Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes

Abstract

Background & Aims The pro-inflammatory chemokine CXCL10 is induced by HCV (hepatitis C virus) infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. Methods CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. Results Huh7 human hepatoma cells expressing both TLR3 and RIG-I 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. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r2=0.88, p=<0.001). Conclusions While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures.

Direct, Interferon-Independent Activation of the CXCL10 Promoter by NF-κB and Interferon Regulatory Factor 3 during Hepatitis C Virus Infection

“…Although type I and type III IFNs induce formation of the ISGF3G complex that binds ISREs (1, 50), we previously observed no contribution of these cytokines to CXCL10 induction in immortalized cell lines, as described above (44). We hypothesized that one or more IRFs may be directly binding to the CXCL10 promoter.…”

“…We recently showed that both TLR3 signaling and RIG-I signaling are required for CXCL10 induction in hepatocytes during early HCV infection but that the initial induction did not require type I or type III IFNs (44). Therefore, in this study we sought to identify the transcription factors activated downstream of TLR3 and RIG-I that contribute to HCV-mediated CXCL10 induction.…”

“…We next evaluated the response of these constructs during HCV infection in TLR3 ϩ /RIG-I ϩ Huh7 hepatoma cells, which also express both RIG-I and TLR3 but are readily susceptible to virus infection in vitro (44). TLR3 ϩ /RIG-I ϩ Huh7 cells were transfected as described above and infected 48 h later with HCV JFH-1 (MOI, 1.0).…”

“…5B) to observe the effects of blocking interferon signaling. The neutralization efficacy of these reagents was demonstrated previously (44). Interferon-neutralized samples were normalized for cell viability as described above.…”

“…As the CXCL10 promoter contains both putative ISREs and putative STAT-binding sites, it responds to all 3 types of IFN and is considered an ISG (11,17). However, neutralization of type I and type III IFNs was previously shown to have no effect on CXCL10 production in primary human hepatocytes and hepatoma cells expressing functional TLR3 and RIG-I during early HCV infection (44). It has yet to be determined if IRFs play a role in CXCL10 induction independently of the action of type I and type III IFNs.…”