The Regulation Mechanism Of Toll-like Receptors In The Maintenance Of Immune Tolerance Of Rumen Epithelium

The immune regulatory network,which responds to the change of microbial community and subsequently maintains the tolerance of rumen epithelium to the symbiotic microorganisms,is existed in the rumen epithelium.However,to date,the regulatory mechanism of this network in the maintenance of immune tolerance has not been known.The Toll-like receptor(TLR)is one kind of pattern recognition receptors(PRRs),which has been most intensively studied in the single gastric animals.By inducing or inhibiting the inflammatory responses in the gastrointestinal tract,they maintain the homeostasis of gastrointestinal tract after the combination of specific microbial components.So far,it has been found that 6 members of TLR family are continuously expressed in the luminal surface of rumen epithelium in ruminant animals.In this study,by using 16S rRNA gene sequencing,we firstly investigated the structure of rumen microbiota in goats fed with 10%and 31%NFC diet,respectively.Subsequently,by using quantitative PCR,we studied the expressions of six TLRs and five genes taht located in the downstream of TLR signaling pathway.Then,by the combination of above data,we run the statistical tests to analyse the correlations of microbes and TLR scripts.By this process,we aimed to know the regulatory mechanism of TLRs in the maintenance of immune tolerance of rumen epithelium.1.The effects of non-fibre carbohydrates(NFC)on the rumen microbiotaSix goats(Boer x Yangtze river,delta white,aged 4 months)were randomly assigned into two groups:MNFC group(31%NFC,n=3)and LNFC group(15%NFC,n=3),and fed with respective diets for 4 weeks.Ruminal fluids were taken in the end of dietary experiment.Microbial DNA was extracted from the samples and then V3V4 region was used for 16S rRNA gene sequencing on Illumina Miseq platform.In rumen microbiota,72 genera belonging to 16 phyla were detected in LNFC group,and 107 genera belonging to 21 phyla were detected in MNFC group,respectively.In MNFC group,Bacteroidetes,Firmicutes,Proteobacteria and Verrucomicrobia were the most abundant phyla,which accounted for 81.5%,12.6%,1.2%and 1.1%,respectively.In LNFC group,Bacteroidetes,Firmicutes,Verrucomicrobia,Synergistetes,Proteobacteria and Lentisphaerae were the most abundant phyla,which accounted for 72%,14.4%,4.4%,3.5%,1.2%and 1.2%,respectively.Comparied with the diversity of LNFC group,MNFC group showed the significant increase.Comparied the relative abundance of detected bacteria between the groups,in MNFC group,Verrucomicrobia was most significantly expanded,and some opportunistic pathogens,namely Rikenella,Anaeroplasma,and Olsenella,were significantly decreased.2.The responses of TLRs to the change of rumen microbiotaIn the end of dietary experiment,rumen samples were taken immediately after the slaughter and total RNAs was extracted from the samples.Quantitative PCRs were applied to compare the expressions of six TLRs and five key genes in the downstream of TLR Signaling pathway.It was found that,in MNFC group,the significantly increased expressions of TLR1,6,10 were associated with the significantly decreased expressions of pro-inflammatory cytokines interleukin(IL)-1?,IL-6,and anti-inflammatory cytokine IL-10.By the analysis of the correlations between the relative abundance of microbes and differentially expressed TLRs,it was found that the increased abundance of commensal bacteria in Verrucomicrobia subdivision 5 was contributed to the upregulation of TLR10 expression.3.The effects of expanded microbial clusters caused by NFC on the metabolism of rumen and transportation of rumen epitheliumIn order to investigate the functions of expanded microbial clusters in MNFC group,we measured the concentration of rumen SCFAs,the major metabolites of rumen microbiota,as well as ten kinds of SCFA-absorption related genes in the rumen epithelium.It was found that the concentrations of SCFAs in MNFC group were significantly higher than that of LNFC group.Meantime,the expressions of genes related to SCFA absorption were also significantly higher in MNFC group than that of LNFC group.These results indicated that the expanded microbial culsters in MNFC group would promote the fermentation of rumen and transportation of rumen epithelium,being the characteristics of commensal bacteria in gastrointestinal tract.In conclusion,our results indicated that NFC-induced expansion of rumen microbiota promoted the immune tolerance of rumen epithelium by enhancement of the intensity of TLR10 signaling.Moreover,the newly established equilibrium benefited to rumen fermentation and rumen transport of energy substances into the blood.