Project 2: Dissection of Type-III Interferon Signaling in iPSC-Derived Hepatocytes

项目2 -项目摘要 III型干扰素（IFN），也称为λ IFN（IFN L），是粘膜免疫系统先天免疫的关键调节剂。 和上皮表面。IFNL基因座的单核苷酸多态性（SNPs）与IFNL的发生率相关。 急性感染期间丙型肝炎病毒（HCV）的自发清除以及治疗后治愈的可能性 慢性感染。从HCV研究中获得的见解促使了使用IFNL的临床试验， HCV，还有B型肝炎（HBV）和丁型肝炎病毒感染。IFN信号传导的调节已经出现， 是艾滋病治疗策略的一个有吸引力的组成部分。虽然HCV感染现在很容易用抗病毒药物治愈， 而艾滋病病毒则不然，现有的治疗方法是抑制性的，是终身的。虽然I型和III型干扰素 信号通路共享多种信号蛋白，我们对这些通路如何交叉的理解， 临床上的沟通是不完整的。考虑到在免疫过程中调节IFN信号传导的兴趣， 治疗慢性病毒感染，有必要进一步了解保护性，病理性， IFNs的作用使用临床样本和体外模型，我们先前表明肝脏IFNL 表达与HCV感染期间的炎症相关，并在HCV的抗病毒抑制期间下降， 这与治疗结果相关的免疫力变化相平行。我们现在寻求扩大我们的 I型和III型IFN如何相互作用和平衡的机制见解。根据我们的初步数据， 我们相信III型IFN信号传导通过以下途径对宿主I型IFN应答施加负控制： IFN信号负调节因子的诱导。我们提出，IFN-III信号传导的效率是 受IFNL受体（IFNLR 1）的典型和非典型亚型的相对表达的影响。 因为rs368234815位点的二核苷酸多态性赋予产生IFNL 4的差异能力 与HCV临床结果相关，我们还预测能够产生IFNL 4的细胞 诱导增强的III型IFN应答，其抑制I型IFN信号传导并抑制病毒清除。到 克服在细胞系和原代人肝细胞中进行的研究的局限性，我们将使用诱导型 多能干细胞（iPSC）衍生的肝细胞作为模型系统。iPSC可以被遗传操纵， 长期培养，分化为肝细胞样细胞，支持HCV和HBV感染，并具有完整的 干扰素信号通路，因此非常适合测试我们的假设。在目标1中，我们将测试 假设通过IFNLR 1的信号传导，以及典型和非典型同种型的相对表达， 在功能上影响I型IFN信号传导的性质和幅度。在目标2中，我们将检验以下假设： rs368234815多态性调节产生IFNL 4的能力， 先天性肝细胞对病毒感染的反应。这些发现将具有广泛的相关性， 对影响肝脏的其他慢性感染（如HBV和HIV）的IFN信号传导进行治疗性调节 并通过感染性和非感染性机制引起慢性炎症。

Project 2 – Project Summary Type-III interferons (IFNs), also called lambda IFNs (IFNLs), are key modulators of innate immunity at mucosal and epithelial surfaces. Single-nucleotide polymorphisms (SNPs) at IFNL loci associate with odds of spontaneous hepatitis C virus (HCV) clearance during acute infection and the likelihood of cure upon treatment of chronic infection. Insight gained from studies of HCV has prompted clinical trials using IFNL to treat not only HCV, but also hepatitis B (HBV) and hepatitis D virus infections. Modulation of IFN signaling has emerged as an attractive component of HIV cure strategies. While HCV infection is now readily curable with antivirals, HBV and HIV are not, and available treatments are suppressive and lifelong. Although type-I and type-III IFN signaling pathways share multiple signaling proteins, our understanding of how these pathways intersect and communicate in the clinical context is incomplete. Given the interest in modulating IFN signaling during the treatment of chronic viral infections, it is essential to further understand the protective, pathologic, and relational roles of IFNs. Using clinical samples and in vitro models, we previously showed that hepatic IFNL expression correlates with inflammation during HCV infection and declines during antiviral suppression of HCV, which parallels changes in immunity that correlate with treatment outcome. We now seek to expand our mechanistic insight of how type-I and type-III IFNs interact and are balanced. Based on our preliminary data, we believe that type-III IFNL signaling exerts negative control over the host type-I IFN response through induction of negative regulators of IFN signaling. We propose that the efficiency of IFN-III signaling is influenced by the relative expression of canonical and non-canonical isoforms of the IFNL receptor (IFNLR1). Because a dinucleotide polymorphism at the rs368234815 locus imparts differential capacity to produce IFNL4 ligand and correlates with HCV clinical outcomes, we also predict that cells capable of IFNL4 production induce a heightened type-III IFN response that dampens type-I IFN signaling and inhibits viral clearance. To overcome limitations of studies conducted in cell lines and primary human hepatocytes, we will use inducible pluripotent stem cell (iPSC) derived hepatocytes as a model system. iPSCs can be genetically manipulated, cultured long-term, differentiated into hepatocyte-like cells, support HCV and HBV infection, and have intact IFN-signaling pathways, and thus are extremely well-suited to test our hypotheses. In Aim 1, we will test the hypothesis that signaling through IFNLR1, and relative expression of canonical and non-canonical isoforms, functionally impacts the nature and magnitude of type-I IFN signaling. In Aim 2, we will test the hypothesis that the rs368234815 polymorphism, which modulates the capacity to make IFNL4, is responsible for heterogeneity in the innate hepatocyte response to viral infection. These findings will have broad relevance when considering therapeutic modulation of IFN signaling for other chronic infections, such as HBV and HIV, that impact the liver and gut and cause chronic inflammation via mechanisms that are both infectious and non-infectious.