Children Gut Flora Of Different Vitamin A Levels Impacts Intestinal Mucosal Barrier Function In Germ-free Mice

Objective:To explore the effects of different vitamin A levels fecal microbiota on intestinal mucosal barrier function of germ free mice after transplantation with children fecal with vitamin A normal and vitamin A deficiency.Methods:The feces of children with vitamin A normal(VA≥1.05mmol/L)and vitamin A deficiency(<0.70 mmol/L)werecollected from a kindergarten of Chongqing,and were respectively transplanted intothegermfreemice(GF mice)toestablish VAN-FMT and VAD-FMT mice.After 28 days of FMT,HE staining was used to observe the pathological changes of mice’s small intestine and colon tissues;Western blotting was detected the expression levels of Occludin and Claudin-1 in the small intestine tissues;ELISA kits was tested the concentrations of serum DAO,IgA and SIgA in cecum feces;Flow cytometry was used to evaluate the percentages of dendritic cells(DCs)in the Peyper Patch,B cells in the mesenteric lymph nodes(MLN),intraepithelial lymphocytes(IELs),T lymphocytes in the lamina propria(LPLs),Treg cells and T cells in the spleen.And 16S rRNA gene sequencing was analyzed colonic mucosal flora in the mice.Results:During the experiment,all the mice in each group were normal and the differences of weight gain were not significant among the three groups after the FMT for 4 weeks.HE staining found that the intestinal mucosa and colonic mucosa of the VAN-FMT mice were more mature compared with those of the GF and VAD-FMT mice,and there were a large number of inflammatory cells infiltrating in the colonic mucosa and submucosa of the VAD-FMT mice.The expression levels of Occludin and Claudin-1 in the VAN-FMT group were significantly higher than those in both GF control and VAD-FMT groups(P<0.01,P<0.001),and only Occludin protein expression level in the VAD-FMT group was significantly increased compared with that in GF group(P<0.01).Serum DAO level in the VAN-FMT mice were statistically decreased compared with those in the VAD-FMT and GF mice.The concentration of IgA in the VAN-FMT group was significantly higher than that in the GF control group(P<0.05);there was no significant difference in the concentration of IgA between the GF mice group and the VAD-FMT group.Meanwhile,no significant differences were observed in serum SIgA concentration among the three groups.The percentages of B lymphocyte in MLN of the VAN-FMT group were significantly decreased compared with those of the VAD-FMT group(P<0.05)and GF group(P<0.001),and the B lymphocytes ratio in MLN of the VAD-FMT group was also significantly lower than that of GF mice group(P<0.05).The percentages of DCs in Peyper Patch of the VAN-FMT group were statistically increased compared with those of the VAD-FMT(P<0.001)and GF two groups,while the DCs ratio in Peyper Patch of the VAD-FMT group was significantly lower than that in the GF mice group(P<0.05).There were no statistical differences in the percentages of CD8~+TCRγδ~+T lymphocyte in intestinal interepithelial lymphocyte among the three groups.The proportions of CD4~+T lymphocyte in lamina propria of both the VAN-FMT group(P<0.001)and VAD-FMT group(P<0.001)were significantly higher than that of the GF control group,while there was no significant difference in the percentages of CD4~+T lymphocyte between the VAN-FMT group and VAD-FMT group.The percentages of CD8~+T lymphocyte in LPLs had no significant differences among the three groups.The percentages of CD4~+CD8~+double positive T lymphocytes of lamina propria in the VAN-FMT group were significantly increased compared with those in the VAD-FMT(P<0.05)and GF two groups(P<0.01),but significant difference was not found in the later two groups.The results of 16S rRNA analysis showed that the composition of colonic mucosal microbiota was similar between the VAN-FMT and VAD-FMT groups,and there were no differences in ACE,Chao,Shannon and Simpson diversity index in both VAN-FMT group and VAD-FMT group(P>0.05).Firmicutes and Bacteroides were the most abundant bacteria in the VAN-FMT and VAD-FMT groups,and Proteobacteria was significantly increased in the VAN-FMT mice(P<0.05).At genus level,Bacteroides was the most abundant bacteria in the VAN-FMT group(P<0.05),Helicobacter(P=0.03)and Lachnospiraceae(P=0.005)were increased significantly in the VAN-FMT group.Staphylococcus was the most abundant bacteria in the VAD-FMT group.LEfSe analysis displayed that 29 species in the VAN-FMT group were significantly different in VAN-FMT group,and 10 species in the VAD-FMT group.Conclusion:The fecal microbiota transplantation of children VAN intestinal microbiota remarkably improves the intestinal development of GF mice,upregulates the expression levels of intestinal tight junction-related proteins Occludin and Claudin-1 to enhance intestinal epithelial barrier function.Meanwhile,the VAN-FMT decreases persentage of B lymphocytes in MLN,induces the maturation of dendritic cells in Peyper Patch to promote the B cells homing from MLN to intestinal tract and IgA secretion,also increases the ratios of CD4~+,CD4~+CD8~+T cells in the intestinal lamina propria.These data suggests that VAN intestinal microbiota induces gut innate mucosal immune activity in the GF mice through dominant bacteria Bacteroides,while opportunistic pathogens Staphylococcus is dominant in the VAD intestinal microbiota,and Prevotella is increased,which lead to intestine of VAD-FMT mice shows inflammation and gut mucosal immune regulation is decreased in the GF mice.