RESUBMISSION -R01 AI120622 –Restoration of Immunity and Function with DAA Treatment in HCV infection

项目摘要 慢性HCV感染困扰着全世界1.8亿人。大多数感染者 与HCV发展持久性，表明HCV成功地颠覆了先天性和适应性免疫 应答数学模型已经预测出， HCV相关并发症（例如，肝硬化，肝细胞癌）在未来几十年的人口 年龄虽然肝细胞占肝脏内总细胞群的大部分，但非肝细胞群占肝脏内总细胞群的大部分。 肝实质细胞已被越来越多地认为在慢性炎症中起中心作用， HCV感染的并发症。尽管直接作用抗病毒药物（DAA）的发展导致了 几乎奇迹般的治愈率[与干扰素（IFN）为基础的治疗相比]，一些重要的问题 仍然没有答案，需要进一步研究。在何种程度上是恢复免疫力的患者治愈 DAA以及对肝脏内多种细胞类型之间的串扰的影响 微环境？HCV治疗对炎症、纤维化发展和进一步发展有什么影响 肝脏失代偿这些问题的答案仍然难以捉摸， 其他肝病。在这里，我们将定义肝脏和外周先天性巨噬细胞的重建的广度和速度。 DAA治疗慢性HCV感染诱导的获得性免疫。我们还将确定 DAA介导的病毒根除如何影响HCV特异性CTL的表观遗传重编程。大型队列 已经入组的患者，包括既往DAA治疗失败且有证据表明 将根据治疗前病毒水平和纤维化阶段对耐药相关变异体进行系列研究。 接下来，我们将描述肝细胞和非实质细胞之间复杂的通讯 （NPC）在HCV感染和DAA介导的病毒清除的影响。我们假设外泌体 在与外泌体的细胞起源错综复杂地联系在一起的细胞间通讯中发挥重要作用， 以及生物环境;外泌体可以穿梭HCV和生物活性分子， 肝癌细胞对各种NPC。我们将研究如何纯化外泌体来源于HCV感染和 DAA治愈的肝细胞在枯否细胞（KCs）、肝窦内皮细胞中差异性地触发反应 （LSEC）和HSC。我们将全面表征HCV暴露后的外泌体含量， 确定哪些成分，包括microRNA，放大或减弱炎症或纤维化 途径。如果成功的话，这些研究将为介导 病毒清除、残余炎症和肝损伤（纤维化），并在以下方面具有广泛的相关性： 快速变化的HCV领域，可能开辟新的研究途径， 全球范围内流行的肝病。

PROJECT SUMMARY Chronic HCV infection afflicts 180 million individuals throughout the world. The majority of individuals infected with HCV develop persistence, suggesting that HCV successfully subverts innate and adaptive immune responses. Mathematical models have projected a massive increase in the number of patients developing HCV-related complications (e.g., cirrhosis, hepatocellular carcinoma) in the next few decades as the population ages. Although hepatocytes comprise the majority of the total cell population within the liver, the non- parenchymal liver cells have been increasingly recognized as playing central roles in chronic inflammation and complications from HCV infection. Although the development of direct-acting antivirals (DAAs) has led to nearly miraculous cure rates [compared to interferon (IFN)-based therapy], a number of important questions remain unanswered that demand further study. To what extent is immunity restored in patients cured with DAAs and what are the effects on cross-talk between the multiple cell types within the hepatic microenvironment? What effect does HCV cure have on inflammation, fibrosis development, and further hepatic decompensation? The answers to these questions have remained elusive and have implications for other liver disease. Here, we will define the breadth and pace of reconstitution of hepatic and peripheral innate and adaptive immunity induced by DAA therapy in patients with chronic HCV infection. We will also determine how DAA-mediated viral eradication affects epigenetic reprogramming of HCV-specific CTLs. A large cohort of already-enrolling patients, including those who have previously failed DAA therapy and have evidence of resistance-associated variants, will be serially studied according to pre-treatment viral level and fibrosis stage. Next, we will characterize the complex communication between hepatocytes and nonparenchymal cells (NPCs) in HCV infection and the impact of DAA-mediated viral elimination. We hypothesize that exosomes play important roles in intercellular communication that is intricately linked to the cell origin of the exosomes, as well as the biologic milieu; exosomes can shuttle HCV and biologically active molecules from infected hepatoma cells to a variety of NPC. We will examine how purified exosomes derived from HCV-infected and DAA-cured hepatocytes differentially trigger responses in Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs) and HSCs. We will comprehensively characterize the exosomal content following HCV exposure and determine which components, including microRNAs, either amplify or attenuate inflammatory or fibrotic pathways. If successful, these studies would provide novel insights into the mechanisms mediating viral clearance, residual inflammation, and liver injury (fibrosis) and have broad-reaching relevance in the rapidly changing field of HCV, potentially opening up new avenues of research into this highly prevalent liver disease worldwide.