The Role And Mechanism Of MyD88 In Ulcerative Colitis By Regulating Intestinal Microbiota

Backgrounds and objectives:Toll-like receptors（TLRs）are crucial for host to recognize and defend against microbial infection.Myeloid differentiation factor 88（MyD88）is the key signal transduction molecule for TLRs.TLRs/MyD88 signaling pathway can participate in intestinal immune regulation,tissue mucosal repair and intestinal flora regulation during microbiota-host interaction.However,the role and mechanism of MyD88 in the pathogenesis of intestinal inflammation are not fully understood.This study aims to investigate the role of MyD88 in the pathogenesis of intestinal inflammation and discuss the potential mechanism.Methods:The C57BL/6J female mice were randomly divided into H₂O+PBS/WT group,DSS+PBS/WT-DSS group,H₂O+TJ5 group,DSS+TJ5 group.The MyD88 knockout mice were randomly divided into MyD88^-/-group and MyD88^-/--DSS group.Mice were administrated 3%DSS for 7 days to establish the acute ulcerative colitis model.The severity of colonic inflammation in each group was compared by disease activity index（DAI）,length of colon,histological scoring（HS）and m RNA expression level of inflammatory cytokines.The m RNA expression levels of inflammatory cytokines in intestinal tissue were measured by RT-PCR.The m RNA sequencing analysis is applied to undergo the differential expression analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis.In order to study the impact of intestinal microbiota on the intestinal inflammation and innate immune system,mice were given broad-spectrum antibiotics orally to reduce the intestinal microbiota.Using 16S r DNA sequencing to analyze the intestinal bacterial composition and abundance among groups.Western blot was used to evaluated the expression level of proteins.Results:Compared with the H₂O+PBS/WT group,the DAI,HS and m RNA expression level of inflammatory cytokines in the DSS+PBS/WT-DSS group,DSS+TJ5 group and MyD88^-/--DSS group were increased,and the colon shortened.Compared with the DSS+PBS/WT-DSS group,the DSS+TJ5 group and MyD88^-/--DSS group showed decreased level of MyD88 protein expression and NF-κB activation in inflamed colon.There was no significant difference in the DAI and HS between the DSS+PBS group and the DSS+TJ5 group,as well as between the WT-DSS and the MyD88^-/--DSS group.Besides,no significant difference was found in the m RNA expression level of IL-1β,TNF-αand IFN-γbetween the DSS+PBS group and the DSS+TJ5 group,and between the WT-DSS and the MyD88^-/--DSS group.TGF-β1,EGF,COX-2 and tight-junction protein expression level was not significantly different among four groups,suggesting relatively normal epithelial reconstruction ability in mice with low MyD88 expression as compared to normal control group.Microbial diversity analysis of colonic mucosa showed increased Proteobacteria（22.2%）and decreased Firmicutes（54.3%）in DSS-induced colitis mice compared with control mice.The proportion of Proteobacteria（43.0%）in MyD88 knockout mice was significantly higher than that of WT mice（9.8%）,and meanwhile the proportion of Firmicutes was decreased in MyD88 knockout mice（49.3%）as compared to WT（64.1%）.The m RNA sequencing analysis revealed 56 significantly differentially expressed genes between DSS+TJ5 group and DSS+PBS group.Analysis of the differential expressed genes using the KEGG pathway showed that NOD-like receptor signaling pathways is the major pathway associated with MyD88 inhibition.Treatment of broad-spectrum antibiotics in DSS+TJ5 group mice partially alleviated the severity of colonic inflammation,with the reduced activation of NOD-like receptor signaling pathway.Combined inhibition of MyD88 and NOD-like signal moderately ameliorated DSS-induced colitis.Conclusion:Inhibition of MyD88 significantly reduces NF-κB signaling activation in colon during the acute DSS-induced colitis.Meanwhile,the MyD88 suppression may impair intestinal anti-bacterial immunity,which in turn induces intestinal dysbiosis,NOD-like receptor signaling pathway activation,and intestinal inflammation.