Intrahepatic IRF-3 and NF-kB Signaling and Interferon Responses in Hepatitis C

描述（由申请人提供）：本R21探索性/发展性研究项目侧重于更好地了解丙型肝炎病毒（HCV）与感染者肝脏内宿主先天抗病毒反应系统的相互作用。HCV表达的NS3/4A蛋白酶通过介导两种至关重要的连接蛋白MAVS（也称为IPS-1、VISA或Cardif）和TRIF的蛋白水解来破坏早期先天细胞抗病毒防御，这两种连接蛋白是病毒通过病原体相关分子模式（PAMP）受体、toll样受体3 （TLR3）和视黄酸诱导基因I （RIG-I）启动的途径诱导1型IFN-1/2合成所必需的。强有力的体外证据支持HCV感染破坏这些途径并破坏病毒诱导的IFN合成的能力。然而，同样有力的证据表明，在体内感染hcv的肝脏中，多种ifn刺激基因（ISGs）的合成被强烈诱导。这些相互矛盾的数据提出了关于体外观察与体内HCV感染发病机制相关性的重要问题。我们建议使用荧光半导体量子点（Qdot）探针和多光子（2PE）显微镜在慢性丙型肝炎患者的肝组织切片中准确识别hcv感染的肝细胞，并确定这些信号通路的状态以及hcv感染肝脏中ISG表达的来源。在Specific Aim 1中，我们将使用敏感的直接Qdot偶联探针和2PE显微镜来确定受感染的人肝脏中肝细胞表达的转录因子IRF-3、RelA和NF-kB2 p52的细胞内位置（核与胞质）和激活状态，并确定它们是否以及如何与HCV感染和可检测的细胞内dsRNA的存在相关。在Aim 2中，我们将评估HCV靶向的关键信号转导蛋白（MAVS和TRIF）的肝内表达，以及动态调节IFN和促炎细胞因子合成的宿主细胞蛋白（包括PAMP受体、RIG-I和TLR3，以及2'5'OAS、PKR、TRIM25、RNF125和NLRX1），并确定它们的表达是否因HCV感染而改变。在Aim 3中，我们将定量分析控制HCV复制的代表性isg （PKR, viperin, ISG20， ISG15和ISG56）的肝细胞表达，并确定其与HCV感染，IRF-3和NFkB激活以及浸润组织巨噬细胞和浆细胞样树突状细胞的关系。对实验感染黑猩猩的存档组织的平行研究将检查这些事件如何与感染结果和适应性免疫的发展动态相关。这些研究将为慢性丙型肝炎的发病机制提供重要的新数据，并有助于为未来治疗方法的临床发展提供信息。公共卫生相关性：丙型肝炎病毒是美国肝脏特异性发病率和死亡率增加的原因，也是肝硬化、肝功能衰竭和肝癌的主要感染性原因。这在很大程度上源于它引起长期持续感染的能力，但这种病毒是如何成功逃脱免疫反应并实现这一目标的，目前尚不清楚。在这个项目中，我们将应用分子成像技术的最新进展来直接检查病毒与先天免疫系统信号通路的相互作用，这些信号通路在肝脏内诱导干扰素介导的抗病毒反应。

DESCRIPTION (provided by applicant): This R21 Exploratory/Developmental Research project focuses on developing a better understanding of the interactions of hepatitis C virus (HCV) with host innate antiviral response systems within the liver of infected persons. The NS3/4A protease expressed by HCV disrupts early innate cellular antiviral defenses by mediating proteolysis of two critically important adaptor proteins, MAVS (also known as IPS-1, VISA, or Cardif) and TRIF, that are required for virus induction of type 1 IFN-1/2 synthesis through pathways initiated by the pathogen- associated molecular pattern (PAMP) receptors, Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I). Strong in vitro evidence supports the ability of HCV infection to disrupt these pathways and ablate virus-induced IFN synthesis. Yet equally strong evidence indicates that the synthesis of multiple IFN-stimulated genes (ISGs) is strongly induced within the HCV-infected liver in vivo liver. These conflicting data raise important questions about the relevance of in vitro observations to the pathogenesis of HCV infection in vivo. We propose the use of fluorescent semiconductor quantum dot (Qdot) probes and multiphoton (2PE) microscopy to accurately identify HCV-infected hepatocytes in sections of liver tissue from patients with chronic hepatitis C, and to ascertain the status of these signaling pathways as well as the sources of ISG expression within the HCV-infected liver. In Specific Aim 1, we will use sensitive direct Qdot conjugate probes and 2PE microscopy to ascertain the intracellular location (nuclear vs. cytoplasmic) and thus activation status of the transcription factors IRF-3, RelA and NF-kB2 p52 expressed by hepatocytes in infected human liver, and determine whether and how they are modulated in relation to HCV infection and the presence of detectable intracellular dsRNA. In Aim 2, we will assess the intrahepatic expression of critical signaling adaptor proteins that are targeted by HCV (MAVS and TRIF), as well as host cell proteins that dynamically regulate the synthesis of IFN and proinflammatory cytokines (including the PAMP receptors, RIG-I and TLR3, as well as 2'5'OAS, PKR, TRIM25, RNF125, and NLRX1), and determine if their expression is altered by HCV infection. In Aim 3, we will quantitatively analyze hepatocellular expression of representative ISGs that control HCV replication (PKR, viperin, ISG20, ISG15 and ISG56), and determine how this relates to HCV infection, IRF-3 and NFkB activation, and infiltrating tissue macrophages and plasmacytoid dendritic cells. Parallel studies of archived tissues from experimentally-infected chimpanzees will examine how these events correlate kinetically with infection outcome and the development of adaptive immunity. These studies will provide important new data concerning the pathogenesis of chronic hepatitis C and help to inform the clinical development of future therapies. PUBLIC HEALTH RELEVANCE: Hepatitis C virus is an increasing cause of liver-specific morbidity and mortality in the United States, and the leading infectious cause of cirrhosis, liver failure and liver cancer. Much of this derives from its ability to cause long-term persistent infections, but how the virus successfully escapes immune responses and accomplishes this is not understood. In this project, we will apply recent advances in molecular imaging techniques to directly examine the interactions of the virus with the innate immune system signaling pathways that induce interferon- mediated antiviral responses within the liver.