Interferon β (IFN-β) Production during the Double-stranded RNA (dsRNA) Response in Hepatocytes Involves Coordinated and Feedforward Signaling through Toll-like Receptor 3 (TLR3), RNA-dependent Protein Kinase (PKR), Inducible Nitric Oxide Synthase (iNOS), and Src Protein

Abstract The sensing of double-stranded RNA (dsRNA) in the liver is important for anti-viral defenses but can also contribute to sterile inflammation during liver injury. Hepatocytes are often the target of viral infection and are easily injured by inflammatory insults. Here, we sought to establish the pathways involved in the production of type I interferons (IFN-I) in the response to extracellular Poly(I:C), a dsRNA mimetic, in hepatocytes. This was of interest because hepatocytes are long-lived and unlike most immune cells that readily die after activation with dsRNA, not viewed as a cell with robust anti-microbial capacity. We found that Poly(I:C) lead to the rapid upregulation of the inducible nitric oxide synthase (iNOS), the double-stranded RNA-dependent protein kinase (PKR), and Src. The production of interferon-beta (IFN-β) was dependent on iNOS, PKR, and Src, and was partially dependent on TLR3/Trif. iNOS and Src upregulation was partially dependent on TLR3/Trif but entirely dependent on PKR. The phosphorylation of TLR3 on tyrosine 759 was shown to increase in parallel to IFN-β production in an iNOS and Src-dependent manner and Src was found to directly interact with TLR3 in the endosomal compartment of Poly(I:C) treated cells. Further, we identified a robust NO/cGMP/PKG-dependent feed forward pathway for the amplification of iNOS expression. These data identify iNOS/NO as an integral component of IFN-β production in response to dsRNA in hepatocytes in a pathway that involves the coordinated activities of TLR3/Trif and PKR.