Non-transcriptional activity of IRF3 promotes liver fibrosis and stress granule assembly via a dsRNA-TLR3-dependent pathway in hepatic stellate cells

BACKGROUND AIMS: Interferon regulator factor 3 (IRF3) executes multiple transcriptional and non-transcriptional functions which regulate development of metabolic liver diseases. However, the contribution of the transcriptional and non-transcriptional activities of IRF3 to hepatic fibrosis are unknown. APPROACH RESULTS: Chronic carbon tetrachloride (CCl4) and choline-deficient L-amino-defined-diets both induced fibrosis in C57BL/6J (wild-type) and mice expressing only non-transcriptional IRF3 activity (Irf3S1/S1) but not Irf3-deficient (Irf3-/-) mice. HSC-specific deletion of Irf3 (Lrat-Irf3) also attenuated CCl4-induced fibrosis. Molecular pathways associated with regulation of extracellular matrix were up-regulated during differentiation in primary HSCs from wild-type and Irf3S1/S1, but not Irf3-/-, mice. Fibrogenic gene expression was also reduced in primary HSCs from Irf3-/-, but not Irf3S1/S1, mice upon TGFβ stimulation. IRF3 gene silencing in LX2 cells suppressed TGFβ-induced fibrogenic gene expression and collagen production. Proteomic analysis identified stress granule (SG) associated proteins within the IRF3 interactome, and IRF3 localized to SGs in LX2 cells upon stimulation with poly(I:C) or TGFβ. Mechanistically, differentiation of primary HSCs or stimulation of LX2 cells with TGFβ induced accumulation of endogenous double-stranded RNA (dsRNA), which triggered assembly of IRF3-containing SGs in a TLR3-dependent mechanism. TGFβ stimulated SG assembly in primary HSCs from wild-type and Irf3S1/S1, but not Irf3-/-, mice, and SG were detected in fibrotic human livers. CONCLUSION: The non-transcriptional activity of IRF3 is a key driver of hepatic fibrogenesis. Non-transcriptional activity of IRF3 is activated via accumulation of endogenous dsRNA and TLR3-dependent assembly of IRF3-containing SGs and acts as a central regulator of HSC activation and hepatic fibrogenesis. (250words).