The non-transcriptional activity of IRF3 modulates hepatic immune cell populations in acute-on-chronic ethanol administration in mice

Background & Aims: Interferon regulatory factor 3 (IRF3) is a transcription factor mediating antiviral responses, yet recent evidence indicates that IRF3 also has critical non-transcriptional functions, including activating RIG-I-like receptors-induced IRF-3-mediated pathway of apoptosis (RIPA) and restricting activity of NF-κB. Using a novel murine model expressing only non-transcriptional IRF3 activity (Irf3 ^S1/S1 ), we tested the hypothesis that non-transcriptional functions of IRF3 modulate innate immune responses in the Gao-binge (acute-on-chronic) model of alcohol-related liver disease. Methods: IRF3 and IRF3-mediated signals were analysed in liver samples from 5 patients transplanted for alcoholic hepatitis and 5 healthy controls. C57BL/6, Irf3 ^−/− and Irf3 ^S1/S1 mice were exposed to Gao-binge ethanol-induced liver injury. IRF3-mediated RIPA was investigated in cultured macrophages. Results: Phospho-IRF3 and IRF3-mediated signals were elevated in livers of patients with alcoholic hepatitis. In C57BL/6 mice, Gao-binge ethanol exposure activated IRF3 signaling and resulted in hepatocellular injury. Indicators of liver injury were differentially impacted by Irf3 genotype. Irf3 ^−/− , but not Irf3 ^S1/S1 , mice were protected from steatosis, elevated alanine/aspartate aminotransferase levels and inflammatory cytokine expression. In contrast, neutrophil accumulation and endoplasmic reticulum stress were independent of genotype. Protection from Gao-binge injury in Irf3 ^−/− mice was associated with an increased ratio of Ly6C ^low (restorative) to Ly6C ^high (inflammatory) cells compared to C57BL/6 and Irf3 ^S1/S1 mice. Reduced ratios of Ly6C ^low /Ly6C ^high in C57BL/6 and Irf3 ^S1/S1 mice were associated with increased apoptosis in the Ly6C ^low population in response to Gao-binge. Activation of primary macrophage cultures with Poly (I:C) induced translocation of IRF3 to the mitochondria, where it associated with Bax and activated caspases 3 and 9, processes indicative of activation of the RIPA pathway. Conclusions: Taken together, these data identify that the non-transcriptional function of IRF3 plays an important role in modulating the innate immune environment in response to Gao-binge ethanol exposure, via regulation of immune cell apoptosis. Lay summary: Activation of the innate immune system contributes to inflammation in the progression of alcohol-related liver disease, as well as to the resolution of injury. Here we show that the protein IRF3 modulates the innate immune environment of the liver in a mouse model of alcoholic hepatitis. It does this by increasing the apoptotic cell death of immune cells that promote the resolution of injury.