Effect Of Aire On TH1 Cell Differentiation During Immunosenescence By IL-27

As an important transcriptional regulator to maintain immune tolerance,Aire removes autoreactive T cells by regulating the expression levels of tissue restricted antigens(TRAs)in peripheral tissues,thereby maintaining immune tolerance.Deletions and mutations in Aire cause a rare hereditary disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy,with the classic triad of mucocutaneous candidiasis,hypoparathyroidism,and adrenocortical failure.Similarly,more and more studies have found that with increasing age,the immune system of the elderly ages aging,the level of IFn-γproduced by peripheral blood CD4+T cells in the elderly is higher than that in the young,while the level of IL-4 decreases,suggesting that elderly individuals have the polarization phenomenon of TH1,and its consequences can lead to an increased incidence of autoimmune diseases.We therefore speculated whether Aire could mitigate aging of the immune system by regulating TH1,thereby affecting susceptibility to autoimmune diseases.In order to better study aging,we used a D-gal aging mouse model that is more convenient to perform,has more stable aging performance,and will experience immunosenescence-related manifestations,and previous studies by our group have found that BMDCs transduced with Aire reduce the expression of IL-27,while recent studies have found that IL-27,which is also a member of the IL-12 cytokine family,can promote the differentiation of na?ve T cells into TH1 and bias the internal environment of the body towards a pro-inflammatory state.Therefore,we envisaged whether Aire could regulate IL-27 expression in senescent DC cells,which in turn affected TH1 cell differentiation and ultimately aggravated the immunosenescence performance of D-gal induced aging model mice.In this study,D-gal was mainly used to induce Aire-KO mice and WT mice to establish aging models,to detect whether Aire would affect the immunosenescence performance of D-gal induced aging model mice,and BMDCs were obtained from aging model mice,and then Aire was transduced into BMDC models and co-cultured with na?ve T cells to study the changes of TH1 during the aggravation of immunosenescence performance by Aire through BMDCs in vitro and in vivo,and then to investigate whether Aire affected the differentiation of TH1 by regulating the expression of IL-27,and the specific research contents included:Ⅰ.Effect of Aire on the aging status and TH1 cell differentiation of D-gal-induced aging model mice1.Effect of Aire on the aging status of D-gal-induced aging model miceIn order to clarify the effect of Aire on immune aging in mice,we used D-gal induced aging model to subcutaneously inject D-gal solution and normal saline into the neck and back of Aire-KO mice and WT mice,respectively,by observing the appearance and diet and drinking water,detecting the SOD activity and MDA content in tissues and serum,performing aging-relatedβ-galactosidase staining in spleen and thymus tissues and calculating their organ index,detecting the expression of Ig G and Ig M antibodies in serum and the expression of SASP in BMDCs to evaluate the establishment of the model and studying the role of Aire in the process of model induction.The results showed that both KO+D-gal mice and WT+D-gal mice showed aging characteristics,including dim and rough hair,decreased SOD activity and increased MDA content in tissues and serum,aging and atrophy in spleen and thymus tissues,decreased expression of Ig G and Ig M antibodies in serum and increased expression of some SASP in BMDCs.And the above aging characteristics of KO+D-gal mice were more obvious.2.Effect of Aire on T cells in D-gal-induced aging model miceIn order to detect the effect of Aire on T cells during aging,the spleens of mice in each group were removed,and the changes of na?ve T/memory T and TH1/TH2 in CD4~+T cells in the spleens of mice were detected by flow cytometry.It was found that the levels of na?ve T/memory T in splenic CD4~+T cells were significantly decreased in KO+D-gal mice and WT+D-gal mice,and the levels of na?ve T/memory T in splenic CD4~+T cells were significantly decreased in KO+D-gal mice compared with WT+D-gal mice.These results suggest that Aire may delay the decrease of na?ve T/memory T levels in splenic CD4~+T cells of mice during aging and then delay immunosenescence.However,the levels of TH1/TH2 in the spleen of KO+D-gal mice and WT+D-ga mice increased significantly,and the increase was most significant in the KO+D-gal group,suggesting that Aire may delay the increase of TH1/TH2 levels in the spleen during aging and then alleviate the pro-inflammatory state in immunosenescence.However,its mechanism of action remains to be investigated.Ⅱ.Effect of Aire via IL-27 on TH1 cell differentiation in D-gal-induced aging model mice1.establishment of BMDC model in D-gal-induced aging model mice overexpressing AireIn order to establish the BMDC model of D-gal induced aging model mice induced by D-gal overexpressing Aire,mature BMDCs were obtained and cultured from WT and WT+D-gal mice and transfected with lentiviral vectors encoding Aire and empty(GFP),respectively,and the transfection effects were detected by fluorescence microscopy,flow cytometry and RT-q PCR.The results of each detection method showed that the transfection efficiency was high,indicating that the lentiviral vector encoding Aire had been successfully transduced into BMDCs of aging model mice.2.the effect of BMDCs from D-gal-induced aging model mice overexpressing Aire on TH1 cell differentiationIn order to examine the effect of BMDCs from D-gal induced aging model mice overexpressing Aire on TH1 cell differentiation,BMDCs from D-gal induced aging model mice that had been transfected with lentiviral vectors were co-cultured with na?ve T cells,and the changes of na?ve T/memory T and TH1/TH2 in splenic CD4+T cells of mice in each group were detected by flow cytometry.It was found that the number of TH1 cells in the aging BMDC model group transfected with GFP(GFP-BMDC+D-gal)and the aging BMDC model group transfected with Aire(Aire-BMDC+D-gal)was increased compared with the control group,and the number of TH1 cells in the Aire-BMDC+D-gal group was lower than that in the GFP-BMDC+D-gal group,indicating that BMDCs from D-gal induced aging model mice overexpressing Aire could down-regulate the differentiation of TH1 cells during aging.3.Effect of Aire on IL-27 expression in BMDCs of D-gal-induced aging model miceIn order to investigate the effect of Aire on IL-27 expression in BMDCs of D-gal induced aging model mice,we first detected the expression of IL-27 m RNA in BMDCs of WT control,WT+D-gal model,KO control,and KO+D-gal model mice by RT-q PCR and found that IL-27 m RNA content in BMDCs of WT+D-gal model and KO+D-gal model mice increased compared with the control group and increased more significantly in the KO+D-gal model group.Next,RT-q PCR was continued to detect the expression of IL-27 m RNA in GFP-BMDC,GFP-BMDC+D-gal,Aire-BMDC,and Aire-BMDC+D-gal cells,and it was found that the expression of IL-27 m RNA in GFP-BMDC+D-gal and Aire-BMDC+D-gal cells was increased to varying degrees compared with the control group,and IL-27 m RNA expression was higher in GFP-BMDC+D-gal cells compared with Aire-BMDC+D-gal cells.These results suggest that Aire can down-regulate IL-27 expression in BMDCs during aging,but whether Aire affects TH1 differentiation by regulating IL-27 expression in DCs during aging in mice remains to be further investigated.4.Aire regulates the differentiation of TH1 cells by affecting the expression of IL-27 in BMDCs of D-gal-induced aging model miceIn order to clarify that Aire regulates TH1 by affecting the expression of IL-27 in BMDCs of D-gal-induced aging model mice,we added IL-27 recombinant protein to BMDC cells in the four groups and then co-cultured them with na?ve T cells,and detected TH1/TH2 changes in each group by flow cytometry,and found that exogenous IL-27 could up-regulate the number of TH1 cells in all groups,while TH2 did not show differential changes.These results indicate that Aire can regulate the differentiation of TH1 cells by affecting the expression of IL-27 in BMDCs of aging model mice.In summary,this study confirmed that Aire can affect the aging status and TH1cell differentiation of D-gal induced aging model mice,and Aire can affect the differentiation of Th1 cells in D-gal induced aging model mice by regulating the expression of IL-27,thereby affecting the state of immunosenescence.This study provides a comprehensive understanding of the role and significance of peripheral Aire in the process of immunosenescence,and provides new ideas for relieving(preventing)immunosenescence against TH1 cell differentiation.