Activation-Induced Cytidine Deaminase Deficiency Regulates The Occurrence And Development Of Type 1 Diabetes By Altering The Gut Microbiome And Regulating Immune Function

Aims:Type 1 diabetes(T1D)is an organ-specific autoimmune disease characterized by damage to pancreatic?-cells.The occurrence and development of type 1 diabetes is affected by many factors.Gut microbiota,innate immune system,and acquired immune system all play a regulatory role in the pathogenesis of T1D.Activation-induced cytidine deaminase(AID)is an important DNA editing enzyme in the development and maturation of B cells.AID can mediate the somatic hypermutation and the class switch recombination of B cells.At present,there are reports in the literature that AID deficiency is involved in the pathogenesis of systemic lupus erythematosus,and there are few studies on the impact of AID deficiency on T1D,and there is no research to explore the impact of AID deficiency on the gut microbiome.Therefore,by knocking out the AID gene systemically in non-obese diabetic mice(NOD)models,we observed the onset of diabetes,changes in gut microbiome and changes in immune function in AID-deficient NOD mice.Explore the mechanism through which AID deficiency regulates the occurrence and development of T1D,so as to provide new ideas for the treatment and prevention of T1D.Methods:(1)This study first established an AID knockout NOD mouse model,observed the incidence of type 1 diabetes in mice of this genotype,then observed the lymphocyte type of each immune organ and cell proliferation experiments in vitro in healthy 10-week-old AID-/-NOD mice.Then through adoptive transfer of lymphocytes into Rag-/-NOD mice,the diabetic ability of B cells and T cells in AID-/-NOD mice was further observed.(2)On the basis of establishing the protective effect of AID deficiency on T1D,this study further explored the effect of AID gene knockout on the gut microbiota of NOD mice.We analyzed the composition of gut microbiota from AID-/-NOD mice through 16S RNA sequencing technology.Then we transfer the gut microbiome of AID-/-NOD mice into germ-free NOD mice,by observing the gut permeability,immune function and antibody secretion function of the recipient mice to explore if the microbiota from AID deficiency mice can shape the immune system and affect type 1 diabetes.Results:(1)We observed the spontaneous diabetes in AID^-/-NOD mice and WT NOD mice.Through 30 weeks T1D incidence monitoring,it was found that the AID deficiency significantly delayed the onset of T1D and had a protective effect on T1D.(2)Through the staining analysis of surface antibodies and intracellular factor antibodies,it was found that in AID^-/-NOD mice,inhibited(CD73+)B cells,regulatory(Foxp3+)CD4+T cells,inhibited(PD1+?CXCR5+?CD73+)CD4⁺T cells and Tfh(CXCR5+PD1+CD4+)cells all increased,the anti-inflammatory cytokines-secreting cells increased,inflammatory cytokines-secreting cells decreased.(3)Through the lymphocytes proliferation experiments in vitro and adoptive transfer of lymphocytes to Rag^-/-NOD mice then observed the incidence of T1D,it was found that in AID-/-NOD mice,the antigen-presenting function of B cells decreased significantly and the diabetic effect of B cells and T cells decreased significantly.(4)The gut microbiota composition of AID^-/-NOD mice and WT NOD mice was detected by 16S RNA sequencing technology,it was found that in AID^-/-NOD mice,the?diversity a,?diversity and the proportion of various bacteria was significantly different from the WT NOD mice;and further analysis found that the ratio of Firmicutes and Bacteroidetes and the ratio of Gram+bacteria and Gram-bacteria were significantly increased in AID^-/-NOD mice.(5)By transferring the gut microbiome of AID-/-NOD mice and WT NOD mice into germ-free NOD mice,the gut permeability,lymphocyte surface antibody and intracellular cytokines antibody staining analysis,lymphocytes proliferation experiment in vitro and immunoglobulin detection of gut flush were observed.It was found that in mice with AID-/-NOD gut microbiome,the gut permeability was significantly lower than the control group,the lymphocytes proliferation was significantly lower than the control group,and the secretion of Ig A in the intestine was significantly higher than the control group.Conclusions:(1)AID deficiency protects the development of T1D in NOD mice;(2)AID deficiency can affect the function of B cells,leading to the weakening of the antigen presenting function of B cells,which in turn leads to the weakening of the role of B cells in causing diabetes;AID deficiency can affect the function of T cells,leading to the increase of regulatory CD4⁺T cells and inhibited CD4⁺T cells,leading to an increase in the secretion of anti-inflammatory cytokines and decrease in the secretion of inflammatory cytokines of T cells,which in turn leads to a weakening of the diabetic effect of T-cells;(3)AID deficiency alters the composition of gut microbiome,affects the?diversity,?diversity and the proportion of various bacteria;(4)The gut microbiota of AID^-/-NOD mice reduced the gut permeability and the inflammatory response in the intestines of the recipient mice;(5)The gut microbiota of AID^-/-NOD mice reduced the level of immune response in recipient mice;(6)The gut microbiota in AID^-/-NOD mice increased the secretion of Ig A in recipient mice.