In this issue of Gut,1 Fina et al demonstrate that interleukin 21 (IL21) expression is increased in the intestinal mucosa of patients with active coeliac disease (CD) but not that of treated patients (see page 887). The authors show that IL21 is produced by CD4+ lamina propria T cells, a finding consistent with previous work indicating that this cytokine is exclusively produced by CD4+ T cells. Using a neutralising anti-IL21 antibody, they observed that blocking IL21 activity reduced the increase in interferon γ mRNA induced by gliadin digests in intestinal organ cultures from treated CD patients. Since the anti-IL21 antibody also reduced the induction of mRNA for T-bet, the transcription factor that controls the differentiation of interferon γ-producing CD4+ T lymphocytes (so-called T helper cell type 1 or Th1 lymphocytes), the authors conclude that IL21 is an important contributor to the mucosal Th1 proinflammatory response.1 The mucosal Th1 response in CD is generally ascribed to lamina propria CD4+ T cells that recognise gluten peptides presented by the human leucocyte antigen (HLA)-DQ2/8 molecules at the surface of mucosal dendritic cells. This response accounts for the interplay between the major genetic risk factor, the MHC (major histocompatibility complex) class II genes encoding the HLA-DQ2/8 molecules, and the triggering environmental factor gluten, and is considered instrumental in the pathogenesis of CD enteropathy.2

These results are of particular interest following the first genome-wide association study recently carried out in CD by van Heel et al.3 The latter study has indeed provided convincing evidence that the chromosomal 4q27 region harbouring the genes encoding the IL2 and IL21 cytokines might be involved in CD. In this study based on the use of single-nucleotide polymorphisms (SNPs), several variants were highly significant when analysed in three distinct populations of UK, Dutch and Irish case–control samples. The strongest association with CD was found approximately 24 kb 5′ of the IL21 gene and was highly significant, with a p value of 1.3×10⁻¹⁴ and an odds ratio of 0.63. This study extends the already large list of potential candidate chromosomal regions associated with CD (for a review, see Naluai et al4) and supports the hypothesis that CD is, as is the case for type I diabetes (TID), a classical model of polygenic disease with a small number of genes with large effects, mainly MHC class II genes, and a large number of genes with small effects.5 Moreover, this study suggests for the first time in CD the potential role of IL2, a cytokine important for the homeostasis and function of regulatory T cells, and of IL21, a more recently identified cytokine that is structurally related to IL2 but possesses distinct immune functions.6^–8 Due to extensive linkage disequilibrium, it was however not possible to determine the causal variant associated with CD in the 4q27 region. The association with the 4q27 region has already been observed in TID and autoimmune thyroid Graves disease, suggesting that polymorphism(s) in this region may more generally contribute to autoimmunity.5 Interestingly, 4q27 is syntenic to the idd3 region on murine chromosome 3 that controls the development of TID in NOD mice as well as the sensitivity to several experimental autoimmune diseases.9 No precise mutation was identified, but it was convincingly shown that idd3 regulates IL2 transcription and thereby the function and recruitment of the subset of regulatory CD4+ T cells that express CD25, the high avidity receptor for IL2. Thus, in mice bearing the susceptibility idd3 haplotype, IL2 transcription was twofold less in response to T cell stimulation than in non-susceptible mice, and the number as well as the suppressor activity of CD4+CD25+ T cells were reduced. On the contrary, no change in the transcription of IL21 was observed in mice bearing the idd3 haplotype conferring susceptibility to autoimmunity.9 In active CD, van Heel observed that IL21 transcription was increased while that of IL2 was moderately, albeit significantly, decreased in intestinal biopsies. The authors considered the latter finding paradoxical given the presence of activated T cells, and suggested a possible defect in IL2 transcription in CD. Variable levels of IL2 transcripts (normal or increased) have, however, been reported in biopsies of active CD,10^–13 and IL2 transcription is upregulated in organ cultures from CD patients stimulated by gliadin.12 14 Additional functional data are therefore necessary to delineate the exact impact of the observed polymorphisms on the transcription of either IL2 or IL21. Complementary genetic studies will also be interesting to analyse the implication of the 4q27 region in cohorts of CD patients with an origin distinct from North Europe.

Whatever the possible genetic link between CD and the IL21-encoding gene, the role of IL21 does not seem unique to CD. Thus, the same group of authors that are involved in the study on CD under discussion1 has reported increased IL21 synthesis in the gastric mucosa of patients infected by Helicobacter pylori15 and in the intestinal mucosa of patients with ulcerative colitis or Crohn’s disease, the increase being however more significant in patients with Crohn’s disease.16 In the latter disease, these authors demonstrated that, as in CD, IL21 promoted the Th1 response, as blocking IL21 decreased the expression of interferon γ and T-bet induced in lamina propria T cells by the stimulation of their T cell receptor.16 Therefore, in humans, increased IL21 production may be part of distinct inflammatory intestinal processes irrespective of their aetiology, but perhaps more particularly if they are associated with the local production of interferon γ by CD4+ Th1 cells.

These observations raise a first set of questions on the role of IL21 in the induction of Th1 responses and on the mechanisms that control the production of IL21 by CD4+ T cells. The answer is even less easy to delineate as the situation seems different in humans and in mice. In both species, there is no clear evidence that IL21 alone can polarise the T cell response. In humans, IL21 appears to be secreted by CD4+ T cells already polarised toward a Th1 profile. Accordingly, in Crohn’s disease, Monteleone et al observed that IL21 was induced, as was interferon γ, by IL12, a major Th1-polarising cytokine produced by dendritic cells. IL21 then stimulated an autocrine loop that synergised with IL12 to promote the production of interferon γ by CD4+ T cells.16 In CD, the mechanism(s) that drive(s) the Th1 response and/or the production of IL21 has not been elucidated. The very recent work by Di Sabatino et al argues against the role of classical polarising Th1 cytokines (IL12, IL23 and IL18), but suggests that interferon α, produced by activated plasmacytoid dendritic cells that accumulate in the mucosa of patients with active CD, may be the cytokine responsible for polarising the Th1 response.17 Interestingly, interferon α can upregulate the expression of IL21 mRNA in human T cells stimulated via their T cell receptor.18 In CD, the interplay between IL21 and interferon α may thus amplify the Th1 response. IL21 is however not the only cytokine able to promote the Th1 response in CD. Consistent with previous results,19 Fina et al observed that blocking IL15 was even more efficient than blocking IL21 to inhibit the transcription of interferon γ and of T-bet induced by gliadin digests in intestinal organ cultures from treated CD.1 Finally, in organ cultures, the interferon γ-producing cells targeted by the blocking antibodies cannot be identified. Lamina propria gliadin-specific CD4+ T cells are not the only source of interferon γ in CD, and intraepithelial lymphocytes (IELs), a subset of cytotoxic CD8 effector cells massively expanded in the gut epithelium of CD patients, seem to be the main producer of this cytokine.20 A contribution of IL21 to interferon γ production by CD8+ IELs is likely, as discussed below.

Due to the widespread expression of its receptor, IL21 exerts pleiotropic effects. Besides a role in promoting the CD4+ Th1 response, IL21 may thus make other contributions to the pathogenesis of CD (see box 1 and fig 1). Notably, expression of the IL21 receptor on intestinal fibroblasts and enterocytes was associated with the capacity of IL21 to stimulate the release of metalloproteases15 21 and the epithelial production of CCL20, a chemokine that stimulates the recruitment of lymphocytes and dendritic cells.22 The IL21 receptor is also widely expressed on immune cells. but the consequences of its stimulation are equivocal, either stimulatory or inhibitory depending on the cell type concerned and on the cytokine environment.6 7 Remarkably, the cytokine that has the strongest influence on the immunomodulatory effects of IL21 is IL15, a cytokine recently recognised as a key player in CD enteropathy.23 Their signalling pathways are partially overlapping, the IL21 receptor comprising a specific IL21Rα chain, and the γ_c chain in common with the IL15 receptor. IL21 stimulates the recruitment of the protein tyrosine kinases JAK1 (by IL21Rα) and JAK3 (by γ_c), and the subsequent activation of several transcription factors, mainly signal transducer and activator of transcription (Stat) 1 and Stat3, but also Stat5A/B. IL15 stimulates JAK2 and JAK3 which, in turn, activate Stat5A/B and to a lesser degree Stat3.6 7 The two cytokines exert both opposing and synergistic effects at the interface between innate and adaptive immunity. Rather surprisingly, in vitro studies indicate that IL21, when used alone, has inhibitory effects on the immune response opposite to the effects of IL15 and inconsistent with a role for IL21 in CD. Thus, in marked contrast to IL15, IL21 alone has potent immunosuppressive effects on the maturation of dendritic cells6 7 and downregulates the expression and activation of the NKG2D receptor in human natural killer (NK) and CD8+ T cells.24 These effects are unexpected in active CD. First, active CD is associated with the recruitment of mature intestinal dendritic cells.25 Secondly, induction and activation of NKG2D by IL15 on IELs simultaneously with upregulation of its ligand MIC on enterocytes was suggested to play a key role in the cytolytic attack of the epithelium by IELs in CD.26 27 It is conceivable that these effects of IL21 are overcome in the presence of IL15 and/or other proinflammatory cytokines. Indeed, contrasting with these effects of IL21 alone, experimental evidence suggests that IL21 may synergise with IL15 to promote IEL activation. We and others have previously shown that enterocyte-derived IL15 induces the expansion of normal CD8+ IELs in CD and of their malignant counterpart in refractory coeliac sprue. IL15 also stimulates their production of interferon γ and their granzyme/perforin-dependent cytotoxicity against epithelial cells.11 27 28 In mice, IL21 alone has only moderate effects on CD8 T cells, but IL21 exerts potent synergistic effects with IL15, enhancing their survival, proliferation, production of interferon γ and granzyme/perforin-dependent cytotoxicity.29 IL21 produced by lamina propria CD4+ T cells may therefore be one actor of the necessary but as yet poorly understood dialogue between gliadin-specific CD4+ T cells and IL15-activated IELs that leads to the onset of inflammation and enteropathy. Finally, besides a synergistic effect with IL15 on IELs, IL21 might also act in concert with IL15 to break local mechanisms of immune tolerance. Thus, IL15 inhibits the Smad3 pathway of transforming growth factor β (TGFβ), a key regulatory pathway to avoid intestinal inflammation.19 Secondly, IL21, like IL15, can prevent the suppressive effects of CD4+CD25+ regulatory T cells on CD4+ effector T cells, a second important pathway to control intestinal inflammation (30 and BenAhmed et al, in preparation).

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Figure 1 Hypothetical roles of interleukin 21 (IL21) in coeliac disease (CD) enteropathy. Intestinal lesions in CD are thought to result from the interplay between CD4+ lamina propria T cells that produce interferon γ (IFNγ) in response to gluten peptides presented by human leucocyte antigen (HLA)-DQ2+ antigen-presenting cells (APCs) and intraepithelial lymphocytes (IELs) activated by IL15 and innate natural killer receptors (NKRs). The possible contribution of IL21 to this scheme is depicted using solid and dotted lines to indicate the demonstrated and hypothetical effects, respectively, in CD. Fina et al demonstrate in this issue of Gut (see page 887) that IL21 enhances IFNγ production by lamina propria CD4+ T cells through an autocrine amplification loop.1 IL21 may also amplify the local Th1 response indirectly by inhibiting regulatory T cells (Treg). In addition, IL21 may stimulate the release of chemokines such as CCL20 by enterocytes and of metalloproteases (MMPs) by fibroblasts, and/or cooperate with other locally produced cytokines. IL21 could more particularly synergise with IL15, a cytokine produced by enterocytes and APCs in active CD, and thereby promote the activation of IELs, their production of IFNγ and their cytotoxicity against enterocytes. The possible role of IFNα produced by plasmacytoid dendritic cells (pDCs) in the induction of IL21 and IFNγ by Th1 cells is depicted.

In conclusion, recent genetic and functional data point out to the role of IL21, a cytokine produced by lamina propria CD4+ T cells, in the inflammatory response associated with CD enteropathy. While the contribution of IL21 to the local Th1 response is clearly demonstrated, further studies are needed to delineate whether IL21 is one gene that accounts for the linkage of CD with the 4q27 region and whether its interplay with IL15 may contribute to drive the activation of IELs and jeopardise local immune-regulatory mechanisms.