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 often fatal complication of acute HBV infection. The pathogenesis of this disease is still largely unknown due to the lack of appropriate experimental systems and the difficulties in obtaining liver samples. We recently identified viral and host factors uniquely associated with ALF. 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. We also demonstrated that these germline antibodies with high affinity against hepatitis B core antigen (HBcAg) detected in all ALF cases bind to HBcAg on the cell surface, which may trigger complement-mediated cell lysis, leading to massive liver necrosis (Chen et al. PNAS 2018). Next, to investigate whether a unique humoral immune response is associated with ALF, we examined the entire intrahepatic antibody repertoire by next-generation sequencing (NGS). We found that unlike the controls, the intrahepatic VH repertoires from ALF patients were characterized by a predominant lack of somatic hypermutation and isotype switch from IgM to IgG without somatic mutations. These results are consistent with our previous finding of intrahepatic specific anti-HBcAg antibodies in germline configuration, and confirm a major role of T-cell independent humoral immunity in the pathogenesis of this disease. 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 80% 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 Liver fibrosis plays a critical role in the outcome of chronic viral hepatitis. Patients with liver cirrhosis, the latest stage of liver fibrosis, have the highest risk of developing HCC. Age at time of infection (over 40 years) is an independent risk factor associated with liver fibrosis progression. However, the mechanisms underlying the role of age in liver fibrosis progression remain to be elucidated. We hypothesized that the variability in liver fibrosis progression may reflect the expression of unrecognized genes in patients over 40 years of age. Access to serum and liver specimens from a large series of 54 liver donors provided us with the unique opportunity to investigate this hypothesis by transcriptomics analysis. None of the 54 subjects had markers of infection with hepatitis viruses, a history of heavy alcohol consumption, or histological evidence of liver fibrosis. Comparison between the two groups of normal liver donors identified chitinase 3 like 1 (CHI3L1) as the most upregulated gene with the highest fold change in donors over 40 years. We confirmed that the levels of CHI3L1 were significantly higher in donors over 40 years by PCR in liver and ELISA in serum. This finding is of particular significance because CHI3L1 was shown to play a critical role in lung and kidney fibrogenesis. The mechanism whereby CHI3L1 may induce fibrosis is unknown. Mechanistic studies indicate that recombinant human CHI3L1 promotes proliferation of hepatic stellate cells (HSCs) using the human hepatic stellate line, LX-2. The effect was similar to that induced by platelet derived growth factor, one of the most potent proliferation factors for HSCs. This study demonstrates for the first time that CHI3L1 promotes cell proliferation of HSCs. Expression of this gene is significantly upregulated in liver and serum of normal liver donors over 40 years of age, suggesting a role of CHI3L1 in the increased susceptibility to liver fibrosis progression along with age. B) Immune Landscape of HBV-, HCV-, and HDV-Associated HCC. The success of immune-based therapies in solid tumors highlights the need to expand our knowledge on the immunologic microenvironment of HCC, for which there is no effective therapy. Access to liver samples from 27 well-characterized patients with HCC associated with HBV, HCV, and HDV, gave us the opportunity to study the immunologic landscape. RNA-seq was performed on paired liver specimens (tumor and non-tumor), and a curated list of immune genes was generated and used to analyze differences and similarities among the 3 tumors. All HCC patients showed a dramatic downregulation, over 90%, of immune genes mainly associated with pathways involved in communication between innate and adaptive immune cells and granulocyte adhesion and diapedesis. HCV-HCC was characterized by downregulation of genes related to Th1 and Th2 activation pathways. HBV-HCC showed downregulation of the complement system and acute-phase response signaling pathways, as well as upregulation of pathways related to iNOS signaling and autophagy. Surprisingly, HDV-HCC shared only 7% of differentially expressed immune genes with HBV- and 28% with HCV-HCC, mostly related to mRNA editing (APOBEC) and cytokine signaling pathways. In addition, HDV-HCC showed downregulation of the PRR pathway. The immune profile was validated by immunohistochemistry. Our data demonstrate that viral-related HCC is associated with a dramatic downregulation of the immune response, with differences according to the specific viral etiology. Cytokines seem to play a crucial role in immune cell infiltration. Despite the dependence of HDV on HBV, HDV-HCC showed a distinct immune landscape. C) Genetic heterogeneity of HDV strains associated with HCC. We recently demonstrated that HDV-HCC, unlike other viral HCC, is associated with an enrichment of genes involved in DNA damage and repair (Diaz et al. 2018). Little is known on the genetic heterogeneity of HDV strains associated with HCC and on the abundance and role of edited genomes, encoding large (L)-HDAg, versus unedited genomes, encoding small (S)-HDAg, in pathogenesis. By NGS , we found that one of the most variable sites was the nucleotide controlling S- versus L-HDAg expression, with a significantly lower frequency of edited genomes in tumor than in non-HCC cirrhosis (P=0.026), suggesting that S- and L-HDAg may play a differential role not only in the biology of the virus but also in HCC pathogenesis. By confocal microscopy, HDAg was detected in 3 of 5 tumors, in 4 nontumor tissues and in all livers with cirrhosis. Likewise, HBsAg was detected in only 2 tumors but in all nontumor tissue, along with a significant downregulation of the HBV receptor, NTCP, essential for HDV entry. Consistent with the limited HDAg expression, HDV RNA was lower in tumors than in nontumor tissue or non-HCC cirrhosis. The decreased NTCP expression may explain the limited levels of HDV infection within the tumor.
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