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Pathogenesis of the inflammatory bowel diseases (IBDs) ulcerative colitis (UC) and Crohn’s disease (CD) involve pro-inflammatory changes within the microbiota, chronic immune-mediated inflammatory responses, and epithelial dysfunction. Converging data from genome-wide association studies (GWAS), mouse models of IBD, and clinical trials indicate that cytokines are key effectors of both normal homeostasis and chronic inflammation in the gut. Yet still many questions remain concerning the role of specific cytokines in different IBDs, within distinct regions of the gut, and with regard cellular mechanisms of action. Here we review current and emerging concepts concerning the role of cytokines in IBD, with a focus on immune regulation, T cell subsets, and potential clinical applications.
The two common inflammatory bowel diseases (IBDs) – ulcerative colitis (UC) and Crohn’s disease (CD) – are chronic, relapsing, and debilitating immune-mediated inflammatory disorders that affect the gastrointestinal (GI) tract1-3. Despite impressive advances in IBD research over the last decade, the mechanisms that initiate and sustain inflammation in IBD remain incompletely understood, and treatments remain inadequate. The necessity for improving the diagnosis and treatment of IBD is underscored by the increasing prevalence of IBDs in western countries over the last 30 years, and the rising healthcare costs associated with IBD therapy4,5. Thus, safer and more effective therapies are needed, yet this requires more complete understanding of local immune regulation in the gut.
Several key concepts in IBD pathogenesis have emerged over the past decade; summarized here, each concept is reviewed elsewhere in more detail2,6,7. First, despite their common clinical classification (as IBDs), UC and CD are fundamentally different diseases with unique pathophysiologic features. For example, UC manifests as continuous mucosal inflammation of the colon, whereas CD is predominantly a small bowel disease that presents as “patchy”, transmural inflammation, most commonly in the ileum1,3. Second, both UC and CD are genetically complex diseases, in which hundreds of independent genetic loci contribute to disease susceptibility. Whereas the molecular functions of many IBD-associated genes remain unknown, several genes have been identified that contribute substantially to genetic load (e.g., NOD2, IL23R, IL10); for these, molecular mechanisms have now been described (see below)8-10. Yet even in these instances, the path from initial genetic insult to overt intestinal inflammation is incompletely understood and variable. In other words, IBDs appear to represent a collection of individual pathophysiologies that ultimately manifest similarly in the clinic. This concept is particularly important as it relates to IBD therapy; disease pathogenesis, and thus relevant therapeutic strategies, may ultimately need to be determined on an individual, rather than overarching, basis. For example, the NOD-like receptor (NLR) NOD2 is a cytoplasmic bacterial-sensor required for innate immune activation and preservation of the epithelial barrier8, whereas IL-10 is a cytokine used by Foxp3-expressing CD4+ T regulatory (Treg) cells to maintain immune tolerance towards commensal bacteria in the gut9,11. IBDs associated with NOD2 or IL10 deficiency thus progress from divergent pathogenic mechanisms; the extent to which IBDs driven by different genetic susceptibilities converge to common targetable pathways (e.g., TNFα) is an important area of active investigation.
In addition to genetics, IBDs are strongly influenced by environmental and microbiological factors. The influence of environment on IBD is best illustrated by the disproportionately high prevalence of IBDs in developed countries, and high rates of disease discordance amongst identical twins (50% for CD, 10% for UC)7. High fat diet, smoking, and early exposure to antibiotics have all been implicated as contributing to IBD susceptibility12-14. The best-established link between IBD susceptibility and the environment is the microbiota, a term that refers to the vast community of enteric bacteria that persistently colonize the mammalian GI tract2,6. The hypothesis that gut bacteria regulate IBD has been around since the 1970’s15. However, it wasn’t until recent advances in high-throughput sequencing technologies that the intestinal microbiota in healthy individuals and IBD patients could be systematically characterized. Indeed, it is now well established that the microbiome of IBD patients is distinct from that of healthy individuals16,17. Whether perturbation of the microbiota is a cause or consequence of intestinal inflammation remains controversial. On one hand, microbiota directly regulate intestinal inflammation in several chemically-induced18-20, T cell transfer-induced21, and genetic mouse models22,23 of colitis. In some instances, discreet species of naturally occurring enteric bacteria – termed pathobionts – have been identified that overgrow their niche upon exposure to high fat diet24 or broad spectrum antibiotics25 and promote IBD-like intestinal pathology. On the other hand, the clinical utility of microbiota-directed therapies in IBD, such as antibiotics or fecal transplantation, remains uncertain26-29. Thus, like the genetics of IBD, the precise role of the microbiota generally, and of discrete pathobionts specifically, are likely “personalized” in IBDs. We submit that it is less productive to debate the overarching role of the microbiota in IBDs than to seek out stratified IBD sub-types or individual patients that stand to benefit from microbiota-targeted therapies. For example, CD shows a higher degree of disease concordance between monozygotic twins (~ 50%) compared to UC (~ 10%)7; this may reflect increased genetic (and decreased environmental) contribution in CD. In addition, SAMP1/YitFc mice develop ileitis, even upon re-derivation in germ free environments – albeit somewhat attenuated30,31 – in contrast to models of colitis (e.g., Il10−/−), which are more strictly microbiota-dependent32. Taken together, these emerging concepts in IBD pathogenesis call for continued personalization of IBD therapy. As discussed below, this is likely also the case for biologic modalities targeting cytokine pathways.
Functional annotation of IBD-associated genes implicates several fundamental processes/pathways in disease pathogenesis. These include regulation of intestinal barrier function and antimicrobial activity, endoplasmic reticulum (ER) stress, autophagy, and CD4+ T helper (Th) cell development and function33. Each pathway interacts with the others to coordinate intestinal homeostasis in healthy individuals and to confer risk in IBD patients. Cytokines regulate each of these pathways, thus enabling molecular crosstalk between the epithelium, mucosal immune cells, and the microbiota. Accordingly, cytokines, cytokine receptors, and regulators of cytokine signaling are amongst the most over-represented class of genes associated with IBD (e.g., IL1R1, IL2, IL2RA, IL12B, IL18RAP, IL21, IFNG, IFNGR1, IL10, IL27, IL12R, IL23R)33. Functionally, immune cells isolated from lamina propria of IBD patient biopsies display increased expression of numerous innate and adaptive cytokines compared with cells from uninflamed intestinal tissue (see below). Finally, several anti-cytokine antibodies are now approved and widely used for IBD therapy, including anti-TNF’s (e.g., Infliximab, Etanercept, Adalimumab) and anti-IL-12/23 p40 (i.e., Ustekinumab)34. Collectively, these data have cemented cytokines, not only as key players in IBD pathogenesis, but also as viable therapeutic targets. Much of the recent interest in cytokines and IBD revolves around innate immunity and epithelial function. However, T cells, and in particular CD4+ Th cells, also play a significant role in chronic inflammatory diseases in general and in IBDs specifically. Indeed, these insights into the genetics of cytokine dysregulation in IBD offer a unique opportunity, not only to treat patients but also to understand the basis by which Th cells develop into pro- and anti-inflammatory subsets, perform tissue-specific functions, and contribute to human inflammatory diseases. The ability to target pathogenic Th cell responses in the gut holds enormous therapeutic potential to improve the effectiveness of IBD treatments whilst moving beyond potentially serious side effects associated with systemic immune modulation/suppression.
The precise role of Th cells in IBD remains controversial. On one hand, the major histocompatibility complex (MHC), which dictate the specificity of T cell responses, contributes only modestly to genetic susceptibility in IBDs, and more so in UC than CD33. By contrast, MHC genotype is by far the strongest genetic determinant of other, more “traditional” autoimmune diseases, such as multiple sclerosis (MS), type 1 diabetes (T1D), and celiac disease35-37. Functionally, some acute, chemically-induced mouse models of IBD (e.g., DSS colitis, TNBS colitis, etc.) do not require T cells38,39. On the other hand, many IBD-associated cytokine loci regulate the development and function of intestinal Th cell subsets, particularly IL-17-secreting Th17 cells and Foxp3-expressing Tregs33. In addition, other chronic mouse models of IBD, including T cell transfer-induced colitis40,41, Il10−/− colitis9, and SAMP1/YitFc42 ileitis are Th cell-dependent. In the clinic, T cell-directed therapies have shown mixed results in IBD. For example, the anti-α4/β7 integrin antibody, vedolizumab, which acts to restrict T cell homing to intestinal tissues, is now approved for use in IBD43. Further, the sphingosine 1 phosphate (S1P) receptor antagonist, RPC-1063, which sequesters activated T cells in lymph nodes, met efficacy endpoints in a recent phase II study in UC44. In contrast, several antibodies directed against T cell effector cytokines (e.g., anti-IFNγ, anti-IL-17A, anti-IL13), as well as recombinant IL-10, failed in IBD trials (discussed below). Here again, the precise role of Th cells in IBDs likely varies depending on individual genetic contexts.
Th cells develop from bone marrow precursor cells, are educated in the thymus where they acquire a unique T cell antigen receptor (TCR), and enter peripheral circulation in blood and lymphatic vessels. Naïve Th cells become activated in peripheral lymphoid organs (e.g., spleen, peripheral lymph nodes) upon recognition of cognate antigen presented by major histocompatibility complex class II (MHC-II)-expressing dendritic cells (DCs). Th cell activation induces clonal expansion (i.e., proliferation), as well as differentiation into phenotypically and functionally distinct effector and regulatory subsets depending on cytokines present in the local microenvironment45. Th cells subsets include IFNγ-expressing Th1 cells, IL-4-secreting Th2 cells, Th17 cells that express IL-17A, IL-17F, and IL-22, immunoregulatory “induced” Tregs (iTregs) that express the transcription factor Foxp3, Th22 cells that express IL-22 but not IL-17A/F, IL-9-producing Th9 cells, and T follicular helper (Tfh) cells that express IL-21 and regulate germinal center reactions45. All of these Th cell subsets are characterized by, and function through, polarized expression of key effector or regulatory cytokines (see below). These subsets also differentially express pro-inflammatory chemokine receptors and tissue-homing integrins, which can drive preferential homing of Th cell subsets to distinct tissues. A robust, yet tightly regulated Th cell response is required for host immune protection against microbial pathogens, immune tolerance to host tissues and commensal symbionts, resolution of inflammation, and development of durable immune memory.
CD and UC are associated with distinct Th cell effector cytokine expression. Specifically, small bowel inflammation in CD is generally associated with increased IFNγ and IL-17A expression (indicative of Th1 and Th17 cells respectively), whereas Th2 cytokines (e.g., IL-4, IL-5, IL-13) predominate in UC46. Transcription factors (e.g., STAT4, TBX21/T-bet) and cytokine receptors (e.g., IL-12Rβ2) that promote Th1 cell development are also highly expressed in inflamed intestinal lamina propria from CD patients47. The only two mouse models of CD-like ileitis – SAMP1/YitFc and TnfΔARE – also show a biased Th1-type lymphocytic infiltrate in inflamed mucosa42,48. Intriguingly, recent studies in mice suggest that the Th1/Th17 bias in CD may be the result of a unique microenvironment in the ileum – even in the absence of inflammation – that favors Th1 and Th17 cell development/effector function49,50. Functionally, the pathogenic potential of Th1 cells in IBDs has been demonstrated in a variety of experimental mouse model settings. First, Th cells from Ifng−/−51, Tbx21−/− (T-bet-deficient)52, and Stat4−/−53mice all fail to transfer intestinal inflammation in SCID recipients. Second, fatal colitis in Il10−/− mice is significantly attenuated in the absence of another Th1-inducing cytokine receptor, IL-27R54. Finally, Th17 cells suppress experimental colitis, at least partly, via suppression of Th1 cell development and function55. Collectively, these studies paved the way for development of an anti-IFNγ antibody (e.g., Fontolizumab) for IBD. Surprisingly, Fontolizumab failed to show efficacy in a phase II trial of CD patients, though a significant reduction in serum C-reactive protein (CRP) was observed in Fontolizumab -treated patients56,57. These data suggest that whereas Th1 are involved in CD pathogenesis, targeting a single cytokine secreted by these cells isn’t therapeutically beneficial. Th1 cells are also major producers of TNFα, and thus at least part of the efficacy of the anti-TNFs in CD may be due to regulation of Th1 cells58. Indeed, the notion that therapeutic modalities targeting upstream cytokine signals (e.g., anti-IL-12) or transcription factors (e.g., T-bet) that control an entire effector Th cell transcriptional program may be beneficial over targeting a single effector Th cell cytokine is now a reoccurring theme in immunotherapy.
Th2 cells are classically considered key effectors of airway inflammation in asthma59. However, as noted above, intestinal immune cells from inflamed lamina propria of UC patients also express high levels of Th2-associated cytokines – particularly IL-5 and IL-13 – and the Th2 master transcription factor, GATA-346,60. Intriguingly, one study found that Th2 cells in UC display uncharacteristically low expression of IL-446, perhaps indicating a distinct Th2 subset is involved in UC, or that Th2 cells in UC have an altered state of activation. The role of both Th2 cells generally, and individual Th2 cytokines specifically, in UC remains controversial.
Early studies suggested that IL-4 expression is reduced in intestinal biopsies from UC patients61,62. Further, IL-4 (like IL-10) was shown to suppress pro-inflammatory cytokine expression (e.g., TNFα, IL-6, IL-1β) by UC patient-derived monocytes following stimulation with LPS63. More recent studies seem to agree that IL-4, along with other Th2 cytokines, are in fact increased in mucosal biopsies from UC patients46,64. These new findings coincided with the development of a new chemically-induced acute model of UC featuring rectal installation of the haptenating agent, oxazalone65. Oxazalone-induced colitis presents as an acute, self-resolving form of experimental UC restricted to the distal colon; IL-4 expression by TCRα/β+ Th2 cells is markedly increased in oxazalone-driven colitis, and intestinal pathology in this model is attenuated by administration of neutralizing anti-IL-4 antibodies65. IL-4 produced by oligoclonal TCRβ/β+ Th2 cells is also pathogenic in spontaneous colitis in Tcra−/− mice66. Normally, Th2 cells develop in an IL-4-dependent manner that requires activation of Stat645. Interestingly however, Th2-mediated colitis in Tcra−/− mice occurs even in the absence of Stat6, even though colitis in Tcra−/−Stat6−/− mice is still effectively treated with anti-IL-4 antibodies67. In contrast, Stat6-deficiency is protective in oxazalone-induced colitis68. Th2 cell development also requires the transcription factor Gata-3, and it was recently reported that Gata-3-transgenic (Gata3-Tg) mice develop more severe colitis following DSS treatment compared with Th1-biased (T-bet-Tg) or Th17-biased (RORγt-Tg) mice69.
IL-4 is important for antibody class-switch recombination in B cells, promoting secretion of immunoglobulin (Ig)-E70. Indeed, elevated serum IgE levels have been reported in UC patients71,72, and in oxazalone-induced murine colitis73. Accordingly, B cells contribute to oxazalone-induced colitisin a manner that requires the IL-4 receptor alpha chain (IL-4Rα)73. IL-4 also regulates expression of IL-13 by Th2 cells73, and several studies point out pathogenic functions of IL-13 in the context of colitis. Unlike IL-4, major targets of IL-13 in the intestine are intestinal epithelial cells and fibroblasts74. IL-13 synergizes with TNFα to perturb epithelial barrier function by regulating the expression of genes involved in tight junction formation60. Further, IL-13 receptor (IL-13Rα2) signaling in epithelial cells promotes TGFβ expression, which in turn drives colitis-associated fibrosis75.
IL-5 produced by Th2 cells is a major driver of eosinophil differentiation (in bone marrow) and tissue infiltration76. Whereas the function of IL-5 and eosinophils in UC has long been enigmatic, a recent study by Powrie and colleagues demonstrate that IL-5 and GM-CSF synergize in a mouse model of IL-23-dependent colitis to promote eosinophil activation and eosinophil-dependent tissue damage77. These data are particularly interesting because they demonstrate how cytokines secreted by multiple Th cell subsets (i.e., Th2/IL-5, Th17/GM-CSF) can act in concert to promote mucosal inflammation. Additional innate cytokines, namely IL-25 and IL-33, have been shown to exacerbate chemically-induced murine models of colitis by promoting Th2 differentiation and cytokine expression78,79. Given this preponderance of pre-clinical data implicating Th2 cytokines in UC pathogenesis, strategies have been developed to block Th2 cytokines in UC patients. Disappointingly, Tralokinumab – an anti-IL-13 antibody – failed to meet efficacy endpoints in a UC trial34,80. As with the failure of anti-IFNγ in CD, this result seems to suggest that blocking a single Th2 cytokine is not sufficient to induce a therapeutic response. Anti-IL-4/13 bi-specific antibodies81 are in development for asthma, and show therapeutic effects in Th2-associated colitis82. Thus, it will be of interest to determine if this approach, or perhaps other combinatorial approaches (for example also targeting IL-5 and/or GM-CSF), may be more broadly therapeutic in UC.
Between the 1980’s and the early 2000’s, immunopathogenesis of IBD was viewed solely through the lens of Th1 and Th2 cells. This bimodal view of the immune system has changed drastically in the last 10 years, spurred in large part by the discovery and molecular characterization of IL-17-secreting Th17 cells83. Since 2005, Th17 cells have dominated the discussion of what pathogenic T cells are, both in IBD and in other immune-mediated inflammatory diseases. Th17 cells differentiate from naïve precursors in an IL-6/Stat3-dependent manner that is modulated by auxiliary cytokines (e.g., TGFβ1, TGFβ3, IL-1β, TNFα, IL-21, IL-23; see below) and that requires the orphan nuclear receptor RORγt (gene symbol RORC2)84,85. Effector/memory Th17 cells express a litany of pro-inflammatory cytokines, including IL-17A, IL-17F, and IL-2284. Importantly, unlike Th1-derived IFNγ and Th2-derived IL-4 and IL-5, Th17 cytokines act primarily on non-hematopoietic cells (e.g., epithelial cells, endothelial cells) and incite pro-inflammatory gene expression. Th17 cytokines also enforce epithelial barriers and have important roles in protective immune responses to certain bacterial species (e.g., Mycobacterium) and pathogenic fungi (e.g., Candida albicans)84.
Shortly after their discovery, it became apparent that Th17 cells are important regulators of IBD pathogenesis. First, Th17 cytokines are elevated in inflamed mucosal biopsies from UC and CD patients86,87. Second, antibody-mediated blockade of the Th17-inducing cytokine IL-688, or genetic ablation of Th17-promoting transcription factors (i.e., Stat3 or RORγt/Rorc)89,90, reduces experimental colitis. Third, a large number of polymorphisms in genes involved in Th17 regulation (e.g., IL6ST, JAK2, STAT3, RORC, IL23R, CCR6) are associated with human IBDs33. For these reasons, it was unexpected that a fully humanized anti-IL-17A antibody, Secukinumab, failed to show efficacy in a recent CD trial91. By contrast, Secukinumab induces dramatic therapeutic responses in another chronic inflammatory disease linked to excessive Th17 function, psoriasis92,93. Thus, whereas IL-17A expression can, in principle, promote chronic inflammatory disease, IL-17A specifically and Th17 cells more broadly appear to have tissue-specific functions. As we will see below, Secukinumab’s failure in CD does not necessarily mean that Th17 cells are not important in IBD pathogenesis, but rather suggests the role of Th17 cells in IBD is more complex than first thought.
Several points need to be considered when interpreting the seemingly paradoxical results of the psoriasis and CD Secukinumab trials. First, Th17 cells are not the only immune cell lineage that expresses IL-17A; other lymphocytes (γΔ+ T cells) and lymphoid-like cells (type 3 innate lymphoid cells [ILC3]) are present at mucosal barriers (e.g., gut, lung, skin), and express IL-17A, IL-17F, and IL-22 in response to innate cytokines, such as IL-1β and IL-2394,95. Thus, differential accumulation and function of Th17 cells, γΔ+ T cells, and ILC3s in the gut vs. skin needs to be considered and better understood. Second, functional responses of intestinal epithelial cells and skin keratinocytes to IL-17A signaling need to be considered. Kruger and colleagues have demonstrated that IL-17A synergizes with TNFα to drive pro-inflammatory transcriptional responses in ex vivo-cultured keratinocytes that mimics the transcriptomic profile of inflamed psoriasis skin lesions96. By contrast, IL-17A signaling in intestinal epithelial cells promotes expression of genes involved in tight junction formation97,98. In this way, the “pro-inflammatory” action of IL-17A on the intestinal epithelium appears important to maintain a symbiotic barrier between the microbiota and mucosal immune cells. Finally, it is now clear that Th17 cells express many other cytokines besides IL-17A, and that there are functionally distinct – pathogenic and non-pathogenic – subsets of Th17 cells (see below). In other words, it would seem that it is not IL-17A per se that is pathogenic in IBD, but rather the cellular source.
IL-17A-secreting Th17 cells can be induced in vitro using a variety of cytokines. Whereas Th17 differentiation was first documented in the presence of TGFβ1 plus IL-699, more recent studies demonstrate that these Th17 cells are “non-pathogenic” in vivo; TGFβ1/IL-6-directed Th17 cells fail to induce severe pathology in a mouse model of multiple sclerosis (MS), termed experimental autoimmune encephalomyelitis (EAE). By contrast, TGFβ3 or IL-1β can substitute for TGFβ1 and act in concert with IL-6 (and IL-23) to promote Th17 differentiation; these Th17 cells induce severe EAE in vivo100,101. Flavell and colleagues have shown that Th17 cells in the uninflamed intestine have a transcriptomic signature similar to TGFβ1/IL-6-induced “non-pathogenic” Th17 cells102. There are 2 major distinctions between pathogenic and non-pathogenic Th17 cells. First, pathogenic Th17 cells are unique in that they produce both IL-17A and IFNγ100,101; IL-17A/IFNγ double-producing T cells is a common feature of inflamed intestinal tissue from IBD patients, inflamed joints of rheumatoid arthritis (RA) patients, and psoriatic skin lesions103,104. In contrast, non-pathogenic Th17 cells lack IFNγ, and instead express IL-17A together with IL-10. Second, pathogenic Th17 cells display a unique transcriptional profile compared with non-pathogenic Th17 cells, highlighted by increased expression of the IL-23 receptor (IL23R)100,101. IL23R, together with NOD2, shows the strongest genetic association with human CD33, and pathogenic Th17 cell function in mouse models of IBD, MS, and RA requires IL-23R signaling101,105,106. Other pathogenic Th17-signature genes include Th1-associated transcription factors (T-bet [Tbx21], Stat4), the granulopoietic cytokine GM-CSF (Csf2), granzyme B (Gzmb), and the IL-7 receptor alpha chain (Il7r)100,101. Importantly, most of these pathogenic Th17-signature genes are also genetically associated with human IBD33.
Whereas the notion of distinct Th17 subsets is significant in and of itself, the translational application of this concept is limited without phenotypic biomarkers of corresponding Th17 subsets in humans. The natural killer (NK) cell-associated receptor CD161 (gene symbol KLRB1) was previously found to be expressed on a subset of human CCR6+ Th17 cells; to enrich for IL-17A/IFNγ double-producing T cells; and to be increased in inflamed IBD patient biopsies107. However, CD161-expressing Treg and Th2 cells have also been reported108,109, demonstrating a lack of specificity of CD161 expression for pathogenic Th17 cells. By contrast, expression of the multidrug/xenobiotic transporter, MDR1 (a.k.a., ABCB1, P-glycoprotein) specifically discriminates pro-inflammatory human Th17 cells that are akin to pathogenic Th17 cells in mice110. MDR1 is a plasma membrane-associated drug efflux pump best known for its expression and functions in chemoresistant tumor cells and epithelial cells of the blood-brain-barrier111,112. Within the CD4+ Th cell compartment, MDR1 is selectively expressed by a subset of CCR6+CXCR3+ human memory cells that display mixed Th17/Th1 functions. Notably, MDR1+ Th17 cells display elevated expression of pathogenic mouse Th17-signature genes, and these cells are enriched in inflamed intestinal biopsies from CD patients110. Although there are few, if any, useful anti-MDR1 antibodies, MDR1 expression/function is readily detectable at the single-cell level (by flow cytometry), by efflux of fluorescent small molecules, such as rhodamine 123 (Rh123)113. With these advances, it is now possible to monitor pathogenic Th17 cells in human IBD patients, and perhaps to devise novel therapeutic strategies that target these cells. It is not yet clear if – or how – MDR1 regulates the development or function of pathogenic Th17 cells, though it is notable that: (i) polymorphisms in the gene encoding human MDR1 (ABCB1) have been associated with IBD114; (ii) mice lacking Mdr1a (Abcb1a) develop spontaneous colitis115; and (iii) MDR1 is capable of effluxing many corticosteroid used to treat active flares in IBD116. Interestingly, MDR1-expressing Th17 cells are resistant to the anti-inflammatory actions of several corticosteroids110, which may contribute to steroid refractory inflammation in a subset of IBD patients.
Pathogenic Th17 cells promote tissue inflammation in an IL-23-dependent manner101,105,106. Certainly, regulation of pathogenic Th17 cells could account for at least some of the therapeutic efficacy of Ustekinumab (anti-IL-12/23 p40) in IBD. Another humanized antibody specifically targeting IL-23 p19 (Tildrakizumab) is now in development at Merck. Tildrakizumab induced durable remission in a number of psoriasis patients during a recent phase IIb trial, and was well tolerated117,118. In mice, IL-23 (p19), but not IL-12 (p35), is required for IL10−/− enterocolitis119, as well as T cell transfer-induced colitis105. Thus, there is growing evidence to suggest that Tildrakizumab could soon join the growing list of FDA-approved IBD therapies directed against cytokine networks.
One final cytokine of interest as it relates to pathogenic Th17 cells and mucosal inflammation in IBD is GM-CSF. As noted above, GM-CSF is highly expressed in pathogenic, but not non-pathogenic, Th17 cells100,101. In mice, Th17-driven EAE (the model of MS) requires GM-CSF, and IL-23 induces GM-CSF expression in Th17 cells120. As noted above, Powrie and colleagues reported that IL-23-dependent colitis in mice also involves GM-CSF. Here, GM-CSF produced by Th17 cells acts in concert with Th2-derived IL-5 to promote eosinophil activation in the gut77. Indeed, studies in human IBD patients have noted increased numbers of activated eosinophils in feces, as well as increased levels of eosinophil cationic protein, a marker of eosinophil activation121. As emphasized throughout, these emerging concepts call for combinatorial approaches to target multiple cytokines in order to maximize benefit to patients.
T regulatory cells (Tregs) are critical suppressors of autoimmunity and enteropathies in mice and humans. Tregs come in two flavors, “natural” Tregs (nTregs) that develop in the thymus and “induced” Tregs (iTregs), which differentiate from mature naive T cells in parallel with conventional effector subsets (i.e., Th1, Th2, Th17) in peripheral lymphoid organs122. Both nTregs and iTregs suppress the bystander activation of T cells – and thus contribute to immune tolerance – in a manner that requires FOXP3, a forkhead box transcription factor that is mutated in the autoimmune Scurfy mouse and in human immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) patients122-124. Both Scurfy mice and IPEX patients display enteropathies characterized by diarrhea and mucosal inflammation. Further, purified (CD4+CD25hi) Tregs prevent naïve CD4+ T cell transfer-induced colitis in SCID recipients125.
Whereas nTregs display a TCR repertoire biased for reactivity against auto-antigens126, the majority of Foxp3+ Tregs in the intestine are iTregs that derive from naïve T cell precursors and that develop in response to symbiotic interactions with commensal bacteria127. First, TGFβ induces peripheral iTreg differentiation, but not thymic development of nTregs128, and Tgfb1−/− mice develop lethal autoimmunity and enteropathy129. TGFβ is secreted in a biologically-inactive complex together with latency-associated peptide (LAP), and is activated by the integrins αvβ6 and αvβ8 expressed on epithelial cells and dendritic cells (DCs), respectively130. Interestingly, ablation of β8 integrin (Itgb8) on DCs reduces intestinal Treg numbers and triggers spontaneous colitis akin to Tgfb1−/− mice131. Intracellular TGFβ signaling is complex, and involves several pathways. Whereas TGFβ activates MAPK and PI-3K/AKT/mTOR pathways, canonical TGFβ signaling proceeds via a family of transcription factors named SMADs (similiarity to mothers against decapentaplegic)132. Here, the ligand-bound TGFβ receptor complex leads to phosphorylation-dependent activation of receptor-activated SMADs, or r-SAMDs (r-SMAD2 and r-SMAD3 in T cells), which then form a heterotrimeric complex with common SMAD4 (co-SMAD4). This complex translocates from the cytoplasm to the nucleus and activates gene expression by binding to cognate DNA elements. Intriguingly, some of the genes induced by TGFβ are negative feedback regulators, termed inhibitory SMADs (i-SMADs; including i-SMAD6 and i-SMAD7). i-SMADs suppress chronic TGFβ signaling by competing with the r-SMADs for TGFβ receptor binding and promoting ubiquitin-mediated degradation of TGFβ receptor subunits132. In IBD patients, i-SMAD7 is overexpressed in Teff cells, rendering these cells insensitive to the immunosuppressive actions of TGFβ133,134. Transgenic overexpression of i-SMAD7 in mice blocks Treg-mediated suppression of experimental colitis135, whereas Smad7 antisense oligonucleotides reduce both TNBS- and oxazalone-induced coltiis136. Most importantly, a SMAD7 antisense oligonucleotide has shown promise in a Phase I clinical trial for CD137.
Metabolites derived from commensal bacteria also directly influence iTreg generation in the intestine. For example, short chain fatty acids (SCFAs), including butyrate and propionate, synergize with TGFβ to upregulate Foxp3 expression and Treg suppressive function138. Regulation of iTreg development and function by butyrate and propionate are due to their activity as histone deacetylase (HDAC) inhibitors. On one hand, butyrate and propionate suppress pro-inflammatory cytokine expression in DCs, enabling increased TGFβ-dependent Foxp3 induction. On the other hand, butyrate and propionate prevent deacetylation of Foxp3 itself, which enhances Treg suppressive activity138. In addition to these SCFAs, polysaccharide A (PSA), produced by the prominent human commensal, Bacteroides fragilis (B. fragilis), directly stimulates IL-10 production by iTregs in a toll-like receptor 2 (TLR2)-dependent manner and is able to both prevent and therapeutically treat TNBS-induced colitis in mice139,140.
Whereas these data clearly implicate Tregs in control of mucosal inflammation, whether or not perturbed Treg function is a common feature of IBD remains controversial. Some studies have reported reduced Treg numbers in inflamed mucosal biopsies from IBD patients141; others have found increased FOXP3 expression142,143. In addition, several studies have suggested that the inflammatory cytokine milieu present in inflamed mucosa can act to limit Treg-mediated suppression of bystander effector cells144,145. Other reports suggest that impaired Treg function in IBD patients coincides with elevated IL-17A expression146,147. Although a unifying mechanism of Treg-mediated immune suppression is not known, several molecules have been implicated in the regulation of Treg-dependent intestinal homeostasis122. Chief among these is IL-10; Treg-specific ablation of Il10 is sufficient to induce spontaneous colitis148. Polymorphisms in genes encoding for subunits of the IL-10 receptor (IL10RA, IL10RB) are also associated with human IBD, particularly very early onset colitis149. Surprisingly however, recombinant human IL-10 therapy is not effective as a stand-alone therapy in CD150.
Tregs also express IL-35, a relatively recent addition to the cytokine family. Like most cytokines, IL-35 is a heterodimeric protein, consisting of EBI3 (encodes for IL-27β) and IL12A (encodes for IL-12p35)151. In mice, Ebi3 is a direct transcriptional target of Foxp3, and IL-35 synergizes with TGFβ to upregulate Foxp3 expression and Treg suppressive function152. Importantly, Tregs lacking Ebi3 or Il12a fail to control T cell transfer-induced colitis152. Less is known about IL-35 in human IBD. One study indicated that human FOXP3+ Tregs do not constitutively express IL-35 akin to mouse Tregs153. However, another, more recent study reported increased expression of IL-35 by Tregs and CD20+ regulatory B cells in intestinal biopsies from active vs. inactive UC and CD patients154, perhaps suggesting that increased IL-35 expression in actively inflamed intestinal tissue is a homeostatic response that precedes resolution of inflammation.
Two additional epithelial-derived cytokines, namely IL-18 and the “alarmin” IL-33, have been recently suggested to enforce Treg-mediated control of colonic inflammation155,156. Notably, these cytokines are unique from other Treg-related pathways that systemically boost Treg development and/or function (e.g., TGFβ, IL-10, IL-35, CTLA-4, etc), in that IL-18- or IL-33-driven Treg function appears to have tissue-specificity for the gut. Conceptually, the notion of targeting pro- or anti-inflammatory T cell subsets in discrete tissue microenvironments is preferable over systemic immune modulation; tissue-specific immune regulation could, in principle, get around potentially serious consequences of systemic immune suppression. Both IL-18 and IL-33 are members of the IL-1 family of cytokines, and have historically been considered pro-inflammatory cytokines; each are produced by intestinal epithelial cells in response to infection, inflammation, and tissue damage157. Whereas IL-18 and IL-33 were first reported to regulate Th1 or Th2 cytokine expression, respectively158,159, more recent data suggests they also have important (Treg-dependent) immunomodulatory functions. IL-18 is elevated in inflamed mucosal biopsies from IBD patients160. IL-18, like IL-1β, is synthesized as an inactive precursor polypeptide (pro-IL-18), which is activated by the inflammasome. Active IL-18 is secreted and binds to the IL-18 receptor in a manner that is enhanced by the IL-18 receptor accessory protein (IL-18RAP)157. Polymorphisms in the IL18R1-IL18RAP locus are associated with IBD33, and a recent study in mice shows that although IL-18R signaling is not required for development of Foxp3+ Tregs in the colon, IL-18 acts directly on colonic Tregs to upregulate key immune-regulatory molecules, including IL-10, and is important for Treg-mediated control of experimental colitis155.
Murine Tregs also display elevated expression of ST2 (encoded by Il1rl1) in the colon156. ST2 is a component of the IL-33 receptor (IL-33R), and Schiering et al. recently reported that ST2 expression in Tregs is critical for Treg-mediated control of experimental colitis in mice. IL-33 again synergizes with TGFβ signaling to foster Treg development, and ST2-deficient Tregs fail to accumulate in the inflamed colon, though there is no defect in ST2-deficient Treg persistence in other, systemic tissues (e.g., spleen)156. Importantly, IL-23 acts directly on Tregs in inflamed mucosa to reduce ST2 expression156, thus downregulating this potentially important tolerogenic mechanism.
These cytokine networks have emerged as key players in the development and function of Tregs that may be leveraged clinically to improve IBD therapy. In addition, the universe of CD4+ Th cell subsets continues to expand. Given their relatively recent discovery, far less is known about the role of these “emerging” Th cell subsets in IBD, though at least two of these merit brief discussion, namely Th9 and T follicular helper (Tfh) cells.
A recent study suggests that IL-9-secreting Th cells (Th9 cells) are important in UC. T cells expressing the transcription factor PU.1 (encoded by SPI1) and IL-9 are elevated in patients with ulcerative colitis161. PU.1 regulates gene expression in hematopoietic stem cells via interactions with other transcription factors, including GATA2 and IRF4162. IL-9 signaling stimulates immune cell proliferation and prevents apoptosis through activation of STAT1 and STAT3163. As in IBD patients, IL-9-producing mucosal Th9 cells are increased in oxazolone-induced colitis, whereas Il9−/− mice are protected from colitis following oxazolone treatment161. IL-9 also inhibits wound healing in the intestinal mucosa and impairs barrier function164. Treatment with an IL-9 neutralizing antibody suppresses intestinal inflammation in oxazolone-induced colitis and mice lacking the transcription factors PU.1 are safeguarded from colitis161. IL-9 directly regulates tissue recruitment and inflammatory function of mast cells. Whereas the Th9/mast cell network is now firmly implicated in allergic airway disease165, the function of mast cells in IBD is enigmatic. Nonetheless, these data suggest that Th9 cells could be a novel and attractive therapeutic target in UC. Indeed, a humanized antibody targeting IL-9, Enokizumab (MEDI-528), has been developed and tested (unsuccessfully) in asthma166; given its strong safety profile in humans, future investigational studies featuring Enokizumab in UC could provide important insight into the utility of targeting the Th9 pathway in IBD.
IL-21-secreting T follicular helper (Tfh) cells have also been implicated in IBD pathogenesis. Tfh cells differentiate in a complex and sequential fashion from naïve Th cell precursors, as instructed by a combination of IL-6, IL-21, and IL-12 cytokine signals, and ICOS-dependent costimulation167. Tfh cell development requires sequential activity of the transcription factors achaete-scute homologue 2 (Ascl2) and Bcl6168,169. Once differentiated, CXCR5+ Tfh cells migrate to germinal centers where they facilitate B cell selection and differentiation167. Polymorphisms in the IL21 gene are associated with IBD33. Further, increases in IL-21-expressing CD4+CXCR5+ Th cells have been reported in mucosal biopsies from both CD170 and UC171 patients. Tfh cells are also elevated in inflamed colons of mice treated with either DSS or TNBS, and Il21 ablation is protective in these models171,172. In addition, treatment with an IL-21R/Fc fusion protein reduces disease severity in DSS colitis172. Yet still, and as with Th9 cells, further studies are needed to clarify the putative role(s) of Tfh cells in intestinal homeostasis and inflammation, as well as their relevance in IBD patients.
The past decade has been witness to an explosion in our understanding of mucosal immunology and IBD pathogenesis. Thus, there is reason to expect that the next 10 years will see similarly impressive advances in IBD therapy. As highlighted throughout, translating our understanding of mucosal immunology to improved IBD therapies will require continued integration of functional and genomic data from IBD patients and human tissue samples, with data from experimental mouse models and clinical trials. In addition, the amount of data collected per experiment/clinical trial continues to increase thanks to exciting technological advances in high-content flow cytometry and next-generation sequencing. Finally, additional new chemical entities and biologics currently in pharmaceutical pipelines will provide physicians and scientists with new tools to address previously-inaccessible mechanisms of immune regulation in humans (Table 1). Ultimately, transformational new IBD therapies may not only come in the form of a new therapeutic compound or antibody, but also in better stratifying patients based on genetic and immune-profiling data, in order to tailor existing therapies (e.g., anti-TNFs) to the patients that stand to benefit most from them.
The authors acknowledge financial support from the NIH (NIAID; 1R21 AI119728-01) and Scripps Florida via the State of Florida.
The authors declare they have no conflicts of interest to disclose.