| Backgrounds and objectives Inflammatory bowel diseases (IBDs), which consist of ulcerative colitis (UC) and crohn's disease (CD), are chronic and recurrent inflammatory disorders of gastrointestinal tract. Up to now, the exact cause of IBDs is still unclear. Nevertheless, most scholars believe that IBDs result from complicated inflammation in which genetic, environmental, infectious and immune factors may play important roles. Tumor necrosis factor-associated factors 5 (TRAF5) is a member of TRAFs protein family. As a cellular adaptor, TRAF5 is able to interact with numerous receptors, thus regulating the downstream signaling in the cell. Many studies have shown that TRAF5 can regulate various aspects of Th cell-mediated immune response and involve in the regulation of NF-?B signaling pathway. However, the roles of TRAF5 in the pathogenesis of IBDs are still unknown. In order to figure out the definite roles of TRAF5 in IBDs, we investigated the effect of TRAF5 deficiency on DSS-induced colitis in mice.Methods TRAF5 knockout mice and their wide-type littermates were giving 3% DSS polymers in the drinking water continuously for 7 days. The severity of colitis was assessed through evaluating body weight loss, disease activity index (DAI), shortening of the colon, histological score and MPO activity. The mRNA expressions of Th cells'specific cytokines and transcription factors were identified by Real-Time PCR (Th1 cells:TNF-?, IFN-?, T-bet; Th2 cells:IL-4, GATA-3; Th17 cells:IL-17a, IL-22, ROR-?, ROR-?t). The secretions and expressions of cytokines IFN-?, IL-4 and IL-17a were detected by ELISA and immunofluorescence. Lamina propria mononuclear cells (LPMCs) were isolated, and the percentages of Th cell subpopulations were analyzed by flow cytometry. Total protein of the colon was extracted, and the phosphorylated and total protein levels of p65 and I?B? were examined by Western blot. Furthermore, the nuclear translocation of p65 was detected by immunofluorescence, and the expressions of p65+CD4+ and p65+CK18+ cells were analyzed by immunofluorescence double-labeling.Results During the induction of DSS, TRAF5 knockout mice suffered from greater body weight loss since day 5 in comparison with wide-type mice (day 5:88.13± 3.64% vs.97.04±2.97%,p<0.01; day 6:78.41±3.25% vs.90.04±2.83%,p<0.01; day 7:71.32±3.28% vs.82.98±2.63%,p<0.01). Furthermore, the DAI scores of DSS-induced knockout mice were much higher than those of wide-type mice (day 4: 5.4±1.8 vs.2.9±1.4,p=0.019; day 5:9.4±2.4 vs.4.7±2.6,p=0.003; day 6:11.2± 0.8 vs.9.0±1.1,p=0.001;day 7:11.8±0.6vs.10.4±1.1,p=0.018). Our results also suggested that, in comparison with wide-type mice, DSS-fed knockout mice suffered from greater shortening of the colon (46.9±2.9 mm vs.55.2±2.5 mm,p<0.01), higher histological score (7.50±0.53 vs.5.60±1.07,p<0.01) and stronger MPO activity (1.58±0.62 vs.1.01±0.19 U/g tissue,p<0.05). Real-Time PCR results showed that, compared with levels of wide-type mice, the mRNA levels of IFN-y, IL-4, IL-17a, T-bet an GATA3 were all significantly elevated in the colons of TRAF5 knockout mice after the administration by DSS. Unfortunately, we failed to find any significant difference in the mRNA levels of ROR-a and ROR-yt between DSS-fed wide-type and knockout mice. ELISA and immunofluorescence results indicated that TRAF5 deficiency could significantly enhance the protein expressions of IFN-y, IL-4 and IL-17a in the colons of DSS-induced mice. Flow cytometry data showed increased rates of Th2 cells and IFN-?+IL-17a+CD4+T cells in the colons of DSS-fed knockout mice. In addition, western blots data showed significant higher protein levels of P-p65, T-p65 and P-I?Ba in the colons of DSS-fed knockout mice. Immunofluorescence results indicated that TRAF5 deficiency significantly enhanced the nuclear translocation of p65 in response to DSS. Finally, our results also suggested that the expression of p65+CD4+ cells was significantly elevated in the colons of DSS-induced knockout mice. However, there was no significant difference in the expression of p65+CK18+ cells between knockout and wide-type mice after the administration by DSS.Conclusions Our study demonstrated that adaptor TRAF5 might act as an anti-inflammatory role during the development of DSS-induced colitis. After the administration by DSS, TRAF5 deficiency could significantly enhance the responses of Th2 and IFN-y+IL-17a+CD4+T cells in the mice colon. Furthermore, the activation of NF-?B in colonic CD4+T cells was also enhanced. Taken together, these results suggested that TRAF5 deficiency could significantly exacerbate DSS-induced colitis, most likely through regulating T helper cell-mediated inflammation. Targeting TRAF5 may thus provide a promising strategy in the treatment of human IBDs. |
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