Molecular Mechanisms of Pathogenesis of Acute and Chronic Liver Diseases

National Institute Of Allergy And Infectious Diseases

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Abstract

The Hepatic Pathogenesis Section has developed an extensive program involving basic and clinical research to study 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 huge burden of disease worldwide. 1. Molecular Mechanisms of Pathogenesis of Acute Liver Failure (ALF) and Acute Viral Hepatitis A. Role of Humoral Immunity against Hepatitis B Virus Core Antigen in the Pathogenesis of HBV-associated ALF The pathogenesis of HBV ALF, also known as fulminant hepatitis B, is still largely unknown due to the lack of experimental systems and difficulties in obtaining liver samples. We identified viral and host factors uniquely associated with ALF (Chen et al. 2018). The association of ALF with HBV variants containing precore or core promoter mutations prompted us to investigate archived liver samples from chimpanzees experimentally infected with a precore HBV mutant implicated in human ALF. We found that infection with a precore HBV mutant caused the most severe acute hepatitis B ever seen in chimpanzees infected with HBV with closer resemblance to HBV ALF than to classic acute hepatitis B (Chen et al. 2020). Capitalizing on these findings, we have started a comprehensive study to define the serologic, virologic, genetic, and immunologic profiles of the three main outcomes of HBV infection, i.e., primary acute self-limited hepatitis B, ALF due to primary HBV, and ALF from disease flares of chronic HBV. Preliminary data suggest major differences among the 3 groups of patients, which may help to identify biomarkers that predict the different clinical outcomes. B. Structural Basis for the Extraordinary Affinity of Intrahepatic anti-HBc Germline Antibodies in ALF We demonstrated a major role of the humoral immunity against hepatitis B core antigen (HBcAg), with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinity, and complement deposition. To elucidate the structural basis for the exceptionally high affinity of anti-HBc antibodies produced in the liver of patients with ALF despite their germline configuration, in collaboration with Drs. Tsybovsky and Kwong from the VRC we used negative-stain electron microscopy (NSEM) and cryogenic electron microscopy (cryo-EM) to determine the mode of antigen recognition by such antibodies. Structural analysis showed that antibodies from acute hepatitis interact with the core in a canonical mode, while ALF-derived antibodies establish additional (non-canonical) contacts which may provide a structural explanation for the very high affinity of these germline antibodies. C. Role of hepatitis E virus (HEV) in Drug-Induced Liver Injury The diagnosis of idiosyncratic drug-induced liver injury (DILI) is difficult because of the diverse clinical presentations that mimic other, more common, causes of liver injury. We investigated the incidence, presentation, and outcome of acute and previous HEV infection in a large cohort of patients with DILI. Our data shows that the overall prevalence of anti-HEV declined from 22% in 2004-2011 to 18% in 2012-2019, which is suggestive of a cohort effect (Fontana et al. 2022). This study shows that acute HEV infection accounts for less than 1% of suspected DILI cases in the US and is more frequent in older men. Previous HEV infection is also most commonly seen in older individuals. Thus, clinicians should consider testing for unsuspected acute HEV infection in older adult patients with acute hepatocellular DILI and jaundice. D. Development of a Gerbil Model to Study the Pathogenesis of Acute and Chronic HEV infection in the Immunocompetent and Immunosuppressed Host Infection with HEV usually induces an acute self-limited hepatitis, but it may also cause chronic hepatitis in immunosuppressed patients. The lack of a small animal model of HEV infection has hampered our understanding of pathogenesis of acute and chronic infection. In collaboration with Dr. McGivern, we characterized acute and persistent HEV infection in immunocompetent and immunosuppressed gerbils and examined HEV replication kinetics, immune responses and liver pathology. Mongolian gerbils infected with HEV genotype 3, while on tacrolimus treatment, mounted ineffective immune responses and showed persistent viremia and fecal shedding, consistent with the pattern seen in immunosuppressed HEV infected patients. This model will be useful for studies of pathogenesis during acute and chronic HEV infection. 2. Pathogenesis of Liver Fibrosis Progression, Cirrhosis, and Hepatocellular Carcinoma (HCC): Role of Viral and Host factors HCC is the third leading cause of cancer-related death worldwide, and chronic infection with hepatitis viruses accounts for more than 70% 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. A) Role of Age in Liver Fibrosis Progression Older age at the time of infection with hepatitis viruses is associated with an increased risk of fibrosis progression, but the mechanisms remain unclear. We identified a gene, chitinase 3-like 1 (CHI3L1), as having the greatest age-dependent increase in expression and as being overexpressed in cirrhotic livers. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis (Nishimura et al. 2021). These findings demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression. B. Molecular Signature and Immune Landscape of Viral-Associated HCC Chronic infections with HBV and HCV account for 71% of all HCC cases worldwide. Therapeutic options are limited and ineffective. The increasing use of immune-based therapies in solid tumors highlights the need to expand our knowledge on the immunologic microenvironment of HCC. Taking advantage of a unique collection of liver specimens from well-characterized patients with HBV or HCV-associated HCC, we demonstrated by RNAseq that both tumors are characterized by a predominant gene downregulation; yet, distinctive features differentiate HCV- from HBV-HCC. We found that HCV-HCC is characterized by a downregulation of immune genes, especially T-cell related genes, while the molecular signature of HBV-HCC is characterized by upregulation of genes associated with cell cycle control and monocyte/macrophage activation, and downregulation of genes involved in various cell metabolisms (De Battista et al. 2022). C. Role of HDV in Hepatocellular Carcinoma The mechanisms whereby HDV promotes liver cancer remain elusive. We demonstrated that the molecular profile of HCC-HDV is unique and distinct from that of HBV-HCC (Diaz et al. 2018), which suggests that HBV and HDV promote carcinogenesis by distinct molecular mechanisms (Farci et al. 2021). There is limited information on HDV replication in the tumor, and on the role of small (S) and large (L) delta antigen (HDAg) in HDV pathogenesis. We found that the tumor showed the highest expression of S-HDAg, a finding that was confirmed by NGS, which showed significantly higher frequency of unedited genomes (S-HDAg) in the tumor compared to non-HCC cirrhosis. Since HDV-HCC is characterized by genomic instability, our data raise the possibility that nuclear accumulation of S-HDAg may lead to somatic mutations and genetic instability. These findings have implications for the different role of S- and L-HDAg in HDV pathogenesis.