| There are population-specific cystic lymph node assembly areas of Peyer’s patches(PPs)in the intestinal tissues of Bactrian camels,which play an extremely important role in mucosal immunity of Bactrian camels.However,molecular characterization of immune properties of specific cystic PPs remains unclear.In this study,six healthy young Bactrian camels(3-5 years old)were selected and studied in the following five aspects:(1)Firstly,immunohistochemistry and statistics methods were used to observe the distribution characteristics,rules and distribution density of SIg A and Ig G antibody secreting cells,which are the main effector cells of mucosal immunity in the large intestine of Bactrian camel.The results showed that SIg A and Ig G ASCs were mainly distributed in the lamina propria(LP)of the mucosa of the large intestine,and some were clustered around the intestinal glands.The SIg A ASCs density increased from ileocecal forum to cecum,and decreased from colon to rectum.The density of SIg A ASCs in cecum was the highest,followed by the anterior segment of colon,ileocecal opening,posterior segment of colon and rectum.The density of SIg A ASCs in cecum was significantly higher than that in the other 4 sites(P<0.05).Similarly,the density of Ig G ASCs in the cecum was also the highest and was significantly higher than that in ileocecum,colon and rectum(P<0.05).The results showed that SIg A and Ig G ASCs were mainly distributed in the main effector site of mucosal immunity in the large intestine,and the cecum was the main effector site of mucosal immunity.(2)The structural characteristics,transcriptional activity and transcriptional characteristics of PPs in Bactrian camel’s cecum were studied.A total of 6 PPs samples and 6 non-pps(NPPs)samples were collected from the cecum for histological observation,total RNA extraction and transcriptome sequencing analysis.The results showed that the bottom of the cystic PPs was enlarged and the neck was reduced.Lymphatic tissue was developed and existed in the submucosa in the form of lymphatic aggregation or isolated lymphatic nodules,and the germinal center in the follicular area was obvious.Collagen fibers are distributed between lymph nodes and serve as a scaffold for lymph tissue.There were 9,257 overlapping genes in the two groups,and 3,910 and 174 group specific genes in the PPs and NPPs groups,respectively.A total of 7,568 up-regulated differentially expressed genes and1,266 down-regulated DEGs were obtained by gene significance difference analysis.The upregulation of DEGs with higher differential multiples was mainly related to immune regulation,inflammation and lipid homeostasis.The down-regulated DEGs with higher differential multiples participated in multiple functions.GO enrichment shows that the positive regulation of immune system processes and the positive regulation of immune system processes appear to be most affected by the formation of high enrichment factors.In addition,KEGG enrichment showed that specific immune system related pathways were mainly enriched by significantly higher expression of up-regulated DEGs.There are 9 core genes involved in multiple immune gene interactions in Bactrian camel intestinal immunity.(3)Previous studies have revealed that there are significant differences in immune characteristics between PPs and NPPs,but whether it has an impact on the selection of symbiotic microorganisms is still unknown.PPs and NPPs samples of cecum were collected for amplification of 16 S r DNA and high-throughput sequencing analysis.The results showed that the species richness of PPs group was significantly lower than that of NPPs group.Firmicutes,Bacteroidetes and Proteobacteria were the main phyla,but the abundance ratio among samples varied greatly.At the genus level,Bacillus pumilus,Escherichia coli/Shigella,Bacteroidetes,Intertrophic bacteria and Riyanella were the TOP5 genera in terms of abundance in all samples.There were 19 phyla,75 families and 123 genera in the two groups,but the flora unshared between the groups were non-dominant groups with low abundance.The results indicated that the phyla intertrophic,Myxomelia and Archaea in PPs group were significantly higher than those in NPPs group,while Firmicutes were the most abundant phyla in NPPs group.Bacillus pumilus,Riyanella,Cladella and rumen cocci were biomarkers of NPPs group,while Escherichia coli/Shigella,Intertrophic bacteria,Veronococcus and Methanoblobacter were more dominant in PPs group.(4)Metabolites were extracted from cecum PPs and NPPs samples and untargeted metabolome tests were performed based on LC/MS.The results showed that the overall composition of metabolites in PPs group was significantly different from that in NPPs group.A total of 113 significant differentially expressed metabolites(DAMs)were obtained from the samples of both groups,including 12 DMAs with significant high expression and 101 DMAS with significant low expression in the PPs group.The compounds with significant abundance in the PPs group were Telmisartan,2-piperidone,1-Stearoyl-sn-glycerol 3-phosphocholine and Deoxycarnitine,and accumulated significantly high abundance ratios of NPPs group such as 5-GLC tricin,Icilin,Agnuside and Phenylbenzimidazolesulfonic acid.Deoxycholic acid,cholic acid and lithic acid were high contributors to the major differences between PPs and NPPs groups.The pathways with the TOP15 KEGG enrichment were mainly concentrated in metabolism,biological system and environmental information processing,among which the down-regulated differential metabolites were enriched in a higher proportion.The vitamin B6 metabolism and Fatty acid elongation were the two most enriched of all pathways.(5)The differences between PPs and NPPs were comprehensively analyzed by combining transcriptome,16 S r DNA high-throughput sequencing,and untargeted metabolome.The results showed that there was a strong interaction between the overall composition of microorganisms in each group and the significantly up-regulated metabolites in each group.A cluster containing 7 intestinal metabolites as well as 14 genera produced close associations and produced strong correlations,producing a total of 97 assemblages,indicating a tight interaction between them,and changes in the entire bacterial composition may alter the distribution of metabolites.However,there was no significant correlation between the expression of the whole gene and the metabolites or the intestinal flora,indicating that the changes of the intestinal metabolites or the intestinal flora had no significant influence or change on the gene expression between the samples during the stabilization stage.In this study,the complete cystic PPs structure was observed by the panoramic section scanner,and the immune microenvironment of cystic PPs in Bactrian camel’s cecum was studied by multi-omics method for the first time.As expected,the PPs group expressed more immune-related genes,and enrichment analysis revealed that nine hubs were key genes involved in multiple immune processes.In addition,untargeted metabolism indicates that most compounds of PPs and NPPs are similar in composition;DAM decreased in 88% of PPs,in which primary or secondary bile acids CA,DCA and LCA were major factors leading to group differentiation.In addition,the species richness of PPs is significantly lower,and the biomarker groups contain both probiotics and pathogens,which may form a synergistic effect of the cystic PPs immune microenvironment.There was no significant correlation between the changes of expressed genes and intestinal metabolites or microbial communities,but the changes of intestinal taxa directly led to the changes of metabolites.Our study provides new insights into the molecular characteristics of the immune function of the sacs PPs in Bactrian camels’ cecum. |
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