Molecular Mechanisms of Pathogenesis of Acute and Chronic Liver Diseases

急慢性肝病发病的分子机制

• 批准号: 10272118
• 负责人: Patrizia Farci
• 金额: $ 185.91万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272118
• 关键词: Acute; Acute Hepatitis; Acute Liver Failure; Acute-Phase Reaction; Adhesions; Adult; Affect; Affinity; Age; Age-Years; Aging; Antibodies; Antibody Formation; Antibody Repertoire; Antigens; Archives; Autophagocytosis; B-Lymphocytes; Binding; Biology; Cancer Etiology; Cells; Cessation of life; Characteristics; Chronic viral hepatitis; Cirrhosis; Clinical; Communication; Complement; Complication; Core Protein; Cytokine Signaling; DNA Repair; Data; Dependence; Deposition; Detection; Development; Disease; Down-Regulation; Early Diagnosis; Etiology; Expression Profiling; Failure; Fibrosis; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Heterogeneity; Genome; Goals; Hepatic; Hepatic Fibrogenesis; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B Virus; Hepatitis C virus; Hepatitis Viruses; Hepatitis delta Antigens; Hepatocarcinogenesis; Human; Humoral Immunities; Immune; Immune response; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Variable Region; Immunohistochemistry; Immunologics; Immunotherapy; Incidence; Individual; Infection; Infiltration; Integration Host Factors; Knowledge; Link; Liver; Liver Cirrhosis; Liver Failure; Liver Fibrosis; Liver diseases; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Messenger RNA; Molecular; Mutation; Outcome; Pan Genus; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Persons; Play; Predisposition; Prevention; Primary carcinoma of the liver cells; Production; Recording of previous events; Risk; Risk Factors; Role; Sampling; Severities; Signal Pathway; Signal Transduction; Solid Neoplasm; Somatic Mutation; Specimen; Speed; Syndrome; System; T-Lymphocyte; Techniques; Technology; Terminator Codon; Testing; Therapeutic; Time; Tissues; Translational Research; Tumor Tissue; Up-Regulation; Variant; Viral; Viral Pathogenesis; Viral hepatitis; Virus; Virus Diseases; Work; age related; burden of illness; chronic infection; chronic liver disease; complement system; cytokine; diagnostic biomarker; differential expression; effective therapy; fibrogenesis; gene function; genome editing; granulocyte; high risk; in vitro Model; innovation; insight; intrahepatic; liver injury; liver transplantation; miRNA expression profiling; mortality; mutant; nanomolar; next generation sequencing; novel diagnostics; pathogen; promoter; prophylactic; response; success; transcriptome sequencing; transcriptomics; tumor; tumor-immune system interactions; virus core

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 were highly divergent from wild-type HBV with core (HBcAg) being the most variable region with the G1896A precore stop codon mutation invariably present. 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. Several pairs of IgM and IgG in each patient shared identical variable regions, indicating that isotype switch had occurred without affinity maturation, a characteristic of T-cell independent B-cell response (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). Our results demonstrated that this is a broad phenomenon typically associated with ALF. 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. Thus, this study reinforces and expands our prior findings, providing further evidence that a robust T cell independent B-cell response appears to be a hallmark of HBV-associated ALF (Chen et al. J Viral Hepatitis, 2020). 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. This study demonstrated that although chimpanzees did not develop ALF, 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. Thus, our study suggests that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease (Chen et al. Plos Pathogens 2020, in press). 2. Pathogenesis of liver fibrosis progression, cirrhosis, and hepatocellular carcinoma (HCC): role of viral and host factors HCC is the fourth 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. Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression, but the mechanisms remain unclear. We hypothesized that the pace of liver fibrosis progression may reflect changes in gene expression within the aging liver. To test this hypothesis, we compared gene expression in liver specimens from 54 adult liver donors, including 36 over 40 years old and 18 between 18 and 40 years old. None of them had a history of liver disease or evidence of liver fibrosis. Comparison of gene expression profiles between the two groups of normal liver donors showed that age-related changes affect the expression of liver genes. We are now characterizing the function of these genes in liver fibrogenesis by using primary liver tissue and in vitro models. The results of this study may provide new insights into the the increased susceptibility of aging livers to fibrosis progression. 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 in these tumors. 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 an enrichment of downreguled 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 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 are studying the distribution of S- and L-HDAg in tumor tissue as well as in the surrounding nontumorous tissue and in non-HCC HDV cirrhosis to investigate whether the biology of the virus plays a role in HCC pathogenesis.

肝脏发病机制科的主要目标是对急慢性肝病的发病机制进行转化研究，重点关注病毒性肝炎及其长期后遗症、肝硬化和肝细胞癌（HCC），这些疾病导致了非常严重的疾病。全球范围内的巨大疾病负担。 1.乙型肝炎病毒（HBV）相关急性肝衰竭（ALF）的分子发病机制：针对乙型肝炎病毒核心抗原的体液免疫在ALF发病机制中的作用 HBV 相关 ALF，也称为暴发性乙型肝炎，是急性 HBV 感染的一种罕见但通常致命的并发症。由于缺乏适当的实验系统以及获取肝脏样本的困难，这种疾病的发病机制仍然很大程度上未知。我们最近发现了与 ALF 独特相关的病毒和宿主因素。 ALF 肝脏中检测到的 HBV 菌株与野生型 HBV 高度不同，核心 (HBcAg) 是变异最大的区域，始终存在 G1896A 前核心终止密码子突变。 组合基因和 microRNA 表达谱揭示了显性 B 细胞疾病特征，肝内大量产生 IgM 和 IgG，以种系结构专门针对 HBcAg，具有亚纳摩尔亲和力，与补体沉积相关。每个患者的几对 IgM 和 IgG 共享相同的可变区，表明同种型转换发生时没有亲和力成熟，这是 T 细胞独立的 B 细胞反应的一个特征 (PNAS 2018)。接下来，为了研究独特的体液免疫反应是否与 ALF 相关，我们通过下一代测序 (NGS) 检查了整个肝内抗体库。我们的结果表明，这是一种通常与 ALF 相关的广泛现象。与对照组不同，ALF 患者的肝内 VH 库的特点是主要缺乏体细胞超突变，并且同种型从 IgM 转换为 IgG，但没有体细胞突变。因此，这项研究强化并扩展了我们之前的发现，提供了进一步的证据，证明强大的 T 细胞独立 B 细胞反应似乎是 HBV 相关 ALF 的标志（Chen 等人，J Viral Hepatitis，2020）。 ALF 与含有前核心或核心启动子突变的 HBV 变体的关联促使我们研究来自黑猩猩的存档肝脏样本，这些黑猩猩实验性感染了与人类 ALF 相关的前核心 HBV 突变体。这项研究表明，虽然黑猩猩没有出现 ALF，但感染前 HBV 突变体会​​导致感染 HBV 的黑猩猩中出现最严重的急性乙型肝炎，与 HBV ALF 更相似，而不是典型的急性乙型肝炎。因此，我们的研究表明，前核心 HBV 突变体具有固有的较高致病性，除了特定的宿主因素外，可能在确定急性 HBV 疾病的严重程度方面发挥关键作用（Chen 等人，Plos Pathogens） 2020 年，待出版）。 2. 肝纤维化进展、肝硬化和肝细胞癌（HCC）的发病机制：病毒和宿主因素的作用 HCC 是全球癌症相关死亡的第四大原因，慢性肝炎病毒感染占病例的 80%。肝硬化是 HCC 的最重要的单一危险因素，在 80-90% 的病例中存在。尽管肝癌的主要病因和危险因素已明确，但肝癌发生的分子机制仍不清楚。 A) 年龄在肝纤维化进展中的作用 肝纤维化在慢性病毒性肝炎的结果中起着至关重要的作用。肝硬化（肝纤维化的最新阶段）患者患 HCC 的风险最高。感染肝炎病毒时年龄较大与肝纤维化进展风险增加相关，但其机制仍不清楚。我们假设肝纤维化进展的速度可能反映了衰老肝脏内基因表达的变化。 为了检验这一假设，我们比较了 54 名成年肝脏捐献者的肝脏样本中的基因表达，其中 36 名年龄超过 40 岁，18 名年龄在 18 至 40 岁之间。他们都没有肝病史或肝纤维化证据。两组正常肝脏供体之间基因表达谱的比较表明，年龄相关的变化影响肝脏基因的表达。我们现在通过使用原代肝组织和体外模型来表征这些基因在肝纤维发生中的功能。这项研究的结果可能为衰老肝脏对纤维化进展的易感性增加提供新的见解。 B) HBV、HCV 和 HDV 相关 HCC 的免疫状况。 基于免疫疗法在实体瘤中的成功凸显了我们对 HCC 免疫微环境的认识的必要性，而目前 HCC 尚无有效的治疗方法。获取 27 名患有与 HBV、HCV 和 HDV 相关的 HCC 患者的肝脏样本，使我们有机会研究这些肿瘤的免疫学状况。对配对肝脏样本（肿瘤和非肿瘤）进行 RNA 测序，生成免疫基因的精选列表，并用于分析 3 个肿瘤之间的差异和相似性。所有 HCC 患者的免疫基因均出现显着下调（超过 90%），这些基因主要与先天性免疫细胞和适应性免疫细胞之间的通讯以及粒细胞粘附和血细胞渗出相关的途径相关。 HCV HCC 的特点是与 Th1 和 Th2 激活途径相关的下调基因富集。 HBV HCC 显示补体系统和急性期反应信号通路的下调，以及与 iNOS 信号传导和自噬相关的通路的上调。令人惊讶的是，HDV HCC 与 HBV HCC 的差异表达免疫基因仅有 7% 相同，与 HCV HCC 则有 28% 相同，主要与 mRNA 编辑和细胞因子信号通路相关。此外，HDV HCC 显示 PRR 通路下调。通过免疫组织化学验证了免疫特征。我们的数据表明，病毒相关的 HCC 与免疫反应的急剧下调有关，但根据具体的病毒病因存在差异。细胞因子似乎在免疫细胞浸润中发挥着至关重要的作用。尽管丁型肝炎病毒依赖于乙型肝炎病毒，但丁型肝炎病毒-肝癌表现出独特的免疫景观。 C) 与 HCC 相关的丁型肝炎病毒株的遗传异质性。我们最近证明，HDV HCC 与其他病毒性 HCC 不同，与 DNA 损伤和修复相关基因的富集相关（Diaz et al. 2018）。对于与 HCC 相关的丁型肝炎病毒株的遗传异质性以及编码大 (L)-HDAg 的编辑基因组与编码小 (S)-HDAg 的未编辑基因组在发病机制中的丰度和作用知之甚少。通过NGS，我们正在研究S-和L-HDAg在肿瘤组织以及周围非肿瘤组织和非HCC HDV肝硬化中的分布，以研究病毒的生物学特性是否在HCC发病机制中发挥作用。