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

doi:10.1053/j.gastro.2003.12.005

Gastroenterology

Volume 126, Issue 3, March 2004, Pages 859-872

https://doi.org/10.1053/j.gastro.2003.12.005 Get rights and content

Abstract

$Background & Aims$ : 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 β-galactosidase, and the persistence of infected cells was measured by histochemistry and enzymatic assays. $Results$ : Hepatocytes from the HCV⁺ 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.

Section snippets

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 Jean-Claude Martinou (Université de Genève, Switzerland). Polyclonal anti-GAPDH (glyceraldehydes-3-phosphate dehydrogenase) antibody was a gift from Jean-Marie

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

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

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 HCV-infected chimpanzees suggests that type 1 interferon genes are nonetheless strongly

Acknowledgements

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-François 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

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Virus-related type 2 diabetes is commonly observed in individuals infected with the hepatitis C virus (HCV); however, the underlying molecular mechanisms remain unknown. Our aim was to unravel these mechanisms using FL-N/35 transgenic mice expressing the full HCV ORF. We observed that these mice displayed glucose intolerance and insulin resistance. We also found that Glut-2 membrane expression was reduced in FL-N/35 mice and that hepatocyte glucose uptake was perturbed, partly accounting for the HCV-induced glucose intolerance in these mice. Early steps of the hepatic insulin signaling pathway, from IRS2 to PDK1 phosphorylation, were constitutively impaired in FL-N/35 primary hepatocytes via deregulation of TNFα/SOCS3. Higher hepatic glucose production was observed in the HCV mice, despite higher fasting insulinemia, concomitant with decreased expression of hepatic gluconeogenic genes. Akt kinase activity was higher in HCV mice than in WT mice, but Akt-dependent phosphorylation of the forkhead transcription factor FoxO1 at serine 256, which triggers its nuclear exclusion, was lower in HCV mouse livers. These findings indicate an uncoupling of the canonical Akt/FoxO1 pathway in HCV protein-expressing hepatocytes. Thus, the expression of HCV proteins in the liver is sufficient to induce insulin resistance by impairing insulin signaling and glucose uptake. In conclusion, we observed a complete set of events leading to a prediabetic state in HCV-transgenic mice, providing a valuable mechanistic explanation for HCV-induced diabetes in humans.
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Transgenic mice with liver-targeted expression of the HCV full-length open reading frame (FL-N/35 lineage) develop a spectrum of liver metabolic disorders, including alterations of lipid metabolism [7] and micro- and macro-vesicular steatosis [8]. In addition to reproducing a wide range of pathological features associated with chronic hepatitis C [9,10], males of this lineage are cancer-prone [8]. While this phenotype is perfectly reproducible in different animal colonies [11], some genetic backgrounds, for example the widely used CB57Bl/6 animals, are protected from the HCV viral protein-driven carcinogenesis ([12] and our unpublished data).
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Transgenic mice with hepatocyte-targeted expression of all HCV proteins (FL-N/35 model) and needle biopsies from hepatitis C patients were studied with respect to patterns of lipid deposition in the context of metabolic zonation of the liver lobule.
We report that low levels of viral proteins are sufficient to drive striking alterations of hepatic metabolic zonation. In mice, a major lipogenic enzyme, fatty acid synthase, was redistributed from its normal periportal expression into the midzone of the lobule, coinciding with a highly specific midzone accumulation of lipids. Strikingly, alteration of zonation was not limited to lipogenic enzymes and appeared to be driven by systemic signalling via the Wnt/β-catenin pathway. Importantly, we show that similarly perturbed metabolic zonation appears to precede steatosis in early stages of human disease associated with HCV infection.
Our results rationalize systemic effects on liver metabolism, triggered by a minority of infected cells, thus opening new perspectives for the investigation of HCV-related pathologies.
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They were used in conformation with the European Community Council directive. Animals were bred and housed as previously described [2]. Studies were performed on age-matched 7- to 10-month-old males.
During chronic HCV infection, activation of fibrogenesis appears to be principally related to local inflammation. However, the direct role of hepatic HCV protein expression in fibrogenesis remains unknown.
We used transgenic mice expressing the full length HCV open reading frame exposed to a ‘second hit’ of the fibrogenic agent carbon tetrachloride (CCl₄). Both acute and chronic liver injuries were induced in these mice by CCl₄ injections. Liver injury, expression of matrix re-modeling genes, reactive oxygen species (ROS), inflammation, hepatocyte proliferation, ductular reaction and hepatic progenitor cells (HPC) expansion were examined.
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Hepatitis C virus proteins induce lipogenesis and defective triglyceride secretion in transgenic mice
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The precise role of HCV protein expression in these phenomena is unclear. The transgenic mice used here, which expresses the HCV full-length open reading frame, are a particularly relevant model for studying HCV protein-induced abnormalities, because all of the HCV proteins are produced at the same time at physiologically relevant levels, without infectious virus production (16, 26–28). We have previously reported that two lineages of transgenic mice expressing the HCV full-length open reading frame develop more frequent and more severe hepatocellular steatosis than their nontransgenic littermates (16).
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^☆: 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.

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Basic-liver, pancreas, and biliary tractImpaired clearance of virus-infected hepatocytes in transgenic mice expressing the hepatitis C virus polyprotein☆

Abstract

Section snippets

Materials and methods

HCV gene expression protects hepatocytes from cytotoxic immune response

Discussion

Acknowledgements

Gastroenterology

J Biol Chem

Gastroenterology

Gastroenterology

J Immunol Methods

Immunity

Gastroenterology

Cell

Cell

Trends Cell Biol

J Biol Chem

J Biol Chem

Virology

J Biol Chem

Cell

Cell

Cancer Cell

Eur J Cancer

Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis

J Immunol

Patients with chronic hepatitis C have circulating cytotoxic T cells which recognize hepatitis C virus-encoded peptides binding to HLA-A2.1 molecules

J Virol

Identification of HLA-A3 and -B7-restricted CTL response to hepatitis C virus in patients with acute and chronic hepatitis C

J Immunol

Pathogenesis of chronic hepatitis Cimmunological features of hepatic injury and viral persistence

Hepatology

Natural history of hepatitis C

Hepatology

Antiapoptotic and oncogenic potentials of hepatitis C virus are linked to interferon resistance by viral repression of the PKR protein kinase

J Virol

Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection

J Immunol

Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease

Science

Hepatitis C virus core protein induces hepatic steatosis in transgenic mice

J Gen Virol

The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice

Nat Med

Hepatitis C virus genotype 1b core protein does not exert immunomodulatory effects on virus-induced cellular immunity

J Virol

Hepatitis C virus core and envelope proteins do not suppress the host’s ability to clear a hepatic viral infection

J Virol

Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta receptor modulates the signal pathway of the lymphotoxin-beta receptor

J Virol

Basic-liver, pancreas, and biliary tract
Impaired clearance of virus-infected hepatocytes in transgenic mice expressing the hepatitis C virus polyprotein☆