LAUSR

BACKGROUND AND AIMS Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/NASH. However, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS Several animal models of human liver pathology including diet induced steatohepatitis in male mice and diverse infections in both male and female mice were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analysed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS Identical infection kinetics and pathological outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared to control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed on a high fat diet to induce NASH. Under all conditions tested we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathological conditions tested in this study.