Study On The Expression Of Tim-1 And Its Mechanism In B Lymphocytes In Type 1 Diabetes Mellitus

Background:Type 1 dibetes mellitus(T1DM)is an autoimmune disease mediated by T cells,Recently,it was found that B cells also play an important role in the pathogenesis of T1DM;B cells are capable of secreting the inflammatory factor interleukin-10(IL-10)to maintain immune homeostasis.The subset of B cells secreting large amounts of IL-10 is defined as regulatory B cells(Breg);T cell immunoglobulin and mucin domain(Tim)protein family members were identified to be important regulators of the immune response.Tim-1,a member of the Tim family,was recently found to be expressed in B cells;The expression and specific role of Tim-1 in B cell subsets in T1DM remains unclear.Objective:(1)Comparing the frequency of B cell subsets and the correlation of clinical parameters in T1DM,Type 2 diabetes mellitus(T2DM)and healthy control(HC).(2)Exploring the mechanism of CD19~+Tim-1~+B lymphocytes in the pathogenesis of T1DM.(3)Detecting Tim family molecules and CD4~+T expression profiles of transcription factors in Peripheral blood mononuclear cells(PBMCs)from T1DM.Methods:(1)PBMCs were collected and isolated from the T1DM,T2DM,and HC populations.The expression frequencies of B cell subsets in all participants were detected using flow cytometry,clinical parameters were collected from all populations,and diabetes-related antibody titers in T1DM were detected by radiobinding assay.The clinical factors affecting the altered expression of B cell subsets were analyzed.In addition,the correlations between B cell subsets and the types of autoantibodies was investigated.(2)To explore the role of CD19~+Tim-1~+B lymphocytes in T1DM in three aspects:quantitative level,regulatory level and functional level;Detect the expression of CD19~+B cells in Tim-1 m RNA and protein level from T1DM and HC populations;Changes in CD19~+Tim-1~+B cells were observed by stimulating cell activation and inhibiting signaling pathways.And to examine the response of CD19~+Tim-1~+B cells to external factors;Magnetic bead sorting and co-culture were used to detect the regulatory effect of CD19~+Tim-1~+B cells on the production of inflammatory factors and proliferation of CD4~+T cells.It provides a new direction for exploring the immune markers and intervention targets of T1DM.(3)The m RNA expression levels of Tim family molecules(Tim-1,Tim-3,Tim-4)and CD4~+T cell subset-specific transcription factors(T-bet,GATA3,RORC,FOXP3)in PBMCs from all the all participants were measured by RT-PCR.The expression of Tim family molecules in serum was examined by ELISA.The T1DM population was grouped according to disease duration,antibody titer,and islet function to clarify the characteristics of the population with significant immune imbalance.The correlation between Tim molecules and CD4~+T cell subset in T1DM was compared,and to investigate the possibility that Tim molecule affects the pathogenesis of T1DM by regulating the differentiation of CD4~+T cells.Results:(1)Compared with HC,the frequency of Bregs in the T1DM was significantly decreased(P=0.002),and the frequencies of Tim-1~+Bregs and IL-10~+Bregs were significantly lower than those in the HC group(P=0.02,P=0.009,respectively);Correlation analysis between the frequencies of various B cell subsets and clinical parameters in all participants showed that:the frequency of Tim-1~+Bregs was negatively correlated with FBG(r=-0.25,P=0.01),and positively correlated with FCP(r=0.23,P=0.02);the frequency of IL-10~+Bregs was negatively correlated with FBG(r=-0.22,P=0.03),negatively correlated with Hb A1c(r=-0.20,P=0.05),and positively correlated with FCP(r=0.37,P=0.0001);There was no significant correlation between all B cell subsets and islet-related autoantibody in the T1DM.(2)Compared with HC,the expression of Tim-1 m RNA in B cells of T1DM was significantly decreased(P=0.002),and the expression of CD19~+Tim-1~+B cells was decreased(P=0.039);The ability of B cells in T1DM to upregulate Tim-1 and IL-10 was impaired after activation(P=0.032,P=0.001,respectively),After inhibiting p38 and STAT-3signaling pathways,CD19~+Tim-1~+B cells in T1DM were down-regulated.However,This decline was not as well as in the HC.In addition,the CD19~+Tim-1~+B cells from T1DM could not effectively inhibit the proliferation and production of inflammatory factors of CD4~+T cells(P>0.05).(3)Compared with HC,the expression of Tim-1 in T1DM was significantly increased,and the expression of Tim-3 was significantly decreased(P=0.0355,P=0.0013,respectively),and the expression of RORC in T1DM was higher than that in HC population(P=0.0423);In terms of expression balance,Tim-3/Tim-1,T-Bet/GATA3 and RORC/FOXP3 ratios were altered in T1DM compared to HC(P<0.0001,P=0.0042 and P=0.0066,respectively).Correlation analyses between Tims with T cell-specific transcription factors was performed by Spearman test.the expression of Tim-1 was positively correlated with RORC in the T1DM(P<0.0001).Conclusions:(1)The expression of Bregs,Tim-1~+Bregs and IL-10~+Bregs was decreased in T1DM.Clinical indicators such as blood glucose and pancreatic islet function levels may be related to the imbalance of expression of B cell subsets.The The expression in Bregs in T1DM patients may affect their function of secreting IL-10.(2)The expression frequency of CD19~+Tim-1~+B cells in the T1DM was decreased,and impaired responses of CD19~+Tim-1~+B cells to activated or inhibited signaling pathways.CD19~+Tim-1~+B cells in the T1DM are defective in suppressing CD4~+T cell proliferation and production of IFN-γand IL-17.(3)Altered expression of Tim-1,Tim-3,and RORC in PBMCs of T1DM.Tim-3/Tim-1,T-bet/GATA3,and RORC/FOXP3 ratios were imbalanced in the T1DM,and this imbalance is related to antibody titers,disease duration and islet function.Tim-1 is positively correlated with the RORC m RNA expression and may be involved in the pathogenesis of T1DM.