Study On The Mechanism Of Experimental Colitis In Mice Treated With Bacteroides Fragilis ZY-312

Background and aimDysbiosis of the gut microbiome is closely associated with the pathogenesis of inflammatory bowel disease(IBD).While microbiome-modulating therapy could alter gut microbiota composition,it could improve intestinal barrier functions as well.However,there are few studies on the effect of specific strain on wound healing of epithelial cells after inflammatory damage,which would aggravate the disease if uncontrolled.Bacteroides fragilis(B.fragilis,BF)ZY-312 is a commensal strain of non-toxigenic B.fragilis isolated from a Chinese healthy infant.Our previous work demonstrates the protective role of B.fragilis ZY-312 in many animal models of intestinal diseases including DSS-induced colitis in mice,all of which highlight the potential of Bfragilis ZY-312 in improving intestinal barrier functions.Our study aims to illustrate whether and how the protective effect of B.fragilis ZY-312 on DSS-induced colitis in mice depends on the promotion of wound healing of epithelial cells.Methods1.Mice with colitis induced by DSS received oral gavage of B.fragilis ZY-312,and their body weight changes and stool consistency were recorded.Following blood collection from the ocular vein,mice were sacrificed and colons were collected for isolation of colonic epithelial cell(CEC)and colonic lamina propria(cLP)cells.2.HE staining was used to assess the pathological scores of colon and immunohistochemistry(IHC)was used to analyze the local expression of Ki67 and STAT3 in colon.3.Mice with STAT3 depletion in intestinal epithelial cells were generated by Cre/loxP system and were identified with PCR,western blot and IHC.4.Cell cycle analysis was measured in CEC and MC38 cells by flow cytometry,5.Cell apoptosis of CEC and MC38 cells was detected by means of flow cytometry and fluorescence in situ apoptosis assay.6.Phenotypes of cLP cells were analyzed by flow cytometry.7.Western blot was used to analyze the expression of certain protein in CEC and MC38 cells.8.Bone marrow-derived dendritic cells(BMDC)and splenic T cells were isolated and stimulated with B.fragilis ZY-312 in vitro.9.Enzyme-linked immunosorbent assay(ELISA)was used to dectect the concentration of certain cytokines in mice colon,plasma and supernatants of cell cultures.Results1.B.fragilis ZY-312 promotes wound healing of CEC in DSS-induced colitis mice,as evidenced by enhanced cell proliferation and decreased cell apoptosis.2.Protection of B.fragilis ZY-312 against DSS-induced colitis is dependent on STAT3 in CEC.Phosphorylation of STAT3 was activated in CECs of B.fragilis ZY-312-treated mice.When STAT3 was depleted in intestinal epithelial cells,the protective effect of B.fragilis ZY-312 in colitis was completely abrogated.3.B.fragilis ZY-312 enhances IL-6 and IL-22 levels in colon of mice with DSS-induced colitis.Levels of IL-22 and IL-6 in both plasma and colon homogenates were much higher in B.fragilis ZY-312-treated mice than those in mice with DSS-induced colitis.Moreover,activated cLP cells from normal mice co-cultured with B.fragilis ZY-312(ZY-312-LPS-cLP)in vitro produced larger amounts of IL-6 and IL-22 than LPS-cLP did.4.IL-22 is required for B.fragilis ZY-312-driven STAT3 activation in MC38 cells,thus promoting cell survival in vitro.ZY-312-LPS-cLP supernatant containing IL-22 upregulated the expression of p-STAT3 and inhibited the activation of caspase-3 in MC38 cells.Beside,cell proliferative and anti-apoptotic capabilities of MC38 cells were enhanced by ZY-312-LPS-cLP supernatant in the context of TNF-αmediated cell death.Blockage of IL-22 in ZY-312-LPS-cLP supernatant impeded cell survival.5.B.fragilis ZY-312 promotes IL-22 production in cLP T cells in mice.CD4+RORγt+IL-22+ cells instead of ILC3 were enriched in cLP T cells in mice treated with B.fragilis ZY-312.6.B.fragilis ZY-312 regulates the differentiation of CD 103+dendritic cells in mice colon.CD 103+dendritic cells instead of CD11b+F4/80+ macrophages were enriched in cLP cells in mice treated with B.fragilis ZY-312.7.IL-6 production by BMDC is intensified by B.fragilis ZY-312 stimulation in vitro.B.fragilis ZY-312 co-cultured with BMDC was capable of enhancing the production of IL-6 via the activation of IL-6/JAK2/STAT3 positive feedback loop in vitro.8.Il-6 from BMDC mediates B.fragilis ZY-312 driven IL-22 production by T cells in vitro.Supernatant of B.fragilis ZY-312 and BMDC co-culture system,containing high level of IL-6,was able to stimulate IL-22 production by both cLP and spenic T cells.It was only when IL-6 was neutralized that the IL-22-inducing effect was suppressed.Further,B.fragilis ZY-312 failed to stimulate IL-22 production by T cells in the absence of BMDC,which highlighted the necessity of BMDC for IL-22 stimulation.ConclusionB.fragilis ZY-312 promotes wound healing of colonic epithelial cells by modulating the IL-6/IL-22/STAT3 axis to protect mice from DSS-induced colitis.