O031 TLR3 and RIG-I sensing of HCV infection by hepatocytes leads to interferon-independent CXCL10 induction

Introduction Chronic hepatitis C is characterized by a persistent hepatic inflammatory response and the recruitment of immune effector cells to the liver by pro-inflammatory chemokines. The chemokine CXCL10 is induced by HCV infection in vitro and in vivo , and is correlated with the outcome of Interferon (IFN)-based therapies. Therefore, we investigated how sensing of HCV infection by the pathogen recognition receptors (PRRs) TLR3 and Retinoic Acid Inducible Gene 1 (RIG-I) led to expression of CXCL10 in hepatocytes. Methods Primary human hepatocytes were infected in vitro with the HCV clone JFH-1. CXCL10 production was measured via real-time RT-PCR, Luminex Bead Array, and immunofluorescence. Type I and type III interferon induction was measured directly by real-time PCR and indirectly by IFIT1 (ISG56) activation. CXCL10 production was also measured in response to RIG-I-specific (Sendai Virus) and TLR3-specific (polyI:C) stimuli as well as siRNA knockdown of RIG-I and TLR3 in PH5CH8 hepatocyte cultures. Transcription factors involved in CXCL10 induction were identified using CXCL10 promoter-Luciferase reporter constructs. Experiments were also performed in Huh7 hepatoma-derived cell lines that expressed either one, both, or neither PRR. Results CXCL10 mRNA and protein expression were detected following HCV infection of both human hepatoma cell lines and primary human hepatocytes. siRNA knockdown of TLR3 and RIG-I protein abrogated CXCL10 induction in response to PRR-specific agonists. Huh7-derived cells expressing both TLR3 and RIG-I produced maximal CXCL10 mRNA with negligible induction of type I or III IFN. Furthermore, neutralization of type I and type III IFN did not impact CXCL10 induction during HCV infection while IFIT1 activation was completely abolished. Immunofluorescence studies revealed a direct positive correlation between intracellular HCV core and CXCL10 protein expression. Finally, TLR3 and RIG-I-specific agonists activated CXCL10 transcription in a NF- κ B-dependent manner. Conclusion Collectively, the data suggest that CXCL10 induction during HCV infection derives directly from PRR signaling to NF- κ B activation and is marginally influenced by paracrine/autocrine IFNs. Direct induction via both TLR3 and RIG-I pathways may indicate the existence of uncharacterized crosstalk within the canonical innate immune signaling cascade and may reveal host-directed drug targets for anti-inflammatory therapies.