Therapeutic Effect Of Bone Marrow DCs Transduced With Aire On Type 1 Diabetes Mellitus In NOD Mice

The autoimmune regulator(Aire)is an important transcription factor involved in immune tolerance.In the central immune system,Aire is mainly expressed in medullary thymic epithelial cells(m TECs)and dendritic cells(DCs),promotes ectopic expression of many tissue-restricted antigens(such as insulin),to prevent the occurrence of autoimmune diseases(such as type 1 diabetes)by inducing central immune tolerance.In addition,Aire also express in the peripheral immune system.In the periphery,Aire is mainly expressed in lymph node stromal cells and DCs.The study found that,the RNA transcripts encoding Aire were detected in mouse and human DC subsets in vitro,but Aire expression level in DCs was low.The pathogenesis of T1 D is that insulin self-reactive T cells destroy islet ? cells,reduce insulin secretion,develop a series of clinical manifestations such as hyperglycemia,at the same time,chronic inflammation leads to ? cell-specific effector T cells(Teff)which mediate islet inflammation.The development of the disease has further aggravated the condition.In addition,the onset of T1 D is also affected by the dysfunction of regulatory T cells(Tregs).The current clinical treatment of T1 D is mainly depended on insulin administration to maintain blood glucose levels in patients.However,there are many adverse reactions in the long-term injection of insulin,there are many adverse reactions associated with drug treatment.For example,the use of metformin as an adjuvant treatment of T1 D leads to a transient reduction in Hb A1 c,long-term use cannot maintain blood glucose levels.Therefore,it requires more effective clinical treatment for T1 D.The study confirmed that under the control of Aire,thymic epithelial cells participate in transcription of all members of the insulin gene family,indicating that Aire plays an important role in preventing the pathogenesis of T1 D,and the management of Aire genes may contribute to the effectiveness of prevention and treatment of autoimmune diseases including T1 D.As people continue to deepen research on DCs,they find that DCs play a pivotal role in the immune system.As an important bridge between innate immunity and adaptive immunity,they play an important role in immune homeostasis and immune response.DCs were regarded as important immune modulators.On the one hand,DCs are capable of initiating immunity against foreign antigens.On the other hand,DCs maintain their tolerance to self-tissue antigens.DCs play an important role in thymus-negative selection and induce the production of Tregs by expressing a series of TRAs and participate in maintaining central tolerance.The migration of peripheral DCs to the thymus gland is a key mechanism for central tolerance of peripheral tissue antigens.In non-lymphoid tissues,migratory DCs continue to migrate through the afferent lymphatic vessels to draining lymph nodes under steady state,maintaining peripheral tolerance.However,there are many types of DCs.The ability of each DC subset to induce and promote immune tolerance is still unknown and needs further investigation.Studies have shown that ectopic expression of Aire can promote the expression of TRAs for the treatment of EAE.It has also been confirmed recently that the use of DCs to treat EAE has significantly improved the condition.Therefore,we plan to use Aire and DCs to induce immune tolerance and combine them to treat T1 D.We first transduced Aire to bone marrow-derived DCs,evaluated their tolerance by detecting the expression of related molecules and their effects on T cell subsets and autoreactive T cell apoptosis in vitro.Furthermore,it was transplanted into mice with T1 D by tail vein injection to observe its effect on the condition of diabetes and explain the possible mechanism of its therapeutic effect in NOD mice,seeking a more effective treatment method for T1 D.Specific research includes:? Aire-overexpressing BMDCs are tolerogenic in vitroThe immature DCs have the property of inducing immune tolerance,which is characterized by low levels of surface co-stimulatory molecules and MHC-II molecules.Therefore,we investigated the effect of Aire on the expression of costimulatory molecules,MHC-II,and TRAs in BMDCs.Then,we examined the effect of Aire-BMDCs on the differentiation and development of CD4+T cell subsets and insulin self-reactive T cell proliferation and apoptosis.1.Effect of Aire on BMDCs tolerance(1)Expression of tolerogenic molecules in Aire-overexpressing BMDCsImmature DCs are immunologically tolerant,and their surface costimulatory molecules(CD40,CD80,CD83,and CD86)and MHC-II molecules have low expression levels.To observe the effect of Aire on the expression of tolerance molecules in BMDCs,we examined the expression of costimulatory molecules and MHC-II molecules after stimulated by LPS,CD40 L,and Poly(I:C)in GFP-BMDCs and Aire-BMDCs,respectively.The results showed that under the stimulation of LPS,CD40 L and Poly(I:C),the costimulatory molecules and MHC-II molecules in Aire-BMDCs were reduced to different degrees compared with GFP-BMDCs,suggesting that Aire could inhibit the maturation of BMDCs to maintain its tolerance.(2)The effect of Aire-overexpressing BMDCs on the expression of TRAsIn the thymus,Aire can induce the expression of various peripheral tissue-restricted antigens(TRAs)and participate in central immune tolerance.To determine the effect of Aire on the expression of TRAs in BMDCs,the expression of several T1D-associated TRAs was examined by RT-q PCR,including insulin 2(Ins2),insulinoma-2(IA-2),glutamic acid decarboxylase 65/67(GAD65/67),insulin-like growth factor 2(IGF-2),islet-specific glucose-6-phosphatase related protein(IGRP),and chromogranin A(CGA).The results showed that,compared with GFP-BMDCs,several T1D-associated TRAs expressions were increased in Aire-BMDCs.These results suggest that Aire can participate in maintaining immune tolerance by promoting the expression of TRAs in BMDCs.2.Effect of Aire-BMDCs on differentiation of T lymphocyte subsets and insulin self-reactive T cells(1)The effect of Aire-overexpressing BMDCs on T-cell subsets differentiationIn addition,DCs can also participate in immune tolerance by affecting the differentiation of different types of CD4+T cell subsets.We observed the effect of Aire-BMDCs on the differentiation and development of na?ve CD4+T cells.First,the na?ve CD4+T cells were sorted by magnetic bead sorting,and the purity of the cells was 96.3%.The isolated cells were then co-cultured with GFP-BMDCs/Aire-BMDCs stimulated with LPS,CD40 L or Poly(I:C)for 24 h.Subsequent differentiation of CD4+T cell subsets was measured by flow cytometry.The results showed that Aire-BMDCs promoted the differentiation of na?ve CD4+T cells into Th2 and Tregs,but inhibited the differentiation of na?ve CD4+T cells into Th1,Th17 and Tfh cells in the LPS-stimulated group;Aire-BMDCs promoted na?ve CD4+T cells into Tregs in the CD40L-stimulated group;in Poly(I:C)stimulated groups,Aire-BMDCs inhibited the differentiation of na?ve CD4+T cells into Th1 and Tfh cells.These suggested that Aire-BMDCs can promote the differentiation of na?ve CD4+T cells into Tregs and Th2 cells,inhibit the differentiation of na?ve CD4+T cells into Th1,Th17 and Tfh cells,which maintain the tolerance.(2)Effect of Aire-overexpressing BMDCs on proliferation and apoptosis of insulin autoreactive T cellsIn the thymus,m TECs and DCs can recognize and bind autoreactive T cells through presenting TRAs,induce their apoptosis and maintain central immune tolerance.To investigate whether Aire-BMDCs also have a similar effect,we used insulin to treat GFP-BMDCs/Aire-BMDCs for 1h and then co-culture with mouse splenocytes for 48 h to examine the proliferation and apoptosis of T cells in vitro.The results showed that Aire-BMDCs can promote the proliferation and apoptosis of insulin-reactive T cells compared with GFP-BMDCs,indicating that Aire can enhance the clearance of autoreactive T cells by BMDCs and participate in the maintenance of immune tolerance.Above all,Aire-overexpressing BMDCs inhibit the expression of costimulatory and MHC-II molecules,promote the expression of several T1D-associated TRAs,induce differentiation of na?ve CD4+T cells into Th2 and Tregs,and inhibit the development of Th1,Th17 and Tfh cells,and promote the proliferation and apoptosis of insulin-reactive T cells.These indicate that Aire-overexpressing BMDCs are tolerogenic under normal conditions.? Transplantation of Aire-overexpressing BMDCs on type 1 diabetes in NOD miceAfter observing the tolerance of Aire-BMDCs in vitro,we further transplanted Aire-overexpressing BMDCs into already-onset diabetic NOD mice and observed their therapeutic effects on T1 D.1.Effect of Aire-BMDCs on the symptoms and survival of NOD mice(1)The effect of Aire-BMDCs on the condition of NOD miceIn order to investigate whether Aire-overexpressing BMDCs can exert immune tolerance in vivo,we observed the effect of transplanted Aire-BMDCs on T1 D status in already-onset NOD mice.NOD mice are set to be 4 groups.The groups were: normal saline(NS group),GFP-BMDCs(GFP group),Aire-BMDCs(Aire group)and Aire/Ins-si RNA-BMDCs(si RNA group).After the onset of NOD mice,BMDCs were injected for treatment.The course of treatment was five times,once every two days.During the treatment period,blood glucose of mice was continuously monitored.After the end of treatment,the changes of insulin autoantibody(IAA)were detected by ELISA.The inflammatory infiltration of pancreatic tissues in NOD mice was observed by HE staining.The results showed that blood glucose levels were lower in the Aire group than in the NS group,the GFP group,and the si RNA group in the NOD mice;the IAA levels in the Aire group were significantly lower than those in the NS group,the GFP group,and the si RNA group;pancreatic islet destruction in the Aire group alleviate.The islet structure is complete compared to the control group.These results indicate that Aire-overexpressing BMDCs can alleviate the condition of T1 D in NOD mice and play a therapeutic role.(2)The effect of Aire-BMDCs on the survival rate of NOD miceIn the late stages of development of T1 D in NOD mice,persistently elevated blood glucose leads to serious consequences that will eventually lead to death.Therefore,in order to understand the effect of Aire-BMDCs on the survival rate of NOD mice,we observed the survival of 4 groups of NOD mice 40 days after treatment(all mice in the NS group died).As a result,it was found that the survival rate of the NOD mice in the Aire group was higher at the 40 th day,while the survival was lower in the NOD mice in the NS group,the GFP group,and the si RNA group.This shows that Aire-BMDCs can delay the condition of NOD mice and prolong their survival period.2.Effect of Aire-BMDCs on T lymphocyte subsets and autoreactive T cells in NOD mice(1)The effect of Aire-BMDCs on the differentiation of T lymphocyte subsets in NOD miceTo initially explain the effect of Aire-BMDCs on the therapeutic mechanism of T1 D in NOD mice,whether it is an immune tolerance effect that affects the subpopulation of CD4+T cells.At the end of NOD treatment,we used flow cytometry to detect CD4+T cell subsets in four groups of NOD mice.The results showed that the number of Th1,Th17 and Tfh cells in the Aire group was lower than those of the control group,and the production of Th2 and Treg increased.The results showed that Aire play a therapeutic role by altering the status of CD4+T cell subsets in NOD mice.(2)Effect of Aire-BMDCs on autoreactive T cells in NOD miceIn order to further investigate the mechanism of Aire-BMDCs in the treatment of NOD mice,whether it can also affect autoreactive T cell apoptosis and exert immune tolerance.Similarly,we used flow cytometry to further examine the apoptosis of self-reactive CD4+T cells from splenocytes of NOD mice after treatment.The results showed that the apoptosis of CD4+T cells in Aire group was significantly higher than that of other groups.The results indicate that Aire-BMDCs protects pancreatic ?-cells and promotes further progression of the disease by promoting apoptosis of autoreactive CD4+T cells in NOD mice.In summary,the results showed that transplanting Aire-BMDCs can significantly reduce blood glucose levels in NOD mice,reduce IAA content,reduce islet tissue damage,and prolong the survival period of NOD mice.Exploration of the mechanism deeply revealed that Aire-BMDCs induced the immune tolerance by promoting the production of Th2 and Tregs cells in NOD mice,inhibiting the production of Th1,Th17 and Tfh cells,promoting autoreactive CD4+T cell apoptosis,and alleviating the disease in NOD mice.