Autoimmune Regulator Mediates Immune Tolerance By Regulating Perforin In BMDCs Through TLR7/8

Immune tolerance is an important function to maintain the homeostasis.Type 1 diabetes(TID)is an autoimmune disease in which autoreactive T cell tolerance is disrupted and attacks pancreatic β-cells leading to dysregulation of blood glucose.Autoimmune regulator(AIRE),as a transcriptional regulator,plays a key role in maintaining immune tolerance.Aire deficiency or mutation will lead to autoimmune polyendocrinopathy candidiasis ectodermal dystrophy(APECED),including TID.This indicates that exploring the mechanism of Aire induced autoimmune T cell tolerance is of great significance for elucidating some autoimmune diseases,and it is still necessary to further study the mechanism of Aire regulating immune tolerance in combination with the latest progress.At present,it is believed that immature dendritic cells(imDCs)play an immunosuppressive role on T cells.Our previous studies have shown that Aire expression in DCs can maintain the immature state of DCs,and participate in peripheral tolerance by inducing T cell anergy,Tregs differentiation and secreting immunosuppressive factors.Therefore,studying the relationship between DCs and T cells will help to explain the mechanism of Aire inducing autoreactive T cell tolerance through DCs.Previous studies have found that imDCs express perforin and clear CD8+T cells through perforin,which provides new insights for the role of DCs in inducing autoreactive T cell tolerance.And previous studies have found that TLR7/8 is a key molecule that mediates the killing of CD8+T cells by imDCs through perforin.In addition,we found that the overexpression of Aire in DC cell line DC2.4 could upregulate the expression of TLR7 and TLR 8.These results suggest that the mechanism of autoreactive T cell tolerance induced by Aire may be further clarified by exploring the effects of TLR7/8 and perforin pathways in DCs on autoreactive T cell tolerance.In order to further analyze the role of Aire as a transcription factor in peripheral tolerance,we introduced transcriptome sequencing and used Aire overexpression/knockout BMDCs and T1D model NOD mice as research objects to explore the role of Aire in inducing immune tolerance of autoreactive T cells through DC cells.In order to achieve the above purpose,we designed the following research:Ⅰ.Aire promotes perforin expression in BMDCs through TLR7/81.The expression of perforin in BMDCs knockout of Aire was decreased BMDCs were obtained from bone marrow cells of Aire knockout mice(B6.129S2Airetm1.1Doi/J)and co cultured with GM-CSF and IL-4.The expression of perforin was significantly decreased after Aire knockout by QRT PCR and FACS.2.The BMDCs cell model overexpressing Aire was successfully established BMDCs were obtained from bone marrow cells of Balb/c mice aged 6-8 weeks by the same method as above.BMDCs were transduced with lentiviral vector encoding Aire,and the Aire overexpression BMDCs cell model(Aire-BMDCs)was successfully constructed.3.The activation of TLR7/8 promotes the expression of perforin in Aire BMDCsTo further clarify the relationship between Aire and the expression of perforin in BMDCs,qRT-PCR and FACS results showed that the expression of perforin in Aire overexpressing BMDCs(Aire-Ctrl)was up-regulated;And the expression of perforin was further increased after stimulation with TLR7/8 agonist-R848(AireR848).Ⅱ.Transcriptome analysis of BMDCs overexpressing Aire1.RNA-seq showed that TLR7/8 activated the transcriptome of Aire BMDCs differentlyIn order to comprehensively analyze the role of Aire as a transcription factor at the gene level,we introduced transcriptome sequencing(RNA-seq).PCA analysis and anosim group similarity analysis showed that Aire overexpression and TLR7/8 activation made BMDCs significantly different at the transcriptome level.2.RNA-seq showed that TLR7/8 activated multiple related pathways in Aire-BMDCs were significantly enrichedThe differential expression analysis of samples in each group showed that the expression difference of perforin between GFP-BMDCs and Aire-BMDCs stimulated by R848 was the largest.Therefore,KEGG enrichment analysis of the differentially expressed genes showed that TLR,MAPK and NF-κB signaling pathway,apoptosis and necrosis,and T1D were significantly enriched.Ⅲ.Aire up regulates the expression of perforin in BMDCs by regulating TLR7/8MyD88/MAPK pathway1.Aire promotes the expression of TLR7/8-MyD88 signaling pathway molecules in BMDCsThe results of qRT-PCR and Western blot showed that TLR7/8 and its signaling pathway molecules(MyD88,IRAK4,TRAF6,NF-κB.IRF5 and MAPKs)were significantly up-regulated;The expression of TLR7/8-myd88 and its downstream molecules were down regulated after Aire knockout.These results indicate that Aire can promote the expression of TLR7/8 and its signaling pathway molecules in BMDCs.2.Aire promotes tlr8 transcription in BMDCsThe dual luciferase reporter gene assay showed that the luciferase activity of TLR8 promoter was significantly increased in Aire-BMDCs,while the luciferase activity of TLR7 promoter was not significantly different,indicating that Aire directly acts on the TLR8 promoter and regulates the transcription and expression of TLR8.The immunofluorescence results also confirmed this point,and it was found that the fluorescence of TLR8 in Aire-BMDCs was stronger.3.Antagonizing TLR7/8 or inhibiting its downstream molecules leads to the decrease of perforin expression in Aire-BMDCsIn order to clarify the molecular mechanism of Aire affecting the expression of perforin in BMDCs,we first antagonized TLR7/8 with CU115,and found that the expression of perforin in Aire-BMDCs decreased significantly.Then by inhibiting the key molecules downstream of TLR7/8 signaling pathway,such as MyD88 and MAPK molecules,the results showed that the expression of perforin in AireBMDCs decreased significantly after inhibiting MyD88,and the expression of perforin in Aire BMDCs also decreased after inhibiting MAPKs.These results indicate that Aire regulates the expression of perforin in BMDCs through the MyD88 dependent pathway through TLR7/8.Ⅳ.Aire affects T cell tolerance by up regulating perforin in BMDCs1.BMDCs overexpressing Aire up regulate the tolerance of T cells induced by perforinIn order to determine the effect of the expression of perforin in BMDCs induced by Aire on T cell tolerance,we used the co culture system of BMDCs and allogeneic T cells as the basis to investigate the proliferation,IFN-γ secretion,exhaustion and apoptosis of T cells by FACS.The results showed that TLR7/8 agonist could promote the inhibition of the proliferation of CD4+T cells and CD8+T cells by Aire-BMDCs(Aire-Ctrl).It can also inhibit IFN-γ in CD8+T cells,has no impact on the production of IFN-γ in CD4+T cells.There was no significant effect on the expression of TIGIT,Tim-3,PD-1,LAG-3 and other depletion related molecules on CD4+T cells and CD8+T cells.TLR7/8 agonist stimulation of AireBMDCs also promoted the apoptosis of CD8+T cells,but had no effect on the apoptosis of CD4+T cells.2.BMDCs knockout of Aire lead to decreased apoptosis of CD8+T cellsThe apoptosis of CD8+T cells induced by BMDCs knockout of Aire was significantly decreased,which further confirmed the key role of Aire in the apoptosis of CD8+T cells induced by BMDCs.3.Aire-BMDCs mediate the apoptosis and proliferation of CD8+T cells through perforinIn order to determine whether BMDCs overexpressing Aire induce apoptosis of CD8+T cells by perforin,When BMDCs and CD8+T cells were co cultured on 0.4μm aperture transwell plates(both were not in contact),the apoptosis of CD8+T cells was reduced.These results suggest that the apoptosis may be related to the direct contact between cells,which is consistent with the characteristics of perforin mediated killing.Furthermore,the expression of perforin in BMDCs was selectively silenced by RNA interference technology.The results showed that the apoptosis effect of perforin on CD8+T was significantly reduced,indicating that perforin is a key molecule mediating the apoptosis of CD8+T induced by BMDCs overexpressing Aire.In the same way,the inhibitory mechanism of BMDCs overexpressing Aire on the proliferation of CD4+T cells and CD8+T cells was explored.The results showed that the proliferation of CD8+T cells increased after silencing perforin,but CD4+T cells did not change,indicating that the inhibition of CD4+T cell proliferation was not through the mechanism of perforin.Ⅴ.Aire-BMDCs mediate apoptosis of islet autoreactive CD8+T cells in T1D mice by up regulating perforin1.Aire-BMDCs did not affect the apoptosis of total CD4+T cells and CD8+T cells in T1D miceThe changes of T1D were evaluated by blood glucose,serum insulin autoantibody(IAA)level and islet tissue integrity.The results showed that the blood glucose and IAA levels of mice with adoptive transfer of TLR7/8 activated Aire-BMDCs(Aire-R848)were significantly decreased,and the islet structure was the most complete.After silencing the expression of perforin(Aire-R848-RNAi group),it was found that the therapeutic effect on T1D mice was reduced.These results suggest that BMDCs overexpressing Aire can promote the therapeutic effect of TID in NOD mice through perforin,and may also mediate tolerance in vivo through the mechanism of perforin.2.Aire-BMDCs induce apoptosis of islet autoreactive CD8+T cells in T1D mice by up regulating perforinThe apoptosis of CD4+T cells/CD8+T cells in spleen and/or pancreatic draining lymph nodes(PLNs)of mice in each group was analyzed by FACS.The results showed that the apoptosis of CD4+T cells and CD8+T cells did not change significantly.3.Aire BMDCs did not affect the expression of CD8+T cell depletion molecules in T1D miceIn order to accurately analyze the interaction between adoptively transferred BMDCs and islet autoreactive CD8+T cells during T1D treatment in NOD mice,MHC/epitope tetramer/PE(InsB/tetramer:H-2Kd(LYLVCGERL))was labeled with fluorescence,and insulin B15-23 was specifically recognized.Islet autoreactive T cells were detected by FACS.In spleen and PLNs,we found that Aire-BMDCs stimulated by TLR7/8 agonist-R848 could promote the apoptosis of InsB15-23 specific CD8+T cells.When perforin was silenced,the apoptosis of InsB15-23 specific CD8+T cells decreased.This suggests that Aire overexpressing BMDCs promote the apoptosis of islet autoreactive CD8+T cells through perforin.4.Aire-BMDCs alleviate the T1D condition of NOD mice by up regulating perforinTo observe the effect of Aire-BMDCs on the depletion of autoreactive CD8+T cells through perforin in vivo.The expressions of depletion related molecules PD-1,Tim-3 and LAG-3 on total CD8+T cells and InsB 15-23 specific CD8+T cells in spleen and PLNs of mice in each group were analyzed.The results showed that there was no significant change in the expression of depletion related molecules on total CD8+T cells and autoreactive CD8+T cells in spleen and PLNs.These results suggest that the expression of perforin in BMDCs induced by Aire may not affect the depletion of autoreactive CD8+T cells and induce peripheral tolerance.In conclusion:1.Aire directly acts on the TLR8 promoter region and promotes the transcription of TLR8 in BMDCs,up regulates the expression of TLR8,and regulates the expression of perforin in BMDCs by affecting the TLR7/8-MyD88/MAPK pathway.2.In vitro studies confirmed that Aire mediated apoptosis and inhibited proliferation of CD8+T cells by up regulating the expression of perforin in BMDCs,thus playing an immune tolerance role.3.In vivo studies have confirmed that BMDCs overexpressing Aire can induce the apoptosis of islet autoreactive CD8+T cells through perforin,promote the peripheral tolerance of T cells,play a therapeutic role on NOD mice with T1D,and significantly alleviate the disease of NOD mice with T1D.This study revealed that Aire expressed in BMDCs can induce perforin expression in DCs through TLR7/8-MyD88/MAPK pathway,indicating that Aire affecting TLR7/8MyD88 signaling pathway is a new mechanism to regulate perforin expression in DCs.It was proposed that perforin was a new effector molecule for Aire to induce peripheral immune tolerance through DCs.And it was confirmed that BMDCs overexpressing Aire could effectively treat T1D in NOD mice through perforin,which provided an experimental basis for the treatment of autoimmune diseases by inducing autoreactive T cell tolerance.