These authors contributed equally to this work.
Hepatitis C virus infection enhances TNFα-induced cell death via suppression of NF-κB†
Article first published online: 26 JUL 2012
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 56, Issue 3, pages 831–840, September 2012
How to Cite
Park, J., Kang, W., Ryu, S.-W., Kim, W.-I., Chang, D.-Y., Lee, D. H., Park, D. Y., Choi, Y.-H., Choi, K., Shin, E.-C. and Choi, C. (2012), Hepatitis C virus infection enhances TNFα-induced cell death via suppression of NF-κB. Hepatology, 56: 831–840. doi: 10.1002/hep.25726
Potential conflict of interest: Nothing to report.
- Issue published online: 28 AUG 2012
- Article first published online: 26 JUL 2012
- Accepted manuscript online: 20 MAR 2012 12:00AM EST
- Manuscript Accepted: 6 MAR 2012
- Manuscript Received: 26 OCT 2011
- Research Program for New Drug Target Discovery. Grant Numbers: 2010-0020471, 2010-0030075
- National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology
- Korea Advanced Institute of Science and Technology Future Systems Healthcare Project from the Ministry of Education, Science, and Technology
Hepatitis C virus (HCV) infection results in liver injury and long-term complications, such as liver cirrhosis and hepatocellular carcinoma. Liver injury in HCV infection is believed to be caused by host immune responses, not by viral cytopathic effects. Tumor necrosis factor-alpha (TNF-α) plays a pivotal role in the inflammatory processes of hepatitis C. TNF-α induces cell death that can be ameliorated by nuclear factor kappaB (NF-κB) activation. We investigated the regulation of TNF-α signal transduction in HCV-infected cells and identified HCV proteins responsible for sensitization to TNF-α-induced cell death. We studied the effect of HCV infection on TNF-α signal transduction using an in vitro HCV infection model (JFH-1, genotype 2a) with Huh-7 and Huh-7.5 cells. We found that TNF-α-induced cell death significantly increased in HCV-infected cells. HCV infection diminished TNF-α-induced phosphorylation of IκB kinase (IKK) and inhibitor of NF-κB (IκB), which are upstream regulators of NF-κB activation. HCV infection also inhibited nuclear translocation of NF-κB and expression of NF-κB-dependent anti-apoptotic proteins, such as B-cell lymphoma—extra large (Bcl-xL), X-linked inhibitor of apoptosis protein (XIAP), and the long form of cellular-FLICE inhibitory protein (c-FLIP). Decreased levels of Bcl-xL, XIAP, and c-FLIP messenger RNA and protein were also observed in livers with chronic hepatitis C. Transfection with plasmids encoding each HCV protein revealed that core, nonstructural protein (NS)4B, and NS5B attenuated TNF-α-induced NF-κB activation and enhanced TNF-α-induced cell death. Conclusion: HCV infection enhances TNF-α-induced cell death by suppressing NF-κB activation through the action of core, NS4B, and NS5B. This mechanism may contribute to immune-mediated liver injury in HCV infection. (HEPATOLOGY 2012;56:831–840)
Hepatitis C virus (HCV) is an enveloped hepatotropic virus with a positive-sense RNA genome. The 9.6-kb genome encodes one large polyprotein that is cleaved into 10 viral proteins: core, envelope protein (E)1, E2, p7, nonstructural protein (NS)2, NS3, NS4A, NS4B, NS5A, and NS5B.1, 2 HCV infection tends to progress to chronic hepatitis, which is often complicated by liver cirrhosis and hepatocellular carcinoma (HCC).3 Thus, HCV represents a serious, worldwide public health problem.4
Cellular and molecular mechanisms responsible for liver injury in HCV infection remain poorly understood. Because HCV infection has no cytopathic effect, liver injury is considered to be induced by host immune responses.5-7 Especially, T-cell responses are known to be responsible for both liver injury and viral clearance in HCV infection. Histological studies have demonstrated that enhanced apoptosis of hepatocytes is a common feature of HCV-infected livers, and the abundance of infiltrating T cells suggests a crucial role for T cells in the apoptosis of hepatocytes.8-10 In T-cell-mediated hepatocyte killing, perforin, Fas ligand, and tumor necrosis factor-alpha (TNF-α) are major effector molecules.8, 11 TNF-α is produced not only by immune cells, but also by hepatocytes,12 and systemic TNF-α levels increase during HCV infection.13 In several ways, TNF-α plays a pivotal role in the inflammatory processes of chronic hepatitis C (CHC) and hepatocyte death.14
Many biological effects of TNF-α are mediated by the transcription factor, nuclear factor kappaB (NF-κB). Under resting conditions, NF-κB forms a complex with the inhibitor protein, inhibitor of NF-κB (IκB), thereby blocking the nuclear import of NF-κB. The binding of TNF-α to its receptor induces the phosphorylation of IκB kinase (IKK) through recruitment of TNF receptor-associated death domain protein (TRADD), TNF receptor-associated factor 2 (TRAF2), and receptor-interacting protein kinase (RIP) to the cytosolic portion of the TNF-α receptor. Phosphorylated IKK, in turn, phosphorylates IκB, inducing IκB degradation and, eventually, NF-κB translocation from the cytosol to the nucleus.15, 16 Upon TNF-α stimulation, the expression of anti-apoptotic proteins, anti-oxidants, inflammatory chemokines, and negative module IκB are under the control of NF-κB.17-19 In addition to its role in the NF-κB pathway, TNF-α also activates c-Jun N-terminal kinase (JNK), which contributes to TNF-α-induced cell death by multiple mechanisms.20 In many cell types, TNF-α-induced cell death depends on the contextual ability of the cell to maintain the activation of either cytoprotective NF-κB or pro-apoptotic JNK.21, 22
Viral infection often alters NF-κB signal-transduction patterns. Hepatitis B virus (HBV)-induced NF-κB activation is well defined.23 Of the HBV-encoded proteins, HBx activates NF-κB by acting on two distinct NF-κB inhibitors, IκB-α and p105.24, 25 In contrast, regulation of NF-κB activity in HCV-infected cells is poorly understood; studies under unphysiological conditions involving forced expression of HCV proteins have yielded inconsistent and conflicting data.12, 26-35 Recently, cell-culture models of HCV infection have been established in human HCC cell lines using JFH-1-based full-length genomes.36-38 This system provided an opportunity to address many aspects of the HCV life cycle and host-virus interactions, including cross-talk with the host signal-transduction system.
In the present study, we investigated the effect of HCV infection on TNF-α-induced cell death and TNF-α signal transduction in Huh-7 and Huh-7.5 cells using an in vitro JFH-1 HCV infection model. Furthermore, we identified the HCV proteins responsible for the regulation of TNF-α signal transduction.
Materials and Methods
Cell Culture and Reagents
Huh-7 and Huh-7.5 cells were provided by Apath (Brooklyn, NY). Antibodies specific for IKK, phospho-IKK, phospho-IκB, JNK, phospho-JNK, X-linked inhibitor of apoptosis protein (XIAP), cellular-FLICE inhibitory protein (c-FLIP), and FLAG were purchased from Cell Signaling Technology (Beverly, MA). Antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, p65, and horseradish-peroxidase–conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Human recombinant TNF-α was acquired from R&D Systems (Minneapolis, MN). The NF-κB inhibitor, SN50, was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). The JNK inhibitor, SP600125, was purchased from Calbiochem (La Jolla, CA). Recombinant HCV protein core, NS3, NS4, and NS5B were obtained from ViroGen (Watertown, MA). The caspase-3 substrate, Ac-DEVD-AMC, was purchased from Calbiochem.
HCV Preparation and Infection.
The JFH-1 strain (genotype 2a) of HCV was produced by transfecting Huh-7.5 cells with linearized RNA from a plasmid encoding the full genome of JFH-1 HCV (provided by Apath). Huh-7.5 cells were transfected with DMRIE-C reagent (Invitrogen, Carlsbad, CA) using in vitro–transcribed JFH-1. After RNA transfection, cell-culture supernatants at the peak of HCV production were used to infect naïve Huh-7.5 cells. HCV-infected Huh-7.5 cells were passaged, and cell-culture supernatants with the highest HCV production were selected as described previously.39 The selected HCV supernatants were filtered (0.45 μm) and frozen at −70°C until use. Naïve Huh-7 and Huh-7.5 cells were infected with HCV supernatants at a multiplicity of infection (MOI) of 0.01. Cells were subcultured every 3.5 days. At the time of subculture, a portion of the cells was permeabilized and immunostained with an anti-HCV core antibody (Affinity BioReagents, Golden, CO) and FITC-anti-mouse immunoglobulin (Ig) (BD Biosciences, San Jose, CA) to determine the percentage of HCV-infected cells. When >80% of cells were infected, cells were used for TNF-α treatment and further analyses.
The HCV RNA Replicon System.
Huh-7.5 cells carrying the full-length H77 (genotype 1a) replicon were maintained in complete Dulbecco's modified Eagle's medium (DMEM), supplemented with 1 g/L of G418 (A.G. Scientific, San Diego, CA). For elimination of HCV RNA, cells were maintained in complete DMEM, supplemented with 10 μg/L of interferon-beta (IFN-β) instead of G418. After HCV became undetectable, HCV-cured cells were maintained in complete DMEM without IFN-β and G418.
Cell-Death and Viability Assays.
After treatment with TNF-α for 24 hours, cells were washed twice with phosphate-buffered saline (PBS), trypsinized, suspended in binding buffer, and stained with propidium iodide (PI) and Annexin V/allophycocyanin (Pharmingen, San Diego, CA). The stained cells were analyzed on an LSR II flow cytometer (BD Biosciences). A water-soluble tetrazolium (WST)-1 assay was also performed to measure cell viability and cell death. Huh-7 and Huh-7.5 cells were seeded in 24-well plates, and WST-1 reagent (Nalgene, Rochester, NY) was added to each well. After incubation for 2 hours at 37°C in a 5% CO2 incubator, absorbance was measured at 450 nm by using a microplate reader (Bio-Rad, Richmond, CA). A lactate dehydrogenase (LDH) release assay (Promega, Madison, WI) was also carried out according to the manufacturer's protocol.
Cell lysates were separated by standard 10% glycine/sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed with antibodies against IKK, IκB, JNK, B-cell lymphoma—extra large (xL), XIAP, c-FLIP, FLAG, GAPDH, and β-actin. Blottings were developed using enhanced chemiluminescence (AbFrontier, Seoul, Korea). Images were captured and band intensities were quantified by the Kodak Image Station (Eastman Kodak, Rochester, NY).
Cells grown in a four-well chamber slides were fixed with 4% paraformaldehyde in PBS for 15 minutes, permeabilized with 0.15% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 15 minutes, and blocked with 1.5% bovine serum albumin (BSA) for 1 hour. Slides were then incubated with polyclonal anti-p65 or anti-HCV core antibody. After washing with PBS, slides were incubated with FITC or rhodamine-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology). Slides were observed under a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).
Subcellular Fractionation and Measurement of DNA-Binding Activity of NF-κB.
Huh-7.5 cells were harvested and fractionated into nuclear and cytoplasmic fractions using a nuclear/cytosol fractionation kit (BioVision, Mountain View, CA), according to the manufacturer's protocols. NF-κB activity was monitored using an enzyme-linked immunosorbent assay (ELISA)-based colorimetric TransAM NF-κB p65 kit (Active Motif, Carlsbad, CA), containing a 96-well plate with immobilized oligonucleotides encoding an NF-κB consensus site (5′-GGGACTTTCC-3′). The amount of immobilized NF-κB was determined by colorimetric reaction and absorbance at 450 nm.
Electrophoretic Mobility Shift Assays.
For the binding reaction, 5 μg of nuclear extract was incubated at room temperature for 30 minutes with probe in binding buffer containing 10 mM of Tris-Cl (pH 7.5), 100 mM of KCl, 1 mM of dithiothreitol, 1 mM of ethylene diamine tetraacetic acid, 0.2 mM of phenylmethanesulfonyl fluoride, 1 g/L of BSA, and 5% glycerol. For competition and supershift experiments, nuclear extracts were pretreated with a 100-molar excess of cold oligonucleotide or 1 μg of NF-κB (p50) antibody (Santa Cruz Biotechnology) for 30 minutes before the addition of the labeled probe. Reaction mixtures were analyzed in a 6% polyacrylamide gel and by autoradiography. The sequence of the oligonucleotide used as the probe was 5′-AGTTGAGGGGACTTTCCCAGGC-3′.
Liver tissues of 5 patients with HCV-associated HCC were included in the present study. During the surgical resection of tumor, nontumorous HCV-infected tissues were obtained and frozen at −70°C for RNA extraction. Part of these samples was dissected, formalin-fixed, and paraffin-embedded for immunohistochemistry (IHC). These specimens were provided by the National Biobank of Korea (PNUH, Busan, Korea). Six liver tissues without viral hepatitis were also included in the study. These tissues were obtained during operations, such as cholecystectomy, adrenalectomy, and partial liver resection for intrahepatic duct stones, under the approvement of the institutional review board (Daejeon St. Mary's Hospital, Daejeon, Korea) and the agreement of the patients. Paraffin-embedded tissues were used for IHC to evaluate the expression of XIAP, c-FLIP, and Bcl-xL.
Extraction of RNA, Complementary DNA synthesis, and TaqMan Real-Time Polymerase Chain Reaction.
Total RNA was isolated from liver tissues using the RNeasy Mini Kit (Qiagen, Valencia, CA). Complementary DNA (cDNA) was synthesized from 800-1,000 ng of total RNA with the First-Strand cDNA Synthesis Kit (Marligen Biosciences, Ijamsville, MD). TaqMan real-time PCR was performed in duplicate to determine mRNA levels of Bcl-xL, XIAP, and c-FLIP using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). Target mRNA levels were normalized to an endogenous reference (β-actin).
Cloning of HCV Genes and Transfection.
Genes for individual HCV proteins (i.e., core, E1, E2, NS2, NS3/4A, NS4B, NS5A, and NS5B) were amplified by PCR from a plasmid encoding the full genome of JFH-1 HCV. PCR products were then digested with restriction endonucleases and ligated into the pCMV-3Tag-3A plasmid vector (Stratagene, La Jolla, CA). The nucleotide sequence of each HCV gene was confirmed by DNA sequencing. Transient transfection was carried out using Lipofectamine 2000 (Invitrogen), and transfection efficiency was assessed by immunoblotting for FLAG-tag.
NF-κB Reporter Assay.
Cells were transfected with the luciferase reporter plasmids containing NF-κB responsive elements using Lipofectamine 2000. The pRL-CMV vector (Promega) was used as a control reporter for normalization. Twenty-four hours post-transfection, cells were treated with TNF-α for 6 hours. Cells were lysed, and luciferase activity was determined using the dual-luciferase assay system (Promega), according to the manufacturer's instructions. Luminescence was measured with a Wallac multilabel counter (PerkinElmer Wallac, Gaithersburg, MD).
IKK Activity Assay.
IKK activity was measured using the CycLex IKK-α/β assay kit (MBL International, Woburn, MA), which is a single-site–binding immunoassay. Plates are precoated with a substrate corresponding to recombinant IκB-α, which contains two serine residues that are phosphorylated by IKK-α and IKK-β. We used a peroxidase-coupled anti-phospho-IκB-α S32 monoclonal antibody as a reporter molecule in a 96-well ELISA format.
Data are presented as the mean ± standard error of the mean (SEM). Level of significance for comparisons between two independent samples was determined using Mann-Whitney's test. Groups were compared by one- or two-way analysis of variance (ANOVA), with Tukey's post-hoc test or Bonferroni's post-hoc test applied to significant main effects.
HCV Infection Sensitizes Cells to TNF-α-Induced Cell Death.
In the present study, we focused on TNF-α-induced cell death and studied the effect of HCV infection on TNF-α-induced cell death using an in vitro JFH-1 (genotype 2a) HCV infection model. After HCV infection of Huh-7 and Huh-7.5 cells, the percentage of HCV-infected cells was determined periodically by immunocytochemistry (ICC) and flow cytometry with anti-HCV core immunostaining. When >80% of cells were infected (Fig. 1A), cells were plated in new culture vessels and treated with TNF-α for 24 hours. TNF-α induced significant cell death in HCV-infected cells, whereas its effect on noninfected cells was marginal, as determined by WST-1 assay (Fig. 1B). This result was confirmed by flow cytometry after PI and Annexin V staining (Fig. 1C) and by LDH assay (Fig. 1D). Enhanced cell death was observed not only in JFH-1 HCV-infected cells, but also in JFH-1 HCV RNA-transfected cells (Fig. 1E) and in cells harboring the H77 (genotype 1a) HCV RNA replicon (Fig. 1F). Taken together, these results indicate that HCV RNA replication, and its protein expression, makes infected cells vulnerable to TNF-α-induced cell death.
HCV Infection Suppresses TNF-α-Induced NF-κB Activation.
TNF-α-induced cell death is regulated by intracellular signaling pathways, such as the NF-κB and JNK pathways. To clarify the roles of the NF-κB and JNK pathways in TNF-α-induced cell death, we evaluated cell death after TNF-α treatment with or without pretreatment with specific inhibitors. SN50 (a NF-κB-specific inhibitor) pretreatment sensitized Huh-7 and Huh-7.5 cells to TNF-α-induced cell death, whereas SP600125 (a JNK-specific inhibitor) rescued cells from cell death. Moreover, SP600125 abolished TNF-α-induced cell death, even in the presence of SN50 (Fig. 2A). Titration studies of SP600125 and SN50 showed that this event occurs in a dose-dependent manner (Supporting Fig. 1A). Without TNF-α treatment, SP600125 and SN50 did not affect cell viability (Supporting Fig. 1B).
Next, we investigated whether HCV infection regulated the activation of the NF-κB and JNK pathways upon TNF-α treatment. When the NF-κB pathway becomes activated, IKK and IκB is phosphorylated, followed by the nuclear translocation of NF-κB. Immunoblotting analysis revealed that TNF-α-induced NF-κB activation was significantly attenuated in HCV-infected cells, as evidenced by the reduced phosphorylation of IKK and the very brief phosphorylation of IκB, contrasting with the increased phosphorylation of IKK and IκB in uninfected cells during the course of TNF-α treatment. Of note, the phosphorylation of JNK was not influenced by HCV infection (Fig. 2B and Supporting Fig. 1C).
We examined the nuclear translocation of NF-κB, a consequence of IκB phosphorylation and degradation. ICC analysis of p65, a major component of the NF-κB complex, revealed that TNF-α-induced translocation of NF-κB was reduced in HCV-infected cells, as evidenced by the diffuse staining pattern of p65 throughout the cytosol and nucleus upon TNF-α treatment (Fig. 3A). We also examined the DNA-binding activity of NF-κB in an ELISA-based colorimetric assay. TNF-α treatment markedly increased the DNA-binding activity of p65, a response that was significantly suppressed by HCV infection (Fig. 3B). These data were confirmed by electrophoretic mobility shift assay (EMSA) (Fig. 3C).
HCV Infection Suppresses TNF-α-Induced Expression of Anti-apoptotic Proteins.
Next, we investigated the expression of NF-κB-dependent anti-apoptotic proteins, including Bcl-xL, XIAP, and c-FLIP. Immunoblotting analysis showed that TNF-α-induced expression of Bcl-xL, XIAP, and the long form of c-FLIP (c-FLIPL), which are well-known anti-apoptotic proteins, was markedly lower in HCV-infected cells. Eventually, caspase-3 was highly activated by TNF-α in HCV-infected cells (Fig. 4A). Augmented activation of caspase-3 in HCV-infected cells was confirmed by the enzyme activity assay of caspase-3 (Fig. 4B). Expression of anti-apoptotic genes was also studied in HCV-infected livers by IHC and quantitative real-time PCR. Compared to livers without viral hepatitis, HCV-infected livers expressed markedly lower protein and mRNA levels of Bcl-xL, XIAP, and c-FLIP (Fig. 4C,D), supporting the results from our in vitro study. Collectively, these data indicate that HCV infection suppressed the TNF-α-induced expression of anti-apoptotic proteins through the inhibition of NF-κB activation and enhanced TNF-α-induced cell death.
HCV Core, NS4B, and NS5B Inhibit TNF-α-Induced NF-κB Activation.
We sought to identify which HCV proteins were responsible for the inhibition of TNF-α-induced NF-κB activation through cotransfection of plasmids encoding each viral protein with a luciferase reporter plasmid containing NF-κB-responsive elements. Expression of each viral protein was confirmed by FLAG-tag immunoblotting (Supporting Fig. 2A). First, we investigated whether HCV proteins regulated baseline NF-κB activity without TNF-α treatment, and found that NS4B and NS5A significantly increased baseline NF-κB activity (Supporting Fig. 2B). Next, we examined the role of each HCV protein in the regulation of TNF-α-induced NF-κB activation. At 24 hours after cotransfection, cells were treated with TNF-α for an additional 6 hours and NF-κB activation was determined by luciferase activity. TNF-α-induced NF-κB activation was significantly inhibited by core, NS4B, and NS5B in a gene-dosage–dependent manner (Fig. 5A). The kinase activity of IKK was also significantly reduced by transfection of core, NS4B, and NS5B (Fig. 5B). Note that IKK activity was remarkably decreased by incubation with recombinant HCV core, NS4, and NS5B (Supporting Fig. 2C,D), implying that core, NS4, and NS5B might suppress NF-κB activity through direct interaction with IKK. We also investigated TNF-α-induced NF-κB pathway activation after cotransfection of plasmids carrying the core, NS4B, and NS5B genes. TNF-α-induced phosphorylation of IKK and IκB was profoundly attenuated in cells cotransfected with the three plasmids (Fig. 5C). Importantly, transfection of individual plasmids carrying the core, NS4B, and NS5B genes enhanced TNF-α-induced cell death (Fig. 5D). The effects of core, NS4B, and NS5B on NF-κB activity, IKK activity, and TNF-α-induced cell death were further enhanced by cotransfection of plasmids carrying all three of these genes.
We showed that HCV infection enhanced TNF-α-induced cell death through suppression of NF-κB activation by the action of core, NS4B, and NS5B. This mechanism may contribute to immune-mediated liver injury in hepatitis C by sensitizing HCV-infected hepatocytes to TNF-α-induced cell death (Fig. 6). HCV infection made the infected cells vulnerable to TNF-α-induced cell death by suppressing TNF-α-induced NF-κB activation and the subsequent expression of NF-κB-dependent anti-apoptotic proteins, such as Bcl-xL, XIAP, and c-FLIPL. Down-regulation of such anti-apoptotic genes were also observed in HCV-infected liver. The effect of HCV infection was also recapitulated with the transfection of plasmids carrying the HCV core, NS4B, and NS5B genes, indicating that the core, NS4B, and NS5B proteins are responsible for the suppression of NF-κB activation. In addition, cell death was enhanced in JFH-1 HCV RNA-transfected cells and in cells harboring the H77 HCV RNA replicon. These results provide insight into the mechanism by which HCV infection alters intracellular events relevant to liver injury and the pathogenesis of hepatitis C.
Multiple HCV proteins interact with host proteins involved in intracellular-signaling pathways. In the case of the NF-κB pathway, core, NS3, NS4B, and NS5A are known to regulate the activity of NF-κB. However, these previous studies investigated the effects of individual HCV proteins by transfection of each HCV genes, not by actual HCV infection.12, 26-35 Such experiment settings were not sufficient to show the comprehensive effect of HCV infection and produced inconsistent results, including inhibition of TNF-α- and Fas-mediated apoptosis by HCV core protein. For the current study, on the other hand, we adopted the in vitro JFH-1 HCV infection system that became available in 200536-38 to study the role of HCV proteins in the setting of actual infection. The value of the HCV infection model in pathophysiologic studies is demonstrated by the fact that HCV infection suppresses TNF-α-induced NF-κB activation, despite the stimulatory effect of NS5A (Fig. 5A). The NF-κB-suppressive role of core, NS4B, and NS5B overruled the NF-κB-enhancing role of NS5A in the HCV infection system, resulting in enhanced TNF-α-induced cell death. Intriguingly, decreased nuclear translocation of NF-κB was reported in HCV-infected liver in a previous study using a chimeric SCID/Alb-uPA mice model.40
Given that NF-κB inhibition facilitates cell death, our results are consistent with previous studies. Various HCV proteins induce apoptosis, and HCV infection sensitizes human hepatocytes to TRAIL–induced apoptosis in a caspase-9-dependent manner.41-43 Moreover, HCV infection induces apoptosis through the caspase-3-dependent pathway.44 In contrast, NS5A is involved in anti-apoptotic effects mediated by NF-κB activation in Huh-7 cells.45 Of interest, the role of NS4B in the regulation of NF-κB activity is influenced by the cellular microenvironment. NS4B increases baseline NF-κB activity in the absence of TNF-α; however, the same protein suppresses NF-κB activity in the presence of TNF-α. The mechanism by which NS4B regulates NF-κB activity needs to be elucidated in future molecular studies.
HCV is known to efficiently evade the intracellular host defense system in various ways. Therefore, we questioned whether HCV has an advantage in viral replication by the inhibition of TNF-α-induced NF-κB activation. From the viewpoint of viral persistence, TNF-α-induced cell death is not advantageous to the virus. Increased cell death in HCV-infected cells may hamper viral replication. Instead, we suggest that this mechanism may help HCV to escape from proinflammatory responses triggered by NF-κB. NF-κB controls the expression of many inflammatory proteins, including chemokines and adhesion molecules. Thus, suppression of TNF-α-induced NF-κB activation may enhance HCV persistence by limiting inflammation and further immune responses. At the same time, however, suppression of NF-κB activation also disrupts the balance between the survival and death of infected cells and sensitizes infected cells to TNF-α-induced cell death.
In the current study, we demonstrated that HCV infection enhanced TNF-α-induced cell death through the suppression of NF-κB activation by the action of core, NS4B, and NS5B. In the HCV-infected liver, TNF-α might be one of the major cytokines. TNF-α can be secreted by a range of immune cells, including T cells, NK cells, and monocytes and macrophages. Without HCV infection, TNF-α activates both signaling pathways (i.e., the anti-apoptotic NF-κB pathway and the proapoptotic JNK pathway), resulting in a cellular response that reflects a balance between survival and death. In HCV infection, however, TNF-α-induced NF-κB activation is suppressed by core, NS4B, and NS5B, and HCV-infected hepatocytes become vulnerable to TNF-α-induced cell death. This mechanism may explain the cause of liver injury in hepatitis C, which often progresses to liver cirrhosis and HCC.
Additional Supporting Information may be found in the online version of this article.
|HEP_25726_sm_SuppFig1.tif||3074K||Supporting Information Figure 1.|
|HEP_25726_sm_SuppFig2.tif||9944K||Supporting Information Figure 2.|
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