Hepatitis C Virus Is a Weak Inducer of Interferon Alpha in Plasmacytoid Dendritic Cells in Comparison with Influenza and Human Herpesvirus Type-1

Abstract

Plasmacytoid dendritic cells (pDCs) are responsible for the production of type I IFN during viral infection. Viral elimination by IFN-α-based therapy in more than 50% of patients chronically infected with hepatitis C virus (HCV) suggests a possible impairment of production of endogenous IFN-α by pDCs in infected individuals. In this study, we investigated the impact of HCV on pDC function. We show that exposure of pDCs to patient serum- and cell culture-derived HCV resulted in production of IFN-α by pDCs isolated from some donors, although this production was significantly lower than that induced by influenza and human herpesvirus type 1 (HHV-1). Using specific inhibitors we demonstrate that endocytosis and endosomal acidification were required for IFN-α production by pDCs in response to cell culture-derived HCV. HCV and noninfectious HCV-like particles inhibited pDC-associated production of IFN-α stimulated with Toll-like receptor 9 (TLR9) agonists (CpG-A or HHV-1) but not that of IFN-α stimulated with TLR7 agonists (resiquimod or influenza virus). The blockade of TLR9-mediated production of IFN-α, effective only when pDCs were exposed to virus prior to or shortly after CpG-A stimulation, was already detectable at the IFN-α transcription level 2 h after stimulation with CpG-A and correlated with down-regulation of the transcription factor IRF7 expression and of TLR9 expression. In conclusion, rapidly and early occurring particle–host cell protein interaction during particle internalization and endocytosis followed by blockade of TLR9 function could result in less efficient sensing of HCV RNA by TLR7, with impaired production of IFN-α. This finding is important for our understanding of HCV-DC interaction and immunopathogenesis of HCV infection.

Hepatitis C Virus Fails To Activate NF-κB Signaling in Plasmacytoid Dendritic Cells

“…Peripheral blood mononuclear cells (PBMCs) from healthy anonymous donors were obtained from the national blood services (Etablissement Francais du Sang [EFS], Marseille, France). pDCs purified from PBMCs as described previously (4,11,13,31) yielded levels of purity from 75% to 95%, with a contamination of less than 5% myeloid dendritic cells. Isolated pDCs were cultured in RPMI 1640 supplemented with 10% fetal calf serum and antibiotics.…”

“…Cell culture-derived HCV (HCVcc) virions were prepared in Huh7.5 cells (2) (kindly provided by APATH L.L.C.) on the basis of plasmid pJFH-1 displaying mutations F172C and P173S in core, and N534K in E2 (5), as described previously (4,11,13,31). The ultracentrifuged virus purified through a cushion of 20% sucrose was resuspended in RPMI 1640 medium to obtain a 1,000-fold-concentrated virus suspension.…”

“…The titers of HCVcc genomecontaining virus particles were determined routinely with quantitative reverse transcription-PCR (RT-PCR) as described previously (4,11,13,31) using the primer RTU1 (25) for cDNA synthesis, primer pair UTR2 and RTU2 (25) for PCR amplification, and HCV JFH-1 RNA prepared in vitro with T7 polymerase as a standard.…”

“…Several reports have shown that exposure of pDCs from healthy donors to HCV particles results in no or only weak production of type I IFN and cell differentiation (4,7,11,13,31). A recent report has shown that pDCs exposed in direct cell-to-cell contact with HCV-infected hepatoma cells, unlike those exposed to cell-free HCV virions, produce large amounts of type I IFN via TLR7 signaling (35).…”

“…4B). We have previously shown that inhibition of TLR9-mediated production of IFN-␣ in pDCs by HCV particles is more efficient if the priming with the cell-free virus precedes stimulation with TLR9 agonist, in comparison with the simultaneous treatment (4,7,11,13,31). Therefore, we investigated the effect of priming of pDCs with HCV-infected Huh7.5 cells followed 2 h later by stimulation with CpG-A or CpG-B on production of TNF-␣ or IL-6 (Fig.…”