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Sanz-Garcia C1,*, Roychowdhury S1,*, Chattopadhyay S2, McMullen MR1, Sen GC2, Nagy LE1

Departments of 1Pathobiology and 2Molecular Genetics, Center for Liver Disease Research, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
* These authors contributed equally to this work

Background and Aims: Increased exposure of hepatic macrophages to lipopolysaccharide (LPS) and activation of TLR4 are important contributors to alcoholic liver disease (ALD). Interferon regulatory factor 3 (IRF3) is a master regulator of host responses to viral infection, but is also activated via the TLR4-MyD88-independent pathway. Upon activation, IRF3 is phosphorylated and translocated to the nucleus where it initiates a specific transcriptional program. Recently, two non-transcriptional functions for IRF3 have been identified: 1) a novel pro-apoptotic function termed RIG-I-induced IRF3-mediated pathway of apoptosis (RIPA) and 2) an interaction with the p65 subunit of NFκB that prevents p65 translocation to the nucleus. While IRF3 has been implicated in the progression of ethanol-induced liver injury, the transcriptional versus non-transcriptional functions of IRF3 have not been studied yet. Here we made use of IRF3-deficient mice (IRF3 KO), as well as a novel knock-in of a mutated IRF3 that cannot translocate to the nucleus and only retains the non-transcriptional functions of IRF3 (nt-IRF3 KI).
Methods: Wild-type C57BL/6, IRF3-KO and nt-IRF3 KI mice were allowed free access to an ethanol containing Lieber-deCarli diet (up to 32% ethanol, 25d) or pair-fed control diets. Measures of liver injury were assessed.
Results: Chronic ethanol feeding increased hepatic steatosis, plasma ALT and AST activities, expression of inflammatory cytokines and hepatocyte apoptosis in wild-type mice. IRF3 KO mice had reduced hepatic steatosis and hepatocyte apoptosis, but expression of TNFα protein, as well as ALT and AST concentrations, were still increased in response to ethanol feeding. In contrast, nt-IRF3 KI mice had reduced TNFα protein, ALT/AST and hepatic steatosis. Interestingly, while hepatocyte apoptosis was reduced in nt-IRF3 KI mice, apoptosis of cells with the morphologic appearance of hepatic macrophages was increased.
Conclusion: Taken together, these data suggest that there are distinct transcriptional and non-transcriptional functions of IRF3 in chronic ethanol-induced liver injury. Interestingly, increased macrophage apoptosis in the nt-IRF3 KI mice was associated with a protection from ethanol-induced inflammatory responses, suggesting a potential protective role for the non-transcriptional activities of IRF3 in the progression of chronic ethanol-induced liver injury.