Nature Reviews Immunology | AOP, published online 22 April 2014; doi:10.1038/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 (TH1)-, TH2- or TH17‑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 ErlangenNürnberg, Kussmaul Campus for Medical Research, 91054 Erlangen, Germany. e‑mail: [email protected] uk‑erlangen.de doi:10.1038/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 IBD3. 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 inflammation4,5. 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 clinic1. Studies in recent years have identified a major role of both genetic and environmental factors in the pathogenesis of IBD1,2 (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 IBD3,6. 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 neoplasias2,7,8. 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 1990s9,10. However, the functional relevance of the observed changes in terms of the clinical activity

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REVIEWS 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 inflammation3. 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 inflammation3,4,11. Additional evidence from genetic studies has suggested a crucial role for cytokines and cytokine-producing immune cells in IBD patho­genesis. 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 (TH) 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 pathogenesis12. 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 transplantation13. 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 disease17. 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 IBD1,2,18,19. 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 disease21. 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 TNF22. 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 IBD23. 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

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REVIEWS

Phase I: pre-disease stage

Genetic factors • Antimicrobial peptides • Autophagy • Handling of bacteria • Chemokines • Cytokines

Environmental factors • Microorganisms • Diet • Infections • Stress • NSAIDs • Appendectomy • Smoking • Antibiotics

Initiating triggers Bacteria

Impaired barrier function Microbial product Translocation of microbial products Phase II: acute intestinal inflammation

Phase III: chronicity or resolution

Phase IV: tissue destruction and complications

Immune cell activation Production of pro-inflammatory mediators Failure of regulatory mechanisms

Macrophage Effector T cell Activation of effector cells Chronic inflammation TReg cell

Fibrosis, stenosis, abscess, fistula, cancer and extra-intestinal manifestations

Figure 1 | Conceptual framework for the pathogenesis of IBD.  Genetic and Nature Reviews | Immunology 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; TReg cell, regulatory T cell.

Necroptosis A programmed form of necrotic cell death that is regulated by receptor-interacting protein kinase 1 (RIPK1) and RIPK3.

in rabbits25, TNF blockade (but not IL‑1 blockade) was effective in treating chronic dextran sodium sulphate (DSS)-induced colitis in mice26. 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 disease27— 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 inflammation29.

IL‑6 and IBD. IL‑6 production by lamina propria macrophages and CD4+ T cells is increased in experimental colitis and in patients with IBD30,31 (FIG. 2). In particular, CD14+CD33+CD68+CD163low 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 cells30. 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 IBD30,34. 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 disease35. 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 TNF3,33,36. 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) activation36–40 (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

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REVIEWS Lumen

IEC

Lamina propria

Bacteria

Goblet cell

NK cell IL-18 ILC1 ILC3

• IFNγ • IL-17A • IL-17F • IL-22

• Homeostasis • Inflammation

• IFNγ • IL-6 • TNF

Crohn’s disease

ILC1

IFNγ ↑ T-bet

IL-12 TH1 cell

Macrophage

• IL-6 • IL-23 • TNF

↑ GATA3

TH2 cell ↑ RORγt

Antimicrobial peptides, such as defensins

Neutrophil TH17 cell Retinoic acid, TGFβ

↑ FOXP3

Paneth cell CD103+ DC

• IL-5 • IL-6 • IL-13 • TNF

?

Ulcerative colitis

• IL-17A • IL-17F • IL-21 • IL-22

• Homeostasis • Ulcerative colitis • Crohn’s disease

• IL-10 • TGFβ

Resolution

TReg cell

Figure 2 | Cytokines in the pathogenesis of IBD.  In patients with inflammatory bowel disease (IBD) and in Nature Reviews experimental mouse models of colitis, pro-inflammatory and anti-inflammatory cytokines have been shown| Immunology 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 (TH1), TH2 and TH17) and regulatory T (TReg) 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 TH1 cell differentiation and

activation in Crohn’s disease22,45. Similarly, these cells produce large amounts of IL‑23 (which is composed of the IL‑12p40 subunit and a p19 subunit; the p19 sub­unit is also known as IL‑23 subunit-α) in patients with Crohn’s disease46; this cytokine perpetuates local TH17 cell responses and suppresses regulatory T (TReg) 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 antibodies11,47–50. 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 disease51,52. 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 inflammation47,48.

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REVIEWS Source

Target cell

Macrophage

Endothelial cell

Adipocyte

Fibroblast

Activation, pro-inflammatory cytokine production and suppression of regulatory macrophages

IL-1, IL-6 and TNF IEC

TNF

Consequence Angiogenesis and hypervascularization

Macrophage

DC

T cell

Effector mechanism

Paneth cell

IEC: MLCK Paneth cell: RIPK1 and RIPK3

IEC: cell death and impaired barrier function Paneth cell: necroptosis

TIMP1

MMP-induced tissue destruction and impaired migration

TRAF2 and NF-κB

Apoptosis resistance, cell survival and IL-6 synthesis

Myofibroblast

Effector T cell

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 Nature Reviews | Immunology 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.

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 T H1‑type cytokine production and the expansion of TReg 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 APCs54,55. However, other investigators have found that IL‑27 has an anti-inflammatory effect or no effect in models of colitis56–58. 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 oligodeoxy­nucleotides 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 TReg 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 mice60. However, treatment with recombinant IFNβ1a was safe but had no therapeutic benefit in patients with steroid-refractory ulcerative colitis16, which suggests that an IFNβ-based approach is not ideally suited for IBD therapy. By contrast, a CpG-containing oligo­ nucleotide was recently used to successfully treat several patients with steroid-resistant ulcerative colitis61, 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.

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REVIEWS 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 colitis62. 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 colitis63. 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 disease64. 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 disease65. 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 LYN67. Moreover, IL‑22 is produced by neutrophils, DCs, γδ T cells and effector αβ T cells in experimental colitis68–70. 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 colitis68,71,72. However, the pro-inflammatory effects of IL‑22 were recently noted in innate immune-mediated colitis73, 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 apoptosis30,36. 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.

TH1 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 controls74,77–79 (FIG. 2). Furthermore, TH1 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 disease80,81. Taken together, these observations indicate that TH1 cells are present in the intestinal lamina propria of patients with Crohn’s disease, as T‑bet and STAT4 are key regulators of TH1 cell differentiation82–84. STAT4 deficiency in T cells protected mice from experimentally induced colitis, whereas the overexpression of STAT4 exacerbated colitis, which suggests that TH1 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 mice4, treatment with the IFNγ-specific antibody fontolizumab had no such effect in patients with Crohn’s disease20, suggesting that the targeting of a single TH1‑type cytokine might not be effective clinically. TH2 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 TH2‑type cytokines IL‑5 and IL‑13 (REFS 74,87,88) and to express the TH2 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 TH2 cells74. 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 TH2 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 colitis89. 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 colitis90. 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 colitis91. 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 colitis92.

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REVIEWS TH17 cell-associated cytokines. TH17 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‑23–TH17 cell pathway in patients with IBD (for example, IL‑23R, IL‑12p40, JAK2, STAT3 and CCR6), various studies have analysed the expression of TH17‑type cytokines in the mucosa of patients with IBD95,96. These studies have shown that there is increased production of TH17 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). TH17 cells also produced IL‑26 and some IFNγ97,98. TH1‑derived IL‑21, APC-derived IL‑23 and TNF-like protein 1A (TL1A; also known as TNFSF15) were shown to induce or perpetuate TH17‑type cytokine production in IBD34,35,99. Furthermore, mucosal T cells from patients with IBD expressed the T H17 cellassociated surface markers CD161 and IL‑23R, and the TH17 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, TH17‑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 fibroblasts101–103. These findings suggested that TH17‑type cytokines may induce tissue destruction in IBD. Consistent with this, the increased expression of the TH17 cell-associated cytokine IL‑26 has been noted in patients with Crohn’s disease and this cytokine augmented pro-inflammatory cytokine production98. TH17 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 activation72. However, other studies have shown that another T cell subset (namely, the TH22 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 disease104. 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 colitis105. 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 colitis107. To date, clinical targeting of TH17 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 TH cell subsets has a crucial role in shaping disease in mouse models of IBD49,89,105,108. However, given the marked plasticity shown by colitogenic effector T cell subsets109, their multifaceted pattern of cytokine production and the disappointing results from anti-IFNγ and anti‑IL‑17A clinical studies21,110, 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 TReg 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)111–113 (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 TReg cells, fail to suppress pro-inflammatory cytokine production by APCs and effector T cells, and spontaneously develop chronic intestinal inflammation113–116. TReg cells are themselves important targets of IL‑10, and IL‑10R signalling in TReg cells leads to the activation of the transcription factor STAT3 (REF. 117). Subsequent studies have revealed that the overwhelming majority of mucosal TReg cells express the key transcription factor forkhead box P3 (FOXP3), however mucosal TReg cells that lack FOXP3 have been identified in the small intestine118. Free fatty acids derived from the normal microbiota regulate the size and function of the inducible intestinal TReg cell pool and protect against experimental colitis in a free fatty acid receptor 2 (FFAR2)-dependent manner 119. TReg 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 proteins120–122. 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 suppression120,121. 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 TReg 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 GWAS12, treatment with recombinant IL‑10 was not an effective therapy in patients with Crohn’s disease11. Additionally, no defect in regulatory cells was found in the majority of patients with IBD. In fact, it was found that TReg 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

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REVIEWS a moderate expansion of TReg 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 TReg cells to patients with Crohn’s disease was well tolerated and had dose-related anti-inflammatory clinical effects125. However, further controlled studies are required to fully explore the therapeutic potential of TReg cells and TRegcell-derived cytokines in patients with IBD.

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 metallo­proteinase inhibitor 1)38. Thereby, anti-TNF agents can specifically block matrix remodelling and tissue destruction in 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, adipo­cytes and stromal fibroblasts — can also produce pro-inflammatory cytokines in IBD37,40. 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

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 IBD19. 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 Tumour

IEC

Bacteria

IL-6 • TNF • IFNγ

TH1 cell

IL-22 IFNγ

IL-6

TH2 cell

ILC1

• IL-6 • TNF

TNF

• IL-6 • TNF IL-6 TNF

IL-22

• IL-6 • IL-27 TNF

TH1 cell

• IL-21 • IL-22

TH2 cell

IL-6

TH17 cell

DC Macrophage

TH17 cell DC

• IL-6 • TNF

Fibroblast

IL-1

Macrophage Neutrophil

Figure 4 | The crucial role of cytokines and epithelial cells on the battlefield: mucosal healing and cancer in IBD. Naturecytokines Reviews during | Immunology Intestinal epithelial cells (IECs) are exposed to numerous pro-inflammatory and anti-inflammatory 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; TH cell, T helper cell; TNF, tumour necrosis factor. 8 | ADVANCE ONLINE PUBLICATION

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REVIEWS 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 disease126. 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 TH1‑type cytokine responses and to induce TH2‑like immune reactions. Furthermore, IL‑33‑induced neutrophil influx during chronic inflammation was shown to reduce the translocation of pathogenic bacteria across the damaged epithelium127. 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 IBD129.

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 controls130. The functional relevance of IL‑37 was suggested by studies using transgenic expression of human IL37 in haematopoietic cells in mice131. 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 IECs37,132. 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 TH1 cell differentiation via STAT4 activation in T cells, stimulates TH1-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

TH1 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 TH17 cells

IL‑27

Macrophages

Exerts pro-inflammatory effects by inducing T cell activation and TH1-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 TH1-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; TH cell, T helper cell; TNF, tumour necrosis factor.

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REVIEWS

Bispecific antibody

Tocilizumab

Soluble IL-6 receptor

Infliximab, adalimumab, certolizumab and golimumab

Ustekinumab

Target more than one cytokine IL-6 γc subunit

βc subunit

gp130

IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, IL-21R

Tofacitinib

IL-12

IL-6R, IL-11R, LIFR, OSMR

IL-3R, IL-5R, GM-CSFR

JAK1, JAK2, TYK2

JAK1, JAK3

IL-23

IL-12Rβ2

TNFR

IL-12R, IL-23R

JAK2

Soluble TNF

Membranebound TNF

TNFR1, TNFR2

JAK2, TYK2

TRAF2

STAT

Activation

P

P

Translocation

STAT

P

STAT

STAT

Dimerization P

P

T cells in Crohn’s disease: Pro-inflammatory STAT1, STAT3 and STAT4 gene expression 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 Nature are shown. In IBD, the Reviews | Immunology 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. βc, cytokine receptor common subunit-β; γc, 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 cytokines8,133–136 (FIG. 4). Cytokines activate tumour cell proliferation, expansion and survival through the activation of intracellular signalling molecules, such as STAT3 and NF‑κB134,137. 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 cancer138. Blockade of IL‑1β activity was shown to reduce tumorigenesis in mice by impairing macrophage-dependent IL‑6 secretion138. 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

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REVIEWS Table 2 | Selected cytokines or cytokine signalling events as therapeutic targets in IBD Cytokine or Disease signalling molecule

Advantages as a target

Disadvantages as a target

Developmental stage Therapeutic agent

Limited or no efficacy in clinical trials

Clinical trials (Phase II)

Recombinant IFNβ

IFNβ

Ulcerative colitis

Immunoregulatory cytokine targeting regulatory cells

IFNγ

Crohn’s disease

Plausible bioactivity seen in studies Limited efficacy in clinical using mouse models and in ex vivo trials studies using cells from patients with IBD

Clinical trials (Phase II)

Fontolizumab

TNF

Ulcerative colitis and Crohn’s disease

Central pro-inflammatory cytokine in IBD pathogenesis

Increased infection risk (lung infection and tuberculosis)

Approved for some indications in IBD and routinely used in the clinic

Infliximab, adalimumab certolizumab and golimumab

TGFβ

Crohn’s disease

Targeting SMAD7 restores the TGFβ-sensitivity of cells

Long-term toxicity and effects unclear

Clinical trials (Phase I)

SMAD7 antisense oligonucleotides

IL‑6R

Crohn’s disease

Plausible bioactivity seen in studies using mouse models and in ex vivo studies using cells from patients with IBD

Response only seen in Clinical trials (Phase II) subgroups of patients; effects on mucosal healing unclear

IL‑10

Crohn’s disease

Plausible bioactivity seen in studies Limited or no efficacy in using mouse models clinical trials

Clinical trials (Phase II)

Recombinant IL‑10

IL‑11

Crohn’s disease

Immunostimulatory cytokine

Limited or no efficacy in clinical trials

Clinical trials (Phase II)

Recombinant IL‑11

IL‑12 and IL‑23

Crohn’s disease

Plausible bioactivity in mouse models and in ex vivo studies using cells from patients with IBD

Response of subgroups of patients only

Clinical trials (Phase II-II)

Ustekinumab and briakinumab (ABT874)

IL‑13

Ulcerative colitis

Plausible bioactivity in some mouse models and on human epithelial cells ex vivo

Efficacy currently unknown

Clinical trials (Phase II)

Anrukinzumab and tralokinumab

IL‑17A

Crohn’s disease

Plausible bioactivity in some models of inflammation

Aggravation of disease and effects on tissue homeostasis

Clinical trials (Phase II)

Secukinumab

JAK3

Ulcerative colitis (Crohn’s disease)

Targeting of several key cytokines simultaneously

Long-term toxicity unclear Clinical trials (Phase II) and not effective in a pilot study in Crohn’s disease

Tocilizumab

Tofacitinib

IBD, inflammatory bowel disease; IFN, interferon; IL, interleukin; JAK, Janus kinase; TGFβ, transforming growth factor‑β; TNF, tumour necrosis factor.

been shown to increase their production of IL‑6 via the activation of NFATC2 (REF. 140). IL‑6 and the soluble IL‑6R were shown to activate the proliferation of tumour cells via the transcription factor STAT3 in this model8,134,135. The inhibition of IL‑6 signal transduction via IL‑6R‑specific antibodies or gp130‑Fc fusion proteins has been shown to inhibit tumour growth. Similarly, IL‑21‑specific anti­ bodies and neutralization of IL‑22 function has been found to suppress tumorigenesis, thus highlighting the potential relevance of anti-cytokine strategies for the treatment of colitis-associated neoplasias141–143. However, anti-cytokine agents are currently not considered as a standard therapy for patients with colitis-associated neoplasias, and clinical studies will be necessary to elucidate the potential of cytokine blockers in these patients.

Perspectives on cytokine-based therapy in IBD Cytokines seem to have a major role in driving intestinal inflammation, local complications (such as colorectal cancer), and systemic and extra-intestinal manifestations in patients with IBD (TABLE 1). Consistent with this, proinflammatory cytokines such as TNF are currently the

key target for IBD therapy. In addition, novel agents that target cytokines or cytokine signalling cascades are currently being tested in clinical trials, suggesting that cytokine blockade will remain a crucial field of interest for IBD therapy (FIG. 5; TABLE 2). However, anti-cytokine therapies (such as antibodies specific for TNF, IL‑12 or IL‑23) and cytokine signalling blockers (such as tofacitinib) only seem to have beneficial clinical effects in certain subgroups of patients1,51,52,144. This may reflect the fact that cytokine networks in the inflamed mucosa are more complex than previously appreciated and that they are subject to multiple layers of regulation by microbial, genetic and immunological factors. A key feature of the mucosal cytokine network is its dynamic fluidity and ability to traverse spatial boundaries, and it is likely that the blockade of a single cytokine in patients with IBD may lead to the development of alternative compensatory pro-inflammatory cytokine pathways. Furthermore, the pathological mechanisms that drive mucosal inflammation are likely to differ between patients, which would explain why the targeting of a single pro-inflammatory cytokine is not effective in many cases.

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REVIEWS Nanobodies Therapeutics that are based on the heavy-chain antibodies found in camels and llamas. Similarly to conventional antibodies, they have heavy-chain variable and constant regions but they lack light-chain domains.

Cytokine function in patients with IBD may be affected by the location and the type of inflammation, by immune cell plasticity, by different pathogenetic mechanisms or by the changing cytokine production patterns that occur during the course of the disease. Several recent observations suggest that all of these factors need to be considered. In fact, a shift from a TH1‑type response to a mixed TH1- and TH17‑type response was frequently noted during the course of Crohn’s disease in a recent study 145. Furthermore, with regard to disease location, recent studies have shown that Crohn’s disease colitis is associated with either a TH1- or a TH17‑type cytokine profile, whereas a mixed TH1- and TH17‑type response was noted in ileal Crohn’s disease 146. In addition, TReg cells producing TH17‑type cytokines were recently identified in a subgroup of patients with Crohn’s disease147. Finally, marked differences in mucosal cytokine responses between Crohn’s disease and ulcerative colitis have been identified3. Collectively, these factors highlight the complexity of the mucosal cytokine network and suggest that anti-cytokine approaches that target a single pro-inflammatory cytokine will have major limit­ ations in terms of offering an effective therapy for all clinical subgroups of IBD. On the basis of these findings, the design of selective targeted therapy for individual patients with IBD would be highly desirable to increase the number of patients that respond to these therapies and to minimize the potential side effects. However, this approach would require the identification of serum markers or the detailed analysis of cytokine levels in the inflamed intestine. A recent study has used fluorescently labelled TNF-specific antibodies and in vivo imaging to determine the frequency of immune cells that express membrane-bound TNF in the mucosa of patients with Crohn’s disease148. It was found that the presence of high numbers of immune cells expressing membrane-bound TNF predicted a positive clinical response to subsequent therapy with TNFspecific antibodies. Patients in which few immune cells expressed membrane-bound TNF showed little or no response to this type of therapy, suggesting that they have TNF-independent gut inflammation. These observations indicate that in vivo imaging of cytokine responses may

Danese, S. & Fiocchi, C. Ulcerative colitis. New Engl. J. Med. 365, 1713–1725 (2011). Baumgart, D. C. & Sandborn, W. J. Crohn’s disease. Lancet 380, 1590–1605 (2012). 3. Strober, W., Fuss, I. J. & Blumberg, R. S. The immunology of mucosal models of inflammation. Annu. Rev. Immunol. 20, 495–549 (2002). 4. Powrie, F. et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1, 553–562 (1994). This groundbreaking study defines cytokines as therapeutic targets in chronic intestinal inflammation. 5. Neurath, M. F., Finotto, S. & Glimcher, L. H. The role of Th1/Th2 polarization in mucosal immunity. Nature Med. 8, 567–573 (2002). 6. Ruffolo, C. et al. Subclinical intestinal inflammation in patients with Crohn’s disease following bowel resection: a smoldering fire. J. Gastrointestinal Surg.14, 24–31 (2010). 7. Peyrin-Biroulet, L., Loftus, E. V. Jr., Colombel, J. F. & Sandborn, W. J. Long-term complications, 1. 2.

be useful for the development of individualized therapy and for the optimization of anti-cytokine approaches. However, additional prospective studies are needed before this concept may enter clinical practice. An alternative approach to the optimization of response rates in IBD would be the use of multi-cytokine blockers that simultaneously inhibit several cytokines or JAK–STAT cytokine signalling pathways (FIG. 5). Examples include JAK inhibitors, such as tofacitinib, that block various pro-inflammatory cytokine signalling cascades. Tofacitinib blocks the JAK3 and JAK1 signalling pathways that are downstream of the receptors of IL‑2, IL‑4, IL‑7, IL‑9, IL‑15 and IL‑21, and this drug has recently been tested in initial clinical studies where it has shown efficacy in ulcerative colitis but not in Crohn’s disease144,149. Furthermore, one may envision that bispecific dual variable-domain antibodies150 — that target two specific pro-inflammatory cytokines at the same time (for example, TNF plus another pro-inflammatory cytokine) — could have substantial potential for future therapy of IBD. Finally, although the treatment of IBD with recombinant anti-inflammatory cytokines has not been successful so far, new approaches for the optimized delivery of such cytokines to the surface of the inflamed mucosa should be explored. For instance, the topical delivery of IL‑27‑producing bacteria (Lactococcus lactis) was recently shown to suppress experimentally induced colitis through the induction of IL‑10 production by host T cells56. In addition, lactobacilli that produce TNF-specific nanobodies were recently successfully tested for the luminal therapy of experimentally induced colitis151, which suggests that the topical delivery of cytokine blockers may open new avenues for optimized therapy of IBD. This approach may increase local drug concentrations and reduce the systemic concentrations that are associated with side effects. Anti-cytokine therapies involving TNF-specific agents form an important cornerstone of clinical therapy in both Crohn’s disease and ulcerative colitis1,2. New cytokine targets, optimized delivery systems and personalized medicine may pave the way to more effective clinical approaches that target the expression or function of pro-inflammatory and anti-inflammatory cytokines in patients with IBD.

extraintestinal manifestations, and mortality in adult Crohn’s disease in population-based cohorts. Inflamm. Bowel Dis. 17, 471–478 (2011). 8. Becker, C. et al. TGF-β suppresses tumor progression in colon cancer by inhibition of IL‑6 trans-signaling. Immunity 21, 491–501 (2004). This study identifies cytokines as a link between inflammation and tumour growth in experimental models of colitis. 9. Ebert, E. C., Wright, S. H., Lipshutz, W. H. & Hauptman, S. P. T‑cell abnormalities in inflammatory bowel disease are mediated by interleukin 2. Clin. Immunol. Immunopathol. 33, 232–244 (1984). 10. Mitsuyama, K., Sata, M. & Tanikawa, K. Significance of interleukin‑6 in patients with inflammatory bowel disease. Gastroenterol. Japon. 26, 20–28 (1991). 11. Neurath, M. F., Fuss, I., Kelsall, B. L., Stuber, E. & Strober, W. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J. Exp. Med. 182, 1281–1290 (1995). 12. Jostins, L. et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 491, 119–124 (2012).

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Acknowledgements

The research of M.F.N. is supported by the Clinical Research Group (CEDER) of the German Research Council (DFG), the DFG Collaborative Research Centers 643 and 796, The German Cancer Aid organization, the United European Gastroenterology Research Prize, the DFG Graduate School of Excellence School in Advanced Optical Technologies, the Excellence program of the Friedrich-Alexander-Universität Erlangen-Nürnberg (Ludwig Demling Center), Germany, and the Interdisciplinary Center for Clinical Research (IZKF) and ELAN (Fonds für Forschung und Lehre) programmes of the University Erlangen-Nürnberg, Germany.

Competing interests statement

The author declares competing interests: see Web version for details.

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Cytokines in inflammatory bowel disease.

Cytokines have a crucial role in the pathogenesis of inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, where they co...
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