Molecular Mechanisms of Pathogenesis of Acute and Chronic Liver Diseases

National Institute Of Allergy And Infectious Diseases

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Abstract

The major goal of the Hepatic Pathogenesis Section 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 Pathogenesis of Hepatitis B Virus (HBV)-Associated Acute Liver Failure (ALF): Role of Humoral Immunity against Hepatitis B Virus Core Antigen in the Pathogenesis of ALF HBV-associated ALF, also known as fulminant hepatitis B, is a rare but very severe complication of acute HBV infection with an exceptionally high fatality rate before the advent of liver transplantation. The pathogenesis of this disease is still largely unknown due to the lack of appropriate experimental systems and the difficulties in obtaining liver samples. To investigate the molecular pathogenesis of this disease, we performed a comprehensive study of both liver tissue and serum samples from ALF patients using cutting-edge technologies. This study allowed us to identify viral and host factors uniquely associated with ALF, and to compare the results with those of classic acute hepatitis B in chimpanzees. HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and microRNA expression profiling revealed a dominant B-cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, associated with complement deposition. Thus, HBV-ALF appears to be an anomalous T-cell-independent, HBV core-driven B-cell disease, which results from the rare and unfortunate encounter between a host with an unusual B-cell response and an infecting virus with a highly mutated core antigen. In contrast to classic acute hepatitis B where liver damage appears to be T-cell mediated, this study demonstrates a major role of the humoral immunity in the pathogenesis of HBV-associated ALF, which may open new avenues for the diagnosis and treatment of this dramatic disease. 2. Molecular Pathogenesis of Hepatitis Viruses-Associated HCC HCC is the third leading cause of cancer-related death worldwide, and chronic infection with hepatitis viruses accounts for over 60% of cases. Cirrhosis is the single most important risk factor for HCC being present in 80-90% of the cases. Although the major etiologic agents and risk factors for HCC are well defined, the molecular mechanisms of hepatocarcinogenesis remain unclear. Hepatitis C virus (HCV) A) Restricted HCV replication in malignant hepatocytes of patients with HCC correlates with disrupted localization of the HCV-entry cofactors claudin1 (CLDN1) and occludin (OCLN). Whether HCV promotes HCC indirectly, through chronic inflammation, fibrosis and liver regeneration, or directly, in a manner similar to other oncogenic viruses, is still unknown. We recently demonstrated that HCV replication is severely impaired within HCC tissue in vivo, suggesting that malignant hepatocytes express factors or, more likely, have lost expression of factors that regulate HCV infection and/or replication. The mechanisms responsible for this restriction are presently unknown. To elucidate the nature of such factors, we performed transcriptomics analysis in multiple liver specimens spanning the entire liver of patients with HCV-associated HCC. The analysis identified 1,115 differentially expressed genes with a large preponderance (76%) of downregulated genes within the tumor, most of them involved in cell-mediated immune responses. Another feature of the malignant hepatocytes was downregulation of transmembrane genes, most of which were also downregulated in hepatoma cell lines. However, none of the four major HCV-entry cofactors, SR-B1, CD81, CLDN1 and OCLN, was differentially expressed between the tumor and the nontumorous tissue, as also confirmed by real-time PCR analysis, thus ruling out a role of downregulation of these entry cofactors in the restriction of HCV replication within the tumor. Next, we investigated the pattern of expression of the 4 major HCV-entry cofactors in primary liver tissue from patients with HCV-associated HCC by confocal microscopy. Strikingly, we found that the TJ proteins CLDN1 and OCLN showed an abnormal distribution within the tumor tissue, appearing clumpy and aggregated compared to the typical linear pattern lining the cellular membranes observed in the surrounding nontumorous tissue. These differences were specific for CLDN1 and OCLN, as both SR-B1 and CD81 displayed typical linear distributions both within and outside the tumor. B) TACSTD2 is among the most downregulated genes in HCV-associated HCC and interacts with the HCV-entry cofactors CLDN1 and OCLN in Huh7.5 cells. We identified tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a novel host co-factor for HCV entry that interacts with the HCV co-receptors CLDN1 and OCLN, and is involved in the regulation of their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 regulates the phosphorylation and intracellular localization of CLDN1 and OCLN in Huh7.5 cells at the post-transcriptional level. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect. HCV-entry assays using genotype 1a and 1b HCV pseudoparticles demonstrated that the inhibitory effect occurs at the level of viral entry. These results identify TACSTD2 as a novel host cofactor for HCV entry, which may have relevance for the pathogenesis, treatment and prevention of HCV infection. Hepatitis D Virus A) Molecular Signature of Hepatitis D Virus (HDV)-Associated Hepatocellular Carcinoma. HDV causes the least common but most severe and rapidly progressive form of hepatitis, leading to cirrhosis in about 80% of cases. Although HCC develops in a high proportion of patients with HDV infection, there are limited data on the molecular mechanisms of HDV-induced hepatocarcinogenesis, a small defective RNA virus that depends on HBV for assembly and transmission. It is also unknown whether HCC is an effect of the underlying cirrhosis or a direct oncogenic effect of HDV, or a cumulative oncogenic effect of HBV and HDV, given the vital dependence of HDV on HBV. Transcriptomic profiling was performed using laser capture-microdissected malignant and non-malignant hepatocytes, tumorous and non-tumorous liver tissue from patients with HDV-HCC, and liver tissue from patients with non-HCC HDV cirrhosis. HDV-HCC was also compared with HBV-HCC alone and HCV-HCC. HDV malignant hepatocytes were characterized by an enrichment of up-regulated transcripts associated with pathways involved in cell cycle/DNA replication, damage and repair. Activation and co-regulation of genes critically associated with DNA replication, damage, and repair point to genetic instability as an important mechanism of HDV hepatocarcinogenesis. This specific HDV-HCC trait emerged also from the comparison of the molecular pathways identified for each hepatitis virus-associated HCC. Despite the dependence of HDV on HBV, these findings suggest that HDV and HBV promote carcinogenesis by distinct molecular mechanisms.