inhibitory factor receptor; OSMR, oncostatin-m-specific receptor subunit-β; TRAF2, TNFR-associated factor 2; TYK2, tyrosine kinase 2. STAT signalling and thus favour mucosal healing. The balance between these types of cytokines seems to be crucial for wound healing in experimental colitis and in IBD, and thus offers potential for therapeutic intervention. This cytokine balance also seems to be important for preventing the uncontrolled proliferation of IECs that can lead to colitis-associated cancer. Cytokines in colitis-associated colon cancer Patients with IBD have an increased risk of developing colitis-associated colorectal cancer. Some of the established risk factors for this are the extent and the duration of the disease as well as the number of flares, which suggests that uncontrolled inflammation can drive tumorigenesis. Studies in patients with IBD-associated cancer and in murine models of colitis-associated cancer have shown that activated neutrophils, fibroblasts, DCs, macrophages and T cells are present in tumour tissue and seem to control tumour growth via the production of soluble mediators, including cytokines 8, (FIG. 4). Cytokines activate tumour cell proliferation, expansion and survival through the activation of intracellular signalling molecules, such as STAT3 and NF κb 134,137. The functional relevance of cytokines has been demonstrated in experimental carcinogenesis. Specifically, recent studies have addressed the functional consequences of inhibiting pro-inflammatory cytokines such as IL 1, IL 6 and TNF. Neutrophils were found to produce IL 1 in experimentally induced colitis and in patients with colitisassociated colorectal cancer 138. Blockade of IL 1β activity was shown to reduce tumorigenesis in mice by impairing macrophage-dependent IL 6 secretion 138. Additionally, augmented levels of TNF were detected in experimental models of colitis-associated colorectal cancer, and TNF blockade was found to suppress tumour growth in the azoxymethane (AOM)- and DSS-induced model of colitis-associated colon cancer 139. Furthermore, T cells in experimental colitis-associated colorectal cancer have 10 ADVANCE ONLINE PUBLICATION