The Effect And Mechanism Of BMDC Overexpressing AIRE On T-bet~+ CXCR3~+ Treg Cells

The deletion and mutation of AIRE can cause autoimmune polyendocrine syndrome type 1,and its clinical manifestations include type 1 diabetes,autoimmune hepatitis and Hashimoto’s thyroiditis and other multi-organ autoimmune diseases.Studies have confirmed that AIRE is closely related to the maintenance of immune tolerance.In central tolerance,on the one hand,AIRE is mainly expressed on m TECs and DCs,which can eliminate itself by regulating the expression of restricted antigens in peripheral tissues Reactive T cells.In peripheral tolerance,AIRE is mainly distributed in the periphery:DCs,monocytes/mcrophages and lymph node stromal cells.Its functions are mainly to eliminate peripheral autoreactive T cells;regulate the maturity of antigen-presenting cells;induce T cells tolerance and so on.As a group of cells with immunosuppressive function,Treg mainly matures in the thymus and migrates to peripheral immune organs and tissues,playing an important role in maintaining normal peripheral immune tolerance.studies have shown that the proportion of Treg cells in the thymus of AIRE knockout mice is significantly lower than that of wild-type mice.Research on AIRE in autoimmune diseases caused by humans also found that Treg subgroups are damaged,suggesting that Tregs defects may be related to the lack of AIRE,but the specific regulatory mechanism is still unclear.The previous study of our research group found that the AIRE-transduced BMDCs was injected into NOD mice through the tail vein,and the results of flow cytometry showed that the number of Tregs increased significantly.We also co-cultured BMDCs from AIRE knockout mice and WT mice with initial CD4⁺T cells.Flow cytometry found that the number of Tregs in AIRE knockout mice was significantly less than that in the wild group.It is suggested that AIRE may affect the production of Treg through DCs.Recently,it has been reported in the literature that T-bet⁺CXCR3⁺Treg plays an important role in maintaining homeostasis,inhibiting local inflammation,and controlling autoimmune diseases.A significant enrichment of CXCR3⁺Tregs in the pancreas,pancreatic draining lymph nodes and systemic lymphatic organs of NOD mice was found,especially in the pancreas.Literature studies also show that the lack of T-bet⁺CXCR3⁺Treg leads to a significant increase in the probability of diabetes in NOD mice,and a significant increase in pancreatitis symptoms.This shows that T-bet⁺CXCR3⁺Treg can suppress autoimmune diseases.In addition,it is reported in the literature that IL-35 secreted by DC can act on na?ve CD4⁺T cells to promote the differentiation of T-bet⁺CXCR3⁺Treg.Therefore,we envisioned whether AIRE could affect the T-bet⁺CXCR3⁺Treg by regulating the expression of IL-35 in DCs,and exert its inhibitory effect on the autoimmune diabetes of NOD mice.This topic mainly uses AIRE-overexpressing BMDCs co-cultured with initial CD4⁺T cells or transferred to NOD mice to study the changes in T-bet⁺CXCR3⁺Treg during the treatment（prevention）of T1D by AIRE through BMDC in vivo and in vitro.And then explore whether AIRE can affect the differentiation of T-bet⁺CXCR3⁺Tregs by regulating the expression of IL-35.The specific research content includes:1.The effect of BMDC overexpressing AIRE on NOD mouse T-bet⁺CXCR3⁺Treg cells1.1 Establishment of a BMDC model overexpressing AIREIn order to establish a BMDC model that overexpresses AIRE,the bone marrow of wild mice was isolated to induce DCs,and they were transfected with empty（GFP）and AIRE-encoding lentiviral vectors,respectively,and detected by fluorescence microscopy,RT-qPCR and flow cytometry Transfection effect,the results show that the transfection efficiency in each detection method meets the requirements,indicating that the AIRE lentiviral vector has been successfully transduced to BMDC.1.2 To detect the effect of BMDC overexpressing AIRE on the T1D condition of NOD miceIn order to clarify the role of BMDC overexpressing AIRE in the TID condition of NOD mice,the BMDCs transfected with the empty group（GFP-BMDC）and the lentiviral vector encoding AIRE（AIRE-BMDC）were transplanted separately to the NOD mice by tail vein injection.they are divided into control group（GFP-BMDCs）,prevention group/treatment group（AIRE-BMDCs）,and the normal saline injection group is set as the blank control group（NS-BMDCs）.At 12,14 and 16 weeks（prevention group）and every other day（treatment group）after injection,the blood glucose and insulin autoantibody levels of mice were tested to monitor the progress of NOD mice.The results showed the blood glucose and autoantibody levels of the AIRE-BMDCs transfected group They are all lower than the control group,indicating that BMDC overexpressing AIRE can prevent or treat the onset of T1D in NOD mice,but the specific mechanism remains to be explored.1.3 Detect the effect of BMDC overexpressing AIRE on T-bet+CXCR3+Treg cells in NOD miceIn order to detect the effect of BMDC overexpressing AIRE on T-bet⁺CXCR3⁺Tregs of NOD mice,the pancreas,spleen,and pancreatic draining lymph nodes of each group of NOD mice were taken out,and the changes of T-bet⁺CXCR3⁺Tregs in each tissue were detected by flow cytometry.It was found that the number of T-bet⁺CXCR3⁺Tregs in the AIRE-BMDC group was higher than that in the control group,indicating that BMDC overexpressing AIRE may affect the production of T-bet⁺CXCR3⁺Tregs in NOD mice,but its mechanism of action needs further study.2.Study on the effect of AIRE on T-bet⁺CXCR3⁺Treg cells and its mechanism2.1 The effect of BMDC overexpressing AIRE on the differentiation of T-bet⁺ CXCR3⁺TregIn order to detect the effect of AIRE-overexpressing BMDC on the differentiation of T-bet⁺CXCR3⁺Treg,First,we used flow cytometry to detect the difference in the number of T-bet⁺CXCR3⁺Treg cells in the spleen,pancreas and pancreatic draining lymph nodes in AIRE^-/-mice and WT mice,and found that AIRE^-/-mice T-bet⁺CXCR3⁺Treg were lower than WT mice,suggesting that the lack of AIRE will affect the production of T-bet⁺CXCR3⁺Treg.Next,BMDC transfected with lentiviral vector was co-cultured with initial CD4⁺T cells,and the changes of T-bet⁺CXCR3⁺Treg in each group were detected by flow cytometry.It was found that the number of T-bet⁺CXCR3⁺Treg cells in the AIRE-transfected BMDC group was higher than that in the control group,indicating that BMDC overexpressing AIRE can promote the differentiation of T-bet⁺CXCR3⁺Treg.2.2 Study on the effect of AIRE on the expression of IL-35 in BMDCsIn order to study the effect of AIRE on the expression of IL-35 in BMDC,the expression of IL-35 mRNA in GFP-BMDCs and AIRE-BMDCs were detected by RT-qPCR,ELISA was used to detect the expression of IL-35 protein in the supernatants of the two cells.It was found that the expression of IL-35 in the AIRE-BMDCs group was higher than that in the control group regardless of the RNA level or the protein level,indicating that AIRE can promote the expression of IL-35 in BMDC,but whether AIRE regulates IL-35 in DC-The effect of 35 expression on the differentiation of T-bet⁺CXCR3⁺Treg needs further study.2.3 Clarify that AIRE regulates the differentiation of T-bet+CXCR3+Treg by affecting the expression of IL-35In order to clarify whether AIRE regulates T-bet⁺CXCR3⁺Treg by affecting the expression of IL-35,we antagonize or interfere with IL-35 in BMDC overexpressing AIRE,and then study its effect on T-bet⁺CXCR3⁺Treg from two parts in vitro and in vivo.The first is the in vitro part.The BMDC overexpressing AIRE is divided into experimental group and control group.The IL-35 gene in the experimental group is silenced by si RNA.The control group is treated with unrelated random control si RNA.The two groups were co-cultured with initial CD4⁺T cells,and the number of T-bet⁺CXCR3⁺Treg cells in each group was detected by flow cytometry.The results showed that the number of T-bet⁺CXCR3⁺Treg in the experimental group was lower than that in the control group.The NOD mice were further used for in vivo verification,and were also divided into the experimental group control group.The above-mentioned silenced IL-35 or control si RNA overexpressing AIRE BMDC were transplanted into NOD mice,Flow cytometry detected the changes of T-bet⁺CXCR3⁺Treg cells in the pancreas,pancreatic draining lymph nodes,and spleen tissues in the two groups.The results showed that the number of T-bet⁺CXCR3⁺Treg cells in each tissue in the experimental group was lower than that in the control group.The results of the above two parts of in vivo and in vitro experiments It shows that AIRE can regulate the production of T-bet⁺CXCR3⁺Treg by affecting the expression of IL-35 in DC cells.In summary,this study confirmed that AIRE can regulate the differentiation of T-bet⁺CXCR3⁺Treg,and that this process is mediated by promoting the expression of IL-35.BMDC overexpressing AIRE can regulate the expression of IL-35 and affect NOD mice.The differentiation of T-bet⁺CXCR3⁺Treg in the body affects the occurrence and development of T1D.This study provides a new entry point for understanding the role of AIRE in peripheral immune tolerance and for regulating the differentiation of T-bet⁺CXCR3⁺Treg to treat T1D and other autoimmune diseases.