Impaired clearance of virus-infected hepatocytes in transgenic mice expressing the hepatitis C virus polyprotein

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Impaired Clearance of Virus-Infected Hepatocytes in Transgenic Mice Expressing the Hepatitis C Virus Polyprotein OLIVIER DISSON,* DELPHINE HAOUZI,* SOLANGE DESAGHER,‡ KIM LOESCH,§ MICHAEL HAHNE,* ERIC J. KREMER,* CHANTAL JACQUET,* STANLEY M. LEMON,§ URSZULA HIBNER,* and HERVE´ LERAT* *Institut de Ge´ne´tique Mole´culaire, Centre National de Recherche Scientifique, Unite Mixte de Recherche 5535, IFR 122, Montpellier, France; ‡Centre National de Recherche Scientifique, Unite Propre de Recherche 2580, Montpellier Cedex, France; and §The University of Texas Medical Branch, Galveston, Texas

Background & Aims: Multiple molecular mechanisms are likely to contribute to the establishment of persistent infection by hepatitis C virus (HCV). The aim of this work was to study the evasion of cell-mediated antiviral immune responses in transgenic mice with livertargeted expression of the hepatitis C viral genome. These mice develop steatosis and hepatocellular carcinoma and constitute a murine model of chronic HCV infection. Methods: Mice of the FL-N/35 lineage were infected with replication-deficient adenoviral vectors encoding ␤-galactosidase, and the persistence of infected cells was measured by histochemistry and enzymatic assays. Results: Hepatocytes from the HCVⴙ transgenic mice are deficient in eliminating an adenoviral infection, despite an apparently normal T-cell response. The defect in adenoviral clearance was associated with resistance of transgenic hepatocytes to apoptosis induced by Fas/ APO1/CD95 death receptor stimulation, a major pathway of cell killing by cytotoxic T lymphocytes. The attenuation of Fas-mediated apoptosis observed in the murine model was associated with a reduced abundance of Bid, a BH3-only member of the Bcl-2 family of apoptosis regulators. Conclusions: Our results suggest that viral evasion of cell-mediated immune responses leading to apoptotic death of hepatocytes may contribute to viral persistence. Such a mechanism might also contribute to the development of liver cancer in HCV.

lthough abundant evidence indicates that infection with HCV (HCV), a hepatotropic human flavivirus, elicits a robust cellular and humoral immune response,1–3 the immune system typically fails to eliminate the virus from the liver. As a result, most acute infections lead to long-term persistence of the virus.4 Continued replication of HCV within hepatocytes is associated with chronic necroinflammatory liver disease, which often leads to progressive fibrosis and, over a period of many years, cirrhosis and in some patients hepatocellular carcinoma.5 Many aspects of the pathogenesis of this disease

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are not well understood, including the mechanisms underlying the development of fibrosis and cancer. As important, although there are data suggesting that HCV may have evolved strategies to confound both innate and adaptive immune responses to the infection,6 – 8 much remains to be learned of the mechanisms by which this positive-strand RNA virus evades the host immune response to establish persistence in the majority of infected persons. Prior efforts to understand the pathogenesis of this medically important infection have been thwarted by a lack of useful animal models. Transgenic mice expressing either a high abundance of the HCV core protein or much lower levels of the complete viral polyprotein have been shown to be at risk for hepatic steatosis and hepatocellular carcinoma, 2 common features of chronic hepatitis C in humans.9 –11 Thus, these aspects of the pathogenesis of this disease may be caused, at least in part, by the direct expression of HCV proteins within the liver. Although these studies have been helpful in understanding some aspects of the pathogenesis of HCV-associated liver disease, relatively little has been learned from transgenic animal models concerning the mechanisms by which the expression of HCV proteins confounds the human immune response to the infection. Transgenic mice with intrahepatic expression of either the core protein or all of the structural proteins of the virus do not appear to be immunologically impaired, as they are fully competent in their abilities to eliminate hepatocytes infected with a recombinant, replicationdeficient adenovirus.12,13 These negative data are important because they refute a series of in vitro and biochemAbbreviations used in this paper: HCV, hepatits C virus; PCR, polymerase chain reaction; rBid, recombinant Bid; TNF-␣, tumor necrosis factor ␣. © 2004 by the American Gastroenterological Association 0016-5085/04/$30.00 doi:10.1053/j.gastro.2003.12.005

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ical studies that suggest that the HCV core protein may play a role in viral persistence, either by virtue of its potential to interact with the intracellular domains of members of the tumor necrosis factor receptor family, such as TNFR1,14 –16 or through other modulating effects on the host immune system.17–19 On the other hand, Blindenbacher et al.20 recently reported that transgenic mice expressing a 32 amino acid segment of ␣1antitrypsin fused to the HCV polyprotein were impaired in their ability to control an intrahepatic lymphocytic choriomeningitis virus infection. Here, we present evidence suggesting that mice expressing a transgene that encodes the entire viral polyprotein in a liver-specific fashion are significantly impaired in their ability to eliminate adenovirus-infected hepatocytes. These data provide evidence from an animal model that the expression of HCV proteins results in a functional impairment of the intrahepatic immune response, a phenomenon that has long been speculated to exist but difficult to directly show. The transgenic mice we studied, derived from the FL-N/35 lineage, express the viral polyprotein at a low abundance,11 as do most patients with chronic HCV infection.21 We show that expression of the transgene is associated with downmodulation of Bid, a BH3-only proapoptotic member of the Bcl2 family, and that the transgenic hepatocytes are partially resistant to Fas-induced apoptosis.

Materials and Methods Antibodies anti-murine Fas (Jo2 clone) and anti-cytochrome C (6H2-B4 clone) were from PharMingen (San Diego, CA), the polyclonal anti-caspase 3 was from Cell Signaling Technology (Beverly, MA), and the polyclonal anti-caspase 8 antibody was from Chemicon (Temecula, CA). Goat anti-Bid was from R&D Systems (Abingdon, UK) or a gift from JeanClaude Martinou (Universite´ de Gene`ve, Switzerland). Polyclonal anti-GAPDH (glyceraldehydes-3-phosphate dehydrogenase) antibody was a gift from Jean-Marie Blanchard (IGM, Montpellier, France). Purified recombinant murine Bid protein was a gift from Jean-Claude Martinou (Universite´ de Gene`ve, Switzerland). Recombinant Fas ligand was from Alexis (San Diego, CA), Z-VAD-fluoromethylketone was from BioVision (Palo Alto, CA), CaspACE fluoroisothiocyanate-VAD-fluoromethylketone (Z-VAD-fmk-FITC) was from Promega (Madison, WI), and tumor necrosis factor ␣ (TNF-␣) was from PeproTech (Rocky Hill, NJ). Cycloheximide, actinomycin D, lonidamine, dimethyl sulfoxide, dexamethasone, and insulin were from Sigma-Aldrich (St. Louis, MO). Collagenase was from GibcoBRL (Gaithersburg, MD).

Animals C57BL6 mice transgenic for the full HCV open reading frame (FL-N/35 lineage11) were bred and maintained ac-

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cording to the institutional guidelines. Age-matched 2–10month-old males were used. Young mice (2–3 months old) were used for adenoviral infections.

Primary Cultures Hepatocytes were isolated by portal vein perfusion of collagenase.22 Freshly isolated hepatocytes were cultivated in 6-well plates in DMEM (GibcoBRL) supplemented with 10% fetal calf serum (GibcoBRL), 0.1 ␮mol/L dexamethasone, 5 ␮g/mL insulin, and 5% (vol/vol) dimethyl sulfoxide at 37°C and 5% CO2.

Apoptosis Assays In vivo. Jo2 was injected intravenously (1 ␮g/g bodyweight). Mice were sacrificed 2 hours postinjection. Liver biopsies were either frozen in liquid nitrogen, frozen in optimal cutting temperature (OCT) compound, or formalin fixed. Paraffin-embedded liver sections were stained with Hoechst 33258, and apoptotic (condensed or fragmented) nuclei were scored in random fields using epifluorescence microscopy. Ex vivo. Primary hepatocytes were treated with Jo2 antibody, cross-linked recombinant Fas ligand, TNF-␣, or lonidamine, in the presence of cyclohexamide (10 ␮g/mL) for Jo2 and Fas ligand or actinomycin D (1 ␮g/mL) for TNF-␣. Cells were harvested 22 (Jo2, Fas ligand, lonidamine treatments) or 36 hours later (TNF-␣ treatment) and either deposited on slides by centrifugation or lysed for biochemical analyses. Apoptosis was quantified by epifluorescence microscopy after incubation with either Hoechst 33258 (10 ␮mol/L, 10 minutes, 20°C) or with FMK-VAD-FITC (5 ␮mol/L, 20 minutes, 20°C). Where appropriate, the cell count in the presence of the protein or RNA synthesis inhibitors was considered as 100% survival.

Quantitative Polymerase Chain Reaction Total RNA was purified from frozen livers pulverized in liquid nitrogen using RNAqueous isolation kit (Ambion, Austin, TX). Complementary DNA was synthesized from 500 ng of RNA using the SuperScript II RT kit (GibcoBRL). Polymerase chain reaction (PCR) was performed with a LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany) using the LC Fast-Start DNA SYBRgreenI reagents (Roche Diagnostics) according to the manufacturer instructions except that 4 mmol/L of MgCl2 was used. Primer sequences were 5⬘-CGGAGGAAGACAAAAGGAAC-3⬘ (Bid sense), 5⬘TGGAAGACATCACGGAGCAA-3⬘ (Bid antisense), 5⬘GCTCACTGGCATGGCCTTCCGTGT-3⬘ (GAPDH sense), and 5⬘-TGGAAGAGTGGGAGTTGCTGTTGA-3⬘ (GAPDH antisense), and annealing temperatures were 63°C and 70°C, for Bid and GAPDH amplifications, respectively.

Immunoblotting Cell lysates were prepared from frozen livers pulverized in liquid nitrogen. Lysates were fractionated into cytosoluble, mitochondrial, or membrane fractions by centrifugation and

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protein concentration was assessed using the BCA assay (Perbio, Helsingborg, Sweden) according to the manufacturer instructions. Cellular fractions or crude extracts were separated on SDS-polyacrylamide gels, blotted on polyvinylidene fluoride membranes (Millipore, Bedford, MA), and detected using enhanced chemiluminescence system (Perbio).

Bid Complementation Assay Mouse liver mitochondria were isolated from tissue homogenized at 4°C in 300 mmol/L sucrose, 5 mmol/L 2-[(2hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES) pH 7.2, and 0.2 mmol/L EGTA in the presence of protease inhibitors. The nuclei were removed by centrifugation at 760g for 10 minutes at 4°C; the mitochondria were collected by centrifugation at 10,000g for 10 minutes at 4°C and resuspended in the same buffer at the protein concentration of 10 mg/mL and used immediately. The cytosolic fractions were prepared in the same buffer and were cleared by an additional centrifugation at 100,000g for 45 minutes at 4°C and kept at ⫺80°C until used. The assay was performed in 50 ␮L final volume (20 mmol/L HEPES pH 7.4, 10 mmol/L KCl, 2.5 mmol/L MgCl2 , 1 mmol/L EDTA, 1 mmol/L EGTA, 250 mmol/L sucrose, 1 mmol/L DTT, 10 mmol/L succinate, 0.7 mmol/L ATP, 1 mmol/L dATP, 10 mmol/L phosphocreatine, 37.5 mg/mL creatine kinase, supplemented with protease inhibitors) with 35 ␮g cytosolic protein, intact mitochondria (50 ␮g protein), 10 ng recombinant caspase 8 (R&D), and 2 ng recombinant His-tagged Bid at 37°C for 30 minutes. The mitochondria were removed by centrifugation at 10,000g for 10 minutes and the cytosol assayed for released cytochrome c by Western blotting.

Flow Cytometry Freshly isolated hepatocytes were incubated 30 minutes at room temperature with 1 ␮g/106 cells of fluorescein isothiocyanate (FITC)-coupled anti-Fas antibody. Data were acquired using a FacScan flow cytometer (Coulter, Hialeah, FL) and analyzed with CellQuest software (Becton-Dickinson, San Jose, CA).

Adenovirus Infections and ␤-Galactosidase Assays Mice were infected with 2.5 ⫻ 109 plaque-forming units (pfu) of AdCMV␤gal, an adenoviral ␤-galactosidaseencoding vector23 by intravenous injections into the tail vein. Liver biopsies were collected from mice 3 and 21 days postinfection. Frozen sections of 8 ␮ were stained for ␤-galactosidase activity and counterstained with neutral red. Crude protein extract from 0.5 ␮g of liver powder was tested for ␤-galactosidase activity using luminescent ␤-gal assay (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer’s instructions.

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Isolation and Analysis of Intrahepatic Lymphocytes Intrahepatic mononuclear cells were prepared essentially as described by Watanabe et al.24 Briefly, livers were pressed through a 100-micron nylon mesh into the complete culture medium. After washing twice in phosphate-buffered saline (PBS) containing 5% fetal calf serum, cells were purified by centrifugation through Ficoll Isopaque cushion, rinsed in PBS, and counted. Cells were then incubated 30 minutes at 4°C, with an anti–CD3⑀-CY (Pharmingen), anti–CD8-PE (Sigma), anti–CD4-CY (Pharmingen), or anti–CD25-PE (Immunotech) antibodies. Rat IgG2a-CY and PE were used as isotype controls (Pharmingen and Immunotech, respectively). After several washes, cells were analyzed using a FacScalibur flow cytometer (Coulter, Hialeah, FL) and analyzed with CellQuest software (Becton-Dickinson, San Jose, CA).

Interferon ␥ Assay Splenocytes (5 ⫻ 105 cells), purified as described for intrahepatic lymphocytes, were cultured in the presence or absence of a 30 ␮g/mL of a synthetic peptide octamer ICPMYARV, representing a ␤-galactosidase epitope in the H-2b haplotype. The stimulation was carried out as described by Mercier et al.25 Culture supernatant (50 ␮L) were assayed for interferon ␥ (IFN-␥) secretion using the mouse enzyme-linked immunosorbent assay IFN-␥ detection kit from Pierce Endogen (Rockford, IL), following the manufacturer’s instructions.

Survival Assays Primary hepatocytes were seeded into 96-well plates. After apoptosis induction, cell viability was assessed using XTT colorimetric test (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions.

Statistical Analysis Results are expressed as arithmetic mean ⫾ standard error of the mean and were analyzed using the Student–Fisher t test.

Results HCV Gene Expression Protects Hepatocytes From Cytotoxic Immune Response FL-N/35 transgenic mice express the entire polyprotein of HCV under control of the murine albumin promoter/enhancer within a C57/BL6 genetic background (Figure 1A). The level of expression is very low, requiring the use of reverse-transcriptase (RT)-PCR to detect transgenic RNA transcripts, but it is nonetheless associated with the development of hepatic steatosis and hepatocellular carcinoma in male animals.11 Thus, both the abundance of viral proteins and the phenotypic features of these transgenic animals are similar to what is observed in many patients

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Figure 1. HCV⫹ mice mount a normal cell-mediated immune response to adenoviral infection. Eleven HCV⫹ mice and 11 control littermates were injected with 2.5 ⫻ 109 pfu of ␤-galactosidase encoding adenovirus. Each animal was subjected to a liver biopsy 2 days postinfection and groups of animals (3 animals at day 3, 7, and 21; 2 animals at day 14) were sacrificed at times indicated. (A) Schematic representation of the transgene in the FL/N-35 transgenic mice.11 (B) Mononuclear cells were purified from livers of transgenic (solid lines and open squares) and control mice (dashed lines and open squares) as described in the Materials and Methods section and counted. (C) Subpopulations of intrahepatic mononuclear cells were analyzed by flow cytometry according to the surface expression of CD3 (dotted), CD3 and CD4 (hatched), or CD3 and CD8 (open bars) markers. (D) Splenocytes isolated from HCV⫹ (solid line) or control littermates (dotted) at day 7, 14, or 21 postinfection were stimulated by a ␤-galactosidase– derived peptide epitope. The secretion of ␥-interferon was assayed by enzyme-linked immunosorbent assay. AU, arbitrary units.

with chronic hepatitis C.5,21 As expected, there is no immune response against the transgenic proteins in these mice, and the animals do not develop any evidence of hepatic inflammation. Because these mice express the entire complement of viral proteins, unlike HCV transgenic animals that have been studied previously for potential impairment of intrahepatic immune responses,12,13 we assessed the influence of transgene expression on their ability to eliminate

an intrahepatic viral infection. Age- and sex-matched transgenic and control animals were infected with AdCMV␤gal, a replication-deficient, E1/E3-deleted, adenovirus vector-expressing ␤-galactosidase. Three days postchallenge, before any significant infiltration of the liver by immune cells (Figure 1B), the amount of ␤-galactosidase activity, determined by histochemical analysis of sections from small liver biopsies (Figure 2A, upper panels) or by direct enzymatic assay of tissue extracts

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Table 1. HCV Transgenic and Control Mice Mount Equivalent Cell-Mediated Immune Response to Adenoviral Infection

Spleen weight (g)

Intrahepatic CD3⫹ cells (⫻106) Intrahepatic CD3⫹CD25⫹ cells (⫻106)

Day

Control

HCV Transgenic

0 7 14 21 0 7 14 21 0 7 14 21

0.09 0.25 ⫾ 0.03 0.08 ⫾ 0.005 0.08 ⫾ 0.006 0.22 42.4 ⫾ 10.7 9.41 ⫾ 0.67 9.02 ⫾ 0.50 0.009 2.19 ⫾ 0.27 0.18 ⫾ 0.06 1.0 ⫾ 0.06

0.07 0.24 ⫾ 0.02 0.09 0.08 ⫾ 0.01 0.19 35.8 ⫾ 0.24 12.22 8.6 ⫾ 0.15 0.007 2.16 ⫾ 0.11 0.34 1.26 ⫾ 0.30

NOTE. HCV⫹ mice and control littermates were injected with 2.5 ⫻ 109 pfu of ␤-galactosidase encoding adenovirus. Animals were sacrificed at indicated times postinfection and analyzed, noninfected mice were used as day ⫽ 0 controls. One to 2 mice per condition were used at d ⫽ 0 and d ⫽ 14, 3 mice were used for samples at d ⫽ 7 and d ⫽ 21. Where appropriate, results are presented as mean ⫾ standard error of the mean.

(Figure 2B), was similar in both transgenic and nontransgenic animals. Lesser amounts of ␤-galactosidase were expressed in the spleen, with no differences evident in transgenic and nontransgenic animals (Figure 2B). Thus, transgenic and nontransgenic mice appear to be equally permissive for infection and expression of ␤-galactosidase after challenge with this defective recombinant adenovirus. To characterize the cellular immune responses to infection with the adenovirus, groups of animals were sacrificed at days 3, 7, 14, and 21 postinfection. Both the HCV transgenic and control mice showed a robust T-cell response to the adenovirus (Table 1 and Figure 1B–D). There was a dramatic transient increase in spleen size (Table 1), accompanied by a more moderate increase in the weight of the liver which at earlier time points was likely related to massive intrahepatic infiltration by mononuclear, largely CD3⫹ cells (Figure 1B, C). The absolute numbers and proportion of CD4⫹ and CD8⫹ cells were similar in the intrahepatic lymphocyte populations recovered from transgenic and control animals (Figure 1C), as was the appearance of a T-cell activation marker, CD25 (Table 1). Furthermore, there was no significant difference in the cellular response to a specific adenoviral antigen, as shown by the measurement of IFN-␥ production by splenocytes stimulated in vitro by a ␤-galactosidase– derived peptide (Figure 1D). We conclude from these results that the presence of the HCV transgene does not impair the T-cell proliferative response to the adenoviral infection.

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Next, we studied the efficiency of clearance of adenovirus-infected hepatocytes by evaluating the persistence of ␤-galactosidase– expressing hepatocytes, assayed by histochemical staining of tissue sections (Figure 2A) or quantified by a luminometric assay applied to liver extracts (Figure 2B, C). Three days postinfection, 40%– 50% of the hepatocytes were ␤-galactosidase positive in both transgenic and nontransgenic control animals (Figure 2A). As indicated earlier, enzymatic activity assays confirmed that levels of ␤-galactosidase expression were not influenced by the transgenic status of the animals. However, there were marked differences between the transgenic and nontransgenic animals when tissues obtained 21 days postinfection were examined for clearance of the infected cells. Histochemical examination (Figure 2A) showed that the transgenic animals were significantly impaired in their ability to eliminate the adenovirus-infected, ␤-galactosidase– expressing hepatocytes from the liver. These results were confirmed by direct assay of ␤-galactosidase activity in liver extracts, which were indistinguishable in HCV⫹ and control samples at days 3 and 7 postinfection; differences were statistically insignificant at day 14, but became marked by day 21 (P ⬍ 0.05) (Figure 2B, C). In striking contrast, there was no difference in the levels of ␤-galactosidase expression in the spleens of the HCV transgenic and nontransgenic control mice at all time points (not shown), including day 21 (Figure 2B). Because the expression of the HCV transgene in these mice is under control of the liverspecific murine albumin promoter enhancer, these data strongly suggest that the transgene expression confers resistance to the cytotoxic T-cell–mediated immune response directed against adenovirus-infected cells. The HCV Transgene Renders Hepatocytes Resistant to Fas-Induced Apoptosis Fas-mediated immune responses play a predominant role in the elimination of adenovirus-infected hepatocytes.26 Hepatocytes are particularly sensitive to Fas receptor ligation and intravenous injection of high doses of Fas agonistic antibody provokes massive hepatocyte apoptosis and liver hemorrhage.27 Animals deficient in different components of the Fas signal transduction pathway are resistant to this treatment.28 –30 To assess Fas-mediated responses, we injected Jo2, an agonistic anti-Fas antibody intravenously into HCV transgenic and control mice and sacrificed the animals 2 hours later. Histological staining of liver sections revealed major destruction of hepatic architecture in the control mice, whereas the transgenic livers were much less affected (Figure 3A). Apoptotic cells, identified by altered nuclear morphol-

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Figure 2. HCV⫹ mice are defective in the clearance of adenovirusinfected hepatocytes. (A) ␤-galactosidase staining was performed on liver sections to qualitatively assess the level of infection in situ. Adenovirus-infected hepatocytes are visible in blue. Forty to 50% hepatocytes stain positive for ␤-galactosidase at day 3 both in transgenic and control samples. Positive cells are readily detectable at day 21 postinfection in the HCV⫹ livers; they are absent from the control samples. The bar represents 100 ␮. (B) Crude protein extracts from HCV⫹ (hatched) and control (empty) livers and spleens were assayed for ␤-galactosidase activity at day 3 and 21 postinfection. Results are presented as mean ⫾ standard error of the mean. (C) ␤-galactosidase activity was measured in crude extracts from of HCV⫹ (solid) and control (dashed) mice sacrificed at day 3, 7, 14, or 21 postinfection. The results are normalized to ␤-galactosidase activity measured in biopsies taken at day 2 postinfection and presented as mean ⫾ standard error of the mean. The data in B and C come from 2 independent experiments.

ogy, were significantly more abundant (P ⬍ 0.05) both in the periportal and in the centrolobular areas of livers from control mice compared with HCV transgenic animals (Figure 3B). Primary hepatocyte explant cultures prepared from the livers of transgenic mice were also resistant to Fasinduced apoptosis (P ⬍ 0.01, Figure 3C). In these ex vivo experiments, we counted cells labeled with Z-VADFITC, a fluorescent peptide that specifically binds activated caspases. These results suggested that the observed

cell death was indeed caused by apoptosis. Stimulation of the Fas pathway both in vivo (data not shown) and in the primary hepatocyte cultures gave rise to both delayed and weaker caspase 3 activation in the HCV⫹ compared with the control hepatocytes (Figure 3D). This was not because of an altered expression of caspase 3 because the unprocessed procaspase 3 was present at the same levels in the livers of all animals (Figure 3E). The diminished response of transgenic hepatocytes to Fas stimulation was not unique to the antibody used in

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Figure 3. HCV gene expression protects hepatocytes from Fas-induced apoptosis. Agonistic anti-Fas antibody (Jo2) was injected into the tail vein of 4 HCV transgenic and 3 control mice. Animals were sacrificed 2 hours postinjection, and the histology of fixed, paraffin-embedded liver sections was analyzed by H&E staining (A). Panels 1 and 2: control mice; panels 3 and 4: HCV transgenic mice. Liver destruction and hemorrhage are severe in the control samples. Darker staining cells with condensed nuclei, typical of apoptotic morphology, are visible at higher magnification (panel 2). Scale bars ⫽ 20 ␮. (B) Sections of control (empty) and transgenic (hatched) livers were stained with the Hoechst 33258 dye. Highly condensed, apoptotic, and normal nuclei were counted in randomly chosen fields. Bars are mean ⫾ standard error of the mean. (C) Primary cultures of 8 transgenic (hatched) and 4 nontransgenic (empty) hepatocytes were treated with Jo2. Twenty-two hours later, cells were stained for activated caspases with Z-VAD-FITC and for nuclear morphology with Hoechst 33258. Apoptotic and normal cells were scored in randomly chosen fields. Bars are mean ⫾ standard error of the mean. (D) Caspase 3 activation was assayed by immunoblotting in cell lysates prepared from primary hepatocyte cultures stimulated by anti-Fas antibody for the times indicated. (E) Crude protein extracts from 2 HCV⫹ and 2 control primary hepatocyte cultures were analyzed by Western blotting using a polyclonal anticaspase 3 antibody recognizing the unprocessed procaspase. The amount of protein loaded on the gel was normalized using an anti-GAPDH antibody. A representative Western blot is shown.

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Figure 4. Apoptosis resistance of primary HCV transgenic hepatocytes is specific to the Fas pathway. Primary hepatocyte cultures from 4 HCV transgenic (solid or hatched) and 4 control (dashed or empty) mice were treated with indicated concentrations of anti-Fas antibody Jo2 (A), recombinant Fas ligand (B), TNF-␣ and, as positive control, Jo2 (0.1 ␮g/mL) (C) and lonidamine (D). Cell viability was assayed by a colorimetric XTT test in A, B, and D. Apoptosis was quantified in C by fluorescence microscopy of cytospin slides labeled with the Hoechst 33258. All results are presented as mean ⫾ standard error of the mean of at least 3 measurements.

these experiments because treatment of cells with increasing concentrations of either the anti-Fas antibody or recombinant Fas ligand gave rise to differential survival of transgenic versus control hepatocytes (Figure 4A, B). To determine the specificity of apoptosis inhibition in the HCV⫹ hepatocytes, we assayed their response to TNF-␣. TNF receptor 1 (TNFR1) belongs to the same family of death receptors as Fas and its stimulation in the presence of protein or RNA synthesis inhibitors also leads to hepatocyte apoptosis. These results were of particular interest, because the HCV core protein, which has been incriminated in the development of liver cancer in transgenic mice,9 –11 has also been suggested to interact with and modulate signal transduction through TNFR1 and related receptor molecules.14 –16 However, there was no difference in the response of the transgenic and control hepatocytes to TNF-␣, even though hepatocytes from the same primary cultures displayed a clear differential response to Fas stimulation (Figure 4C). Both Fas and TNFR1 signal apoptosis via the “extrinsic” pathway, leading from death receptors to effector caspases activation via the recruitment and activation of the apical caspase 8. In some cell types, including hepatocytes, this signal transduction is amplified by the “intrinsic” pathway relying on changes in mitochondrial membrane permeability.31 Expression of the HCV core

protein has been associated with mitochondrial injury and cytochrome C release to the cytoplasm, and, consistent with these results, HCV transgenic mice develop oxidative stress and accumulate abnormal levels of lipid peroxides on sublethal challenge with carbon tetrachloride.32,33 However, the mitochondrial induction of apoptosis was not altered in the FL-N/35 transgenic hepatocytes because direct stimulation of the release of apoptogenic factors from the mitochondria by lonidamine34 generated an identical response in transgenic and control hepatocytes (Figure 4D). The Cytoplasmic Abundance of Bid Is Decreased in HCV Transgenic Hepatocytes In an effort to identify the molecular defect in the apoptotic response of the HCV transgenic hepatocytes to Fas stimulation, we assayed different steps of this signal transduction pathway. We found no difference either in the amount of Fas present at the cell surface or in the truncated form of Fas mRNA or protein or in the recruitment, cleavage, and early activation of procaspase 8 or the apical caspase of the Fas pathway, when we compared control versus transgenic hepatocytes (data not shown). Hepatocytes have been classified as type II cells35 in which the weak initial caspase 8 activation is amplified

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Figure 5. HCV gene expression inhibits the Fas pathway at the level of mitochondrial cytochrome c release. (A) Primary hepatocytes isolated from HCV⫹ and control mice were cultured in the presence or absence of Jo2. Twenty-two hours later, the cells were collected, lysed and the mitochondrial (M) and cytosoluble (C) fractions were separated. The release of cytochrome c from the mitochondria was assayed by immunoblotting. (B) Fractionated liver extracts from 4 HCV⫹ (hatched) and 3 control (empty) animals treated in vivo Jo2 were analyzed by immunoblotting. The cytoplasmic release of cytochrome c was quantified by densitometry and normalized to the level of expression of GAPDH in the cytosol. Results are presented as mean ⫾ standard error of the mean.

by interactions of pro-apoptotic Bcl2 family proteins with mitochondria.36,37 Active caspase 8 cleaves Bid, the truncated form of which translocates to mitochondria, recruits Bax or Bak, and leads to the release of apoptogenic factors, including cytochrome c, from the inner mitochondrial space.38 Cytochrome c participates in the formation of an apoptosome, a multiprotein complex that recruits and activates caspases 9 and 3. We found that the cytoplasmic relocalization of cytochrome c after Fas stimulation of HCV transgenic hepatocytes was inhibited by at least 50%, both ex vivo (Figure 5A) and in vivo (Figure 5B) compared with nontransgenic controls. This finding was particularly interesting because caspaseindependent permeabilization of the mitochondrial membrane with lonidamine efficiently killed both transgenic and control hepatocytes (Figure 4D). These results suggest a defect in the upstream effector of the caspase 8 –mediated mitochondrial permeability change, namely the protein Bid. The truncated, active form of Bid is unstable39 and difficult to quantify. Thus, disappearance of the fulllength protein from the cytoplasm is often used as an indicator of Bid’s cleavage. Preliminary to analyzing the

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expression of Bid, we determined the basal abundance of Bid transcripts in transgenic and control livers. The abundance of Bid-specific mRNA, measured by quantitative RT-PCR, was identical in transgenic and nontransgenic mouse livers (Figure 6A). However, in HCV transgenic liver, the basal level of Bid protein was significantly reduced (P ⬍ 0.01, Figure 6B, C). In contrast, there was no difference in the abundance of Bid in kidneys and in spleens from these transgenic animals compared with nontransgenic control mice, consistent with the liver-specific expression of the HCV transgene in these animals. Thus, the resistance to Fas-induced apoptosis in the transgenic hepatocytes is associated with a significant, posttranscriptional, liver-specific reduction (⬃60%) in the level of Bid expression. We reasoned that if the decreased Bid expression were instrumental in lowering the sensitivity to Fas stimulation in transgenic hepatocytes, the defect in the apoptotic signal transduction should be overcome by complementation of cells with recombinant Bid (rBid). Because attempts to re-establish normal physiological levels of Bid in cultured primary hepatocytes were unsuccessful, we evaluated the effects of Bid complementation in an in vitro assay. Intact mitochondria, prepared from a control mouse liver, were incubated with cytosolic extracts prepared from either control or HCV transgenic mouse livers. Addition of low levels of activated caspase 8 led to increased mitochondrial membrane permeability with the control extract, as evidenced by cytochrome c release, whereas the transgenic cytosol had no effect on the integrity of the mitochondria (Figure 6D). Importantly, this functional difference was evident despite a rather minor difference in the abundance of endogenous Bid in the control versus transgenic cytosolic as detected by immunoblotting in this particular experiment (1.3-fold difference after normalization with GAPDH). Complementation with rBid, which increased the effective Bid concentration in the control extract by a factor of 2–3, resulted in a comparable degree of mitochondrial cytochrome c release with both transgenic and control cytosolic extracts (Figure 6D). In the absence of active caspase 8, rBid did not cause any leakage of cytochrome c. Thus, under these in vitro conditions, Bid appears to be a limiting factor in apoptotic signal transduction initiated by active caspase 8, the downstream component of the Fas-signaling pathway.

Discussion The mechanisms by which HCV evades the immune system to establish and maintain a persistent infection in most human hosts remain poorly understood.4

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Figure 6. Down-regulation of Bid expression in the livers of the HCV transgenic mice. (A) Expression of Bid mRNA in 7 control (empty) and 6 HCV⫹ (hatched) livers was measured by quantitative RT-PCR and normalized to the GAPDH mRNA expression levels. Results are presented as mean ⫾ standard error of the mean. (B, C) Liver, kidney, and spleen extracts from 10 HCV⫹ (hatched) and 10 control (empty) mice were analyzed by immunoblotting. A representative Western blot with anti-Bid and anti-GAPDH antibodies is shown in B. A densitometric analysis of Bid expression in livers, normalized to the GAPDH expression level, is shown in C. Results are presented as mean ⫾ standard error of the mean. (D) Bid complementation assay: cytosolic fractions from HCV⫹ and control hepatocytes were incubated with intact mitochondria in the presence or absence of recombinant, active, caspase 8, and recombinant Bid (rBid). Release of cytochrome c from the mitochondria was measured by Western blot of the cytosolic supernatant. The total content of cytochrome c is shown in the first lane, which corresponds to the mitochondrial fraction.

It is likely that multiple mechanisms contribute to viral persistence. Strong in vitro evidence supports the disruption of both interferon signaling pathways and effector functions by the viral proteins NS3/4A and NS5A.6,8 However, global mRNA profiling of HCVinfected chimpanzees suggests that type 1 interferon genes are nonetheless strongly induced and that clearance of the infection is associated with the expression of IFN-␥–stimulated genes and genes associated with the adaptive immune response.40 Although the propensity of the virus to undergo mutation and the associated high degree of genetic diversity that is evident among different HCV strains have been proposed as factors in virus persistence,41,42 they are as likely to result from, rather than be the cause of, the inability of an activated immune system to eliminate the virus infection from the liver.43 Here, we have shown that transgenic mice expressing the

complete polyprotein of HCV have a molecular defect in the Fas pathway that renders the transgenic hepatocytes resistant to Fas-induced apoptosis. This defect, which is associated with a reduction in the abundance of Bid within the cytoplasm of transgenic hepatocytes (Figure 6), impairs the ability of the transgenic animals to eliminate hepatocytes infected with and expressing ␤-galactosidase encoded by a recombinant adenovirus. This defect is present despite what appears to be a normal proliferative T-cell response to the adenovirus vector that is marked by infiltration of the transgenic liver with both CD4⫹ and CD8⫹ lymphocytes (Figure 1, Table 1). Interestingly, the defect is specific to the liver because the efficiency with which virus-infected cells were cleared from the spleen was not affected by the presence of the transgene (Figure 2B). This is consistent with the liver-specific expression of the transgene, which

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is under control of the murine albumin promoter enhancer.11 These data strongly support the conclusion that the in vivo antiviral cellular immune response is equivalent in the transgenic and control animals. Furthermore, they argue against the possibility of a spurious alteration of target cell sensitivity to cytotoxic T-cell attack because of the chromosomal location of transgene integration. These results sharply contrast to previously published data delineating the immune competence of transgenic mice with liver-specific expression of either the core protein alone, or all the structural proteins, of HCV.12,13 Thus, although the precise mechanism leading to downregulation of Bid in the FL-N/35 transgenic lineage remains uncertain, it is likely that either the non-structural proteins of HCV, or the full complement of the viral proteins, are necessary for the immunomodulatory effect we observed. Significant reductions in the level of Bid expression in HCV⫹ hepatocytes may have far-reaching pathological consequences. Bid is a target of both caspase 8 and granzyme B,44 2 proteases involved in cell-mediated cytotoxicity. Thus, it is likely that the reductions in Bid contribute directly to the protection afforded to the transgenic hepatocytes against a cytotoxic attack stimulated by recombinant adenovirus. In hepatocytes, Fasinduced apoptosis is strictly dependent on caspase-mediated cleavage of Bid, a proapoptotic BH3-only member of the Bcl-2 family: Bid⫺/⫺ mice are resistant to liver destruction caused by the injection of an agonistic antiFas antibody.45 In agreement with these results, we found that a decrease in the abundance of cytoplasmic Bid correlated with resistance of the transgenic hepatocytes to Fas-mediated apoptosis. This suggests that in hepatocytes Bid is a limiting factor in this apoptotic signal transduction pathway, a hypothesis strongly supported by the in vitro complementation study (Figure 6D). We detected no significant variation in the expression patterns of several other members of the Bcl2 family (data not shown). Remarkably, the immune evasion displayed by the transgenic hepatocytes appears to be at least as efficient as that reported for Fas-ligand– deficient (gld) mice challenged with a ␤-galactosidase– expressing adenovirus.26 Although the role of Fas in clearance of adenoviral infections is well known, its participation in elimination of human hepatocytes infected with HCV is uncertain. However, because the Fas pathway is implicated in a wide range of cell-mediated immune responses,46 it is tempting to speculate that the novel mechanism of immune evasion described here might operate in conjunction with previously described immunomodulatory responses to diminish viral clearance and

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thus contribute to the establishment of persistent HCV infection in humans. In particular, expression of the NS5A protein has been suggested to decrease apoptosis by disruption of protein kinase R (PKR) function.6 Similarly, impaired interferon-␣ signaling, as reported recently in an HCVtransgenic mouse line,20 or NS3/4A disruption of interferon regulatory factor 3 activation8 may also contribute to reduced clearance of infected cells. Many different viral infections affect apoptotic signal transduction pathways.47,48 Previous reports are conflicting concerning the effects of HCV proteins on the regulation of apoptosis.16,48,49 Many of the model systems that have been studied previously have involved the expression of only a single viral protein, however, or the overexpression of HCV proteins at abundances far higher than would be expected in infected patients. In contrast, the transgenic mice that we studied11 closely mimic the situation observed in patients with chronic hepatitis C. The HCV transgene encodes the complete viral polyprotein, the viral gene expression is weak, and, most importantly, the mice develop hepatocellular carcinoma late in their lives. These mice are therefore a good model of non–immune-mediated pathology relevant to human disease. Our results confirm previously published data, which showed that acute, high-level expression of a subset of HCV proteins protects murine hepatocytes from Fasinduced apoptosis.50 However, no molecular defect was identified in that study, possibly because long-term effects could not be analyzed because of the inherent toxicity of the experimental system. Many regulators of death receptor signaling pathways have been described,51 a large number of which function upstream of the recruitment and/or activation of the apical caspase 8. We detected no alteration in caspase 8 activation in the HCV⫹ hepatocytes, arguing strongly that the HCV transgene in our mice exerts no major effect on the initial steps of the Fas pathway. The molecular events leading to a reduction in Bid abundance in the transgenic hepatocytes remains to be determined, although the absence of changes in Bid mRNA abundance suggests that this is regulated posttranscriptionally (Figure 6A). Importantly, the abundance of Bid is not reduced in Huh7 cells supporting the autonomous replication of genome-length HCV RNAs52 (Li K., unpublished data, 2003). It is interesting that the transgenic HCV⫹ hepatocytes displayed unaltered sensitivity to TNF-␣ (Figure 4C), even though the apoptotic-signaling pathways downstream of Fas and TNFR1 receptors are closely related.31 As in the case of Fas stimulation, treatment with TNF-␣

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results in a reduced cytochrome c release from mitochondria in HCV⫹ hepatocytes (O.D., H.L., and U.H., 2003, unpublished data). Although hepatocytes are type II cells35 and require the mitochondrial signaling with respect to the Fas pathway, our findings suggest that they may behave like type I cells when signaling through TNFR1. Deregulation of apoptosis is a prerequisite for cancerous transformation.53 Alone, however, defects in apoptotic signal transduction pathways are generally insufficient for tumor development. The weak association of cancer with defects in Fas/FasL signaling54,55 is probably due to a deficient immune response. The initial stages of oncogenesis are typically characterized by an increased sensitivity to apoptotic stimuli.56 However, in some cases, inhibition of cell death, although not oncogenic in itself, creates a permissive environment for subsequent cellular transformation.57 This may well be the case with respect to the hepatocarcinogenesis observed in our transgenic mice.11 Indeed, the CD95/Fas pathway of apoptosis induction was significantly compromised in the transgenic hepatocytes well before detectable oncogenic transformation. Although the defect we have identified in the Fas pathway may contribute to the development of cancer in these transgenic mice since male mice in this lineage develop hepatic tumors at relatively high frequency,11 we would predict that one or more viral proteins may also promote deregulation of cellular proliferation. Alternatively, oxidative stress, engendered by expression of the core protein,32,33 may lead to cellular DNA damage, thereby setting the stage for malignant transformation. In this context, different oncogenes, including viral gene products, target down-regulation of Apaf-1 and caspase 9, two components of the mitochondrial apoptosis signaling pathway.58,59 Moreover, decreased Apaf-1 expression was detected in at least one highly malignant type of tumor.60 It is tempting to speculate that a diminished apoptotic response associated with HCVinduced Bid down-regulation might participate in creating conditions permissive for hepatocarcinogenesis. This argument is strengthened by the observation that decreased Bid expression is also associated with HBVrelated hepatocellular carcinoma.61 Clearly, the mechanisms by which these 2 viruses cause Bid down-regulation are likely to differ, but the fact that both target the same member of the Bcl2 family supports the physiological relevance of these observations. The demonstration of Bid down-regulation in the HCV transgenic hepatocytes opens new avenues for designing future therapies of hepatitis C and possibly ap-

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proaches to preventing or treating HCV-related hepatocellular carcinomas. Specific activation of apoptosis in tumor cells is actively pursued for cancer therapy.62 Our results suggest that in the case of chronic HCV infection complementation of Bid might prove an interesting strategy to explore.

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Cordon-Cardo C, Lowe SW. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 2001;409:207–211. 61. Chen GG, Lai PB, Chan PK, Chak EC, Yip JH, Ho RL, Leung BC, Lau WY. Decreased expression of Bid in human hepatocellular carcinoma is related to hepatitis B virus X protein. Eur J Cancer 2001;37:1695–1702. 62. Kaufmann SH, Gores GJ. Apoptosis in cancer: cause and cure. Bioessays 2000;22:1007–1017. Received December 20, 2002. Accepted December 4, 2003. Address reprint requests to: Urszula Hibner, Ph.D., Institut de Ge ´ne ´tique Mole ´culaire, CNRS UMR 5535, 1919 Route de Mende, F-34293 Montpellier cedex 5, France. e-mail: [email protected]; fax: (33) 4-67-04-02-31. Supported by INSERM, CNRS, ANRS, the French Ministry of Research “Programme de Recherche Fondamentale en Microbiologie, Maladies Infectieuses et Parasitaires” (UH), and the National Institute of Allergy and Infectious Diseases through the Hepatitis C Cooperative Research Center U19-AI40035 (S.L.). Dr. Lerat was a recipient of fellowships from Ligue Nationale contre le Cancer and ANRS. We are grateful to Dr. Jean-Claude Martinou and Sylvie Montessuit for the gift of recombinant murine Bid and 224A anti-Bid antibody, Dr. Antonio Freitas for the fluorochrome-conjugated anti-CD3, anti-CD4 and anti-CD8 antibodies. We thank Eric Joffre for expert assistance with animal experiments, Dr. Jean-Franc¸ois Emile for help with histological analyses, Audrey Ceschia for help with quantification of apoptosis, and Francis Bodola for quantitation of adenovirus DNA in liver tissues. We are especially grateful to Caroline Desnevre, Ste ´phanie Mercier, and Naomi Taylor both for their help and for precious reagents for the analysis of the antiadenoviral immune response.