Potential conflict of interest: Dr. Rice owns stock in Apath, LLC.
Hepatitis C virus (HCV) replication in primary liver cells is less robust than that in hepatoma cell lines, suggesting that innate antiviral mechanisms in primary cells may limit HCV replication or spread. Here we analyzed the expression of 47 genes associated with interferon (IFN) induction and signaling following HCV infection of primary human fetal liver cell (HFLC) cultures from 18 different donors. We report that cell culture-produced HCV (HCVcc) induced expression of Type III (λ) IFNs and of IFN-stimulated genes (ISGs). Little expression of Type I IFNs was detected. Levels of IFNλ and ISG induction varied among donors and, often, between adapted and nonadapted HCV chimeric constructs. Higher levels of viral replication were associated with greater induction of ISGs and of λ IFNs. Gene induction was dependent on HCV replication, as ultraviolet light-inactivated virus was not stimulatory and an antiviral drug, 2′-C-methyladenosine, reduced induction of λ IFNs and ISGs. The level of IFNλ protein induced was sufficient to inhibit HCVcc infection of naïve cultures. Conclusion: Together, these results indicate that despite its reported abilities to blunt the induction of an IFN response, HCV infection is capable of inducing antiviral cytokines and pathways in primary liver cell cultures. Induction of ISGs and λ IFNs may limit the growth and spread of HCV in primary cell cultures and in the infected liver. HCV infection of HFLC may provide a useful model for the study of gene induction by HCV in vivo. (HEPATOLOGY 2011;)
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Between 130 and 170 million persons worldwide are persistently infected with hepatitis C virus (HCV).1 Chronic HCV infection can lead to cirrhosis, hepatocellular carcinoma, and liver failure and is a leading indication for liver transplant. HCV persists in part because of failures in innate and immune-mediated control of viral replication and spread.2, 3 A better understanding of HCV/host interactions is urgently needed in order to develop improved therapies. Our ability to study HCV-host-virus interactions has been hampered by the lack of robust in vitro systems that faithfully replicate viral growth and spread in the liver. Until recently, most studies of HCV infection and replication have been performed in derivatives of a hepatoma cell line (Huh-7) that were selected for their ability to support high-level HCV replication in culture. These transformed cells may not reproduce the more complex environment in the infected liver. Indeed, they may work so well for in vitro studies because of defects in innate antiviral pathways.4, 5 Furthermore, studies based on Huh-7 derivatives may not address the impact of host genetic diversity on the outcome of HCV infection.
Using cell lines, many groups have reported that HCV infection can block innate antiviral mechanisms within infected cells (reviewed3). The NS3-4A protease of HCV cleaves the signaling adapter interferon (IFN)β promoter stimulator (IPS-1),6-8 and it is believed that this cleavage prevents IPS-1-dependent signal transduction needed to stimulate IFNα/β expression. Furthermore, NS3-4A can cleave the adapter TRIF and thereby interfere with signaling by Toll-like receptor 3 (TLR3), an endosomal double-stranded RNA (dsRNA) receptor.9 In a hepatoma line, HCV infection induced transient interferon regulatory factor (IRF)-3 activation, but the activation was not sustained.10 In HEK293 kidney cells, ectopic NS3-4A expression reduced virus-stimulated activation of IFN (α, β, λ) and chemokine promoters, suggesting that HCV-infected cells might be deficient in IFN induction and response.11 Much less is known about the degree to which HCV influences innate antiviral mechanisms within the infected liver. In contrast to the robust spread of cell culture-derived HCV in cell lines, primary liver cells support lower levels of HCV infection in vitro12 and in vivo.13, 14 Furthermore, IFN-stimulated genes (ISGs) are highly expressed in liver biopsies during acute13, 15 and chronic HCV infection,14, 16, 17 even in tissues lacking detectable IFNα/β expression.13, 16-18 ISG expression may produce an antiviral state restricting HCV spread in vivo.14, 19, 20 The cellular source of IFNs in the HCV-infected liver has not been identified. Also important, many of the hundreds of genes known as ISGs are directly inducible by viral proteins and nucleic acids in the absence of IFN.21
Genome-wide association studies have revealed important roles for the λ IFNs in control of HCV infection and responses to antiviral therapy (reviewed22). There are three human IFNλs: IL29 (IFNλ1), IL28A (IFNλ2), and IL28B (IFNλ3). IL28A and IL28B are closely related in sequence and promoter structures. Analysis of the IL29 and IL28α/β promoters suggests that these genes are differentially regulated, with IL29 controlled in a manner similar to IFNβ, and IL28α/β control more similar to IFNα.23 All three IFNλs are inducible by viral infection.24 Hepatocytes, including those in the HCV-infected liver, express the IFNλ receptor.25 IFNλ activates a transcriptional program resembling that of IFNα/β in liver cells and inhibits HCV replication in cell lines as well as primary fetal liver cells.25-29
Recent advances in the field have included the development of cell culture-adapted HCV (HCVcc) chimeras with useful properties,30-34 and of cell-based reporters permitting real-time visualization of HCV infection.35 New systems permit prolonged culture of untransformed human hepatocytes that retain their differentiated phenotype and HCV susceptibility over weeks in vitro12, 36. Here we have taken advantage of these advances to evaluate the impact of HCV infection on gene expression in primary human fetal liver cell (HFLC) cultures. Our results demonstrate that HCV infection activates expression of IFNλ and a number of ISGs. Both viral and host factors influence the level of gene induction. IFNλ levels produced after HCV infection are sufficient to inhibit HCV replication in primary liver cells, and an inhibitor of IFN receptor signaling enhances HCV replication even in the absence of added IFNs. These results suggest that endogenous IFNs may limit the spread of HCV in primary liver cells.
The preparation of HFLC is detailed in the accompanying article by Andrus et al.36 Briefly, deidentified human fetal liver tissue (16-22 weeks gestation) was procured through Advanced Bioscience Resources (Alameda, CA) or the human fetal tissue repository of the Albert Einstein College of Medicine (New York, NY). The Rockefeller University Institutional Review Board exempted this use of fetal liver tissue from review. Fetal gestational ages are listed in Supporting Table S1. Liver cells were isolated by collagenase and DNase I digestion, and hepatoblasts enriched relative to hematopoietic cells and other small cell populations by a series of low-speed spins and sedimentation at 1g. For some experiments, hepatoblasts were further enriched by centrifugation through lymphocyte separation medium (Cellgro, Manassas, VA) as described.37 Hepatoblasts were plated at 1 × 105/cm2 in 24- or 48-well collagen I-coated plates (Biocoat-I, BD Biosciences, Bedford, MA), cultured overnight, and washed extensively to remove nonadherent cells. After overnight plating, cultures contained predominantly cells with hepatoblast morphology. Cultures were maintained in Hepatocyte Culture Medium (BD Biosciences) with medium aspiration and replacement every 2 days (or less, as shown for each experiment).
We used two chimeric HCVcc constructs, each encoding a fully infectious HCV (genotype 2a) with different advantages. Jc1FLAG2(p7nsGLuc-2A) (Jc1G)33 is a J6/JFH chimeric genome encoding a Gaussia princeps luciferase (GLuc) reporter between p7 and NS2. Infected cells secrete GLuc, allowing measurement of viral replication by sampling the supernatant. Clone 234 is a J6/JFH chimeric genome selected by serial passage in Huh-7.5 cells. This HCVcc bears 12 mutations that increase infectious titers, and does not include a reporter. Infectious titers of both Jc1G and Clone 2 were determined by titration on Huh-7.5 cells using the TCID50 (50% tissue culture-infectious dose) method.38
HCV-Dependent Fluorescent Reporter (HDFR).
The construction of a modified HDFR with hepatocyte-specific expression is detailed in the Supporting Information.
Monitoring Infection in HFLC.
On the third or fourth day after plating, HFLC (1 × 105/cm2) were infected with 3 × 106 TCID50 units/well of HCVcc. Inocula were left in place for 6 hours and then removed with three washes (500 μL) of Williams E media without serum. Jc1G replication was monitored by measurement of secreted GLuc,33 quantitation of HCV RNA using the EraGen MultiCode-RTx method (EraGen Biosciences, Madison, WI),39 and assessment of HDFR translocation.35 Because HCVcc Clone 2 does not encode a reporter, we used RNA quantitation and HDFR translocation to monitor Clone 2 infection.
RNA was prepared using the Qiagen RNeasy mini kit (Valencia, CA) and yields determined by absorption spectroscopy using a NanoDrop (NanoDrop Products, Wilmington, DE). Five μL of RNA was used as a template for reverse transcription using SuperScript III (Invitrogen, Carlsbad, CA) and random hexamers. Five μL of 10-fold-diluted cDNA was used as in a SYBR Green qPCR assay (Applied Biosystems, Carlsbad, CA) on the LightCycler 480 Real-Time PCR System (Roche Applied Sciences, Indianapolis, IN). Primer pairs were selected using the Harvard Medical School Center for Computational and Integrative Biology Primer Bank website (http://pga.mgh.harvard.edu/primerbank/), and are listed in Supporting Table 2. Expression was normalized to that of the housekeeping gene RPS11 and to expression in uninfected cultures from the same donor at the same timepoint.
Ultraviolet (UV) Inactivation.
Five mL of virus, in one well of a six-well plate, was placed on a 1.5-inch platform and exposed to 82 mJoule of UV light (GS Gene Linker, Bio-Rad, Hercules, CA). UV-inactivated virus was noninfectious as determined by titration on naïve Huh-7.5 cells.
Drugs and Cytokines.
2′C-methyladenosine (2′CMA), an inhibitor of HCV's NS5B polymerase,40 was generously provided by Drs. D. Olsen and S. Carroll (Merck Research Laboratories, West Point, PA) and used at 2.5 μM. This is >80× the previously reported IC50 for this drug using J6/JFH HCVcc and Huh-7.5 cells30; however, for unknown reasons 2′CMA is less effective in HFLC than in Huh-7.5 cells. Pyridone-6 (Calbiochem, San Diego, CA), a pan-Janus kinase (JAK) inhibitor,41 was used at 1 μM. Pyridone-6 and 2′CMA were replaced at each media change. Recombinant human IFNβ (produced in CHO cells), IFNλ1 and IFNλ2 (produced in E. coli), were purchased from PeproTech (Rocky Hill, NJ). Cytokine endotoxin levels were <1 EU/μg. Polyinosinic:polycytidylic acid (p(I:C)) was purchased from GE Healthcare (Piscataway, NJ) and used at 5 μg/mL.
IL29 was measured by enzyme-linked immunosorbent assay (ELISA) (eBioscience, San Diego, CA) according to the manufacturer's instructions. The linear range of this assay was 31-1,000 pg/mL. CXCL10 and CXCL11 ELISAs (R&D Systems, Minneapolis, MN) were performed according to the manufacturer's instructions. The linear ranges of these assays were 15-500 pg/mL (CXCL10) and 125-4,000 pg/mL (CXCL11).
IL28B Single Nucleotide Polymorphism (SNP) Genotyping.
The IL28B rs12979860 SNP42 was genotyped by sequencing following PCR amplification of a 429-basepair region using the following primers: forward, 5′-cgcttatcgcatacggcta-3′; reverse, 5′-gggaccgctacgtaagtcac-3′.
Real-time PCR data were acquired on the Roche Light Cycler 480 (software release 1.5.0 SP3) and analyzed using the 2-ΔΔC(T) method.43 HCV RNA was quantitated with MultiCode-RTx Analysis Software v. 1.1.5d from EraGen. Graphing and statistical analysis were performed with Prism 5.0d (GraphPad Software, San Diego, CA). All data are presented as the mean ± SD of duplicate cultures.
HCVcc Infection in HFLC Cultures.
We used two chimeric HCV viruses to monitor HFLC infection: Clone 234 and Jc1G.33 Limited and variable replication of both viruses was detected over 24-96 hours (Fig. 1). UV irradiation of the virus inoculum blocked replication without significantly reducing amounts of input viral RNA (Fig. 1A). Replication was sensitive to the nucleoside analog 2′CMA, an inhibitor of the HCV RNA-dependent RNA polymerase NS5B (Fig. 1B). In side-by-side comparisons, Clone 2 RNA levels were often significantly higher than those of Jc1G (P < 0.05, repeated measures analysis of variance [ANOVA]) (Fig. 1C). HCV replication was also demonstrated by nuclear translocation of the HDFR35 in infected cells (Supporting Fig. S1A,B). Using the HDFR system, we confirmed that Clone 2 infection of HFLC is more robust than Jc1G, and that inhibiting replication with 2′CMA reduced the signal from the reporter. Replication of Jc1G could also be demonstrated by measurement of secreted GLuc (Fig. S1C; also see accompanying article by Andrus et al.36).
Gene Induction in HCVcc-Infected HFLC Cultures.
HCVcc replication in HFLC was less robust than in hepatoma cell lines. Additionally, as reported in the accompanying article by Andrus et al., paramyxovirus V proteins that interfere with IFN induction and signaling enhanced HCV replication in HFLC. Therefore, we hypothesized that innate antiviral mechanisms in primary cells limited HCV replication and/or spread. We used quantitative RT-PCR approaches to measure expression of 47 genes associated with IFN signaling and innate antiviral responses (Table S2) in HCVcc-infected HFLC. HCVcc infection stimulated the expression of many of these genes, notably including IL29, certain chemokines, and several ISGs (Table 1). The experiments shown represent the two patterns of gene expression observed when comparing Clone 2-stimulated to Jc1G-stimulated gene expression in cells from the same liver: greater gene induction by Clone 2 than by Jc1G (three of four experiments) and similar gene induction by both viruses (one of four experiments). Time courses for induction of selected genes by both Clone 2 and Jc1G are shown in Fig. S2. The experiments shown compare Clone 2 and Jc1G-stimulated gene induction in HFLC from the same livers; messenger RNA (mRNA) levels are compared with uninfected control cultures for each timepoint. IL29 mRNA was transiently induced (Fig. S2A). IL29 expression was more frequently detected than was IL28B expression, consistent with the differential control of these genes.23, 24 Robust increases in CXCL10, CXCL11 (Fig. S2A), and ISGs such as Viperin, IFITM1, IFIT2, MX2, and OAS2 (Fig. S2B) were observed within 24-48 hours of infection. Low-level IFNβ induction was observed in response to Clone 2 in two of 18 experiments; no induction of IFNα, IFNγ, or IFNω was observed in any experiment (out of six experiments). The synthetic dsRNA analog, p(I:C), stimulated expression of IFNs and ISGs in HFLC; exogenous IFNβ and IL29 each stimulated expression of ISGs (Fig. S3). Gradient-enriched hepatoblasts prepared as described37 demonstrated a further increase in HCV-stimulated gene expression (Fig. S4A). After HCVcc infection, gradient-purified HFLC cultures were primed to respond to p(I:C) stimulation with increased expression of IL29, IL28B, IFNβ, and IFITM1 when compared with uninfected cultures (Fig. S4B).
Table 1. Genes Induced Following HCV Infection in HFLC
Cytokines and Chemokines
Results of 4 analyses on two independent livers are shown. Liver AB-080510 and AE-102910 were each infected with both Clone 2 and Jc1G. Values shown represent the peak fold increase in mRNA levels for each gene, relative to uninfected cultures from the same liver at the same time point. See Figure S2 for kinetics of gene induction. Expression of IFNα1, IFNω, IFNγ, IL1β, IL6, IL8, MDK, MOV10, DDIT4, c15orf8, and c8orf4 was not detected at any time point. In three of four comparisons, the relative gene induction in response to Clone 2 was dramatically higher than that in response to Jc1G, as shown for AB-080510. In one of four comparisons (AE-102910, shown here), high levels of gene induction were observed in response to both Jc1G and Clone 2.
Interferon Stimulated Genes
Gene Induction Is Influenced by HCV Replication and the JAK Signaling Pathway.
In general, greater gene induction was seen in cultures supporting higher levels of viral replication. In three of four comparisons, gene induction was greater in cultures infected with Clone 2 than with Jc1G (Table 1; Fig. S2). IL29 was reproducibly induced at the RNA and/or protein level in HFLC infected with Clone 2 (seven of eight donors); in contrast, Jc1G stimulated detectable IL29 mRNA expression in 3 of 14 donors (P < 0.01, Fisher's exact test). Notably, Clone 2 typically has higher specific infectivity than does Jc1G, meaning that the same number of infectious units of the latter typically contain more copies of viral RNA. In contrast to replicating HCVcc, UV-irradiated HCV stimulated little if any gene expression (Fig. 2A). Gene induction by HCVcc was reduced in the presence of 2′CMA (Fig. 2B). IFNs signal through activation of JAK and Tyk kinases.44 The pan-JAK inhibitor, Pyridone-6, enhanced HCVcc replication in HFLC, and reduced or delayed the induction of some ISGs (but not IFNλ) during early timepoints of infection (Fig. 2C,D). At later timepoints, ISG expression was increased in Pyridone-6-treated cultures that demonstrated enhanced HCV replication.
Expression of IL29 and Chemokine Proteins in HCVcc-Infected Cultures.
We performed ELISAs to confirm the expression of selected genes following HCVcc infection of HFLC (Fig. 3). In the experiment shown, HCV RNA persisted but did not increase during culture (Fig. 3A). IL29 mRNA expression was transient, with 45-fold induction at 24 hours, and IL29 protein peaked after the mRNA peak (Fig. 3B). Maximal IL29 protein levels in five experiments averaged 211 ± 110 pg/mL (range, 64-388 pg/mL). IL29 protein peaked at day 2 after infection in 3/5 experiments, but peaks at day 1 and day 4 after infection were observed in one experiment each. A >10,000-fold induction of CXCL10 mRNA was observed beginning 24 hours after the start of infection; CXCL10 protein was detectable as early as 24 hours, with maximal levels detected 48-96 hours after infection. In two experiments, CXCL10 protein peaked at an average of 2,300 pg/mL (range, 2,000-2,500 pg/mL). Similarly, CXCL11 mRNA was induced >5,000-fold over uninfected cultures beginning 24 hours after infection, and CXCL11 protein was readily detected beginning 48 hours after infection. In two experiments, CXCL11 protein peaked at an average of 3,800 pg/mL (range, 300-4,000 pg/mL).
IFNs Inhibit HCVcc Infection in HFLC.
Because IL29 protein was detected in HCV-infected cultures (Fig. 3), we evaluated the effects of IL29 on HCV replication in HFLC (Fig. 4). We measured the GLuc reporter to monitor Jc1G infection. As a control, IFNβ inhibited HCVcc infection in a dose-dependent fashion, and the inhibitory effect of 10U IFNβ was diminished in the presence of Pyridone-6. Recombinant IL29 inhibited HCV-dependent luciferase expression at 100 pg/mL, a level below that detected in HCV-infected HFLC cultures. This IL29-mediated inhibition of HCV replication was reversed in the presence of Pyridone-6. Recombinant IL28A also inhibited Jc1G replication in HFLC, and the inhibitory effect of 1 ng/mL IL28A was blocked in the presence of Pyridone-6. Pyridone-6 did not reverse inhibition by higher doses of IFNs. These results extend previous reports that Type III IFNs (at higher levels) can inhibit HCV replication in hepatoma cells and primary liver cells,26-29 and suggest that such inhibition is dependent on JAK activity. They also suggest that endogenous IFNs may contribute to the low HCV infection efficiency in HFLC.
HCV Replication, but Not IL28B Genotype, Correlates with IFNλ and ISG Induction.
In genome-wide association studies, single nucleotide polymorphisms close to and within the IL28B locus have been found to correlate with spontaneous clearance versus persistence of untreated HCV infection, and with sustained virologic control versus nonresponse in HCV (genotype 1) patients treated with pegylated IFNα plus ribavirin.22 In light of the varied outcomes in HCVcc-infected cultures shown here, we performed IL28B SNP rs12979860 genotyping42 on the HFLC used in these assays (subject to the availability of cells for DNA preparation). The CC genotype at rs12979860 is associated with increased likelihood of spontaneous recovery from HCV infection, whereas CT and TT genotypes are associated with increased risks of persistent infection and treatment failure.22 As shown in Fig. 5, the rs12979860 genotype did not predict the ability of HFLC to support robust HCV replication, nor did it correlate with levels of IL29, CXCL10, or CXCL11 induction. Instead, the levels and kinetics of gene induction correlated most closely with the levels and kinetics of HCV replication.
In vitro studies of virus/host interactions in HCV infection have until recently been restricted largely to derivatives of a hepatoma cell line selected for their ability to support high-level HCV replication. These cell lines may not accurately reproduce the complex host/virus interactions occurring in vivo. In an accompanying article (Andrus et al.36), we report that HFLC supports persistent HCV infection in vitro. Here, we demonstrate that HCV infection of primary liver cells stimulates expression of a number of potential antiviral genes, including IL29 (IFNλ1), but not IFNα or (in 16/18 experiments) IFNβ. IL29 protein was produced in quantities sufficient to inhibit HCV infection of HFLC. Pyridone-6, a JAK inhibitor that blocks the antiviral effect of IL29 (Fig. 4), enhanced HCV replication even in the absence of exogenous IFNs (Figs. 2, 4), suggesting that endogenous IFNs may decrease HCV replication in these cultures. The level of gene induction varied between donors, suggesting that host polymorphisms influence the response to HCV infection. For some donors, gene induction also varied depending on the infecting virus. Induction of some ISGs was reduced or delayed by inhibition of JAK signaling. Together, these results indicate that HCV infection of HFLC can serve as a valid in vitro model for studying innate immune responses to hepatotropic viruses.
The precise functions of many ISGs remain to be determined.21 However, recent work demonstrates that specific ISGs including RIG-I, MDA-5, IRF1, and IRF7 each mediate significant antiviral activities against HCV and other viruses.44 A common feature of these genes is their potential to stimulate downstream expression of other ISGs. All four of these ISGs are induced in HCV-infected HFLC. Therefore, we hypothesize that expression of these and other ISGs in HFLC may limit HCV replication in these cultures. Other genes, such as CXCL10 and CXCL11, may serve in vivo to recruit immune cells to the site of infection. These chemokines are highly expressed in HCV patients (reviewed46, 47).
Our observation of transient IFNλ production following HCV infection, and the ability of the JAK inhibitor, Pyridone-6, to increase HCV replication in HFLC even without added IFNs suggest that IFNλ may contribute to control of HCV replication and gene induction in HFLC and in vivo. Notably, in HCV patient liver biopsies, levels of HCV RNA—but not of IFNβ mRNA—correlated with ISG expression.14 Thus, factors other than IFNα/β may modulate intrahepatic ISG expression.
Although genetic variation at the IL28B locus is associated with outcomes of HCV infection and treatment,22 the mechanisms behind this association are poorly understood. IL28B SNP genotype did not correlate with IL28A/B expression levels in HCV patient liver biopsies.48 Furthermore, the associations between IL28B and HCV infection may be weaker for subjects infected with HCV genotype 2 or 3 than those infected with genotype 1.22 The JFH strain on which so much in vitro HCV research depends is of genotype 2. As other HCV culture systems are developed it will be important to learn how HCV isolates representing other HCV genotypes interact with IFN systems.
HCV replicates less efficiently in primary HFLC cultures than in Huh-7.5 cells. Furthermore, the ability of these mixed cell populations to support HCV infection and spread varies between different donors. These observations highlight important differences between primary tissue and highly selected cell lines. HCV may infect only a minor population of hepatocytes in vivo.13, 14, 49 ISG expression is readily detected in the liver during acute and chronic HCV infection, even when type I IFN expression is not.13-18 This study demonstrates that a similar gene expression profile is observed during acute HCV infection in HFLCs. It has been proposed that IFNs induce an antiviral state in most uninfected hepatocytes in vivo, thereby limiting the number of cells capable of supporting infection.19, 20 This may benefit HCV by restricting antigen levels, delaying recognition by the adaptive immune system,2 and preventing immune-mediated liver damage. In the infected liver, transient, localized lessening of ISG expression may provide HCV with a steady supply of susceptible hepatocytes for new infectious events.
We thank S.M. di Vittorio, C. Murray, and J. Sable for expert administrative assistance and laboratory management; we thank W.T. Barry, M.E. Castillo, B.R. Flatley, M. Holz, S. Pouzol, S. Shirley, and A. Webson for technical assistance.