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)，又称lambda IFN(IFNLS)，是粘膜天然免疫的关键调节剂 和上皮性表面。IFNL基因座的单核苷酸多态(SNPs)与家族性心脏病的发病几率相关 丙型肝炎病毒(丙型肝炎病毒)在急性感染期间的自发清除和治疗后治愈的可能性 慢性感染的症状。从丙型肝炎病毒研究中获得的洞察力促使临床试验使用IFNL不仅治疗 丙型肝炎病毒，但也有乙肝(乙肝)和丁型肝炎病毒感染。干扰素信号的调制已经出现 这是艾滋病毒治疗战略的一个有吸引力的组成部分。虽然丙型肝炎病毒感染现在很容易通过抗病毒药物治愈，但乙肝病毒 和HIV并非如此，现有的治疗方法是抑制和终生的。虽然I型和III型干扰素 信号通路共享多个信号蛋白，我们对这些通路如何相交和 在临床环境中的沟通是不完整的。考虑到在细胞周期中调节干扰素信号的兴趣 治疗慢性病毒感染，有必要进一步了解保护性、病理学和 IFN的关系作用。利用临床样本和体外模型，我们先前证明了肝脏IFNL 表达与丙型肝炎病毒感染期间的炎症和抗病毒抑制期间的下降相关， 与治疗结果相关的免疫力变化是平行的。我们现在寻求扩大我们的 对I型和III型IFN如何相互作用和平衡的机械性洞察。根据我们的初步数据， 我们认为，III型IFNL信号通过以下途径对宿主I型干扰素反应施加负面控制 干扰素信号负调控因子的诱导。我们认为，干扰素-III信号的效率是 受IFNL受体(IFNLR1)规范和非规范亚型相对表达的影响。 因为rs368234815基因座上的二核苷酸多态赋予了产生ifnl4的不同能力。 配体与丙型肝炎病毒临床结局相关，我们还预测能够产生IFNL4的细胞 诱导高水平的III型干扰素应答，抑制I型干扰素信号转导，抑制病毒清除。至 克服在细胞系和原代人类肝细胞中进行的研究的局限性，我们将使用诱导剂 多能干细胞(IPSC)来源的肝细胞作为模型系统。IPSCs可以被基因操控， 长期培养，分化为肝细胞样细胞，支持丙型肝炎病毒和乙肝病毒感染，并有完整的 干扰素信号通路，因此非常适合测试我们的假说。在目标1中，我们将测试 假设通过IFNLR1的信号，以及规范和非规范异构体的相对表达， 在功能上影响I型干扰素信号的性质和大小。在目标2中，我们将检验假设 Rs368234815多态是异质性的原因，rs368234815多态调节产生ifnl4的能力。 在先天性肝细胞对病毒感染的反应中。这些发现将具有广泛的相关性，当考虑 干扰素信号对影响肝脏的其他慢性感染，如乙肝病毒和艾滋病病毒的治疗调节 和肠道，并通过感染和非感染的机制引起慢性炎症。

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.