The Role And Mechanism Of ST2 In Cardiac Allograft Chronic Disease Of Murine Cardiac Transplantation

Background:Although advances in immunosuppression regimens and improved overall survival,chronic rejection is the leading cause of mortalityafter heart transplant.Cardiac allograft chronic disease is a major vexing problem in the long-term survival of the cardiac transplant recipient.The mechanisms for cardiac allograft chronic diseaseare not fully elucidated.The ST2receptor is a member of the IL-1 receptor family.IL-33 as the ligand of ST2 is a member of the IL-1 family of cytokines.In stead state,IL-33 is localized in the nucleus of cells.Upon tissue damage,it is releasedextracellularly to play an immunomodulatory role in the extracellular space.ST2 is expressed on Th2 cells,macrophages and regulatory T cells（Treg）.ST2 receptor is constitutively expressed by a broad range of cardiac myocytes,vascular smooth muscle cells and vascular endothelial cells.At present,the function and mechanism of ST2 in cardiac graft chronic disease remains unclear.This study focused on the function of ST2 in cardiac graft chronic diseaseof heart transplantation and further explored the mechanisms.Objective:This study is made to analyze the roles of ST2 in cardiac graft chronic disease,which might shed light on unveiling novel mechanisms for the development of cardiac graft chronic disease after cardiac transplantation.Metheds:1.In experiment,we investigated the roles of ST2 on allograft or recipient in cardiac graft chronic disease respectively using MHC-mismatch murine chronic cardiac allograft rejection model.1.1 For heterotopic cardiac transplantation models,C57BL/6（B6）and bm12 mice（B6（C）-H2-Ab1bm12/KhEgJ[H-2bm12]）mice were used both as donors and recipients,respectively.In single MHCII mismatched models,bm12 mice were used as donors and B6 mice were used as recipients.Meanwhile,the ST2^-/- mice received bm12 donors’hearts as the experimental group.Donors and recipients switch roles in turning.This is an established mice cardiac transplantation model of chronic allograft rejection without immunosuppressive treatment.1.2 Combinedheterotopic cardiac transplantation were performed in the bm12 recipient mice from the wild-type C57BL/6 and the C57BL/6ST2^-/-donor mice.After cardiac transplantation,allograft impulse was assessed by daily abdominal palpation.2.Histological analysis of allograft sections2.1 HE and Masson staining to test cardiac allograft vasculopathy and allograft vasculopathy fibrosis.2.2 Immunohistochemical staining to detect immune cells which infiltrated in allograft vascular.3.Flow cytometry to detect the T cells and its subpopulation,B cells and macrophages.4.One-way mixed lymphocyte culture to evaluate donor specific T cell alloreactivity.Results:1.Successful establishment of the isograft heart transplantation.The cold high-potassium solution（cardioplegia solutions）were administered in graft of syngeneic transplantation,which were no obvious myocardial injury within 5 hours and all the isograft could resume beating normally.2.Successful establishment of the heterotopic cardiac allograft transplantationof model of chronic rejection,harvested cardiac allograftsamples which reflected chronic pathological changes such as cardiac graft vasculopathy（CAV）,myocardial tissue damage and fibrosis.3.We found that recipients ST2 deficiency significantly exacerbated allograft vascular occlusion,myocardial damage and fibrosis,accompanied by increased infiltration of macrophages,T cells and lymphangiogenesis.In contrast,allografts ST2 deficiency resulted in decreased infiltration of macrophages,T cells,B cells,lymphangiogenesis and thus alleviated vascular occlusion,myocardial damage and fibrosis of allografts.3.1 ST2 deficiency in recipients markedly increased infiltration of macrophages,B cells within intima and T cells in the middle membrane of the allografts.3.2 Deficiency of ST2 in allogrft significantly diminished the number of T cellswithin intima and macrophages,B cells and T cells in areas around the middle membrane of arteries.3.3 There wereno significant changes in tertiary lymphoid tissue formation in ST2deficiency of allografts and recipients respectively.3.4 Co-culture experiments showed that allograft ST2 deficiency attenuates allo-reaction T cells proliferation.4.The development trend of cardiac allograft chronic disease in the single heart transplantation was different from combined heart transplantation in the bm12recipient mice from thewild-type C57BL/6 and the ST2^-/-donor mice.However,in this setting,allograft ST2 deficiency significantly exacerbated cardiac allograft chronic disease.Conclution:1.The cold hyperpotassium solution may play a protective role in the heart transplantationduring arrest and ensures the isograft to survive in vitro for a long time.2.These findings indicatethat allografts or recipients ST2 oppositely affected cardiac allograft chronic diseases,which show that the expression of ST2 in cardiac resident cell aggravates cardiac allograft chronic diseases and ST2 in recipient immune cell alleviates it.3.The allografts or recipients ST2 oppositely affected cardiac allograft chronic diseases by altering immune cell infiltration,cardiac graft lymphatic formation.4.The technique of combined heart transplantation in mice is feasible.Combined heart transplantation is different from single-heart transplantation in pathology.Thus,single-heart transplantation can’t be replaced by combined heart transplantation.