The recent discovery and characterization of T helper 17 cells (Th17) and their signature cytokines (IL-17) represents a hallmark in T-cell immunobiology by providing a new distinctive pathway for the communication between adaptive and innate immunity. From the six members of the IL-17 cytokine family presently known, at least two have evident proinflammatory qualities and are involved in several chronic inflammatory disorders, including inflammatory bowel disease (IBD). IL-17A and IL-17F are abundantly found in inflamed IBD mucosa, suggesting their pivotal role in IBD. However, the precise implication of IL-17 cytokine family members in IBD pathogenesis and the mechanisms regulating their secretion are incompletely understood. Importantly, recent findings suggest that beyond IL-17 production-Th17 cells may secret a plethora of other effector cytokines such as IL-21, IL-22, and IL-9- which is in part induced by its own IL-9 production. However, the use of anti-IL-17 therapeutic strategies in experimental models of chronic inflammation results in disease-ameliorating effects suggesting their potential use in IBD patients. In this review article we discuss the latest findings on the role of Th17 cells and IL-17 family members in IBD immunopathology, as well as research perspectives. (Inflamm Bowel Dis 2011;)
Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD), which are defined by an abnormal and exacerbated immune response against mucosal constituents and components of the luminal flora in individuals with genetic predisposition. Importantly, proinflammatory T cells and their secreted cytokines are the main effectors in induction and perpetuation of the intestinal inflammation.1–4 While treatment strategies aim at suppression and modulation of this aberrant immune response,5 the focus and exigency of basic mucosal immunological research is to contribute to a growing understanding of inflammatory and signaling pathways.
In this context, beyond the classical and largely accepted dichotomic view where CD is linked to a predominant T helper cell (Th1) immune response (e.g., interferon-gamma [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin [IL]-12) and in UC dominate Th2 cytokine effects (IL-5, IL-13),1, 6, 7 CD and UC may have common endstage pathways and cytokines, such as IL-6, IL-21, and IL-23.8, 9
In addition to the classical Th1/Th2 pathways, a distinctive pathway has been discovered, namely, IL-17 signaling, by identification of two novel CD4+ T-cell subsets: Th17 cells producing IL-17,10, 11 and Th1/Th17 cells producing both IL-17 and INF-γ.12, 13 Furthermore, very recently IL-9 was discussed to be an effector cytokine of Th17 cells and even postulated to be produced by a separate T-cell population, namely, Th9 cells.14, 15 In IBD, IL-17 as well as their source, mainly highly differentiated Th17 cells, are abundantly found in the inflamed intestinal mucosa.16, 17 Other chronic inflammatory and autoimmune disorders like rheumatoid arthritis,18 multiple sclerosis,19 systemic sclerosis,20 systemic lupus erythematosus,21 and asthma22 share similar features regarding the overexpression of IL-17 and the occurrence of Th17 cells.
Since the discovery of Th17 cells and the IL-17 cytokine family, there have been several review articles dealing with the role of Th17 cells and IL-17 in a general manner, or arguing the pivotal relevance of Th17 cells in IBD. The focus of the present review is to summarize the present knowledge regarding both the role of the pleiotropic IL-17 cytokine family members and Th17 cells within IBD immunopathology and to point out several possible lines of investigation in order to reveal and understand the complete functional implication of IL-17 in IBD.
Th17 CELLS AS PRIMARY SOURCE OF THE IL-17 CYTOKINE FAMILY MEMBERS
As mentioned above, the discovery and characterization of the new lineage of effector CD4+ T cells, Th1710 was a hallmark in immunology, which ultimately led to a revision of the Th1/Th2 paradigm in IBD. Th17 cells seem to have evolved as a defending strategy of the adaptive immune system highly specialized in protecting the host against pathogenic bacteria, fungi, and other microbes—plausibly filling the “immune gap” which is not well covered by the spectrum of Th1 and Th2 immunity.23 Primary studies showed differences between the development of Th17 in experimental models and humans, but recent studies demonstrated no major deviations.24, 25 Th17 cells originate from naïve CD4+ T cells in the presence of TGF-β and IL-6 (see Fig. 1).26–28 Th17 cell differentiation does not require IL-17. The amplification and stabilization of Th17 is provided by IL-21 and IL-23.27–29 At the same time, the retinoic orphan receptor gamma(t) (RORγt) has been identified as the master key regulator and transcription factor of Th17 cell differentiation,30, 31 in which RORγt is found to be expressed elevatedly in lamina propria T cells of CD patients.32 Th17 cell development is significantly influenced also by the intestinal microbial flora33 and the same microflora is necessary for IL-17A-producing CD4+T cells in lamina propria of the colon.34
Th17 cells are characterized by RORγt35 and IL-23R-expression.26 Recently, CD161 was described as a potential surface marker of disease driving Th17 cells in IBD.36 Moreover, a recent publication postulates that Th17 subsets might be distinguished by their specific capacity, namely, “pathogenic” or “nonpathogenic.” Pathogenic Th17 cells are characterized by the production of IFN-γ and also by the expression of several surface markers such as IL-18 receptor 1 and CXCR3.37
However, one of the principal features of Th17 cells is the production of IL-17 cytokines.10, 26, 29, 38 Besides being produced by Th17 cells, the members of the IL-17 cytokine family are secreted by various other cells like γσ-T cells24, 25 NK T cells, NK cells, cytotoxic CD8+ cells,39, 40 neutrophils,41 eosinophils, fibroblasts, macrophages,42 monocytes,30 etc. The IL-17 cytokine family includes six members so far: IL-17A (the founding member, called IL-17), IL-17-B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F.23, 43–45 Initially, IL-17 (IL-17A, the founding member) was isolated from a murine CTL hybridoma in an attempt to identify CTL-associated transcripts, and was called CTLA-8.46 Within the IL-17 family, IL-17E (IL-25) is not produced by Th17 cells, but by Th2 cells,47 and is probably involved, besides induction of Th2 cytokines, and chemokines, especially in the allergic immune response. So far, the crystal structure of only one member of the IL-17 family has been completely defined: for IL-17F, classifying it in the so-called cysteine knot protein family.48 IL-17 family members have no sequence homology with other presently known mammalian proteins, and therefore it was considered a distinctive cytokine family, probably one of the first cytokine lines, since it was identified in an ancient jawless fish.49 Within this distinctive cytokine family, IL-17A and IL-17 F are 50% identical regarding amino acid structure, while IL-17B, IL-17C, and IL-17D have lower homology. In humans, IL-17E (IL-25) has the lowest homology, ≈16% from the primary protein structure.49
CELLULAR TARGETS AND FUNCTIONAL ROLE OF THE IL-17 CYTOKINE FAMILY MEMBERS
IL-17A and IL-17F are the most intensely studied members of the cytokine family. Both IL-17A and IL-17F are able to induce the expression of several proinflammatory cytokines and chemokines in diverse cells, because of the widespread expression of the corresponding receptor IL-17RA.50
The principal proinflammatory effects of IL-17A and IL-17F are mediated by the induction of TNF-α, IL-1β, chemokines (CXCL8, CXCL1, CXCL10), GM-CSF, G-CSF, IL-6, and metalloproteases.38, 51–54 Several studies demonstrated evident proinflammatory effects on a wide range of cellular targets like epithelium,55 endothelium, osteoblasts, monocytes/macrophages,51–53 and fibroblasts.23, 50 Importantly, the functional feature of IL-17A and IL-17F is especially the recruitment, activation, and migration of neutrophils.43, 56 This aptitude provides the explanation for this new coordination and signaling pathway over both adaptive and innate immunity.57 While IL-17A and F may recruit a neutrophil immune response, IL-17E (IL-25) recruits eosinophils and basophils. This happens by the induction of the expression of RANTES (CCL5) and Eotaxin 1 (CCL1), which is also important for Th2-cytokine expression, IL-5, IL-13, and probably IL-4 expression.47, 58 Furthermore, IL-17A activates NF-κB and MAPK pathways.55, 59, 60
Additionally, it has been proven that mice with IL-17 deficiency have decreased antibody production and antigen-specific T-cell activation.61, 62 Several other proinflammatory cytokines amplify some of the effects of IL-17 A and F, especially TNF-α and IL-1β. At the same time IL-17B and C are important activators of TNF-α and IL-1β expression.50 In addition, IL-17A contributes to the spontaneous formation of germinal centers of lymph follicles, driving autoimmune response before they increase production of pathogenic autoantibodies in autoimmune BXD2 mice expressing more IL-17. Blocking IL-17 signaling disrupts CD4+ T-cell and B-cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced germinal center B-cell development and humoral responses.63
OTHER EFFECTOR CYTOKINES OF Th17 CELLS (IL-21, IL-22, IL-9)
IL-21 is a cytokine from the IL-2 cytokine family, produced by activated T cells and NKT cells but not by antigen-presenting cells. Among the T cells, the maximum amount of IL-21 is produced by Th17 cells,64 in comparison with Th1 and Th2. IL-6 is a potent inducer of IL-2165 and the IL-21 expression depends on STAT3 but not on RORγt. Regarding its function, IL-21 regulates the CD4+ T-cell differentiation into Th17 cells. By amplifying the Th17 cells response, IL-21 acts as a positive autoamplification loop,64, 66, 67 similar to IFNγ in Th1 and IL-4 in Th2 cells. Interestingly, high IL-21 expression levels were described in CD,68 predominating in lamina propria lymphocytes.69 The important functional role of IL-21 in gut inflammation is also confirmed by the fact that the neutralization of IL-21 in cultured lamina propria mononuclear cells previously extracted from CD patients reduces the STAT3 and T-bet expression resulting in an inhibition of IFNγ.68 Thereby, IL-21 may be proinflammatory in a double-track fashion, both on Th17-mediated inflammation and by perpetuating Th1 mucosal immune response in IBD.68
IL-22 belongs to the IL-10 cytokine family and is produced by differentiated Th17 cells (apparently) in response to IL-23,70 but it also requires the triggering effect of IL-23R and expression in its target cells. TGF-β represses IL-22, and also IL-23R.71 Enhanced IL-22 expression was described in the serum and intestine of active CD patients,72–74 and also in UC but with a clear predomination of the expression in active CD.74 High IL-22R levels were also detected in intestinal epithelial cells.72 IL-22 is likely to have rather ambivalent functions, meaning both pro- and antiinflammatory, as follows: IL-22R activation drives STAT3 and the elevated transcription of IL-8 and TNF-α.72 Further proinflammatory cytokines and chemokines were upregulated over NF-κB activation in IL-22-stimulated colonic myofibroblasts.74 On the other hand, IL-22 also has an intestinal barrier protective role, by inducing the expression of protective defensins,72 and high levels of epithelial protective antimicrobial peptides.75, 76
Another interesting aspect of Th17 immunity is the production of IL-9, as coexpressed cytokine during Th17 differentiation in vivo.77, 78 Human Th17 cells have also been shown to produce IL-9 in vitro.15 In memory T cells, the classical Th17-promoting cytokine IL-1β and IL-21 enhance also IL-17 and the production of IL-9. The exact role of IL-9 in IBD is unclear yet. IL-9 may have rather ambivalent functions mediating immunosuppression, but at the same time promoting the (proinflammatory) expansion of mast cell population, as principal target cells of IL-9.
FUNCTIONAL RELEVANCE OF Th17 CELLS AND IL-17 IN INTESTINAL INFLAMMATION
In IBD, IL-17 as well as their source, mainly highly differentiated Th17 cells, are abundantly found in the inflamed intestinal mucosa.16, 17
Recently, the IL23R gene has been identified as an IBD susceptibility gene that regulates Th17 cytokine expression.79 IL-17A is produced also in the healthy gut,76 but high levels of IL-17A mRNA expression was found in inflamed IBD mucosa16 and elevated IL-17F mRNA in active CD.79 It has also been reported an upregulation of IL-17 in active CD and UC.80 Additional data demonstrated higher IL-17 production in inflamed IBD mucosa (in organ culture biopsies) compared to uninflamed IBD mucosa and controls.17 The principal proinflammatory effects of IL-17A and IL-17F are mediated by the induction of TNF-α, IL-1β, chemokines (CXCL8, CXCL1, CXCL10), GM-CSF, G-CSF, IL-6, and metalloproteases.38, 51–54 IL-17A and F have a highly pathogenic role also in experimental models of IBD, as in murine colitis (e.g., trinitrobenzenesulfonic acid [TNBS]-induced). Further, IL-17R knockout mice were significantly protected against TNBS-induced weight loss, colonic inflammation, and local macrophage inflammatory protein-2 induction.81 Signaling via IL-17R is also crucial in acute TNBS colitis.82 As indirect evidence, Th17 cells—as the main IL-17 producers—are at least as efficient and powerful in transferring colitis into RAG knockout mice as Th1 cells.83 Furthermore, IL-17F aggravates dextran sodium sulfate (DSS)-induced colitis while IL-17F-knockout mice are relatively protected.84
Focusing on the transcriptional regulation of IL-17 responses, RORγt has been identified as a key master regulator of Th17 cells. RORγt is most likely a necessary transcription factor which is sufficient for Th17 development and differentiation.85, 86 Furthermore, a crucial role of RORγ-expressing Th17 cells in chronic intestinal inflammation was described, by the fact that RORγ controls IL-17A and IL-17F production, and therefore exerts a highly pathogenic role in gut inflammation.81 As a confirming fact, mRNA expression of the human ortholog of RORγt, RORC87 was found overexpressed in lamina propria CD4+ T cells both in UC and CD patients.88 It has also been shown that interferon regulatory factor (IRF) 4 selectively regulates cytokine gene expression in chronic intestinal inflammation, including IL-17A.89, 90 Recent data show that IRF4 is involved in experimental autoimmune encephalomyelitis and that lack of IRF4 protects mice from autoimmune encephalomyelitis.91 Recent data strongly suggest that IRF4 directly activates IL-17A through direct promoter binding (Mudter et al, Inflamm Bowel Dis, in press).
Taken together, the main implication of IL-17A and IL-17F in intestinal inflammation consists of the perpetuation of the inflammation by a supplementary enhancement of proinflammatory cytokines and chemokines. On the other hand, it seems that there is a complex and highly active interplay between Th17 cells and intestinal epithelial cells (IEC) which leads to a perpetuation of intestinal inflammation, since IEC express not only IL-17R but also receptors for IL-22 and IL-2632, 55 (Fig. 2). Beyond enhancing the inflammatory response, IL-17A also mediates protective effects on the gut epithelial barrier by modulating tight junctions via claudins92 and upregulating the expression of antimicrobial peptides.931
After stimulation by Th17 cytokines, IEC release a wide range of proinflammatory cytokines and chemokine, e.g., CXCL8 for neuthrophil chemotaxis and CCL20 for attracting Th17 cells and dendritic cells, further amplifying the gut inflammation (Fig. 2). Recent data show also a protective function for IL-17A in T-cell-mediated intestinal inflammation.94 In this sense, there are also controversial or discrepant data. Awasthi and Kuchroo95 showed that IL-17A directly inhibits Th1 cells and thereby suppresses development of intestinal inflammation. Ogawa et al96 reported that anti-IL-17A monoclonal antibody treatment aggravates DSS-induced colitis.
CONCLUSIONS AND PERSPECTIVES
IL-17A and IL-17F are two proinflammatory cytokines within the IL-17 family that are produced also by Th17 and Th1/Th17 cells, and both of them are pathogenic in IBD (especially in CD). Both cytokines directly contribute to a perpetuation of intestinal inflammation through complex mechanisms, including the proinflammatory effects on IEC/epithelial barrier and other effectors (neutrophils, macrophages, lymphocytes, and dendritic cells). Clinical trials focusing on the therapeutic effects of anti-IL-17 antibody (AIN457) in CD are currently under way.97
Future studies will have to clarify also the role of other less-studied IL-17 cytokine members in IBD, as well the immune architecture of the mutual positive or feedback stimulation in vivo, in order to reveal soft spots for new blocking agents/antibodies. Another therapeutic approach in IBD might be the intracellular inhibition of RORγ and IRF4, because IRF4 seems to have a major role in controlling IL-17-dependent experimental colitis by a direct binding to IL-17 promoter and induction of RORγt levels and IL-17 gene expression.91, 98
More detailed studies will have to clear up the discrepancy of some experimental data regarding the role of IL-17A and F in experimental colitis models.
In the paradox context where an immunosuppressive cytokine (TGF-β) is needed for the differentiation of Th17 cells with potent proinflammatory effects, the most difficult challenge will be the identification of the factor(s) like genes, pathogens, and/or intestinal allergens that trigger at the same time IL-21 production and also TGF-β secretion, both vital for Th17 differentiation.
In the same way, the shifting and plasticity between Th17 and regulatory T cells in the intestine has to be addressed in future projects. The scope of these studies would be to identify the key regulator(s) for “repairing” an initiated proinflammatory Th17 response by converting Th17 cells into regulatory T cells, including the reestablishment of immune tolerance and in the end leading to an efficient and long-term inhibition of intestinal inflammation.