J. Goswami1, R. How1, Q. Du2, S. Kimura2, D. Geller1,2 1University Of Pittsburgh Medical Center,Department Of Surgery,Pittsburgh, PENNSYLVANIA, USA 2Thomas E. Starzl Transplantation Institute,Department Of Surgery, University Of Pittsburgh School Of Medicine,Pittsburgh, PENNSYLVANIA, USA
Introduction: Necroptosis is a highly regulated form of cell death that is thought to be triggered by TNFα when caspase-8 is inhibited. Unlike apoptosis, necroptosis does not lead to fragmentation of DNA. Necroptosis is thought to be dependent on a complex involving receptor-interacting protein 1 kinase (RIPK1), RIPK3, caspase 8, and FADD. RIPK3 is involved in necroptosis while RIPK1 is involved in both apoptosis and necroptosis. Previously, we have shown that interferon regulatory factor 1 (IRF-1) contributes to liver transplant I/R injury by increasing apoptosis. However, the role of IRF-1 in necroptosis has not been examined. Our goal in this study is to determine if there is a role of necroptosis in liver transplant I/R injury and to characterize the role of IRF-1 in necroptosis.
Methods: Orthotopic murine liver transplant (OLTx) was performed with B6 wild-type (WT) or IRF-1 knockout (KO) donor livers to isograft B6 recipients. Cold ischemia time was 24 hr. Serum was collected at 1, 3, and 6 hr after liver transplant reperfusion for serum transaminases to assess liver injury. Protein was isolated and western blot performed for RIPK1and RIPK3.
Results: OLTx using IRF-1 KO donor liver isografts resulted in markedly diminished liver I/R injury compared to WT as we have previously shown (graph). Hepatic RIPK3 protein expression was induced 1 to 3 hr after OLTx and returned to baseline by 6 hr (figure). RIPK3 expression was not observed in isografts that received IRF-1 KO donor livers. Interestingly, the necroptotic form of RIPK1 (78 kDa protein) was mildly increased after OLTx in an IRF-1 independent manner, while the apoptotic form of RIPK1 (40 kDa protein) was not induced.
Conclusion: These findings demonstrate activation of the necroptosis pathway with rapid induction of RIPK3 protein expression during OLTx I/R injury. Moreover, we have identified that induction of hepatic RIPK3 in the liver transplant setting is IRF-1-dependent. Further studies are needed to better define the mechanism of IRF-1 mediated induction of RIPK3 and necroptosis. Inhibition of the necroptotic pathway by blockade of RIPK proteins may be a novel strategy to ameliorate liver transplant I/R injury.
