A novel multi-organ male model of alcohol-induced acute-on-chronic liver failure reveals NET-mediated hepatocellular death which is prevented by RIPK3 inhibition

Background and aims: Alcohol abuse is the most frequent precipitating factor of acute-on-chronic liver failure (ACLF). We aimed at developing an alcohol-induced ACLF model and dissecting its underlying molecular mechanisms.

Methods: ACLF was triggered by a single alcohol binge (5g/Kg) in a bile duct ligation (BDL) liver fibrosis murine model. Liver, kidney and brain tissues and behavior were assessed in mice. Livers from sclerosing cholangitis patients with/without ACLF were also evaluated.

Results: In advanced fibrosis induced by BDL, an alcohol binge induced features of ACLF including significant liver damage, systemic inflammation (increased endotoxin and pro-inflammatory cytokines) and hepatocyte dysfunction compared to BDL alone. ACLF was associated with extrahepatic manifestations including increased BUN and creatinine, impaired coagulation, and features of encephalopathy. We discovered significantly increased neutrophil count and neutrophil extracellular traps (NET) in the liver, kidney and brain in murine ACLF. Livers from ACLF mice showed increased pyroptosis (gasdermin D) and necroptosis (RIPK3), when compared to BDL. In vitro, cell-free NETs were induced by alcohol and/or bile acids and triggered pyro-/necro-ptotic death in hepatocytes. NETosis, pyroptosis and RIPK3 activation were validated in human livers with ACLF. Moreover, pharmacological inhibition of necroptosis with a RIP3 inhibitor ameliorated inflammation, NETs, and liver fibrosis, improving multi-organ ACLF pathophysiology.

Conclusions: Our novel ACLF model triggered by alcohol binge mimics key features of pathophysiology and multi-organ impairment in human ACLF. Our results indicate that neutrophil infiltration and NETs contribute to hepatocyte cell death via pyroptosis and necroptosis in ACLF, identifying RIPK3 as a potential therapeutic target.