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HIV Proteins Accelerate Liver Disease; HIV Increases HCV Replication in a TGF-ß1-Dependent Manner
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Gastroenterology March 2008
Wenyu Lin_, Ethan M. Weinberg_, Andrew W. Tai_, Lee F. Peng_, Mark A. Brockman, Kyung-Ah Kim_, Sun Suk Kim_, Carolina B. Borges_, Run-Xuan Shao_, Raymond T. Chung_
_ Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
Partners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts
Received 17 October 2007; accepted 17 December 2007. published online 11 January 2008.
"We speculate that HIV indirectly regulates HCV replication and, ultimately, the fibrogenic actions of HCV by augmenting TGF-ß1, and perhaps other fibrogenic molecules, in HCV-infected hepatocytes. The finding that persistence of HIV viremia in patients undergoing antiretroviral therapy is associated with liver fibrosis progression is consistent with our observation that HIV could directly contribute to hepatic fibrosis and increased HCV levels through induction of TGF-ß1.34 We therefore conclude that HIV and gp120 enhance HCV persistence through up-regulated TGF-ß1. This unique mechanism suggests an attractive target for the development of treatments that interrupt HIV enhancement of viral replication. Further study is warranted to determine the intermediary pathways and protein interactions responsible for these effects on HCV replication."
ABSTRACT
Background & Aims: Human immunodeficiency virus (HIV) coinfection increases hepatitis C virus (HCV)-related progression of hepatic fibrosis, increases HCV persistence, and decreases response rates to interferon-based anti-HCV therapy. It has remained unclear how HIV, a nonhepatotropic virus, accelerates the progression of liver disease by HCV.
Methods: We explored the possibility that circulating HIV and/or its proteins contribute to the pathogenesis of HCV through engagement of extracellular coreceptors on hepatocytes.
Results: In this study, we found that inactivated HIV or gp120 increases HCV replication and enhances HCV-regulated transforming growth factor (TGF)-ß1 expression in both a replicon and an infectious model of HCV. This proviral effect of HIV and gp120 on HCV replication is neutralized by antibodies to CCR5 or CXCR4. However, HIV and gp120 did not alter type I interferon-mediated signaling in these HCV models, indicating that HIV regulates HCV replication through an alternative mechanism. Interestingly, we found that human TGF-ß1 also enhanced HCV replication. The effect of HIV on HCV replication was blocked by a neutralizing antibody to TGF-ß1, indicating that its effects on HCV replication are TGF-ß1 dependent.
Conclusions: These results suggest a novel mechanism by which HIV not only enhances HCV replication but also contributes to progression of hepatic fibrosis.
BACKGROUND
Human immunodeficiency virus (HIV) infects approximately 40 million persons and hepatitis C virus (HCV) infects about 180 million persons worldwide. It is estimated that 5 million persons are coinfected with HIV and HCV.1, 2 The HIV 9.8-kilobase genome encodes at least 9 proteins that include structural (Gag, Pol, Env), regulatory (Tat and Rev), and accessory (Vpu, Vpr, Vif, and Nef) proteins.3 The 160-kilodalton Env protein (gp160) is cleaved by cellular proteases to generate gp41 and gp120. HIV accelerates HCV-related liver disease, whereas HCV does not appear to accelerate HIV disease progression. Coinfection with HCV has become a major cause of morbidity and mortality among HIV-infected persons.4 However, the precise molecular mechanisms by which HIV interacts with HCV to increase HCV persistence and accelerate liver fibrosis have not been fully explored.
It has been shown that both the CD4 receptor and the ß-chemokine receptors CCR5 or CXCR4 are required for HIV entry into host cells.5, 6 Consistent with the fact that hepatocytes lack CD4 receptors, there is no evidence that HIV can enter and infect hepatocytes.1, 6 However, it has been shown that HIV gp120 can bind CCR5 and CXCR4 receptors expressed on the surface of hepatocytes, macrophages, or lymphocytes and alter cell signaling without direct infection of cells.1, 7 The effects of the interaction of HIV envelope glycoprotein with CCR5 or CXCR4 receptors in hepatocytes require further clarification. HIV-HCV coinfection has been associated with a significant increase in expression of transforming growth factor (TGF)-ß1 in serum and in the liver.8 We hypothesized that alterations in the circulating and intrahepatic cytokine environment that accompanies HIV infection, particularly the profibrogenic cytokine TGF-ß1, contribute to the increased HCV replication and accelerated liver fibrosis observed in persons coinfected with HIV and HCV.
In this study, we used both HCV replicons and an infectious HCV model9 to examine the effects of HIV and gp120 on HCV viral replication and TGF-ß1 expression. We show that HIV increases HCV replication through TGF-ß1 independently of its effects on cellular immunity.
Discussion
In this study, we have shown that inactivated HIV and gp120, but not other HIV proteins, consistently increase HCV replication independently of the effects of the cellular immune system. This enhancement of HCV replication is dependent on coreceptor engagement of CXCR4 or CCR5 by gp120/HIV. Several previous reports have suggested that indirect interactions between HIV and HCV could occur within the liver: first, human hepatocytes express CXCR4 receptors and can bind to gp1201, 7; second, gp120 can regulate intracellular gene expression through its binding to cell surface chemokine receptors, such as CXCR4, CCR5, and CCR326; third, gp120 can stimulate cytomegalovirus replication without requiring HIV coinfection of the target cell27; and fourth, gp120 induces apoptosis and proinflammatory cytokine production in hepatocytes, endothelial cells, and lymphocytes.1, 28 Our finding that HIV and gp120 enhance HCV replication through CXCR4 or CCR5 receptors in 2 independent HCV replicon systems and the JFH1 infectious model provides a new avenue to further examine the molecular mechanisms by which HIV and gp120 enhance HCV replication.
IFN-α is the predominant innate antiviral agent responsible for inhibiting HCV replication. In previous reports, we have shown that HCV contributes directly to the suppression of type I IFN signaling by decreasing STAT1 and phospho-STAT1 levels.17, 18 In this study, HIV and gp120 both increased HCV replication but did not reduce STAT1 or ISRE-mediated IFN signaling in either replicon or infectious HCV models. These data suggest that HIV does not influence HCV through the classic type I IFN signaling pathway but that its effects are mediated through an alternative pathway.
In a previous study, we noted that HIV/HCV coinfection was associated with significantly increased TGF-ß1 expression in the liver and circulation compared with HCV monoinfection.8 Another study found that HCV infection is associated with higher serum levels of TGF-ß1.22 Furthermore, increasing levels of hepatic TGF-ß1 may enhance progression of liver fibrosis in patients with HCV.29 HIV and gp120 have also been shown to independently increase TGF-ß1 secretion by T lymphocytes20 and macrophages.30 By using serum-free cell culture conditions, we directly studied the effect of HIV and gp120 on TGF-ß1 expression by hepatocytes and found that HCV infection increased TGF-ß1 in OR6 replicon and JFH1-infected Huh7.5.1 cells. Inactivated HIV and gp120, but not other HIV proteins, enhanced TGF-ß1 expression in both uninfected and HCV-infected hepatocytes. Finally, we found that TGF-ß1 has moderate stimulatory actions on HCV replication, suggesting a positive feedback loop operative in HIV coinfection.
TGF-ß1 is a regulatory molecule with pleiotropic effects on cell proliferation, differentiation, and survival that include fibrosis and immune responses to viral infection.31 TGF-ß1 has been reported to increase respiratory syncytial virus replication by a direct effect of TGF-ß1 on viral replication.24 It has also been shown that TGF-ß1 enhances JC virus replication, possibly through the promotion of Smad binding to JC virus promoter sequences.25 TGF-ß1 has also been shown to enhance macrophage susceptibility to HIV-1 by selectively increasing CXCR4 expression.32 While the effect of TGF-ß1 on HCV replication has not been well characterized, a previous study suggested that TGF-ß suppressed HCV replication through the inhibition of cell growth.33 It appears that the TGF-ß used in the previous report exhibited stronger inhibition on cell growth in a subgenomic replicon than did the TGF-ß1 used in our full-length replicon model. However, another report found that human polymorphisms associated with lower TGF-ß1 promoter activity were associated with an increased likelihood of spontaneous HCV clearance.23 Moreover, the finding that HCV/HIV-coinfected persons have higher TGF-ß1 and HCV RNA levels than persons infected with HCV alone suggests that HIV enhances TGF-ß1 and HCV replication in vivo.8 Our cell culture data show that HIV and gp120 increase HCV replication and TGF-ß1 expression, that TGF-ß1 itself increases HCV replication in replicon and infectious HCV models, and that HIV or gp120 enhances HCV replication through TGF-ß1. We speculate that HIV indirectly regulates HCV replication and, ultimately, the fibrogenic actions of HCV by augmenting TGF-ß1, and perhaps other fibrogenic molecules, in HCV-infected hepatocytes. The finding that persistence of HIV viremia in patients undergoing antiretroviral therapy is associated with liver fibrosis progression is consistent with our observation that HIV could directly contribute to hepatic fibrosis and increased HCV levels through induction of TGF-ß1.34 We therefore conclude that HIV and gp120 enhance HCV persistence through up-regulated TGF-ß1. This unique mechanism suggests an attractive target for the development of treatments that interrupt HIV enhancement of viral replication. Further study is warranted to determine the intermediary pathways and protein interactions responsible for these effects on HCV replication.
Results
HIV gp120 and Inactivated HIV Increase HCV Replication
To test whether HIV proteins have an effect on HCV replication, we incubated a panel of purified recombinant HIV-1 proteins, including SF162 gp120 (SF gp120; CCR5-tropic), CN54 gp120 (CN gp120; CXCR4-tropic), Gag, Rev, and Tat with Huh7-2-3 cells. We consistently found a greater than 2-fold increase in intracellular HCV core protein levels from baseline in the presence of either CCR5-tropic SF gp120 or CXCR4-tropic CN gp120 (P < .01 for comparisons of each gp120 with phosphate-buffered saline [PBS]) (Figure 1A and Supplementary Table 1A; see supplemental material online at www.gastrojournal.org). Other HIV proteins had no effect on HCV core protein levels.
OR6 replicon cells are Huh7 cells that harbor full-length genotype 1b HCV RNA and coexpress Renilla luciferase as a marker of replicon RNA level.12, 13 In previous studies, we successfully used OR6 replicon cells to test small molecule regulators of HCV replication.12, 13 OR6 cells were incubated with each of the HIV proteins for 48 hours to further assess their effects on HCV replication in an independent HCV replication model. In OR6 cells, we also found a greater than 2-fold increase in HCV replication (measured as RLU) from baseline in the presence of gp120 but not other HIV proteins or PBS control (Figure 1B and Supplementary Table 1B; see supplemental material online at www.gastrojournal.org). We then incubated OR6 cells with heat-inactivated CXCR4-tropic HIV (strain NL4-3; 45 ng/mL p24), CCR5-tropic HIV (strain BAL; 35 ng/mL p24), or negative control cell culture supernatants for 48 hours to determine whether HIV itself has an effect on HCV replication. We found that CXCR4-tropic HIV supernatants increased HCV replication by 106%, 79%, and 53% at dilutions of 1:4, 1:16, and 1:64, respectively, compared with negative control supernatant. In contrast, CCR5-tropic HIV supernatants only modestly increased HCV replication (by 48% and 33% at dilutions of 1:16 and 1:64, respectively) (Figure 1C and Supplementary Table 1C; see supplemental material online at www.gastrojournal.org). We also infected Huh7.5.1 cells with JFH1 virus, a fully cell culture-infectious genotype 2a HCV isolate.9, 10 We found that gp120 enhanced HCV replication by 2-fold compared with control treatments in JFH1-infected Huh7.5.1 cells (Figure 1D and Supplementary Table 1D; see supplemental material online at www.gastrojournal.org). We also found that gp120 and inactivated HIV supernatant significantly increased HCV RNA replication compared with control PBS treatments in OR6 replicon cells (Figure 1E and Supplementary Table 1E; see supplemental material online at www.gastrojournal.org).
Anti-CXCR4 and Anti-CCR5 Antibodies Neutralize the Effects of HIV on HCV Replication
To determine the mechanism by which inactivated HIV and gp120 increase HCV replication, we preincubated Huh7-2-3 cells or OR6 cells with neutralizing antibodies against CXCR4, CCR5, or mouse immunoglobulin G (MSIgG). The Huh7-2-3 cells or OR6 cells were then incubated with gp120, inactivated HIV, or other HIV proteins. We found that the increase in HCV levels mediated by SF162 gp120 or inactivated CCR5-tropic HIV was abrogated by preincubation with neutralizing antibody to CCR5; likewise, the increase in HCV levels mediated by CN54 gp120 or CXCR4-tropic HIV was blocked by preincubation with neutralizing antibody to CXCR4 in Huh7-2-3 (Figure 2A and Supplementary Table 2A) and OR6 cells (Figure 2B and Supplementary Table 2B; see supplemental material online at www.gastrojournal.org). These data indicate that HIV and gp120 produce enhancement of HCV replication that is dependent on CXCR4 or CCR5 coreceptor engagement.
HIV and gp120 Do Not Further Block pISRE-Mediated Type I IFN-α Signaling in JFH1-Infected Cells
In an earlier study, we showed that HCV blocked type I IFN signaling by impairing STAT1 and phospho-STAT1 accumulation.17, 18 Using Huh7.5.1 cells stably expressing firefly luciferase under control of an ISRE (pISRE-Huh7.5.1 cells), we analyzed the effects of inactivated HIV and gp120 on the type I IFN-α signaling pathway. We confirmed that HCV blocked ISRE-mediated type I IFN signaling in JFH1-infected cells compared with uninfected cells. However, neither HIV nor gp120 protein produced additional effects on type I IFN signaling either in the presence or absence of HCV (Figure 3A and Supplementary Table 3A; see supplemental material online at www.gastrojournal.org). To assess whether inactivated HIV affected STAT1 protein expression, Huh7.5.1 cells were first infected with day 7 JFH1 infectious supernatant for 6 hours. Inactivated HIV or HIV negative controls were incubated with uninfected or JFH1-infected Huh7.5.1 cells for 48 hours. We found that STAT1 levels were highly reduced in JFH1-infected Huh7.5.1 cells compared with uninfected Huh7.5.1 cells, consistent with previous observations in HCV replicon or core-overexpressing cell lines (Figure 3B). However, inactivated HIV supernatants did not reduce STAT1 expression in either JFH1-infected or uninfected Huh7.5.1 cells (Figure 3B). These results confirm our previous report that HCV blocks type I IFN signaling by reducing STAT1 but indicate that the effects of HIV on HCV regulation are not mediated by type I IFN signaling.
Inactivated HIV and gp120 Proteins Increase TGF-ß1 Expression in HCV Infection Models
We next examined the possibility that HIV regulated HCV replication through IFN-independent pathways. One such pathway is TGF-ß1, which is independently influenced by HCV and HIV infection.20, 21, 22, 23 We first asked whether HCV influences TGF-ß1 levels. There are 20-40 ng/mL of TGF-ß1 in human serum and fetal bovine serum.8, 22 To avoid introducing exogenous TGF-ß1 from culture medium to the experiments, we performed these assays in serum-free medium. Heat-inactivated HIV or HIV proteins were incubated with OR6 cells, cured OR6 cells, JFH1-infected Huh7.5.1 cells, or uninfected Huh7.5.1 cells in serum-free medium for 48 hours. We assessed TGF-ß1 levels in serum-free medium in OR6 cells and compared them with HCV-negative cured OR6 cells, as well as in JFH1-infected Huh7.5.1 cells compared with uninfected Huh7.5.1 cells. We found that TGF-ß1 levels were significantly higher in the medium of OR6 cells compared with cured OR6 cells and in the medium of JFH1-infected Huh7.5.1 cells compared with uninfected Huh7.5.1 cells (Figure 4A and B and Supplementary Tables 4A and B; see supplemental material online at www.gastrojournal.org). Inactivated HIV and gp120 proteins increased TGF-ß1 expression in OR6 cells and JFH1-infected Huh7.5.1 cells (Figure 4A and B and Supplementary Tables 4A and B; see supplemental material online at www.gastrojournal.org). Inactivated HIV and gp120 proteins also modestly enhanced TGF-ß1 expression in cured OR6 cells and Huh7.5.1 cells. In contrast, HIV-negative supernatant or other HIV proteins, including Gag, Pol, Rev, Tat, and Vif, did not increase TGF-ß1 expression in OR6 cells, cured OR6 cells, JFH1-infected Huh7.5.1 cells, or Huh7.5.1 cells (Figure 4A and B and Supplementary Tables 4A and B; see supplemental material online at www.gastrojournal.org). These data indicate that HCV increases TGF-ß1 secretion and that this secretion is enhanced by either HIV or gp120 in OR6 and JFH1-infected cells.
TGF-ß1 Exhibits a Dose-Dependent Effect on HCV Replication in OR6 Cells
TGF-ß1 has been shown to enhance the replication of other viruses, including respiratory syncytial virus24 and human JC virus.25 We next assessed the effects of TGF-ß1 on HCV replication. OR6 cells were incubated with serial dilutions of human TGF-ß1 in serum-free medium for 48 hours. We found that doses of TGF-ß1 up to 8 ng/mL significantly increased HCV replication more than 2-fold in OR6 cells compared with control in serum-free medium in a dose-dependent manner. Higher concentrations of TGF-ß1 did not appear to further increase HCV replication (Figure 5A and Supplementary Table 5A; see supplemental material online at www.gastrojournal.org). However, doses of TGF-ß1 ≥16 ng/mL appeared to be associated with decreased OR6 cell viability (Figure 5B and Supplementary Table 5B; see supplemental material online at www.gastrojournal.org). When normalized for viability, the enhancement of HCV replication by TGF-ß1 was still evident at higher TGF-ß1 concentrations (Figure 5C and Supplementary Table 5C; see supplemental material online at www.gastrojournal.org). These data indicate a dose-dependent enhancement of HCV replication by TGF-ß1. Preincubation of OR6 cells with TGF-ß1 neutralizing antibody completely blocked the observed effects of TGF-ß1 on HCV replication (Figure 5D and Supplementary Table 5D; see supplemental material online at www.gastrojournal.org). In contrast, control immunoglobulin G did not block TGF-ß1-induced HCV replication enhancement.
Neutralizing Antibody to TGF-ß1 Blocks the Enhancing Effect of HIV on HCV Replication
With our findings that inactivated HIV, gp120, and TGF-ß1 enhanced HCV replication and that inactivated HIV and gp120 increased TGF-ß1 expression levels in hepatocytes, we sought to determine whether HIV and gp120 acted through TGF-ß1 to enhance HCV replication. OR6 cells were preincubated with TGF-ß1 neutralizing antibody, PBS, or MSIgG for 1 hour, washed with 1~ PBS, and then incubated with recombinant HIV proteins or inactivated HIV for 48 hours. As seen previously, inactivated HIV and gp120 proteins enhanced HCV replication in OR6 cells in the presence of MSIgG and PBS (Figures 2B and 6 and Supplementary Tables 2B and 6; see supplemental material online at www.gastrojournal.org). However, HIV and gp120 did not increase HCV replication in cells that had been preincubated with TGF-ß1 neutralizing antibody (Figure 6 and Supplementary Table 6; see supplemental material online at www.gastrojournal.org). Taken together, these data provide direct functional evidence that TGF-ß1 enhances HCV replication and that HIV coinfection indirectly regulates HCV replication through the effects of gp120 on TGF-ß1.
Materials and Methods
Cell Cultures and Transfection
Huh7.5.1 cells10 were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. Huh7-2-3 cells (full-length HCV 1b replicon)11 and OR6 cells (a dicistronic HCV replicon encoding full-length genotype 1b HCV RNA and Renilla luciferase)12, 13 were grown in 10% fetal bovine serum/Dulbecco's modified Eagle medium supplemented with 400 g/mL of G418 (Promega, Madison, WI). To monitor the effect of inactivated HIV and gp120 on TGF-ß1 expression in hepatocytes, cells were cultured in UltraCulture Serum-Free Medium with Glutamine (BioWhittaker, Walkersville, MD) with G418 added as described previously. To prepare cured OR6 cells, OR6 cells were treated with 50 ng/mL pegylated interferon (IFN) (Schering Co, Kenilworth, NJ) for 2 weeks. The cured OR6 cells were cultured without IFN for another 2 weeks before the assay. Renilla luciferase assay and Western blot for HCV core protein confirmed the absence of HCV replication in cured OR6 cells. JFH1 (genotype 2a infectious HCV isolate) virus was prepared and used to infect Huh7.5.1 cells as previously reported.9, 10
HIV Stocks
Laboratory-adapted HIV-1 strains were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. Plasmid encoding the CXCR4-tropic virus NL4-3 was used to transfect HEK293T cells, and supernatant virus was propagated using a CEM-derived T-cell line that expresses CD4, CXCR4, and CCR5 (CEM-GXR).14 The CCR5-tropic virus BaL15 was also propagated using CEM-GXR cells. Viral stocks were assayed for HIV-1 p24 using the Alliance p24 Antigen ELISA Kit (PerkinElmer, Waltham, MA).
Effect of HIV-1 Proteins or Inactivated HIV on HCV Replication
Recombinant HIV-1 proteins were obtained through the National Institutes of Health AIDS Research and Reference Reagent Program. HIV-1 proteins used in this study included SF162 gp120 (SF gp120) (CCR5-tropic), CN54 gp120 (CN gp120) (CXCR4-tropic),16 Gag, Pol, Tat, Vif, and Rev. Recombinant proteins were incubated for 48 hours at a final concentration of 10 g/mL with Huh7-2-3, OR6, cured OR6, Huh7.5.1, or JFH1-infected Huh7.5.1 cells. To assess the effects of inactivated HIV on HCV replication in either OR6 cells or the JFH1 infectious model, 100 L of heat-inactivated HIV (56C for 30 minutes) was incubated with OR6 cells (3000 cells/well) or JFH1-infected Huh7.5.1 cells (10,000 cells/well) for 48 hours at 4-fold serial dilutions from 1:4 to 1:64 (11.25 to 0.70 ng/mL p24 for NL4-3; 8.75 to 0.55 ng/mL p24 for BaL). The assay was performed in a 96-well plate. Neutralizing antibodies to CCR5 or CXCR4 (R&D Systems, Minneapolis, MN) were used to verify the dependence of the effects of HIV on HCV through gp120 binding to its cognate coreceptors. Samples for luciferase assay, protein lysates, and supernatant were harvested separately at 48 hours postincubation.
Effect of Human TGF-ß1 on HCV Replication and Cell Viability
Assay
To test the effects of TGF-ß1 on HCV replication, OR6 cells were incubated with serial dilutions of human TGF-ß1 (R&D Systems) in serum-free medium for 48 hours in 96-well plates. Neutralizing antibody to TGF-ß1 (R&D Systems) was used to verify the effect of TGF-ß1 on HCV replication. Mouse immunoglobulin G (Innovative Research, Inc, Southfield, MI) was used as a negative control. Cell viability was monitored by using Cell Titer-Glo Luminescent Cell Viability Assay (Promega).13
HCV Replication
Levels of HCV in Huh7-2-3 cells and JFH1-infected Huh7.5.1 cells were measured by Ortho HCV core antigen enzyme-linked immunosorbent assay (ELISA) (Ortho-Clinical Diagnostics, Inc, Raritan, NJ).17, 18 HCV replication in OR6 cells was determined by monitoring Renilla luciferase units (RLUs) (Renilla Luciferase Assay System; Promega).13
Real-Time Polymerase Chain Reaction Quantification
HCV RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA were measured according to the method of Castet et al.19
Luciferase Reporter Assay
Type I IFN signaling was monitored by a reporter assay system (Promega). The vector pISRE-luc expresses firefly luciferase downstream of the interferon-stimulated response element (ISRE). To test the possibility that HIV and gp120 proteins act to increase HCV replication through blocking type I IFN-α signaling, we developed a stable Huh7.5.1 cell line expressing pISRE-luc under G418 selection (pISRE-Huh7.5.1). ISRE-mediated transcription was studied in the presence of 100 IU/mL pegylated IFN (interferon alfa-2b; Schering Co).
Protein Sample Preparation
Protein samples for ELISA or Western blot were prepared as previously described.17, 18
Western Blot and ELISA
Proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis with precast NuPAGE 4%-12% gels (Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride membranes. The primary antibodies include mouse anti-STAT1, rabbit anti-P-STAT1 (Tyr701) (Cell Signaling Technology, Inc, Beverly, MA), and mouse anti-HCV core (Affinity BioReagents Inc, Golden, CO). The secondary antibodies were horseradish peroxidase-conjugated enhanced chemiluminescence donkey anti-rabbit immunoglobulin G or horseradish peroxidase-conjugated enhanced chemiluminescence sheep anti-mouse immunoglobulin G (Amersham Biosciences, Piscataway, NJ). TGF-ß1 levels in supernatants were measured using the Quantikine Human TGF-ß1 ELISA Kit (R&D Systems).
Statistics
Data analysis was performed using a 2-tailed Student t test with pooled variance. Data are expressed as mean ± SD of at least 4 sample replicates unless stated otherwise. In all analyses and graphs, a value of P < .05 was denoted with *; P < .01 was denoted with **; and P < .001 was denoted with #. Details of mean ± SD and P values for each experiment are reported in the supplementary tables (see supplemental material online at www.gastrojournal.org).
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