| Aire?Autoimmune regulator?plays a role in maintaining central immune tolerance by regulating the ectopic expression of thousands of tissue-restricted antigens?TRAs?in medullary thymic epithelial cells?m TECs?and dendritic cells?DCs?,leading to the deletion of autoreactive T cells or induction of regulatory T cells?Tregs?and preventing autoimmune disease development.Aire mutations result in APECED disease,which is characterized by multiple organ disorders mediated by an autoimmune response.These patients may also develop diabetes,hypoparathyroidism,Addison's disease et al.Autoimmune diseases comprise a group of various immune-mediated disorders characterized by an invasive adaptive immune response to self-components,resulting in pathology and clinical disease such as type 1 diabetes?T1D?,multiple sclerosis,systemic sclerosis,and rheumatoid arthritis.More than 60 autoimmune diseases have been reported.Current clinical therapy is mainly the application of hormones and immune inhibitors.Although such treatments have improved the prognosis of autoimmune diseases,they cannot cure these diseases.A cure for autoimmune disease has become a major clinical challenge.The ideal treatment should involve specialized deletion of autoreactive T cells and induction of stable and lifelong immune tolerance,which may achieve both controlling the immune response to self-components and maintaining resistance to pathogens.DCs are professional antigen-presenting cells?APCs?that are key controllers of innate and adaptive immunity and central regulators of immune tolerance.Recently,more attention has focused on exploring methods for prevention and treatment of autoimmune diseases,which are based on DCs.Fortunately,some therapeutic effects have been achieved and the security has been proved,DC-based immunotherapy may be a new and effective method to establish tolerance.Based on the backgrounds,we tried to establish a new way to induce immune tolerance and explore a new strategy for autoimmune disease treatment.Gene transduction and bone marrow transplantation were employed to generate Aire-overexpressing bone marrow-derived DCs?BMDCs?.We observed the effect of Aire on BMDCs tolerance,then intravenously transplanted Aire-BMDCs into unexplained NOD T1 D mice and streptozotocin?STZ?induced T1 D mice,observing the effect of Aire-BMDCs on the development of T1 D,further exploring the possible mechanism.I.The effect of Aire on the tolerance state of BMDCs Studies have shown that immature DCs can induce immune tolerance by low expression of costimulatory molecules and MHC-II,induction of Tregs and promoting Th1/Th2 drift.DCs in thymus play an important role in negative selection by expressing various of TRAs regulated by Aire.Therefore,we observed the effect of Aire on the expression of costimulatory molecules,MHC-II and TRAs in BMDCs.Furthermore,we investigated the influence of Aire-/--BMDCs?KO-BMDCs?on CD4+T cell subsets differentiation.1.Establishment of Aire-overexpressing BMDCs First of all,we separated mice bone marrow cells,which were successfully induced into BMDCs by GM-CSF?20ng/ml?and IL-4?20ng/ml?for six days.The purity of CD11c+cells was detected by FACS,as high as 84.5%.The lentivirus vector encoding Aire was constructed and high concentration virus was successfully obtained.Then BMDCs was infected by virus for 72 h and the transfection efficiency was 80-90%.The expression of Aire was high in transfected BMDCs detected by RT-q PCR and Western Blot.The Aire-overexpressing BMDCs?Aire-BMDCs?was successfully established.2.The effects of Aire on tolerance related molecules in BMDCs In order to observe whether Aire could influnce the expression of tolerance related molecules in BMDCs,such as costimulatory molecules and MHC-II,the expression of these molecules in WT/KO-BMDCs and GFP/Aire-BMDCs was detected by FACS.Results showed that,Aire-BMDCs expressed lower levels of CD40,CD86 and MHC-II than GFP control group.KO-BMDCs expressed higher levels of CD40,CD80,CD86 and MHC-II than WT-BMDCs.These data indicated that Aire may induce immune tolerance through keeping the low level expression of costimulatory molecules and MHC-II in BMDCs.3.The influence of Aire on TRAs expression in BMDCsTo investigate the influence of Aire on TRAs expression in BMDCs,the expression of TRAs related to multiple autoimmune diseases in GFP/Aire-BMDCs and WT/KO-BMDCs was detected by RT-q PCR.Results showed that,a variety of TRAs expression in Aire-BMDCs were upregulated,such as Ins2,GAD65/67,IA-2,IGRP,Rbp3,Spt1,Nalp5 and Mog;the expression of Ins2,GAD65/67,IA-2,IGRP,RRAD,Rbp3,Spt1,Nalp5,Mog,MUP1 and Lad1 in KO-BMDCs were lower compared to WT-BMDCs.In addition,the effects of Aire on TRAs expression were broadly examined in DC2.4 and RAW264.7 cells,the results were similar to in BMDCs.These results revealed that the ectopic expression of Aire in BMDCs can upregulate the expression of various TRAs and then induce immune tolerance.4.The effects of Aire-/--BMDCs on CD4+T cell subsets differentiation.?1?To observe wether Aire can influence CD4+T cell differentiation through BMDCs,WT/KO-BMDCs,which were unstimulated,stimulated by 1?g/ml LPS and100ng/ml CD40 L for 24 h respectively,were co-cultured with na?ve CD4+T cells?98% purity detected by FACS?,separated by magnetic beads.The dishes were coated with anti-CD3 antibody?5ug/ml?for 3h in advance.After 72 h,CD4+T cells differentiation was detected by FASC.Results showed that in unstimulated condition,KO-BMDCs inhibited the na?ve CD4+T cells to Th2,Treg and promoted them to Th1,Th17,Tfh;In LPS stimulation group,KO-BMDCs inhibited the na?ve CD4+T cells to Treg and promoted them to Th1,Tfh;In CD40 L group,KO-BMDCs inhibited the na?ve CD4+T cells to Treg and promoted them to Th17.In short,Aire can induce Treg and inhibite Th1,Th17 or Tfh.?2?To explore the mechanism of Aire affects BMDCs,the expression of cytokines related to T cells subsets differentiation in WT/KO-BMDCs was further detected by RT-q PCR.,Results showed,the production of IL-4 and TGF-? was decreased,the production of IL-12p35 and IL-6 was increased in KO-BMDCs.These data indicated that Aire may affect CD4+T cells subsets differentiation through cytokine production.From the results,we can conclude that Aire may induce immune tolerance through keeping the low level expression of costimulatory molecules and MHC-II,promoting IL-4 and TGF-?,reducing the production of IL-12p35 and IL-6 in BMDCs.II The effects of transplantation of Aire-overexpressing BMDCs on Type 1diabetesThe vitro studies have shown that Aire in BMDCs have the potentiality to induce immune tolerance by maintaining the low expression of costimulatory molecules and MHC-II,upregulating the expression of TRAs and inducing Treg and Th2 production.Therefore,we further tried to study if the transplanted Aire-BMDCs into T1 D mice could prevent onset of disease,and also to explore the possible mechanism.1.The effects of transplantation of Aire-overexpressing BMDCs on pathogenesis of NOD mice?1?The prevention of T1 D by transplanting Aire-BMDCs in NOD miceTo observe the effects of Aire-BMDCs on T1 D,GFP/Aire/Aire-si RNA-BMDCs were intravenously transplanted into unexplained 4 weeks NOD mice,NOD injected NS were taken as control group.The injection of BMDCs every two weeks,for five times in all.The level of blood glucose and the degree of inflammatory infiltration was detected on 6,8,10,12,14 and 16 weeks,the level of IAA was detected on6,10,12 and 16 weeks NOD.Results showed that the degree of inflammatory infiltration in Aire group was lighter,the production of blood glucose and IAA was lower.At 16 weeks,NS,Aire/Ins-si RNA and GFP group.atttacked T1 D,the Aire group didn't have the disease.The results indicated that transplantation of Aire-BMDCs prevents the onset of T1 D in NOD mice.Also demonstrated that Aire can induce immune tolerance by regulating Ins expression.?2?The mechanism of induction of immune tolerance by Aire-BMDCs.In order to investigate the mechanism of induction of immune tolerance,splenocytes were separated from GFP/Aire/Aire-Ins-si RNA-BMDCs mice at 6,10,12 16 weeks.Results showed that at 10 weeks,the apoptosis rate of autoreactive CD4+T cells in Aire-BMDCs group was high.At 12,16 weeks,the apoptosis rate keep increasing in Aire group.At 16 weeks,the amount of Th1,Th17 and Tfh was lower than control group,the amount of Treg and Th2 was significantly increased.The data indicated that Aire-BMDCs can induce immune tolerance in NOD mice by deleting autoreactive CD4+T cells and induction of Treg and Th2.2.The effects of transplantation of Aire-overexpressing BMDCs on pathogenesis of STZ-T1 D mice?1?The delay of T1 D by transplanting Aire-BMDCs in STZ-T1 D miceTo observed the effect of Aire-BMDCs on T1 D in STZ-T1 D mice,GFP/Aire-BMDCs were transplanted into mice and injected once every two days for a total of four injections.The mice were injected intraperitoneally with streptozotocin?STZ?for induction of T1 D on the second day after injection of BMDCs.The results showed that the blood glucose and IAA of Aire-BMDCs group were significantly lower than those of the control group.Pancreatic tissue HE staining showed that the infiltration of the Aire group was mild and the islet structure was complete.The results indicated that,Aire-BMDCs can relieve STZ-T1 D mice disease,delayed onset.?2?The mechanism of Aire-BMDCs induces immune tolerance in STZ-T1 D miceIn order to study the mechanism of Aire-BMDCs in the induction of immune tolerance in STZ-T1 D mice,the apoptosis of CD4+T cells,the activation state of TCR signaling pathway and CD4+T cell subsets in the spleen of mice were examined by FACS.The results showed that Aire-BMDCs promoted the apoptosis of autoreactive CD4+T cells.The expression of p-ERK and Ca2+ in TCR signaling pathway was decreased after Con A,CD3 and Insulin stimulation for 24 h.Aire induced CD4+T anergy;the number of Th1,Th17 and Tfh in spleen cells of Aire-BMDCs group decreased,the production of Treg increased and Th1/Th2 ratio decreased.Results indicated that Aire-BMDCs can induce immune tolerance by inducing apoptosis and anergy of autoreactive CD4+T cells in STZ-T1 D mice,promoting the production of Treg,promoting the migration of Th2.From the results,we can conclude that transplantation of Aire-overexpressing BMDCs can induce immune tolerance,consequently prevent the occurrence of T1 D in NOD mice,delay the onset of STZ-T1 D mice.In summary,this study investigated the effects of Aire on the tolerance status of BMDCs and the effect of transplantation of Aire-overexpressing BMDCs on the pathogenesis of autoimmune diabetic mice.The results confirmed that Aire-BMDCs can prevent or delay the onset of T1 D by inducing immune tolerance.From the study,a reasonable and effective way to induce immune tolerance have been explored,and that could provide new ideas and new methods for prevention and treatment of autoimmune disease. |
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