Despite recent advances in immunosuppression, patients who have undergone allogeneic cardiac transplantation still suffer from poorly understood chronic graft loss. Previous studies have shown that Tumour Necrosis Factor Alpha (TNFΑ) contributes to cell death by activating apoptotic and newly identified Receptor Interacting Protein 1 and 3 (RIP1/RIP3) mediated necroptotic death pathways. These variations in cell death may be important for graft survival as necroptosis can lead to the release of chemotactic and activating danger molecules which have been shown to activate host immune cells. This pathway has yet to be studied in transplantation.

Our data shows that sirolimus treatment (9 days) markedly prolongs cardiac allograft survival of B6-RIP3 null as compared to B6 wild type donor heart grafts into Balb/c recipients (95 ± 5.8 vs 24 ± 2.6 days p<0.001). In vitro, murine vascular endothelial cell (VEC) cell death is reduced by the RIP1/RIP3 inhibiting small molecule necrostatin-1 (Nec-1) following TNFΑ treatment (68.5% vs 32.8%, PI positive at 48 hours). As well, necrosis and release of the pro-inflammatory danger molecule HMGB1 are attenuated in vivo in RIP3 null heart allografts and in vitro with VEC after RIP1/RIP3 inhibition. Finally, quantitative blinded scoring of necrotic cell death, vascular endothelial cell damage and graft infiltration was attenuated in RIP3 null hearts compared with wildtype allografts (0.8 +/- 0.4 in RIP3 null vs 1.8 +/- 0.4). These data suggest that RIP1/RIP3 contribute to inflammatory injury in cardiac allografts through VEC necroptotic death and the release of danger molecules. The ability of immunosuppression to provide rejection protection or permit tolerance is influenced by the level of cell death and inflammation. We suggest targeting RIP1 and RIP3 mediated necroptosis may be an important therapeutic strategy in solid organ transplants.

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