Intrahepatic innate immunity in hepadnaviral infection in the woodchuck model of hepatitis B

Guy, Clifford Scott (2008) Intrahepatic innate immunity in hepadnaviral infection in the woodchuck model of hepatitis B. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Hepatitis B virus (HBV) causes lifelong chronic liver disease in approximately 400 million people worldwide. The causative factors underlying resolution of HBV infection and the development of chronic hepatitis B are not completely understood, although an inadequate anti-viral immune response is considered to be important in establishing chronic symptomatic infection. In this regard, previous studies have demonstrated the significance of broad strong innate and anti-HBV adaptive immune responses in inhibition of hepadnaviral replication and resolution of acute hepatitis. -- The contribution of the liver to induction and maintenance of peripheral immune tolerance or to decreased immune responsiveness is increasingly recognized. These functions may in part be ascribed to the unique cellular composition of the liver, including a disproportionately high number of innate immune cell subsets and specialized antigen presenting cells. Liver cells, including hepatocytes, also appear to contribute to the immune response by interacting with naïve or activated T cells. -- We hypothesized that hepatocytes can act as effector immune cells and they can directly shape hepatic immune responses and that the natural tolerizing properties of the liver may contribute to an impaired effectiveness of the anti-viral response in hepadnaviral infection. To investigate these possibilities, we utilized experimental hepadnaviral infection in woodchucks infected with woodchuck hepatitis virus (WHV), which represents the closest virological and pathogenic model of human HBV infection and hepatitis B. -- In the first study, we discovered that normal hepatocytes constitutively express CD95 ligand (CD95L) and can cause death of contacted cells via a CD95L-CD95-dependent mechanism. Furthermore, interferon-gamma (IFNγ) and, to a lesser extent, tumour necrosis factor-alpha (TNFα) can enhance hepatocyte CD95L-mediated cytotoxicity. This suggests that the local cytokine environment may modulate the contribution of hepatocytes to liver immunity. Subsequently, we discovered that hepatocytes also express biologically competent perforin capable of cell killing. The ability of hepatocytes to cause cell death by two different but complementary mechanisms, differentially regulated by the cytokine microenvironment, emphasized the role of hepatocytes as cytotoxic effectors while demonstrating for the first time that perforin is expressed by non-immune cells. -- Further investigations revealed that hepatocyte CD95L and perforin-mediated cytotoxicity is significantly augmented during chronic hepadnaviral hepatitis and following recovery from acute infection. This could be a consequence of increased intrahepatic production of IFNγ due to virus-induced liver inflammation and strengthens the possibility that hepatocytes may actively contribute to shaping the local immune environment in hepadnaviral infection. On the other hand, a direct contribution of WHV to augmented hepatocyte cytotoxicity was excluded in a series of in vitro experiments applying woodchuck hepatocyte lines stably expressing WHV genome or its individual genes. -- In a final study, quantitative analyses of intrahepatic hepadnaviral replication and molecular markers indicative of activation of innate and adaptive immune responses, using real-time RT-PCR assays, revealed that hepadnavirus establishes replication almost immediately following exposure to a large, liver pathogenic virus dose and that the innate response is promptly activated. However, this response appears to be insufficient in priming an effective adaptive T cell response capable of early virus elimination. These findings indicate that, in contrast to observations in HBV-infected chimpanzees, hepadnavirus is recognized immediately after invasion by the hepatic innate immune system. -- Taken together, hepatocytes constitutively display cytotoxic capabilities which are susceptible to cytokine-induced augmentation. Hypothetically, this hepatic local cytotoxic activity may constrain priming of adaptive T cells by activated innate immune effectors due to deletion of virus-specific T cells. This paradigm would favour the expansion of virus replication in the liver during the prolonged incubation period typically seen in hepadnaviral infection, prior to the extrahepatic development of effective antiviral T cell responses capable of resolution of acute hepatitis. The relevance of this paradigm to the establishment of chronic hepadnaviral hepatitis will require further investigation.

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