Free fatty acids induce ER stress and block antiviral activity of interferon alpha against hepatitis C virus in cell culture
© Gunduz et al.; licensee BioMed Central Ltd. 2012
Received: 19 October 2011
Accepted: 27 July 2012
Published: 3 August 2012
Hepatic steatosis is recognized as a major risk factor for liver disease progression and impaired response to interferon based therapy in chronic hepatitis C (CHC) patients. The mechanism of response to interferon-alpha (IFN-α) therapy under the condition of hepatic steatosis is unexplored. We investigated the effect of hepatocellular steatosis on hepatitis C virus (HCV) replication and IFN-α antiviral response in a cell culture model.
Sub-genomic replicon (S3-GFP) and HCV infected Huh-7.5 cells were cultured with a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in these cells was visualized by Nile red staining and electron microscopy then quantified by microfluorometry. The effect of FFA treatment on HCV replication and IFN-α antiviral response was measured by flow cytometric analysis, Renilla luciferase activity, and real-time RT-PCR.
FFA treatment induced dose dependent hepatocellular steatosis and lipid droplet accumulation in the HCV replicon cells was confirmed by Nile red staining, microfluorometry, and by electron microscopy. Intracellular fat accumulation supports replication more in the persistently HCV infected culture than in the sub-genomic replicon (S3-GFP) cell line. FFA treatment also partially blocked IFN-α response and viral clearance by reducing the phosphorylation of Stat1 and Stat2 dependent IFN-β promoter activation. We show that FFA treatment induces endoplasmic reticulum (ER) stress response and down regulates the IFNAR1 chain of the type I IFN receptor leading to defective Jak-Stat signaling and impaired antiviral response.
These results suggest that intracellular fat accumulation in HCV cell culture induces ER stress, defective Jak-Stat signaling, and attenuates the antiviral response, thus providing an explanation to the clinical observation regarding how hepatocellular steatosis influences IFN-α response in CHC.
Hepatitis C virus (HCV) infection affects an estimated 180 million people worldwide and is one of the major causes of chronic liver diseases, liver cirrhosis, and hepatocellular carcinoma [1–3]. The standard of care for chronic HCV genotype 1 infection includes a combination of interferon-alpha (IFN-α) , ribavirin and one of the protease inhibitors (either telaprevir or boceprevir). This triple combination therapy has greatly improved the sustained antiviral responses among chronic HCV patients. However, a large percentage of chronic HCV patients are still unable to clear the infection with this regimen. The predictors of sustained virological response (SVR) to interferon based combination therapy have been linked to host genetic factors IL-28B genotypes, viral load, viral genotypes, body weight, stage of liver diseases, obesity, type-2 diabetes mellitus (DM), fibrosis stage and co-infection with human immunodeficiency virus [4–9]. Our understanding of the mechanisms of IFN-α and ribavirin action has significantly increased due to the availability of the HCV cell culture system. We and other groups have shown that IFN-α efficiently inhibits replication of HCV in the cell culture model [10–14]. Therefore, the HCV cell culture model provides an excellent in vitro model system to assess the contribution of a number of host related factors in the mechanisms of IFN-α resistance.
A number of clinical studies have reported that overweight or obese HCV-infected individuals or those with steatosis of the liver are at a higher risk for IFN-α non-responsiveness [15–17]. The prevalence of hepatic steatosis in chronic hepatitis C patients has been reported to vary between 50-80%, and is associated with excessive alcohol drinking, increased body weight, DM and other metabolic diseases . The increased lipogenesis and the free fatty acid (FFA) overflow to hepatocytes have been proposed to be the major cause for hepatic steatosis . Chronic HCV infection also leads to abnormalities of lipid metabolism and insulin resistance, factors that also increase the risk of type-2 DM . There are data supporting the fact that patients with high body mass index have a lower chance of SVR . The molecular mechanisms explaining how the hepatic steatosis and related metabolic liver diseases reduce the SVR of IFN-α are unknown.
Palmitic and oleic acids are the most abundant FFAs in liver triglycerides in patients with nonalcoholic fatty liver disease . This study was carried out to examine the effect of co-culturing the mixture of these two FFAs on HCV replication and IFN-α antiviral response using stable sub-genomic replicon and full-length HCV infected cell cultures. We show that FFA treatment of HCV cell culture induces hepatocellular steatosis and lipid accumulation in a dose dependent manner. Intracellular fat accumulation in HCV cell culture increased the viral replication and partially blocked the antiviral response of IFN-α. We present experimental evidence indicating that intracellular lipid accumulation induces ER stress response and down regulates the IFNAR1 chain of the type I interferon receptor, leading to the creation of defective Jak-Stat signaling and impaired antiviral response of IFN-α against HCV.
Materials and methods
HCV cell culture and chemicals
The stable S3-GFP replicon cell line (HCV2a) was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, sodium pyruvate, nonessential amino acids, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum supplemented with G-418 (1 μg/mL) . Nile red, sodium oleate, sodium palmitate, and fatty acid free bovine serum albumin (BSA) were obtained from Sigma Chemical Co., Saint Louis, MO. Recombinant human IFN-α 2b (Intron A) was purchased from Schering Plough, Kenilworth, NJ. The Huh-7.5 cell line was obtained from the laboratory of Charlie Rice (The Rockefeller University, New York) and maintained in DMEM with 10% FBS.
Protein lysates from S3-GFP replicon cells were prepared after treatment with FFAs. Equal amounts of protein were resolved on SDS-PAGE gels . The antibodies to Stat1, Stat2, p-Stat1 (Y701), p-Stat2 (Tyr690), p-Jak1 (Tyr1022/1023), p-Tyk2 (Tyr1054/1055), total eIF2α, p-eIF2α (Ser51), beta-actin, IRE1-alpha, PKR, PERK, BIP, SOCS-3, anti-mouse IgG, and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling, Beverly, MA. Antibodies to interferon alpha-receptor 2 (IFNAR2) and p-IFNAR1 were purchased from Santa Cruz Biotechnologies, Santa Cruz, CA. The antibody to p-PKR (pT446) was obtained from Epitomics, Burlingame, CA. A mouse monoclonal antibody to IFNAR1 (GB8) was kindly provided by Biogen Idec Inc., Cambridge, MA, USA.
Fatty acid treatment
We used a formulation of FFA mixture at a 2:1 ratio of oleate to palmitate that mimics benign chronic steatosis with low toxicity described by other investigators [22–25]. Briefly, 100 mM palmitate (Sigma catalog No. P-0500) and 100 mM Oleate (Sigma Catalog No. 0–7501) stocks were prepared in 0.1 M NaOH at 70°C and filter sterilized. Five percent (w/v) FFA-free BSA solution was prepared in double distilled water and filter sterilized. A 5 mM stock solution for each fatty acid was prepared in 5% BSA solution in distilled water at 60°C then the mixture was cooled to room temperature. These two FFAs were first mixed together in growth medium in a sterile environment under laminar flow so that the final concentration of FFA was 1 mM and BSA was 1%. S3-GFP cells were cultured in 10-cm dishes to 80-85% confluence and co-cultured with different concentrations of FFA for 24 hours to induce steatosis. At indicated time points, the cells were washed in PBS and cultured in regular growth medium containing 10% FBS.
Nile red staining and microfluorometry
Intracellular fat accumulation in the HCV cell culture was determined by Nile red staining as described . Briefly, hepatocyte monolayers were washed twice with phosphate-buffered saline (PBS) and incubated for 15 minutes with Nile red solution at a concentration of 1 mg/mL in PBS at 37°C. After this treatment, the cell monolayer was washed with PBS and counterstained with a nuclear staining Hoechst dye (H33342, Calbiochem, Darmstadt, Germany) at a concentration of 10 μg/mL prepared in PBS. Cells were then examined under a fluorescence microscope as described previously . The intracellular fat content was quantitated using a microfluorometer (excitation 488 nm and emission 550 nm).
Intracellular fat accumulation in the FFA treated cells was also confirmed by electron microscopy using a standard protocol .
The effect of FFA treatment on the cell proliferation was measured using a MTT colorimetric assay . The assay uses a tetrazolium compound (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) (M5655, Sigma-Aldrich, St Louis, MO), which is reduced intracellularly to formazan by a mitochondrial dehydrogenase enzyme. The percentage of cell viability was determined by comparison with untreated controls.
The effect of FFA treatment on HCV replication and IFNAR1 expression was measured by flow analysis as described previously . Briefly, S3-GFP replicon cells with or without FFA treatment were collected 24 h post- treatment. Intracellular GFP expression was quantified using a flow cytometer (BD LSR II, BD Biosciences). For the analysis of IFNAR1 expression, Huh-7 cells were treated similarly and processed using an IFNAR1 antibody (Epitomics, Burlingame, CA), as per the supplier’s protocol, with the secondary antibody (Alexa Fluor 488-labeled) procured from Invitrogen, CA. Histograms were generated using WinMDI version 2.8 software (Scripps.edu).
Total RNA was isolated from the cells using the GITC method and real-time RT-PCR was carried out as described .
Infectious cell culture model for HCV with a luciferase reporter
Full-length chimeric virus encoding Renilla luciferase (pJFH-ΔV3-Rluc) was kindly provided by Curt H Hagedorn, University of Utah School of Medicine, Salt Lake City . The pJFH-ΔV3-Rluc plasmid was linearized with Xba I, extracted with phenol /chloroform and precipitated with ethanol. HCV-RNA transcripts were prepared in vitro by using T7 RNA polymerase. 1 × 107 Huh-7.5 cells were electroporated with 20 μg of HCV RNA. Transfected cells were cultured in complete DMEM. Infectivity assay was carried out using the supernatants 96 hr post-transfection. Briefly, Huh-7.5 cells were infected with JFH-ΔV3-Rluc virus (MOI 0.1) overnight. On the following day, the infected culture was washed with PBS and then incubated with 10 mL of DMEM with 10% FBS. Infected Huh-7.5 cells were cultured long-term by splitting at a 1:10 ratio at five-day intervals. Replication of HCV in the infected cell culture at each interval was confirmed by measuring the Renilla luciferase activity or the HCV RNA level by RT-qPCR. Renilla luciferase activity was measured in 5μL cell lysate using a luciferase assay kit (Promega) and total protein was measured using the Bradford assay.
The effect of FFA treatment on Jak-Stat signaling and IFN-β (pISRE-Firefly luciferase) promoter activity was examined using a published protocol (21). The ATF6-Luc activity was measured by co-transfecting 0.5 μg of p5XATF6GL3 (Addgene, Cambridge, MA) DNA along with pRL-TK plasmid (0.5 μg) in S3-GFP cells 24 h prior to FFA treatment. Cell lysates were prepared 24 h post FFA treatment and the Firefly luciferase values were normalized to the Renilla luciferase values.
Immunohistochemical staining for HCV-core protein
Infected Huh-7.5 cells were mounted onto a glass slide via the cytospin method. The cells were washed twice with 10 mM PBS pH 7.4 (Sigma-Aldrich, St Louis, MO) for 5 minutes. The cells were fixed in chilled acetone for 15 minutes and then permeabilized by treatment with Reveal Decloaker RTU (Biocare Medical, RV 100) for 25 minutes at boiling point. Slides were then cooled down at room temperature for 25 minutes. Blocking was performed utilizing Background Sniper (Biocare Medical, BS966) for 10 minute at room temperature. The cells were incubated with monoclonal anti-core antibody (Thermo Scientific, Pierce hepatitis C virus core antigen specific mouse monoclonal antibody, Ma1-080) at 1:200 diluted with Da Vinci Green Diluent (Biocare Medical, PD900) for 1 hour at room temperature. Following the primary antibody incubation, the cells were washed 3 times in Tris Buffered Saline (pH 8.0), and incubated with MACH 4 mouse probe (Biocare Medical, UP534) for 10 minutes. After mouse probe treated, the cells were incubated with MACH4 HRP Polymer (Biocare Medical, MRH534) for 30 minutes, and the cells were washed with TBS 3 times. Next, the cells were treated with diaminobenzidine (DAB) chromogen (Dako Cytomation, Carpinteria, CA) for 5 minutes. The slides were counterstained with hematoxylin for 30 seconds and Tacha’s bluing Solution (Biocare Medical, HTBLU) for 30 seconds, dehydrated, mounted and observed by light microscopy.
Microfluorometry assay results, ISRE, ATF6 (activating transcription factor 6), and HCV (pJFH-ΔV3-Rluc) luciferase activity, real time RT-PCR and FACS analysis were compared for significance using the Student’s t test. P values less than 0.05 were considered significant.
FFA treatment induces cellular steatosis in HCV cell culture
Intracellular fat accumulation increases HCV RNA replication
Intracellular lipid accumulation blocks IFN-α antiviral response against HCV
FFA induces ER stress to down regulate IFNAR1 and blocks Jak-Stat signaling
The standard of care for chronic HCV genotype 1 infection includes IFN-α plus ribavirin along with one of the protease inhibitors. However, results of clinical studies indicate that the sustained virologic response of this combination therapy is impaired by viral and host related factors. Viral factors play an important role in the treatment response since patients infected with HCV genotype 1 show poor response as compared to genotype 2 and 3 [32, 33]. In addition to virus genotype, several host-related factors can also affect the outcome of the antiviral therapy including viral load, presence of cirrhosis, age, race, and metabolic diseases such as obesity and diabetes [34, 35]. Obesity is a risk factor resulting in a poor treatment response to both pegylated interferon and pegylated interferon in combination with ribavirin [15–17]. Hepatic steatosis can develop secondary to obesity, DM, alcohol abuse, protein malnutrition, carbohydrate overload, and chronic HCV infection . Hepatic steatosis is also a common histopathological feature of chronic HCV infection that is found in 30-70% of patients [37, 38]. There are reports indicating that HCV infection induces the development of hepatocellular steatosis by blocking the release of very low-density lipoprotein (VLDL) particles from the liver to the circulation . It has been reported by a number of investigators that the presence of hepatic steatosis in patients with chronic HCV infection affects liver disease progression, pathogenesis, and treatment response [39–43]. The mechanisms of the impaired response to interferon-based therapy in the condition of hepatic steatosis are not clearly understood.
We took advantage of the HCV cell culture system established in a liver-derived cell line to study the mechanisms of IFN-α antiviral response in the presence or absence of FFAs. Hepatocellular steatosis was induced in HCV replicon cells with a mixture of saturated and non-statured FFAs. Other investigators have used this FFA cocktail to study the pathogenic mechanism of hepatic steatosis in cell culture. Our results support that FFA treatment can induce steatosis in HCV replicon cells in a dose-dependent manner. High dose FFA treatment in HCV cell culture leads to increased cell toxicity and cell death by apoptosis as reported by others [23, 30]. We show that the FFA at 10–100 μM range increased HCV replication in the infected cell culture supporting data published previously . Our results suggest that intracellular fat accumulation partially blocks IFN antiviral action and viral clearance in replicon and infected cell culture. Published reports from our laboratory and others indicate that cellular Jak-Stat signaling is critical for the successful antiviral response of IFN-α against HCV . Our results provide evidence supporting that FFA treatment of HCV cell culture induces an ER stress response that blocks cellular Jak-Stat signaling by down regulating IFNAR1. As a result, IFN-α induced Stat1, Stat2 phosphorylation, and IFN-β promoter activity was attenuated. Studies by other laboratories, including ours, have shown that ER stress is correlated well with down regulation of IFNAR1 in cell culture models [45–47]. We propose that future studies should be conducted to determine if attenuating the ER-stress response by pharmacological inhibitors or siRNA knockdown improves the antiviral response of IFN-α in the condition of hepatic steatosis by retained expression of IFNAR1.
FG and FA performed major biochemical experiments, participated in the design of the study and wrote the initial draft of the manuscript. PKC, SH and BP did some biochemical experiments. DPB provided critical reagents and revised the manuscript. LAB and SD supervised, helped to design the study and wrote the manuscript.
We thank Mallory Heath for critically reading the manuscript. This work was supported by the NIH grant CA127481 and CA089121. Feyza Gunduz was supported by funds received from the Akdamar Fellowship Program, Department of Gastroenterology and Hepatology, Tulane University Health Sciences Center. Fatma M Aboulnasr was supported by funds received from the Egyptian Ministry of Higher Education, Egypt.
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