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

描述（由申请人提供）：该R21探索性/发展性研究项目的重点是更好地了解丙型肝炎病毒（HCV）与感染者肝脏内宿主先天性抗病毒反应系统的相互作用。HCV表达的NS 3/4A蛋白酶通过介导两种重要衔接蛋白MAVS的蛋白水解破坏早期先天性细胞抗病毒防御（也称为IPS-1、VISA或Cardif）和TRIF，它们是病毒通过病原体相关分子模式（PAMP）受体引发的途径诱导1型IFN-1/2合成所需的，Toll样受体3（TLR 3）和视黄酸诱导基因I（RIG-I）。强有力的体外证据支持HCV感染能够破坏这些途径并消除病毒诱导的IFN合成。然而，同样有力的证据表明，多种IFN-刺激的基因（ISG）的合成强烈诱导体内HCV感染的肝脏。这些相互矛盾的数据提出了重要的问题，体外观察的相关性，体内HCV感染的发病机制。我们建议使用荧光半导体量子点（Qdot）探针和多光子（2 PE）显微镜，以准确地识别HCV感染的肝细胞在慢性丙型肝炎患者的肝组织切片，并确定这些信号通路的状态以及HCV感染的肝脏内ISG表达的来源。在特定目标1中，我们将使用灵敏的直接Qdot偶联探针和2 PE显微镜来确定细胞内位置（细胞核与细胞质），从而确定受感染人肝脏中肝细胞表达的转录因子IRF-3、RelA和NF-kB 2 p52的激活状态，并确定它们是否以及如何与HCV感染和可检测的细胞内dsRNA的存在相关地被调节。在目标2中，我们将评估HCV靶向的关键信号衔接蛋白的肝内表达（MAVS和TRIF），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TLR 3，以及25 'OAS、PKR、TRIM 25、RNF 125和NLRX 1），并确定它们的表达是否因HCV感染而改变。在目标3中，我们将定量分析控制HCV复制的代表性ISG（PKR，viperin，ISG 20，ISG 15和ISG 56）的肝细胞表达，并确定这与HCV感染，IRF-3和NF κ B活化以及浸润组织巨噬细胞和浆细胞样树突状细胞的关系。对实验性感染黑猩猩的存档组织的平行研究将研究这些事件如何与感染结果和适应性免疫的发展在动力学上相关。这些研究将提供有关慢性丙型肝炎发病机制的重要新数据，并有助于为未来治疗的临床开发提供信息。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不断增加的原因，也是肝硬化、肝衰竭和肝癌的主要感染原因。这在很大程度上源于其引起长期持续感染的能力，但病毒如何成功地逃避免疫反应并实现这一点尚不清楚。在这个项目中，我们将应用分子成像技术的最新进展，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.