Toll-like Receptor 3 Modulating Microvascular Endothelial Cell Necroptosis In Transplantation Immunity

BackgroundBefore the era of immunosuppressants,an allogenic organ transplant can result in quick occurrence of immunoinflammatory responses and lead to rejection.Although the game has been revolutionarily changed by accumulation of knowledge on transplant immunology and development of immunosuppre-ssants in the last 50 years,transplantation is still bottlenecked by post-surgical early-stage rejection as well as long-term end-stage the heart disease.However,comparing to liver and kidney transplants,transplanted heart grafts are less potent in self-repairing and functional recovery after transplant injury.This is mainly caused by graft ischemia-reperfusion injury and the immunoinfla-mmatory response after transplant.After transplantation immune inflammatory reaction not only induces programmed cell death,but also resulting in release of cell contents which can subsequently worsen proinflammatory responses and eventually cause organ dysfuctionality and premature rejection.Organ cell death and injury with premature graft loss remain the leading cause of morbidity and mortality in transplant patients.Despite our insights and the therapeutic potential,control of cell death per se has not yet translated to clinically relevant and effective treatment strategies.Therefore,intervention on immune inflammatory response in the process of transplantation has become a top topic in current transplantation research.As a pattern recognition receptor,Toll like receptor 3-TLR3 is expressed in cells or on the surface of the cell,macrophages,dendritic cells and endothelial cells.When binding to endogenous RNA,TLR3 can induce production of inflammatory cytokines and thus play an important role in innate immunity.In addition,previous studies have found that the interaction between RNA and TLR3-mediates a fatal systemic inflammatory response leading to necroptosis,but the mechanism of this inflammatory response that eventually results in necroptosis,but the mechanism of this inflammatory response in early stage heart transplantation and related signaling pathways have not been studied.ObjectiveThe aim of this study is to investigate the roles of TLR3 in the necroptosis of microvascular endothelial cells（MVECs）death.We wish to reveal the key pathogenesis mechanisms of TLR3 mediated MVEC apoptosis and necroptosis and heart transplant rejection.The results will provide rationales for new therapeutic target in the prevention and treatment graft injury and rejection caused by immune inflammatory responses during heart transplantation.Method（1）Primary culture of MVECs.The ventricles of 3-4-week-old mice were collected,and cut and incubated in D-hank digestive buffer solution at 37℃for 40 minutes.Debris was removed with 100 mm nylon mesh filter.The cell suspension was incubated with Dynal M450 beads coated with anti-CD31 for30 minutes,and the magnetic beads was washed 3 times with PBS.CD31⁺cells were cultured in complete EGM-2 medium.The MVECs were immortalized by transfection of origin-defective SV40 DNA vector（Sigma-Aldrich）.MVECs phenotype was confirmed by staining with anti-CD31,CD102 and CD105 then analyzed on flow cytometry.（2）Investigation on the mechanisms of TLR3-mediated MVECs death.The expression of TLR3 on MVECs was detected by anti-TLR3 and flow cytometry.The expression of TLR3 in the heart graft was detected by anti-TLR3 in immunohistochemistry.Apoptosis after exogenous RNA binding to TLR3 in MVECs were quantified by caspase3/7 detection kits.Mi RNeasy kit was used in endogenous RNA extraction from MVECs.MVECs death induced by exogenous dsRNA（Poly I:C）and endogenous RNA were monitored and quantified by IncuCyte Zoom real-time live cell imaging system.（3）The effect of TLR3 on MLKL activation and mitochondrial permeability.In order to identify the down-stream molecures of TLR3-mediated necroptosis,total proteins of wild-type and TLR3 gene konck-out MVEC were extracted,and levels of mixed lineage kinase domain-like protein（MLKL）and its activated form phosphorylated MLKL（pMLKL）were determined by Western blot.Mitochondrial permeability was determined by monitoring mitochondrial loss by Incucyte real-time live cell imaging system after fluorescent Mito Tracker staining.（4）Animal model of heart transplantation.The donor animal was anesthetized with ketamine/xylazine mixture,then the chest was opened after disinfection and aorta and inferior vena cava were exposed,the artery and vein were ligated after heparinization,and the donor heart was obtained by disconnection of blood vessels.Then the recipient was sterilized and anesthetized according to the above steps.The recipient abdominal aorta was anastomosed with the donor aorta end side,and the recipient inferior vena cava was anastomosed with the donor pulmonary artery.The recipients were given immunosuppressant rapamycin（1 mg/kg）for 9 days after heart transplantation to attenuate acute rejection.（5）The effect of TLR3 on the immune inflammatory response of heart transplantation mice.Survival length of TLR3^-/-and wild type grafts after transplantation were monitored.Histological and immunohistochemical analysis were conducted to evaluate cardiac vessel damage including angiogenesis and neointima formation,vascular fibrosis,anti-cleaved caspase-3staining and pMLKL staining.Immunohistochemical analysis and flow cytometry were used in monitoring T cells within the grafts and the spleens of recipents.Results（1）The mechanism of TLR3-mediated MVEC death.We observed whether Toll-like receptor 2（TLR2）,Toll-like receptor 3（TLR3）and Toll-like receptor 4（TLR4）of the TLR family mediate the occurrence of cell death in MVEC.It was confirmed by experiments that TLR2 and TLR4 can not mediate MVEC death,implying a TLR3-specific mechanism.TLR3 is highly expressed in the heart graft and on the surface of wild type MVECs.Polyinosinic acid and polycytidylic acid（poly I:C）can induce MVEC necroptosis and apoptosis.As poly（I:C）induced MVEC necroptosis can be blocked by receptor-interacting protein kinase-1（RIPK1）inhibitor,also known as necrostatin-1（Nec-1）.Hence,TLR3 mediated MVEC necroptosis is RIPK1 dependent.Meanwhile,MVEC RNAs can induce MVEC necroptosis.（2）TLR3 mediated MVEC necroptosis through activation of downstream MLKL,independent of ROS.The occurrence of necroptosis in MVEC may be associated with ROS and MLKL pathway.Reactive oxygen species（ROS）inhibitors（NAC-1 and Tempol）and mitochondrial-specific antioxidant（Mito-Tempo）did not inhibit MVEC necroptosis,suggesting that,ROS are not involved in TLR3-mediated MVEC necroptosis.But treatment by MLKL inhibitor,GW806742X,can inhibit TLR3-mediated MVEC necroptosis.The results of MLKL and pMLKL protein electrophoresis（western blot）showed that the expression of pMLKL decreased significantly in MVECs after the treatment by RIPK1 inhibitor,Nec-1.We also found that pMLKL decreased in TLR3^-/-MVECs and in the TLR3^-/-graft post transplantation.Our results indicated that TLR3 mediated necroptosis is via activation of MLKL.（3）The effect of TLR3 on mitochondrial permeability.Next we studied whether TLR3-induced cell death involves in mitochondrial.Cyclosporine A（CsA）can inhibit cyclophilin D（CypD）-mediated mitochondrial membrane permeabilization to maintain the integrity of mitochondrial.Our study demonstrated that CsA can significantly inhibit MVEC death and maintain the integrity of mitochondrial.Furthermore,CypD^-/-MVECs cells showed less necroptosis.Our study confirmed that TLR3-mediate necroptosis is related to mitochondrial permeability.This is the first report that TLR3 leads to mitochondrial destruction via CypD.（4）The effect of TLR3 on the immune inflammatory response in heart transplantation mice.The survival length of the TLR3^-/-graft after heart transplantation was significantly longer than that of the wild type graft（121±67 days vs 32±6 days,n=7,P=0.002）.Vascular injury（1.9±0.74 vs 0.6±0.89,n=5,P<0.05）,neointimal formation（1.3±0.45 vs 0.5±0.35,n=5,P<0.01）and fibrosis were significantly lower in the TLR3^-/-graft compared to the wild type graft.Meanwhile,immunohistochemical analysis indicated that expression levels of activated caspase-3 and pMLKL in TLR3^-/-graft were lower compared to the wild type graft.FoxP3⁺Treg cells in the TLR3^-/-heart graft and the spleen of the graft recipient were significantly increased,while CD4⁺T proliferation was decreased significantly.Conclusion（1）Exogenous dsRNA and endogenous RNA can bind TLR3 to mediate apoptosis and necroptosis in MVECs via caspase-dependent and RIPK1-dependent pathways respectively.（2）TLR3-mediated MVEC necroptosis is depended on the activation of downstream MLKL,ROS are not involved in MVEC necroptosis.（3）TLR3-induced MVEC necroptosis is through the CypD-regulated mitochondrial permeabilization.（4）TLR3 deletion can attenuate transplant rejection and promote long term graft survival.In conclusion,interactions between TLR3 and RNA induced MVEC apoptosis via a caspases-dependent pathway,and MVEC necroptosis via a MLKL and CypD-dependent pathway MVEC.The lack of TLR3 in donor heart can reduce apoptosis and necroptosis,increase FoxP3⁺Treg cell expression,reduce CD4⁺T proliferation,and favor the long-term graft survival.These findings have provided rationales and new therapeutic targets for the development of TLR3 signaling pathway based treatment strategies,which may effectively block inflammation,apoptosis and necroptosis in the graft and promote the long-term graft survival.