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

项目总结 慢性丙型肝炎病毒感染困扰着全世界1.8亿人。大多数被感染的人 随着丙型肝炎病毒的持续存在，表明丙型肝炎病毒成功地颠覆了先天和获得性免疫 回应。数学模型预测出现的患者数量将大幅增加。 丙型肝炎病毒相关并发症(如肝硬变、肝细胞癌)在未来几十年作为人口 年龄。虽然肝细胞占肝脏内总细胞群的大多数，但非 越来越多的人认识到实质肝细胞在慢性炎症和 丙型肝炎病毒感染的并发症。尽管直接作用抗病毒药物(DAA)的发展导致了 近乎神奇的治愈率[与基于干扰素的治疗相比]，一些重要的问题 仍然没有回答这一需要进一步研究的问题。治愈的患者的免疫力恢复到什么程度 DaaS对肝脏内多种细胞类型之间的串扰有什么影响 微环境？丙型肝炎病毒的治疗对炎症、纤维化的发展以及进一步的 肝脏失代偿？这些问题的答案仍然难以捉摸，并对 其他肝病。在这里，我们将定义肝脏和外周先天重建的广度和速度 DAA治疗慢性丙型肝炎患者诱导的获得性免疫。我们还将确定 DAA介导的病毒根除如何影响丙型肝炎病毒特异性CTL的表观遗传重编程。一大群人 已经登记的患者，包括那些以前未能通过DAA治疗并有证据表明 耐药相关变异，将根据治疗前的病毒水平和纤维化阶段进行系列研究。 接下来，我们将描述肝细胞和非实质细胞之间的复杂联系。 (Npc)在丙型肝炎病毒感染和DAA介导的病毒清除的影响。我们假设外显体 在与外体的细胞起源错综复杂地联系在一起的细胞间通信中发挥重要作用，如 以及生物环境；外切体可以穿梭丙型肝炎病毒和感染病毒的生物活性分子 肝癌细胞可分化为多种鼻咽癌细胞。我们将研究纯化的外切体是如何从感染了丙型肝炎病毒的 DAA治疗的肝细胞在Kupffer细胞(KCs)、肝窦内皮细胞中的差异触发反应 (LSECs)和HSCs。我们将全面表征丙型肝炎病毒感染后的胞外体内容物，并 确定哪些成分，包括microRNA，可以放大或减轻炎症或纤维化 小路。如果成功，这些研究将为调解机制提供新的见解 病毒清除、残留炎症和肝损伤(纤维化)之间具有广泛的相关性 迅速变化的丙型肝炎病毒领域，潜在地开辟了研究这一高度 全球流行的肝病。

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.