Effect of ethanol on innate antiviral pathways and HCV replication in human liver cells
© Plumlee et al; licensee BioMed Central Ltd. 2005
Received: 06 September 2005
Accepted: 02 December 2005
Published: 02 December 2005
Alcohol abuse reduces response rates to IFN therapy in patients with chronic hepatitis C. To model the molecular mechanisms behind this phenotype, we characterized the effects of ethanol on Jak-Stat and MAPK pathways in Huh7 human hepatoma cells, in HCV replicon cell lines, and in primary human hepatocytes. High physiological concentrations of acute ethanol activated the Jak-Stat and p38 MAPK pathways and inhibited HCV replication in several independent replicon cell lines. Moreover, acute ethanol induced Stat1 serine phosphorylation, which was partially mediated by the p38 MAPK pathway. In contrast, when combined with exogenously applied IFN-α, ethanol inhibited the antiviral actions of IFN against HCV replication, involving inhibition of IFN-induced Stat1 tyrosine phosphorylation. These effects of alcohol occurred independently of i) alcohol metabolism via ADH and CYP2E1, and ii) cytotoxic or cytostatic effects of ethanol. In this model system, ethanol directly perturbs the Jak-Stat pathway, and HCV replication.
Infection with Hepatitis C virus is a significant cause of morbidity and mortality throughout the world. With a propensity to progress to chronic infection, approximately 70% of patients with chronic viremia develop histological evidence of chronic liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The situation is even more dire for patients who abuse ethanol, where the risk of developing end stage liver disease is significantly higher as compared to HCV patients who do not drink [1, 2].
Recombinant interferon alpha (IFN-α) therapy produces sustained responses (ie clearance of viremia) in 8–12% of patients with chronic hepatitis C . Significant improvements in response rates can be achieved with IFN plus ribavirin combination [4–6] and pegylated IFN plus ribavirin [7, 8] therapies. However, over 50% of chronically infected patients still do not clear viremia. Moreover, HCV-infected patients who abuse alcohol have extremely low response rates to IFN therapy , but the mechanisms involved have not been clarified.
MAPKs play essential roles in regulation of differentiation, cell growth, and responses to cytokines, chemokines and stress. The core element in MAPK signaling consists of a module of 3 kinases, named MKKK, MKK, and MAPK, which sequentially phosphorylate each other . Currently, four MAPK modules have been characterized in mammalian cells: Extracellular Regulated Kinases (ERK1 and 2), Stress activated/c-Jun N terminal kinase (SAPK/JNK), p38 MAP kinases, and ERK5 . Interestingly, ethanol modulates MAPKs . However, information on how ethanol affects MAPKs in the context of innate antiviral pathways such as the Jak-Stat pathway in human cells is extremely limited.
When IFN-α binds its receptor, two receptor associated tyrosine kinases, Tyk2 and Jak1 become activated by phosphorylation, and phosphorylate Stat1 and Stat2 on conserved tyrosine residues . Stat1 and Stat2 combine with the IRF-9 protein to form the transcription factor interferon stimulated gene factor 3 (ISGF-3), which binds to the interferon stimulated response element (ISRE), and induces transcription of IFN-α-induced genes (ISG). The ISGs mediate the antiviral effects of IFN. The transcriptional activities of Stats 1, 3, 4, 5a, and 5b are also regulated by serine phosphorylation . Phosphorylation of Stat1 on a conserved serine amino acid at position 727 (S727), results in maximal transcriptional activity of the ISGF-3 transcription factor complex . Although cross-talk between p38 MAPK and the Jak-Stat pathway is essential for IFN-induced ISRE transcription, p38 does not participate in IFN induction of Stat1 serine phosphorylation [14, 16–19]. However, cellular stress responses induced by stimuli such as ultraviolet light do induce p38 MAPK mediated Stat1 S727 phosphorylation .
In the current report, we postulated that alcohol and HCV proteins modulate MAPK and Jak-Stat pathways in human liver cells. To begin to address these issues, we characterized the interaction of acute ethanol on Jak-Stat and MAPK pathways in Huh7 cells, HCV replicon cells lines, and primary human hepatocytes.
KeywordsHCV IFN virus-host interactions signal transduction alcohol
Materials and methods
Cells and chemicals
Human hepatoma Huh7 cells were grown in DMEM containing 10% FBS, 1× penicillin, streptomycin, fungizone, 10mM L-glutamine, and 1× non-essential amino acids (all reagents were from Invitrogen; Carlsbad, CA). BB7 cells are derived from Huh7 cells and support the replication of a subgenomic HCV replicon containing a S2204I adaptive mutation in the NS5A gene . FL-Neo cells are a stable Huh7 derived cell line containing a genomic length HCV replicon with the S2204I mutation in NS5A and a P1496L mutation in NS3. BB7 and FL-Neo cells were obtained from Apath, LLC. Subgenomic replicon cell lines 9–13 and 5-15-9-2-3 (referred to as 5–15 in this paper) containing different adaptive mutations [21–23] were kindly provided by Dr. Ralf Bartenschalger. Replicon cell lines were maintained in Huh7 media containing 400 μg/ml of G418 (Calbiochem; San Diego, CA). Primary human fetal hepatocytes were isolated and grown in chemically defined serum free medium as described . Primary hepatocyte cultures were analyzed within 2 days of isolation. Cells were maintained in humidified incubators at 37°C with 5% CO2. Ethanol (AAPER; Shelbyville, KY) at concentrations of 0–200 mM, was added to cells at the same time as IFN-α (Sigma, St. Louis, MO). Relative to untreated cells, ethanol did not induce any cytotoxic or growth inhibitory effects on any of the cell types at any of the doses tested (see Additional File 1). MAPK inhibitors UO126, PD98059, and SB203508, used to inhibit p42/44, MEK1, and p38 MAPK pathways, respectively, were solubilized in DMSO and obtained from Calbiochem. ADH and CYP2E1 inhibitors 4-methylpyrazole (4-MP) and diallylsulfide (DAS) , were obtained from Sigma and solubilized in DMSO. In all experiments, the final concentration of DMSO was below 0.2%, so as to prevent DMSO inhibition of CYP2E1 .
The day prior to transfection, 2 × 105 cells were plated in 12-well tissue culture plates. Endotoxin free plasmid DNA was purified (Endofree kit, Qiagen; Valencia, CA), and was introduced into cells with lipofectamine 2000 according to manufacturer's recommendations (Invitrogen). Transfection efficiency was monitored by including 0.5 μg of plasmid pQ150 (provided by Dr. Jeffery Vieira), which expresses GFP under control of the constitutive EF-1α promoter. Prior to harvesting protein lysates, cells expressing GFP were visualized by fluorescence microscopy and the transfection efficiency calculated by determining the percentage of green cells to total cells. For reporter gene studies, 0.5 μg of the luciferase gene under control of the interferon stimulated response element (ISRE) in plasmid pISRE-luc (ISRE promoter; Stratagene; La Jolla, CA), was transfected into cells in duplicate or triplicate. In certain experiments a dominant negative p38 (p38 AGF) expressing plasmid , provided by Dr. Michael Kracht, was transfected into cells. Twenty-four hours post-transfection, ethanol, either alone or in combination with IFN was added directly to cells. Six hours later, luciferase activity was measured on cell lysates and normalized for transfection efficiency and total protein content.
Western blot analysis
Protein lysates were quantitated by BCA Protein Assay (Pierce; Rockford, IL) and equal amounts (typically 20–30 μg) of total protein was separated on 4–20% SDS-PAGE gels. For detection of phosphorylated Stat1 proteins, Stat1 phosphotyrosine (Y701) and phosphoserine (S727) specific antibodies were used (Zymed-Invitrogen). Total Stat1 was detected using a polyclonal antibody (Zymed or Santa Cruz Biotechnology; Santa Cruz, CA). Total and phosphorylated forms of p42/44 (ERK2/1), and p38 MAPK were detected with specific antisera (Cell Signaling; Beverly, MA). Cytochrome P4502E1 (CYP2E1) was detected using polyclonal rabbit antiserum (provided by Arthur Cederbaum), while alcohol dehydrogenase (ADH) was detected with a mouse monoclonal antibody (AbCam; Cambridge, MA). HCV proteins were detected using random, de-identified HCV infected patient serum, as described . Infected serum was inactivated by adding triton X-100 to 1% prior to use.
The activity of p38 MAPK in Huh7 cells was assessed via kinase assay using a kit (Cell Signaling). Briefly, cell lysates were immunoprecipitated with antibodies that recognize the phosphorylated form of p38 MAPK. After stringent washing, recombinant ATF-2 protein, a substrate for p38, was added to immunoprecipitates and incubated for 30 minutes according to manufacturer's specifications. Phosphorylated protein ATF-2 was detected by western blot.
HCV RNA quantitation
HCV RNA was quantitated by real time RT-PCR, using a modified version of a recent procedure . Total cellular RNA was isolated from replicon cells using a commercial kit (Qiagen). Ten nanograms of RNA was added to wells of a 384 well plate containing the EZ RT-PCR master mix (Perkin Elmer; Wellesley, MA). Samples were run on an ABI HT7900 real time RT-PCR machine. The RT reaction consisted of 50°C for 2 minutes followed by 60°C for 30 minutes. The PCR consisted of an initial denaturation of 2 minutes at 95°C, then 45 cycles of 95°C for 15 seconds followed by simultaneous annealing/extension at 60°C for 1 minute. For each run, dilutions of BB7 plasmid DNA (precisely quantitated using the PicoGreen DNA quantitation kit (Invitrogen)) ranging from 0–107 copies per tube were run in triplicate to generate a standard curve, which served as a reference to calculate HCV RNA copy number based on the cycle threshold (Ct). The HCV RNA copy number is reported as copies per 10 ng total cellular RNA. Additional controls included reactions lacking template as well as RNA from Huh7 cells. For both negative controls, these samples were always negative for HCV RNA.
ADH enzyme assay
Cells were harvested in PBS and whole cell extracts prepared via sonication. Aliquots of protein extracts were mixed with 0.1 M glycine pH 10.0 buffer, 2.4 mM β-nicotinamide adenine dinucleotide, and 33 mM ethanol, and conversion of NAD to NADH+ was monitored with a spectrophotometer at a wavelength of 340 nm. All reagents for the assay were from Sigma. As a positive control, purified human ADH (provided by Dr. Carol Stone) was also run in the assay.
Differences between means of luciferase readings were compared using a Student's T-test. A p-value of <0.05 was considered significant. For western blots, data were analyzed with Image J, a software version of NIH Image for the Macintosh OS × operating system. Changes in protein levels were normalized to control western blots and expressed as fold or percent change relative to controls.
Effect of acute ethanol on Jak-Stat pathway
To investigate this regulation further, we analyzed levels of phosphorylated Stat1 and Stat2, which are obligatory steps for ISRE activation. Stat1 and Stat2 activation involves phosphorylation on conserved tyrosines at amino acid positions 701 and 690, respectively, while phosphorylation of Stat1 also occurs on conserved serine amino acid at position 727 and provides maximal transcriptional activation . Figure 1B depicts the levels of Stat1 S727 (top panel), Stat1 Y701 (second panel), and Stat2 Y690 (third panel), and the total levels of Stat1 protein (fourth panel) in Huh7 cells. Phosphorylation of Stat1 on S727 was induced by IFN-α or 100 mM ethanol. Stat1 Y701 and Stat2 Y690 phosphorylation occurred with IFN treatment, whereas no effect was observed with 100 mM ethanol. The differences in Stat phosphorylation were not due to differences in the amount of Stat1 protein, since total Stat1 protein levels were equivalent (Figure 1B, lower panel). Similar results were also observed for primary human fetal hepatocytes (see Additional File 2) and HCV replicon cells (data not shown).
Effect of acute ethanol on the p38 MAPK pathway
Acute alcohol stimulation of the Jak-Stat pathway involves MAPKs
Effect Of acute alcohol on HCV replicons
Acute alcohol inhibits the IFN-α induced antiviral response towards HCV
Expression of alcohol metabolizing enzymes in human liver cell cultures
In the current study, it was demonstrated that high physiological doses of acute ethanol induces Stat1 serine phosphorylation and ISRE transcription. Given alone, ethanol appears to inhibit HCV replication in several independent replicon cell lines, and this is in part mediated by a Jak-Stat transduced antiviral response. In contrast, in the presence of exogenously added IFN-α, ethanol partially inhibits the antiviral actions of IFN-α, involving inhibition of IFN-α induced Stat1 tyrosine phosphorylation. Analysis of the effects of chronic ethanol administration on basal and IFN-α induced signaling responses is currently in progress.
Recent studies have demonstrated that alcohol abuse may be associated with increased HCV RNA titers in patients . This could be due to an increase in release of HCV RNA from alcohol-damaged hepatocytes, a direct stimulatory effect of alcohol on HCV replication, or modulation of innate and acquired immune responses to HCV. A single published report by Zhang and colleagues found that ethanol stimulates HCV replication in the replicon system , while our data indicate that acute ethanol inhibits HCV replication. There are several explanations for the divergent results. First, different stable replicon cell lines were used in our study as compared to the published study, so it is very likely that both the replicons  and Huh7 cells [30, 31] are genetically different. Second, in Zhang's study, alcohol was added to replicon cells daily, so 48 and 72-hour time points actually received 2 and 3 daily doses of ethanol. This is in direct contrast to our experimental design where a single "shot" of alcohol was given. Nonetheless, chronic ethanol treatment of cells for 3 consecutive days further inhibited HCV replication in our system (data not shown). Third, in our study, the observed effects on the IFN system and HCV replication appeared to be due to the direct action of ethanol, rather than via ethanol metabolism, as reported in the Zhang study . However, the dose of the ADH inhibitor 4-methypyrazole used in Zhang's study was 0.1 μM, 50,000 fold lower than the 5 mM dose used in our study, and the dose used in a seminal study demonstrating the effect of various inhibitors of alcohol metabolism . Further evidence for a direct effect of ethanol for the observed results in our study stems from the observation that all replicon and Huh7 cells expressed low to undetectable levels of ADH and CYP2E1 protein, and ethanol still induced ISRE transcription in the presence of ADH and CYP2E1 inhibitors. The Zhang study did not measure ADH and CYP2E1 protein expression. Furthermore, in our studies, the effects of ethanol on the Jak-Stat pathway occurred at an ethanol concentration of 100 mM, well above that of the Km for ADH (1–5 mM) and CYP2E1 (16 mM) . Moreover, high-dose ethanol has been previously shown to activate IFN-β-dependent antiviral activities , reminiscent of the data reported in the present study. Collectively, our data suggest that ethanol acts directly on cells to modulate hepatocyte signaling pathways.
Exactly how ethanol induces these signaling responses is currently under investigation. Ethanol is known to act on lipids in cell membranes as well as interact directly with membrane proteins [44–47], so it is possible that changes in membrane fluidity (defined as the physical state of the phospholipids in terms of rate and angular motion) induce downstream signal transduction events (Figure 8). In terms of the activation of the Jak-Stat pathway by acute ethanol, it is possible that besides Stat1, other proteins with the capacity to bind ISRE-like sequences are involved in ethanol induced ISRE transcription. A possible candidate is Stat3, since Stat3 is modulated by ethanol . Indeed, preliminary data suggest that Stat3 is also modulated by acute ethanol in our system (data not shown). As for ethanol inhibition of IFN-induced Stat1 tyrosine phosphorylation and antiviral actions, several mechanisms might be operative (Figure 8). Since IFN-β has been shown to modulate plasma membrane fluidity , ethanol might inhibit IFN-α induced changes in membrane fluidity. Other possible mechanisms include ethanol inhibition of IFN-receptor interactions, or induction of negative regulators of the Jak-Stat pathway such as suppressors of cytokine signaling (SOCS) proteins. For example, SOCS-1 inhibits IFN signaling by binding Jaks to prevent Stat phosphorylation . Also of note is the observation that ethanol doses of 1–20 mM did not affect HCV replication (Figure 4A), so it is possible that ethanol-induced blockade of IFN antiviral activity is more relevant in vivo. The data presented herein highlight the complexity, and emphasize the need for further study of the cellular response to acute and chronic alcohol, on innate antiviral signaling pathways and HCV replication.
In conclusion, acute ethanol treatment of Huh7 hepatoma, HCV subgenomic and genomic-length replicon cells, and primary human fetal hepatocytes has multiple effects on innate cellular defense pathways. In particular, high physiological doses of ethanol can activate antiviral responses and inhibit HCV replication, whereas it can also inhibit the IFN-α induced antiviral response against HCV replication. The data suggest that the effects of alcohol on the IFN system are not simply a stimulation or inhibition, but rather reflect highly complex processes involving cross-talk of a number of signaling pathways. The net effect of ethanol likely depends on whether ethanol is given acutely or chronically, the dose of ethanol, and whether alcohol is metabolized or not.
cytochrome P450 2E1
extracellular regulated kinase
hepatitis C virus
interferon stimulated gene factor 3
interferon stimulated response element
janus associated kinase
mitogen activated protein kinase
relative light units
signal transducer and activator of transcription
We thank Michael Austin, John Gallegos, Jacob Glaspey, Jamison Green, Amanda Heitzke, Kristen Miller, Paula McPoland, and Jessica Wagoner for technical assistance, Jeffery Vieira for pQ150, Arthur Cederbaum for CYP2E1 antiserum and helpful advice, Dennis Rasmussen for advice, Carol Stone for recombinant human ADH, Sidney Nelson for recombinant CYP2E1, Apath LLC and Ralf Bartenschlager for HCV replicon cell lines, and Michael Kracht for p38 plasmids. CL and NF are partially supported by NIH grant AI048214. SJP is partially supported by NIH grants AA13301 and DK62187, and the University of Washington Royalty Research Fund.
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