The Role And Mechanism Of CD40-TRAF6 In Acute And Chronic Rejection Of Murine Cardiac Transplantation

Background:Heart transplantation is the best treatment for all kinds of end-stage heart disease at present.The acute and chronic rejection after heart transplantation is the main causes affecting the effects of heart transplantation.CD40-TRAF6 is involved in a variety of chronic inflammatory and fibrotic processes,and its role in solid organ transplantation remains unclear.Therefore,we investigated the role of CD40-TRAF6 interactions in murine cardiac transplantation.Objective:In this study,acute and chronic rejection models of murine cardiac transplantation were conducted.CD40-TRAF6 specific antagonist was used to explore the effects of CD40-TRAF6 on the alloimmune responses of murine cardiac transplantation.To explore whether CD40-TRAF6 inhibitor has protective effects on cardiac allograft vasculopathy.To provide a theoretical and experimental basis for the clinical application of new immunosuppressive drug targets for heart transplantation.Methods:1.To explore the effects of CD40-TRAF6 specific antagonist in murine heart transplantation acute rejection model.Murine cardiac transplantation acute rejection model:Using BALB/C as donors and C57BL/6 mice as recipients.CD40-TRAF6 specific antagonist or DMSO was given after transplantation.Survival time of cardiac allografts was observed,allografts and spleens were collected at fixed time points and then analyzed.2.To explore the role of CD40-TRAF6 in chronic rejection model of murine cardiac transplantation.Chronic rejection model of murine cardiac transplantation with B6.C-2H-2^bm12KHEG as donors and C57BL/6 as recipients.CD40-TRAF6 specific antagonist or DMSO was given after transplantation.Cardiac allografts and spleens were collected and analyzed at fixed time points.3.The effect of CD40-TRAF6 antagonist combined with cyclosporine A on acute rejection model of murine cardiac transplantation.Acute rejection of murine cardiac transplantation model:BALB/C mice as donors and C57BL/6 mice as recipients.CD40-TRAF6 specific antagonist combined with cyclosporine A were given in this model.The cardiac allografts’survival time were observed.Cardiac allografts and spleens were collected at fixed time points.4.Bone marrow derived macrophages（BMDMs）were further cultured in vitro.CD40-TRAF6 antagonist or DMSO or given to explore effects of CD40-TRAF6 inhibition on BMDMs migration and differentiation.Mixed lymphocyte culture in vitro,to explore the possible mechanism of CD40-TRAF6 pathway inhibition.Results:1.Acute and chronic murine cardiac transplantation rejection models were successfully established.Pathological analysis of cardiac allografts harvested at each time point were basically consistent with the process and development of acute rejection and chronic rejection model.2.CD40-TRAF6 specific antagonist prolonged the survival time of cardiac allografts in acute rejection model,and alleviated Th1/Th17 mediated alloimmune responses.3.CD40-TRAF6 specific antagonist alleviated the severity of CAV in the chronic rejection model.The infiltration of CD4+and CD11b+cells in the cardiac allografts were reduced.4.CD40-TRAF6 specific antagonist in combination with cyclosporine A significantly prolonged the cardiac allografts’survival time in the acute rejection model,and alleviated the severity of CAV compared with CTLA-4Ig treatment.5.Inhibition of the CD40-TRAF6 pathway in vitro.（1）Effects on CD4+naive T cells.Inhibition of the CD40-TRAF6 pathway has limited effects on the mixed culture of murine lymphocytes.（2）Inhibition of the CD40-TRAF6 pathway had no significant effects on the proliferation of CD4+naive T cells stimulated by CD3\CD28.（3）Inhibition of the CD40-TRAF6 pathway decreased the expression of the pro-inflammatory phenotype of BMDMs.Conclusion:Inhibition of CD40-TRAF6 alleviated murine cardiac transplantation alloimmune responses.Inhibition of CD40-TRAF6 alleviates CAV in chronic rejection model of murine cardiac transplantation.Inhibition of CD40-TRAF6 combined with Cs A could induce long-term survival of cardiac allografts in acute rejection model.Inhibition of CD40-TRAF6 affected the migration of BMDMs and reduced the differentiation of BMDMs into inflammatory phenotype in vitro.CD40-TRAF6 inhibitor has the potential to be a supplementary drug in clinical heart transplantation,and may become a new target to alleviate CAV.