Molecular Mechanisms of Pathogenesis of Acute and Chronic Liver Diseases

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

The major goal of the Hepatic Pathogenesis Section (HPS) is to conduct translational research on the pathogenesis of acute and chronic liver disease, with a major focus on viral hepatitis and its long-term sequelae, cirrhosis and hepatocellular carcinoma (HCC), which contribute to a very large burden of disease worldwide. 1. Molecular mechanisms of pathogenesis of acute liver failure (ALF). Infection with hepatitis B virus (HBV) is a major cause of ALF. However, only 1% of cases of acute hepatitis B develop ALF, suggesting that besides viral factors, host factors play an important role in the pathogenesis of this disease. The lack of small animal models and the extremely rapid clinical course have seriously hampered pathogenesis studies. Taking advantage of a unique collection of serum and multiple liver samples from patients who underwent liver transplantation (LT), we previously documented an overwhelming B-cell gene signature centered in the liver of ALF patients, with massive accumulation of plasma cells secreting IgM and IgG directed against the hepatitis B core antigen (HBcAg). Extra-lymphatic Ig production has been documented in persistent viral infection and autoimmune diseases, but not, to our knowledge, in acute viral infection. We characterized the IgM and IgG produced in the liver of ALF patients and we found that the majority of VH genes coding for HBcAg-specific intrahepatic IgM and IgG lacked somatic mutations, indicating that HBcAg is the target of germline human antibodies. The presence of both IgM and IgG isotypes of the same germline antibodies in all ALF patients demonstrates that isotype switching occurred without somatic hypermutation. Strikingly, surface plasmon resonance analysis of individual cloned antibodies demonstrated exceptionally high affinities for the cloned homologous HBcAg, despite the germline configuration, with KD values in the subnanomolar range. To elucidate whether these antibodies are uniquely associated with ALF, we studied two chimpanzees infected with HBV who developed acute self-limited hepatitis. Analysis of phage display libraries derived from the liver of the two animals documented the presence of HBcAg-specific IgG, but not of IgM. In contrast to ALF-associated antibodies, all the anti-HBc IgG exhibited somatic hypermutation, with 7 to 38 nucleotide substitutions in their VH genes. Thus, the chimpanzee IgG V genes exhibited mutational patterns consistent with antigenic selection and affinity maturation in a T-cell dependent manner. In fact, both animals showed signs of T-cell mediated anti-HBV immune responses, with IFN gamma and IFN-induced chemokines whose appearance correlated with viral clearance. Moreover, an integrated analysis of mRNA and miRNA showed that the majority of ALF-specific miRNAs target B-lineage-associated genes, whereas only a very small number of miRNA target T-cell genes, in line with the limited T-cell signature detected in ALF. Thus, in contrast to acute hepatitis B, where the liver damage is T-cell mediated, our data suggest that a unique type of humoral immunity against HBcAg plays a primary role in the pathogenesis of HBV-ALF. 2. Determinants of disease progression, fibrogenesis, and viral evolution in chronic hepatitis C. We have identified profibrogenic and proinflammatory chemokines that predict rapid progression of hepatitis C to cirrhosis, providing a new model of HCV-disease pathogenesis and opening new perspectives for the early diagnosis and treatment. We are now attempting to validate these predictive markers in a large cohort of patients with slow and rapid HCV-disease progression. 3. Host and Viral Factors in the Pathogenesis of HCC and search for biomarkers for the early detection HCC is the third leading cause of cancer-related death worldwide. Cirrhosis is the single most important risk factor for HCC being present in 80%-90% of the cases. Chronic infection with hepatitis viruses are responsible for over 80% of HCC cases worldwide. Although the major etiologic agents and risk factors for HCC are well defined, the molecular mechanisms of hepatocarcinogenesis remain unclear. Our aim is to investigate the pathogenesis of HCC by studying both the host and the virus. HCV-associated HCC Over the past two decades, the incidence of HCC has tripled in the United States, and this alarming trend is due primarily to hepatitis C virus (HCV) infection. Whether HCV promotes HCC indirectly through chronic inflammation and fibrosis, or directly through the expression of viral proteins like other human oncogenic viruses, remains to be established. Analysis of the levels of HCV replication and quasispecies distribution within the tumor of patients with HCC can provide insights into the role of HCV in hepatocarcinogenesis and, conversely, on the effect that this cancer has on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from HCC patients who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared to surrounding non-tumorous tissues, whereas no differences were observed in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared to control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in viral population between tumorous and non-tumorous areas, suggesting viral compartmentalization. Overall, the lower the rate of viral replication within the tumor, the higher the degree of both viral diversity and malignant cell proliferation. This study shows that HCC is associated with severe restriction of HCV replication despite high viral genetic diversity, suggesting that the tumor behaves like a viral reservoir, which preserves a diverse viral population by virtue of the high proliferative rate of HCV-infected malignant hepatocytes. HDV-Associated HCC Hepatitis D virus (HDV) is a unique defective RNA virus that causes the most severe and rapidly progressive form of hepatitis, leading to cirrhosis in about 80% of cases. A high proportion of patients die of liver decompensation or HCC unless they receive LT. Although the risk of developing HCC dramatically increases at the cirrhosis stage, there are no data on the molecular mechanisms of HDV-associated hepatocarcinogenesis. Moreover, because of the obligatory link of HDV with HBV, the specific role of HDV in promoting liver cancer remains unknown. We used genomic and molecular techniques to investigate host-virus interactions in patients with HDV-associated HCC who underwent LT. The intrahepatic expression of HDAg was also evaluated. Gene expression profiling was performed on whole liver tissue specimens (WLT, tumor vs. non-tumor) and selected laser capture-microdissected hepatocytes (LCM, malignant vs. non-malignant hepatocytes) from individual livers of 4 patients with HCC. A parallel analysis was performed on multiple liver specimens obtained from the same liver of 11 patients with HBV-associated HCC, using the same methods. The gene expression profiling of patients with HDV-associated HCC was characterized by an enrichment of genes related to DNA repair mechanisms, pointing to genetic instability as an important mechanism in HDV hepatocarcinogenesis. This gene signature was even more evident in microdissected malignant hepatocytes, thus appearing as a specific feature of HDV HCC. By contrast, HBV-associated HCC was characterized by a metabolism switch-off. These data emphasize the different molecular features of HCC associated with different hepatitis viruses, suggesting that each virus may promote carcinogenesis by distinct molecular mechanisms.