Induction and Regulation of CXCL10 in Hepatocytes During Hepatitis C Virus Infection

Abstract

Chronic Hepatitis C affects an estimated 170 million people worldwide and 4 million in the United States. The pro-inflammatory chemokine CXCL10 is induced by hepatitis C virus (HCV) infection <italic>in vitro</italic> and <italic>in vivo</italic>, and is associated with the outcome of interferon (IFN)-based therapies. Since persistent hepatic inflammation can lead to degenerative liver disease, this work sought to evaluate how innate immune sensors of HCV infection (Toll-like receptor 3 [TLR3] and retinoic acid inducible gene I [RIG-I]) contribute to CXCL10 induction in hepatocytes. CXCL10 mRNA and protein were measured in primary human hepatocytes (PHH) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. The contribution of hepatocyte-derived type I and type III IFNs and specific pro-inflammatory transcription factors to CXCL10 induction were also examined. In this study, PHH and immortalized PH5CH8 hepatocytes were confirmed to express functional TLR3 and RIG-I. Specific activation of TLR3 and RIG-I led to CXCL10 induction in a non-synergistic manner, and Huh7 human hepatoma cells expressing both receptors (TLR3+/RIG-I+ Huh7 cells) produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ Huh7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs, suggesting that standard PHH cultures contain non-parenchymal cells (NPCs). Immunodepletion of NPCs eliminated expression of immune and anti- inflammatory markers in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Instead, HCV infection and specific TLR3/RIG-I activation induced binding of NF-κB and IRF3 to the CXCL10 promoter. Together, these data indicate that initial CXCL10 induction in hepatocytes during early HCV infection is independent of hepatocyte-derived type I and type III IFNs, while NPC- and immune cell-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures and <italic>in vivo</italic>. Further elucidation of the regulatory pathways controlling CXCL10 induction may reveal novel targets for host-oriented therapies to reduce chronic inflammation, as well as provide insight into the complex and redundant signaling network of the innate immune system.