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- Materials and Methods
Background: WSX-1, a component of the interleukin (IL)-27 receptor, is a novel class I cytokine receptor with homology to the IL-12 receptor β2 chain. Initially, WSX-1 signaling was reported to play an important role in the promotion of T helper-1 responses, but recent reports have revealed an anti-inflammatory property in WSX-1 signaling. In the present study, we investigated the role of IL-27/WSX-1 signaling in a murine colitis model, dextran sulfate sodium (DSS) colitis, by using WSX-1 knockout (KO) mice. Methods: First, we observed whether WSX-1 KO mice developed colitis spontaneously. Second, we induced DSS colitis in WSX-1 KO and wild-type (WT) mice. Results:WSX-1 KO mice were observed not to develop colitis spontaneously. The severity of DSS colitis was decreased in WSX-1 KO mice in comparison with WT mice in association with a reduced production of interferon-γ, IL-6, and tumor necrosis factor-α by lamina propria mononuclear cells from WSX-1 KO mice and the absence of T-bet expression in the colon from WSX-1 KO mice. Conclusions: This study revealed the inflammatory property of IL-27/WSX-1 signaling in intestinal inflammation. As a result, IL-27/WSX-1 signal pathway may thus be a promising candidate for the therapeutic intervention of human inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.
The pathogenic mechanisms of inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC) have yet to be elucidated despite numerous studies in gastroenterology and mucosal immunology. Recent studies, however, have shown that T helper 1 (TH1) cytokines, such as interleukin-12 (IL-12), IL-18, and interferon-γ (IFN-γ), are upregulated in the gut of patients with CD, thereby demonstrating the activation of TH1 cells to likely be a major pathogenic mechanism in CD.1–5 In contrast, the T cell response pattern in UC is less well defined.
A novel cytokine receptor, WSX-1 was initially identified as a class I cytokine receptor with homology to the IL-12 receptor (IL-12R) β2 chain.6,7 WSX-1 expression is restricted to lymphoid tissues and it tends to be the highest in naïve T cells and natural killer (NK) cells. In an analysis of a Leishmania major infection model, WSX-1 knockout (KO) mice showed susceptibility to L major infection relative to wild-type (WT) mice, associated with an impaired IFN-γ production by naïve CD4+ T cells early in the infection.8 WSX-1 was thus revealed to play an essential role in the initiation of the TH1 response. Recently, IL-27, a newly identified IL-12-related heterodimeric cytokine, was shown to bind to WSX-1.9 IL-27 is composed of Epstein-Barr virus-induced gene 3 (EBI3), an IL-12 p40-related protein, and p28, an IL-12 p35-related protein. Because EBI3 and p28 are coexpressed in antigen-presenting cells (APCs), the source of IL-27, like IL-12, is thought to be APCs. We demonstrated that IL-27/WSX-1 signaling induces the phosphorylation of STAT1 and subsequent T-bet expression, followed by IL-12Rβ2 expression in naïve CD4+ T cells.10 These results indicate that IL-27/WSX-1 signaling plays a critical role in the initial TH1 commitment and such signaling acts before IL-12/IL-12R signaling to provide IL-12 responsiveness in naïve CD4+ T cells. Recent studies, however, have clarified that IL-27/WSX-1 signaling also negatively regulates the inflammatory processes. As a result, studies on Toxoplasma gondii infection,11Trypanosoma cruzi infection,12 and concanavalin A-induced hepatitis13 have demonstrated that WSX-1 plays a role in limiting the intensity and duration of T cell activation because WSX-1 KO mice developed lethal inflammatory diseases because of an overproduction of a group of proinflammatory cytokines. Because the role of IL-27/WSX-1 in intestinal inflammation has not yet been addressed, such pleiotropic characters of the IL-27/WSX-1 pathway prompted us to investigate whether IL-27/WSX-1 signaling plays an inflammatory or anti-inflammatory role in intestinal inflammation.
In the present study, we used an acute colitis model, dextran sulfate sodium (DSS) -induced colitis,14 to examine the role of WSX-1 in the development of intestinal inflammation by taking advantage of WSX-1 KO mice. WSX-1 KO mice developed a milder degree of colitis than WT mice and lamina propria mononuclear cells (LPMCs) from WSX-1 KO mice produced significantly lower amounts of IFN-γ, IL-6, and tumor necrosis factor-α (TNF-α) than those from WT mice. Moreover, the T-bet expression was absent in the colon of WSX-1 KO mice. These results suggest that IL-27/WSX-1 signaling is associated with an exacerbation of acute colonic inflammation.
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- Materials and Methods
This is the first study to clarify the role of IL-27/WSX-1 signaling in intestinal inflammation. We showed that IL-27/WSX-1 signaling is associated with an exacerbation of acute intestinal inflammation. We demonstrated that IL-27 (IL-27 p28 and EBI3) mRNA increased in the colon of DSS-treated WT mice and that DSS-treated WSX-1 KO mice developed milder colitis than did DSS-treated WT mice. These data indicate that IL-27/WSX-1 signaling contributes to an exacerbation of intestinal inflammation in murine DSS colitis. Such an exacerbation seems to be dependent on inducing the T-bet expression and the subsequent IFN-γ production, followed by TNF-α and IL-6 production, as our study showed that LPMCs from DSS-treated WSX-1 KO mice produced significantly lower amounts of IFN-γ, IL-6, and TNF-α than those from DSS-treated WT mice and T-bet expression was reduced in the colon of DSS-treated WSX-1 KO mice. The immunological property of IL-27/WSX-1 signaling is known to be pleiotropic as it can be both inflammatory and anti-inflammatory. That is, IL-27/WSX-1 plays an essential role in the early phase of TH1 differentiation under some conditions,8 whereas it acts as a regulator of the excessive immune reactions under other conditions.11,12 In this study, we, for the first time, clarified that IL-27/WSX-1 signal has some inflammatory property and therefore plays a role in the development of intestinal inflammation, at least in the DSS-induced colitis model. The reason why IL-27/WSX-1 plays such different roles depending on the conditions is still not fully understood, although the differential usage of signal-transducing molecules has been implied.21 In the DSS colitis model, reduced inflammation is associated with less IFN-γ production as seen in an L major infection model.8 We assume that the production of IL-6 and TNF-α and inflammation/tissue damage is secondary to IFN-γ production.
In the present study, MLN cells from both WT and WSX-1 KO mice, which were administered water without DSS, produced high levels of IFN-γ. This seems to conflict with the findings of our previous study,8 but it can be explained as follows. In a previous study, Yoshida et al showed that popliteal lymph node cells of WSX-1 KO mice failed to produce INF-γ in the early phase of L major infection, whereas they produced the same levels of IFN-γ as WT lymph node cells in the late phase.8 In these experiments, CD4+ T cells were stimulated with L major antigen and APCs. In the present study, whole MLN cells were stimulated with anti-CD3 and anti-CD28. It is, therefore, considered that MLN lymphocytes from water-treated WSX-1 KO mice, which have been persistently exposed to luminal antigens, are able to produce IFN-γ just like WT cells at least in the presence of IL-12 produced by the surrounding APC populations. In contrast to MLN cells, LPMCs from water-treated WT and WSX-1 KO mice did not produce any detectable levels of IFN-γ. We consider this difference in IFN-γ production to be the result of the difference in cell types, because MLNs contain effector cells induced by the continuous stimulation with luminal antigens and LPMCs are known to be hyporesponsive to stimulation through T cell receptors. It seems that MLN cells from older water-treated mice (Fig. 1, C; 14 months old) produced much larger amounts of IFN-γ than those from younger water-treated mice (Fig. 4, B; 8 weeks old), probably because MLN cells in the older mice were exposed to luminal antigens for long periods of time. After the administration of DSS, MLN cells from WSX-1 KO mice failed to produce a larger amount of IFN-γ than those from the water-treated mice, whereas MLN cells from DSS-treated WT mice produced significantly higher levels of this cytokine. Similarly, although LPMCs from water-treated mice of both genotypes did not produce detectable levels of IFN-γ, in contrast to MLN cells, LPMCs from DSS-treated WT mice produced significantly higher amounts of IFN-γ than the cells from DSS-treated WSX-1 KO mice. These results indicate generation of colitogenic TH1 cells by the administration of DSS to be attenuated in the absence of IL-27/WSX-1 signaling.
DSS colitis is an established, well-characterized experimental murine colitis model. The administration of DSS dissolved in water to mice has been shown to cause hematochezia, body weight loss, a shortening of the large intestine, mucosal ulcers, and an infiltration of neutrophils.14 The oral administration of DSS activates nonlymphoid cells such as macrophages to release proinflammatory cytokines. Because DSS colitis occurs in mice lacking T cells, B cells, and NK cells,22,23 T cell-mediated immunity is not essential for the induction of DSS colitis. T cell-mediated immunity, more specifically TH1 response, is activated in this model, however, because TH1 cytokines such as IL-12 and IFN-γ are upregulated in DSS colitis.24 IL-12 p35-deficient mice fed DSS have been reported to develop milder colitis associated with reduced IFN-γ and TNF-α production by LPMCs.25 Therefore, TH1-biased T cell-mediated immunity is considered to significantly influence an exacerbation of inflammation in this model. Our results clearly demonstrated that IL-27/WSX-1 signaling played an important role in the exacerbation of DSS colitis by the augmentation of TH1 responses and the subsequent proinflammatory cytokine production.
Sensitivity to DSS varies among mouse strains.26 C57BL/6 mice are one of the sensitive strains to DSS and severe colonic inflammation can be induced using doses of DSS as low as 1% to 3% (w/v). In a pilot study, we administered 3%, 2%, and 1% (w/v) DSS to WT mice and WSX-1 KO mice for 7 days to set a suitable concentration. Severe colitis was induced under each condition. Although we found a lower IFN-γ production of LPMCs from DSS-treated WSX-1 KO mice in comparison with DSS-treated WT mice, the clinical and histological differences between WT and WSX-1 KO mice were less evident (data not shown). We also tried 0.5% (w/v) DSS administration, and observed more obvious differences both clinically and histologically. At this dose of DSS, the development of colitis seemed to be slower than with usual doses, and WT mice began to show clinical manifestations after day 7. As a result, in the present study, we administered a lower dose (0.5%) of DSS for a longer period of time (14 days) than the conventional protocols of DSS-induced colitis. In the high-dose DSS protocol, macrophages may be highly activated and thus produce large amounts of proinflammatory cytokines such as TNF-α and IL-6, and severe intestinal inflammation is induced. The influence of TH1 response may be masked by the severe storm of proinflammatory cytokines. In contrast, in the low-dose DSS protocol, the first activation of macrophages may be weak and intestinal inflammation may be influenced by subsequent TH1 activation more clearly and directly. Some investigators analyze DSS-induced colitic animals in the recovery period. In such experiments, the induction with DSS is followed by an analysis of the recovery period after returning to regular drinking water. We tried this protocol (2% DSS in drinking water for 5 days, followed by drinking water without DSS for 7 days) and did not find any obvious clinical and histological differences between WT and WSX-1 KO mice during the recovery period (data not shown).
In CD, the local immune response is predominantly TH1 and it is reflected by the local release of cytokines such as TNF-α, IFN-γ, IL-12, and IL-18.1–5 Recently, Matsuoka et al reported that the T-bet expression of CD4+ LPMCs in CD increased and the T-bet induction correlated with the IFN-γ production and augmentation of the surface expression of IL-12Rβ2 before IL-12 signaling for enhanced IFN-γ production,27 thus suggesting that T-bet may not only initiate IFN-γ production but also control the responsiveness to IL-12. As we reported previously, IL-27/WSX-1 signaling induces the phosphorylation of STAT1 and leads to T-bet induction, followed by IL-12Rβ2 expression in naïve CD4+ T cells.10 In this regard, it is possible that IL-27/WSX-1 signaling plays an important role in the T-bet induction in CD. As a result, IL-27/WSX-1 presumably plays an important role in a vicious cycle, in which IL-27/WSX-1-mediated TH1 polarization of T cells leads to a greater IFN-γ production followed by more inflammation/tissue damage and the subsequent greater macrophage activation results in an increased IL-27 and IL-12 production in the intestines of CD patients. Larousserie et al demonstrated that EBI3 and p28 were coexpressed in the granulomas infiltrating the intestinal wall or the MLNs of CD patients.28 The local production of IL-27 in the inflamed tissues in CD may thus be a critical mechanism in the initiation of CD. Clinically, anti-human TNF-α monoclonal antibody (infliximab) is effective for many patients with CD refractory to conventional therapy.29 The effectiveness of other cytokine-targeted treatments, for example, a humanized anti-IL-12 monoclonal antibody,30 has been examined in CD. Likewise, IL-27/WSX-1 signaling-targeted treatment may also be a new approach for the treatment of CD.
In UC, another IBD in which the upregulation of TH2 cytokine expression such as IL-4 and IL-5 has been reported,31,32 the T cell response pattern has been less well defined than in CD. In addition, the role of IL-27/WSX-1 signaling is unclear in UC. Mice deficient for EBI3, one of the IL-27 components, showed an impaired ability to mount a TH2 response and such mice were resistant to oxazolone colitis, which resembles human UC.33 Clinically, there have been some reports on EBI3 expression in UC patients. An elevated level of EBI3 expression was reported in mucosal samples of UC compared with CD.34,35 From the results of oxazolone colitis in EBI3-deficient mice, it is possible that the upregulation of EBI3 in UC is related to an exacerbation of intestinal inflammation. As a result, IL-27/WSX-1 signaling may also be important for establishing the immunopathology of UC. Further studies will be necessary to clarify the relevance of IL-27/WSX-1 signaling in UC.
In conclusion, the results in the present study demonstrated that IL-27/WSX-1 signaling is associated with an exacerbation of intestinal inflammation by inducing the T-bet expression followed by the local production of IFN-γ, IL-6, and TNF-α. The modulation of IL-27/WSX-1 signaling therefore appears to be an attractive target for therapeutic intervention in human IBD, such as CD and UC.