Transcriptional and non-transcriptional function of IRF3 in ALD - ABSTRACT Chronic alcohol abuse underlies the pathogenesis of alcohol-associated liver disease (ALD), encompassed by a spectrum of pathologies, ranging from steatosis, to more severe forms of liver injury, including alcohol- associated hepatitis (AH), fibrosis, and cirrhosis. Acute AH has a very high mortality rate, with no effective therapeutic Patients with AH who exhibit stage 3-4 fibrosis have worse prognosis; however, treatment strategies do not include therapy for the resolution of fibrosis, representing an important unmet clinical need. Recruitment of innate immune cells to the liver and the regulation of their phenotype between pro-inflammatory/fibrogenic vs pro-resolution activity, impacts both disease progression and resolution. Hepatic stellate cells (HSC), the primary producer of extracellular matrix in the liver, are also regulated by innate immune pathways. During the previous granting cycle, we discovered that IRF3-dependent pathways, a component of host anti-viral responses, contribute to injury in murine models of ALD, NAFL/NASH and fibrosis. These data led us to ask the fundamental question: what is the mechanism by which metabolic/sterile liver diseases activate anti-viral signaling and can this be leveraged to develop novel therapeutics? We identified perturbations in homeostatic regulation of endogenous dsRNA in both immune cells and HSCs suggesting that endogenous dsRNAs are novel host-derived DAMPs that activate IRF3 signaling and contribute to the pathogenesis of ALD. IRF3 is classically recognized as a transcription factor essential for host anti-viral responses, but also has non-transcriptional activities. Using a novel knock-in mouse expressing an IRF3 protein that only exhibits non-transcriptional functions of IRF3 (Irf3S1/S1), we discovered that 1) IRF3-mediated apoptosis of innate immune cells recruited to the liver in response to Gao-binge ethanol increases inflammation and liver injury and 2) non-transcriptional functions of IRF3 restricted NFκB activity in a murine model of NAFL/NASH. Here we propose to extend our studies into IRF3 functions in pathogenesis of hepatic fibrosis in two specific aims: 1) Determine the contribution of the transcriptional and non-transcriptional IRF3 to murine models of hepatic fibrosis: Using Irf3-/-, Irf3S1/S1 and cell-specific deletions of Irf3, we will test the hypothesis that non-transcriptional activity of IRF3 contributes to recruitment of neutrophils to liver and enhances activation of HSCs in mice exposed to CCl4+ethanol or CDAA models of fibrosis. 2) Discover the cell intrinsic functions of IRF3 in the pathogenesis of fibrosis: Mechanistically, we will identify specific dsRNAs accumulating in cultured HSCs and neutrophils, interrogate perturbations in expression of RNA chaperones and dsRNA recognition molecules critical to IRF3 activation in fibrosis. Further, we will interrogate mechanisms for Irf3-dependent cell- cell interactions in fibrosis using human-iPSC 3-dimensional liver cultures. In summary, our studies will reveal novel cell-specific roles for non-transcriptional activity of IRF3 in hepatic fibrosis, as well as identify potential therapeutic targets that would prevent fibrosis and/or improve the resolution of fibrosis in patients with ALD.
