Autoimmune Regulator Regulates T_FH Cell Differentiation Through DCs And Its Effects On Type 1 Diabetes

Type 1 diabetes(T1D)is an autoimmune disease characterized by inflammatory infiltration of islets and destruction of pancreatic β cells by autoreactive cells.The pathogenesis of T1D is a very complicated process.Dendritic cells(DCs),T cells and B cells play different roles in the β-cell injury of T1D,and the pathogenesis has not been fully elucidated.Autoimmune regulator(AIRE)plays an important role in immune tolerance.The deletion or mutation of AIRE will lead to the occurrence of T1D.In the central immune system,AIRE is mainly expressed on medullary thymic epithelial cells(mTECs),and induces autoreactive T cell apoptosis and regulatory T cell differentiation by regulating the expression of a variety of tissue-restricted self-antigens.In the peripheral immune system,AIRE is mainly expressed in DCs and maintains peripheral immune tolerance by regulating the differentiation of CD4+T cells such as follicular helper T cells(TFH).To explore the role of AIRE can further elucidate the pathogenesis of T1D.TFH cells are crucial in the pathogenesis of T1D.Excessive activation of TFH cells can regulate the activity of autoreactive B cells by secreting IL-21,and promote the production of pathological autoantibodies during the germinal center(GC)reaction,then leading to T1D.DCs participate in the induction of naive CD4+T cells to differentiate into TFH cells.Our previous work found that AIRE-overexpressed bone marrow-derived dendritic cells(BMDCs)(AIRE-BMDCs)had preventive and therapeutic effects in T1D mouse models.To explore the effect of AIRE-BMDCs on the differentiation of TFH cells in T1D mice could further elucidate the role of AIRE in the pathogenesis of T1D.Based on the above background,we took DCs overexpressing AIRE as the research object,combined with in vitro and in vivo experiments,to explore its impact on T1D and TFH cell differentiation.The research content is as follows:Ⅰ.The effect and mechanism of AIRE-overexpressing DCs on TFH cell differentiation in vitro1.Establishment of stably transfected AIRE-overexpressing DCs(1)Establishment of stably transfected AIRE-overexpressing DC2.4 cellsIn order to detect the transfection efficiency and AIRE expression level of DC2.4 cells transfected with PEGFP/C1(C1-1-1)or PEGFP/AIRE(A1-1),Flow cytometry(FACS),inverted fluorescence microscopy,Western blot and RT-qPCR were used,the results showed the transfection efficiency in both cell group reached about 90%and A1-1 cells have a high level of AIRE expression and can be used for subsequent experiments.(2)Establishment of stably transfected AIRE-overexpressing BMDCsWe established AIRE-overexpressing BMDCs(AIRE-BMDCs)and control(GFP-BMDCs).The transfection efficiency and AIRE expression level in these two cells were detected by the same experimental method as above.The results showed that transfection efficiency of both groups was about 85%and AIRE was highly expressed in AIRE-BMDCs,indicating that the BMDCs overexpressing AIRE(AIRE-BMDCs)has been successfully established and can be used in subsequent experiments.2.The Effects of AIRE overexpression on the expression of molecules related to TFH cell differentiation on DCs(1)The Effects of AIRE overexpression on the expression of molecules related to TFH cell differentiation on DC2.4 cellsTo explore whether AIRE can affect the expression of molecules related to TFH cell differentiation on DCs,including ICOSL,IL-27 and IL-6,we selected LPS and poly(I:C)to stimulate DC2.4 cells,respectively.We found that AIRE overexpression and exogenous stimulation had no effect on DC2.4 cell proliferation and apoptosis.At the same time,the results showed that AIRE can inhibit the expression of ICOSL and IL-27 in DC2.4 cells,but has no effect on the expression of IL-6.In addition,Western blot results showed that AIRE suppress the expression of ICOSL by inhibiting the level of IκB,the specific mechanism needs to be further studied.(2)The Effects of AIRE overexpression on the expression of molecules related to TFH cell differentiation on BMDCsIn order to explore the effect of AIRE on the expression of molecules related to TFH cell differentiation on BMDCs,we used the same experimental method as above,and found that AIRE overexpression and exogenous stimulation had no effect on the proliferation and apoptosis of BMDCs.Unlike the DC2.4 cell line,AIRE not only inhibited the expression of ICOSL and IL-27 on BMDCs,but also inhibited the expression of IL-6.We also found that AIRE may inhibit the expression of ICOSL or IL-6 by inhibiting IκB in the NF-κB canonical pathway,and IRF3 in the IRF3/7 signalling pathway may be involved in regulating the expression of AIRE on IL-27 in BMDCs.The results of the DC2.4 cells and BMDCs experiments are not completely consistent.The reason may be that the physiological state and genetic integrity of the DC2.4 cell line are different from the BMDCs,or it may be due to the DC2.4 cells and the BMDCs in this study were derived from mouse strains with different genetic backgrounds.Compared with the DC2.4 cells,the primary cell BMDCs retains more biological characteristics of the original cells in vivo,which can better reflect the growth status of DCs in vivo.Compared with IL-6 and IL-27,AIRE significantly inhibited the expression of ICOSL in BMDCs under inflammatory or viral environment,indicating that AIRE has the most obvious inhibitory effect on ICOSL in DCs,indicating that ICOSL may be a entry point to exploring the effects of overexpression of AIRE in DCs on TFH differentiation in T1D mice.3.The effect and mechanisms of AIRE-overexpressing DCs on the TFH cell differentiation and the production of effector molecule(1)The effect and mechanisms of AIRE-overexpressing DC2.4 cells on the TFH cell differentiation and the production of effector moleculeTo understand whether AIRE-overexpressing DC2.4 cells affect TFH cell differentiation and effector molecule production,we co-cultured A1-1 and C1-1-1 cells with na?ve CD4+T cells.All data showed that AIRE expressed in DC2.4 cells can inhibit TFH cell differentiation and their ability to secrete IL-21.In addition,TFH cell differentiation and IL-21 expression in C1-1-1 and A1-1 group were all increased after adding recombinant ICOSL or IL-27 protein,which suggesting AIRE suppress the differentiation of TFH cells and production of IL-21 by inhibiting the expression of ICOSL and IL-27.(2)The effect and mechanisms of AIRE-overexpressing BMDCs on the TFH cell differentiation and the production of effector moleculeUsing the BMDCs/T cell co-culture system,we found AIRE significantly inhibit the ability of BMDCs to induce the TFH cell differentiation and IL-21 production.The addition of ICOSL,IL-6 or IL-27 recombinant protein effectively promoted the differentiation of naive CD4+T cells into TFH cells and significantly induced TFH cells to secrete IL-21,suggesting that AIRE can inhibit the differentiation of TFH cells and the secretion of IL-21 by inhibiting the expression of ICOSL,IL-6 and IL-27 in BMDCs.Ⅱ.The effect and mechanism of AIRE-overexpressing BMDCs on the differentiation of TFH cells in T1D mice in vivo1.The effect of AIRE-BMDCs transplantation on T1D(1)The preventive effect of AIRE-BMDCs transplantation on T1DAIRE-BMDCs were transferred to 3-4-week-old T1D mouse mode(NOD mice)using tail vein injection.We found that NOD mice transplanted with AIRE-BMDCs significantly reduced blood glucose,islet autoantibodies(IAA)production and infiltration at 12 or 16 weeks of age,while the use of ICOSLG decreased the inhibitory effect of AIRE-BMDCs,indicating that transplantation of AIRE-BMDCs in the early stages of the disease can significantly delay the progression of diabetes,and this process could be inhibited by ICOSL.(2)The therapeutic effect of AIRE-BMDCs transplantation on T1DThe experimental groups were set up as above.BMDCs were transplanted into NOD mice with new-onset type 1 diabetes at the age of 17 weeks.NOD mice transplanted with AIRE-BMDCs had significantly lower blood glucose,IAA,and less inflammation infiltration,it is worth noting that more NOD mice survived in the AIREBMDCs group after the treatment.The application of ICOSLG reduced the efficacy of AIRE-BMDCs in the treatment of autoimmune diabetes.In summary,AIRE-BMDCs transplantation attenuates new-onset type 1 diabetes in NOD mice,and this process could be inhibited by ICOSL.2.The effect and mechanism of AIRE-BMDCs transplantation on TFH cells and effector molecule production in T1D mice(1)The effect and mechanism of AIRE-BMDCs transplantation on TFH cells and effector molecule production in the process of preventing T1DIn the process of transplanting AIRE-BMDCs to prevent the occurrence of T1D in NOD mice,we detected the proportion of TFH cells and IL-21 expression in the spleen,skin draining lymph nodes(sLN)and mesenteric lymph nodes(mLN).The transplantation of AIRE-BMDCs could inhibit the differentiation of TFH cells and the production of effector molecules in NOD mice,and the application of ICOSLG could regulate this inhibitory effect.In addition,we found that the proportion of TFR cells in each group was consistent with that of TFH cells,and the proliferation characteristics of TFR cells are consistent with those of TFH cells,and the ratio of the TFH/TFR cells is not statistically different between the groups,indicating that AIRE mainly affects TFH cell differentiation.(2)The effect and mechanism of AIRE-BMDCs transplantation on TFH cells and effector molecule production in the process of attenuating T1DUsing FACS to detect the frequency of TFH cells in the three secondary lymphoid organs of NOD mice at the end of the transplantation experiment.The results indicate that transplantation of AIRE-BMDCs does indeed inhibit the expansion of TFH cells during the treatment of T1D,and this effect can be inhibited by ICOSL.3.The effect of AIRE-BMDCs transplantation on GC B cells in T1D mice(1)The effect of AIRE-BMDCs transplantation on GC B cells in the process of preventing T1DThe above results indicate that transplantation of AIRE-BMDCs can effectively reduce IL-21+TFH cells,which prompted us to further explore whether GC B cells will also be affected in this process.The results showed AIRE-BMDCs can inhibit the generation of GC B cells in the prevention of T1D,and ICOSL could partially reverse its effects.(2)The effect of AIRE-BMDCs transplantation on GC B cells in the process of attenuating T1DAt the end of the transplantation experiment,we used FACS to detect the GC B cell proliferation in each group of mice.We found that AIRE-BMDCs can inhibit the generation of GC B cells in the onset of T1D mice,and ICOSL has an inhibitory effect on them.In summary,the results indicate that AIRE could inhibit TFH cell differentiation and IL-21 production by inhibiting the expression of TFH cell-associated molecules such as ICOSL on DCs.Transplantation of AIRE-BMDCs can inhibit the proliferation of TFH cells and reduce the production of IL-21 during the prevention or treatment of T1D in NOD mice,thereby inhibiting the development of GC B cells.ICOSL can partly affect the regulation of AIRE-BMDCs on TFH cells in T1D mice.This study provides new ideas for understanding the role and mechanism of AIRE in peripheral immune tolerance,and provides an experimental basis for using AIRE as a target to prevent or treat T1D by regulating TFH cell differentiation and GC B response.