The Role And Mechanism Of HMGB1in Chronic Rejection Of Murine Cardiac Transplantation

Background:Chronic rejection that leads to allograft vasculopathy is the most important cause of cardiac allograft dysfunction. Current molecular mechanisms are still unknown. The role and underlying mechanism of HMGB1, as an established inflammatory mediator in acute rejection, has been clarified, whereas remains poorly understood in chronic rejection.Objective:To explore the role and cellular as well as molecular mechanisms of HMGB1in chronic rejection of murine cardiac transplantation and allograft vasculopathy.Methods:1. Preparation of chronic rejection of murine cardiac transplantation model--B6.C-H-2bm12KhEg mice were used as donors and C57BL/6mice were used as recipients. This is an established murine cardiac transplantation model of MHC class-II mismatched with spontaneous chronic rejection.2. HMGB1antibody blocking treatment--Monoclonal anti-HMGB1neutralizing antibody was injected into recipient intraperitoneally (i.p.),200μg each time, from the day before transplantation, then twice a week till week four after transplantation. Mouse IgG served as isotype controls.3. Detection methods and indices(1) Immunohistochemical staining, immunofluorescence to detect the expression of HMGB1, transforming growth factor beta (TGF-β) in cells which infiltrated in grafts.(2) H&E and EVG staining to test the chronic rejection and cardiac allograft vasculopathy.(3) RT-PCR and Western Blot to detect the expression of HMGB1, TGF-p.(4) ELISA to measure the levels of associated cytokines in sera, tissue homogenates and cell culture supernatants.(5) FAM to detect the T cells reaction, the change of regulatory T cells, the content of allo-antibodies, macrophage, the maturation of dendritic cells (DCs) and its subpopulation.(6) One-way mixed lymphocyte culture to test donor specific T cells response.Results:1. Successful establishment of the abdominal heterotopic heart transplantation model of chronic rejection, harvested the transplanted heart samples which reflected the occurrence, development process of chronic rejection, cardiac allograft vasculopathy (CAV) and myocardial fibrosis.2. HMGB1and TGF-P were increased accompanying with the development of chronic rejection. Blockade of HMGB1with specific neutralizing monoclonal antibody to treat with recipients, could substantially ameliorated chronic rejection-mediated vasculopathy and fibrosis of allograft.3. Anti-HMGB1mAb treated recipients, the effect of chronic rejection associated cells and molecules included--(1) Effects on the innate immune cellsBlockade of HMGB1treatment markedly declined the proportion and number of macrophages and matured DCs in allografts and peripheral lymphoid organs, especially a subpopulation of CDllb+Ly6Chigh mature DCs that share the phenotypes with inflammatory DCs (Inf-DCs).In vitro experiments shown that with the stimulation of independent HMGB1or TGF-β1, murine bone marrow-derived dendritic cells’(BMDC) phenotype matured and were differentiated to similar with Inf-DCs subgroup. At the same time, HMGB1induced matured DCs drove allo-reactive T cells proliferation and Th17and Thl responses.(2) Effects on the T cellsBlockade of HMGBl treatment markedly decreased the donor-specific cell-mediated immune responses such as Thl/Thl7reaction, increased the proportion of Tregs.(3) Effects on the pro-inflammatory factorsBlockade of HMGB1treatment significantly reduced inflammatory cells infiltration in allograft during chronic rejection process and the production of associated cytokines such as, TNF-α, IL-1β and TGF-β.(4) Effects on the pro-fibrotic key molecule TGF-βBlockade of HMGB1treatment declined the expression of TGF-β. In vitro experiments shown that HMGB1had a direct role on promoting TGF-β, it could induce mice cardiac mononuclear cells to secret free active TGF-β directly.(5) Did not affect the donor-specific humoral immune response and complements reaction.Conclusion:1. HMGB1is involved in chronic rejection of murine cardiac transplantation and the development of CAV. Its primary mechanism may be by (1) promoting DCs maturation in heart allograft and recipient’s spleen and drive donor-reactive Th17and Thl responses.(2) promoting inflammatory cells to secrete TGF-β then further accentuating inflammatory response and injury, repair of tissue.2. HMGB1specific neutralizing monoclonal antibody can remarkly ameliorate chronic rejection as well as vasculopathy and fibrosis of allograft.3. The damage associated molecular pattern (DAMP) molecule is also involved in chronic rejection the same as alloantigen.