ABSTRACT
BACKGROUND AND AIM: The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features. METHODS: The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNß, TRAIL, DR4, DR5 and their correlation to viral replication. RESULTS: HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I. CONCLUSION: These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection.
Subject(s)
DEAD-box RNA Helicases/metabolism , Hepacivirus/physiology , Host-Pathogen Interactions , Liver/metabolism , Liver/virology , Toll-Like Receptors/metabolism , Cell Death/drug effects , Cell Line, Tumor , DEAD Box Protein 58 , DEAD-box RNA Helicases/genetics , Down-Regulation/drug effects , Hepacivirus/drug effects , Hepacivirus/metabolism , Humans , Interferon-beta/pharmacology , Liver/cytology , Liver/drug effects , Protein Binding/drug effects , Receptors, Immunologic , Signal Transduction/drug effects , TNF-Related Apoptosis-Inducing Ligand/metabolism , Toll-Like Receptors/genetics , Viral Envelope Proteins/metabolism , Virus Replication/drug effectsABSTRACT
There is limited information on the direct role of the neutralizing antibody responses against hepatitis C virus (HCV) infection or methodologies to study them. Previously we have demonstrated that interleukin-10 (IL-10) administered to chronic hepatitis patients led to a decrease in disease activity, but an increase in HCV viral burden. The mechanism behind this is unknown. The objective of this study was to examine the antibody response in IL-10-treated patients. To establish a neutralization antibody assay, HCV-positive and HCV-negative sera were collected and incubated with HCV strain JFH-1 particles before culture with Huh 7.5 cells. Viral replication was measured a week later by either indirect immunofluorescence assay (iIFA) or real-time reverse transcriptase polymerase chain reaction (RT-PCR). After validation of the methodology, the sera from 30 previously-described subjects of a group previously treated with IL-10 were tested for the neutralization capacity of their antibodies. The amount of total anti-HCV antibody in the sera was also measured by direct staining of HCV full-length replicon cells. With this validated neutralization assay for anti-HCV antibodies we found that HCV-neutralizing antibodies are universally present, but with significantly different titers. In patients who were treated with IL-10, the total anti-HCV antibody titers appear to be constant, but with significantly decreased antibody neutralization activity. Our study validates an assay to quantitatively determine the presence and strength of HCV-specific neutralizing antibodies. We have found that IL-10-treated patients have significantly lower HCV antibodies, but maintain the total anti-HCV antibody titer, suggesting a novel mechanism by which IL-10 treatment increases viral load in patients.
