Alcohol impairs interferon signaling and enhances full cycle hepatitis C virus JFH-1 infection of human hepatocytes

Abstract

Alcohol drinking and hepatitis C virus (HCV) infection frequently coexist in patients with chronic liver disease. There is limited information, however, about the impact of alcohol on host cell innate immunity and full cycle replication of HCV. This study investigated whether alcohol impairs the intracellular innate immunity in human hepatocytes, promoting HCV infection and replication. Alcohol treatment of human hepatocytes before, during and after viral infection significantly enhanced full cycle HCV replication. Alcohol suppressed intracellular expression of type I interferons (IFN-α/β) in human hepatocytes. Investigation of the mechanisms responsible for the alcohol action revealed that alcohol inhibited the expression of the IFN regulatory factors (IRF-5 and IRF-7), and signal transducer and activator of transcription (STAT-1 and STAT-2), the key positive regulators in type I IFN signaling pathway. In addition, alcohol induced the expression of suppressors of cytokine signaling (SOCS-2 and SOCS-3), the key negative regulators of IFN-α/β expression. These in vitro findings suggest that alcohol, through modulating the expression of key regulators in IFN signaling pathway, inhibits type I IFN-based intracellular innate immunity in hepatocytes, which may contribute to the chronicity of HCV infection and the poor efficacy of IFN-α-based therapy.

Fig. 1

Alcohol enhances HCV replication in human hepatocytes. HCV JFH-1-infected Huh7 cells or primary hepatocytes were cultured in the presence or absence of alcohol at indicated concentrations. Alcohol was added to the cell cultures every 24h. Cell lysates and culture supernatants were collected at day 6 postinfection. (A–D) The levels of intracellular or extracellular HCV RNA were determined by real-time RT-PCR. (A) The intracellular HCV RNA in Huh7 cells; (B) The extracellular HCV RNA in Huh7 cell cultures; (C) The intracellular HCV RNA in primary human hepatocytes; (D) The extracellular HCV RNA in primary human hepatocyte cultures. The levels of intracellular HCV RNA (A, C), with normalization to corresponding GAPDH mRNA, are expressed as the fold of control (without alcohol treatment, which was defined as 1). The levels of extracellular HCV RNA (B, D) are expressed as copies/mL. The results (A–D) shown are the mean ± SD of three repeated experiments. All variables in each experiment were tested in triplicate (*, P<0.05, **, P<0.01, alcohol treated vs untreated). (E) Alcohol enhances HCV NS3 protein expression. HCV JFH-1-infected Huh7 cells were cultured in the presence or absence of alcohol (40mM) for 6 days. Total cellular proteins were collected and subjected to Western blot using the antibodies against to HCV NS3 and GAPDH. The numbers in the right panel are the signal intensities of protein bands of western blot shown in the left panel, which are expressed as densitometry scanning units (DSUs).

Fig. 2

Time course effect of alcohol on HCV replication in human hepatocytes. HCV JFH-1-infected Huh7 cells or primary human hepatocytes were cultured in the presence or absence of alcohol (40mM). Alcohol was added to the cultures every 24h. Cell lysates and culture supernatants were collected at different time points as indicated. The levels of intracellular or extracellular HCV RNA were determined by real-time RT-PCR. (A) The intracellular HCV RNA in Huh7 cells; (B) The extracellular HCV RNA in Huh7 cell cultures; (C) The intracellular HCV RNA in primary human hepatocytes; (D) The extracellular HCV RNA in primary human hepatocyte cultures. The levels of intracellular HCV RNA (A, C), with normalization to corresponding GAPDH mRNA, are expressed as the fold of control (without alcohol treatment, which was defined as 1). The levels of extracellular HCV RNA (B, D) are expressed as copies/mL. The results (A–D) shown are the mean ± SD of three repeated experiments. All variables in each experiment were tested in triplicate (*, P<0.05, **, P<0.01, alcohol treated vs untreated).

Fig. 3

Alcohol treatment increases infectious HCV JFH-1 production in human hepatocytes. (A, B) Dose-dependent effect of alcohol on production of infectious HCV. HCV JFH-1-infected Huh7 cells were cultured in the presence or absence of alcohol at indicated concentrations. Alcohol was added to the cell cultures every 24h. Cell lysates and culture supernatants were collected at day 6 postinfection. Intracellular (A) or extracellular (B) HCV infectious titers were determined by serial dilution and immunofluorescence. (C, D) Time course effect of alcohol on production of infectious HCV. HCV JFH-1-infected Huh7 cells were cultured in the presence or absence of alcohol (40mM). Alcohol was added to the cultures every 24h. Cell lysates and culture supernatants were collected at different time points as indicated. Intracellular (C) or extracellular (D) HCV infectious titers were determined by serial dilution and immunofluorescence. The HCV titers (A–D) are expressed as foci form units/mL (FFU/mL) and presented as the mean ± SD of three repeated experiments. All variables in each experiment were tested in triplicate (*, P<0.05, **, P<0.01, alcohol treated vs untreated).

Fig. 4

Alcohol enhances HCV JFH-1 infection of human hepatocytes under different situations. Huh7 cells were cultured in the presence or absence of alcohol for either 24h prior to HCV infection, or simultaneously or 8h postinfection. After HCV infection, the infected cells were then washed 5 times with plain DMEM to remove input HCV and then cultured in the presence or absence of alcohol (40mM) for 6 days. Cell lysates and culture supernatants were collected at day 6 postinfection. The levels of intracellular (A) or extracellular (B) HCV RNA were determined by real-time RT-PCR. The levels of intracellular (C) or extracellular (D) HCV infectious titers were determined by serial dilution and immunofluorescence. (E, F) Effect of long-term alcohol treatment on HCV infection/replication. 1% DMSO (vol/vol) treated-Huh7 cells were cultured in the presence or absence of alcohol (40mM) and the cells were replenished alcohol (40mM) every 24h thereafter. After cultured for 10 days, Huh7 cells treated with or without alcohol were infected with HCV JFH-1. Cell lysates and culture supernatants were collected at day 6 postinfection. The levels of intracellular (E) or extracellular (F) HCV RNA were determined by real-time RT-PCR. The levels of intracellular HCV RNA (A, E), with normalization to corresponding GAPDH mRNA, are expressed as the fold of control (without alcohol treatment, which was defined as 1). The levels of extracellular HCV RNA (B, F) are expressed as copies/mL. The intracellular (C) and extracellular (D) HCV titers are expressed as FFU/mL. The results shown (A–F) are the mean ± SD of three repeated experiments. All variables in each experiment were tested in triplicate (*, P<0.05, **, P<0.01, alcohol treated vs untreated).

Fig. 5

Effect of alcohol on HCV entry of hepatocytes. (A) Effect of alcohol treatment on HCV receptor expression. Huh7 cells were cultured in the presence or absence of alcohol for 4h or 24h. Total cellular RNA extracted from the cell cultures was subjected to the real-time PCR for quantification of HCV entry cellular receptor (CD81, claudin-1, LDLR, SB-RI, and occludin) and GAPDH mRNA. The data, with normalization to GAPDH mRNA, are expressed as the fold of control (without alcohol treatment, which was defined as 1). (B) Effect of alcohol treatment on HCV pseudovirus entry into Huh7 cells. Huh7 cells were cultured in the presence or absence of alcohol at indicated concentrations for 24h, and then infected with NLluc⁺env⁻ virus pseudotyped with HCV E1E2 glycoproteins in the presence or absence of alcohol. Luciferase activities (relative light units, RLU) were measure at 36h postinfection. The data are expressed as the RLU fold of control (without alcohol treatment, which was defined as 1).

Fig. 6

Alcohol suppresses IFN-α/β expression and compromises anti-HCV activity of recombinant IFN-α. (A, B) Alcohol suppresses IFN-α/β expression in Huh7 cells (A) and primary human hepatocytes (B). Human hepatocytes were incubated in the presence or absence of alcohol (40mM) for 4h or 24h. Total cellular RNA extracted from the cell cultures was subjected to the real-time PCR for IFN-α/β and GAPDH mRNA quantification. The data, with normalization to GAPDH mRNA, are expressed as the fold of control (without alcohol treatment, which was defined as 1). (C–G) Alcohol compromises the anti-HCV activity of IFN-α. HCV JFH-1-infected Huh7 cells (at day 3 postinfection) were incubated with or without alcohol (40mM) and/or IFN-α (20U/ml, 100U/mL) for 48 h. The levels of intracellular (C) and extracellular (D) HCV RNA were determined by real-time RT-PCR. The data are expressed as copies/mL. The levels of intracellular or extracellular (F) HCV infectious titers were determined by serial dilution and immunofluorescence. The data are expressed as FFU/mL. The results (A–F) shown are the mean ± SD of three repeated experiments. All variables in each experiment were tested in triplicate (*, P<0.05, **, P<0.01, alcohol treated vs untreated). (G) Total cellular proteins were subjected to Western blot using the antibodies against to HCV NS3 and GAPDH. The numbers in the right panel are the signal intensities of protein bands of western blot shown in the left panel, which are expressed as densitometry scanning units (DSUs).

Fig. 7

Alcohol suppresses the expression of IFN regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) in human hepatocytes. Huh7 cells were cultured in the presence or absence of alcohol (40mM) for 24h. (A) Total cellular RNA extracted from the cell culture was subjected to the real-time RT PCR for IRF, STAT, and GAPDH mRNA quantification. The data, with normalization to GAPDG, are expressed as the fold of control (without alcohol treatment, which is defined as 1). The results shown are mean ± SD of three repeated experiments. All variables in each experiment were tested in triplicate (*, P<0.05, **, P<0.01, alcohol treated vs untreated). (B) Huh7 cells were cultured in the presence or absence of alcohol (40mM) for 72h. Total cellular proteins were subjected to Western blot assay using the antibodies against IRF-3, IRF-5, IRF-7, STAT-1, STAT-2, and GAPDH. The numbers in the right panel are the signal intensities of protein bands of Western blot shown in the left panel, which are expressed as densitometry scanning units (DSUs).

Fig. 8

Alcohol induces the expression of suppressors of cytokine signaling (SOCSs) in human hepatocytes. (A) Huh7 cells were cultured in the presence or absence of alcohol (40mM) for 24h. Total cellular RNA extracted from the cell culture was subjected to the real-time RT PCR for SOCS, protein inhibitor of activated STAT (PIAS), and GAPDH mRNA quantification. The data, with normalization to GAPDH mRNA, are expressed as the fold of control (without alcohol treatment, which is defined as 1). The results shown are mean ± SD of three repeated experiments. All variables in each experiment were tested in triplicate (**, P<0.01, alcohol treated vs untreated). (B) Huh7 cells were cultured in the presence or absence of alcohol (40mM) for 72h. Total cellular proteins were subjected to Western blot using the antibodies against to SOCS-2, -3 and GAPDH. The numbers in the right panel are the signal intensities of protein bands of Western blot shown in the left panel, which are expressed as densitometry scanning units (DSUs).

Fig. 9

Schematic diagram of mechanisms involved in alcohol-mediated suppression of type I IFN pathway in human hepatocytes. Alcohol not only inhibits the expression of the IRF-5 and IRF-7 (the key positive regulators of type I IFN production), but also suppresses the expression of STAT-1 and -2 (two key elements in JAK-STAT pathway). In addition, alcohol induces the expression of SOCS-2 and -3 (the negative regulators of JAK-STAT pathway). Abbreviations: IRF, interferon regulatory factor; ISGF3, IFN-stimulated gene factor-3; JAK, Janus kinase; Mx, myxovirus-resistance proteins; OAS, oligoadenylate synthetase; PKR, protein kinase; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription.