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.

肝发病机理部分的主要目的是对急性和慢性肝病的发病机理进行转化研究，重点是病毒性肝炎及其长期后遗症，肝硬化和肝细胞癌（HCC），这对全球疾病产生了很大的疾病负担。 1。丙型肝炎病毒（HBV）相关的急性肝衰竭（ALF）的分子发病机理：对乙型肝炎病毒核心核心抗原在ALF发病机理中的作用 HBV相关的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是致命的并发症。由于缺乏适当的实验系统和获得肝样本的困难，该疾病的发病机理仍然在很大程度上是未知的。我们最近确定了与ALF唯一相关的病毒和宿主因素。在ALF肝脏中检测到的HBV菌株与野生型HBV高度分歧（HBCAG）是G1896A Precore终止密码子突变的最大区域。 组合基因和microRNA表达谱分析显示出主要的B细胞疾病特征，在种系构型中，肝内的IgM和IgG广泛产生仅针对与补体分数相关的亚洋摩尔亲和力的HBCAG。每位患者中的几对IgM和IgG对共享相同的变量区域，表明同型开关发生了没有亲和力成熟的情况，这是T细胞独立B细胞响应的特征（PNAS 2018）。接下来，为了研究ALF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，这是一个广泛的现象，通常与ALF相关。与对照不同的是，来自ALF患者的肝内VH曲目的特征是缺乏体细胞过度突变，而同型从IgM从IgM转变为IgG，而没有体细胞突变。因此，这项研究加强并扩大了我们先前的发现，提供了进一步的证据，表明强有力的T细胞独立B细胞反应似乎是HBV相关的ALF的标志（Chen等人J Viral Hepatisis，2020年）。 ALF与含有前孔或核心启动子突变的HBV变体的关联促使我们研究了来自黑猩猩的肝样品，该肝样品经过实验感染了与人类ALF有关的precore HBV突变体。这项研究表明，尽管黑猩猩没有发展为ALF，但先前HBV突变体的感染引起的，在用HBV感染的Chimpanzees中看到的最严重的急性肝炎，与HBV ALF相似，而不是经典的急性肝炎。因此，我们的研究具有固有的hBV突变性效果。急性HBV疾病的严重程度（Chen等，Plos病原体2020，印刷中）。 2。肝纤维化进展，肝硬化和肝细胞癌（HCC）的发病机理：病毒和宿主因子的作用 HCC是全球癌症相关死亡的第四个主要原因，肝炎病毒的慢性感染占病例的80％。肝硬化是80-90％的病例中HCC存在的最重要的危险因素。尽管HCC的主要病因和危险因素是明确的，但肝癌发生的分子机制尚不清楚。 a）年龄在肝纤维化进展中的作用 肝纤维化在慢性病毒肝炎的结局中起关键作用。肝肝硬化的患者是肝纤维化的最新阶段，其患者患HCC的风险最高。肝炎病毒感染时的年龄与肝纤维化进展的风险增加有关，但这些机制尚不清楚。我们假设肝纤维化进展的速度可能反映了肝脏内基因表达的变化。 为了检验这一假设，我们比较了来自54个成年肝供体的肝样品中的基因表达，其中包括36岁以上的36岁和18至40岁的18岁之间。他们都没有肝病病史或肝纤维化的证据。两组正常肝脏供体之间基因表达谱的比较表明，与年龄相关的变化会影响肝脏基因的表达。现在，我们通过使用原发性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会提供新的见解，以了解肝脏对纤维化进展的易感性增加。 b）HBV-，HCV-和HDV相关HCC的免疫景观。 基于免疫疗法在实体瘤中的成功凸显了我们对HCC免疫学微环境的了解的必要性，而HCC没有有效的治疗。从与HBV，HCV和HDV相关的27例特征良好的HCC患者中获取肝脏样品，使我们有机会研究这些肿瘤中的免疫学景观。在成对的肝样品（肿瘤和非肿瘤）上进行RNA-Seq，并生成了策划的免疫基因列表，并用于分析3个肿瘤之间的差异和相似性。所有HCC患者的免疫基因均显示出急剧下调，主要与与先天性和适应性免疫细胞以及粒细胞粘附和尿粘附和二尿症之间通信有关的途径相关的免疫基因。 HCV HCC的特征在于与Th1和Th2激活途径相关的下调基因的富集。 HBV HCC显示了补体系统和急性相反应信号通路的下调，以及与iNOS信号传导和自噬相关的途径的上调。出乎意料的是，HDV HCC仅与HBV- HCV HCC共享7％的差异表达的免疫基因，其中HCV HCC与HCV HCC共享，主要与mRNA编辑和细胞因子信号通路有关。此外，HDV HCC显示了PRR途径的下调。免疫特征通过免疫组织化学验证。我们的数据表明，与病毒相关的HCC与免疫反应的显着下调有关，根据特定的病毒病因差异。细胞因子似乎在免疫细胞浸润中起着至关重要的作用。尽管HDV依赖于HBV，但HDV-HCC表现出明显的免疫景观。 C）与HCC相关的HDV菌株的遗传异质性。我们最近证明，与其他病毒HCC不同，HDV HCC与参与DNA损伤和修复的基因富集有关（Diaz等，2018）。关于与HCC相关的HDV菌株的遗传异质性以及编辑的基因组的丰度和作用，编码大型（L）HDAG，与未经编辑的基因组，编码小（S）HDAG，在病原体中编码小（L）HDAG。通过NGS，我们正在研究肿瘤组织以及周围非肿瘤组织和非HCC HDV肝硬化中S-和L-HDAG的分布，以研究该病毒的生物学是否在HCC发病机理中起作用。