Discovery and Characterization of Cellular Factors Modulating Type III Interferon Induction and Signaling

原理：干扰素（IFN）是细胞因子，在抵抗真菌、细菌和病毒病原体方面发挥重要作用。它们在抗肿瘤反应、粘膜免疫和脂质代谢中也发挥积极作用。干扰素的表达在细胞中受到严格调控。我的长期目标是了解细胞蛋白质在细胞中各种IFN的诱导和信号传导的调节中的作用。基于进化相关性，干扰素分为三类，即I型、II型和III型。其中，最近鉴定的组是III型IFN，其包括IFN λ 1-4（IFN-L1-4）。尽管是宿主屏障免疫应答的主要组成部分，但尚未对调节III型IFN诱导和信号传导的细胞蛋白进行系统筛选。我们推测，几种细胞蛋白和途径可能在调节干扰素系统中发挥关键作用。在这项发现授权中，我们建议使用全基因组CRISPR敲除和激活文库筛选以及基于TurboID的邻近标记相互作用组筛选来全面绘制积极或消极调节IFN-L诱导和信号传导的细胞蛋白。目的1将表征在两种PRR（RIG-I和TLR-3）被其同源配体双链RNA激活后调节IFN-L3诱导的细胞蛋白。通过CRISPR基因标记产生的在基因组背景下表达氖绿色荧光蛋白标记的IFN-L3基因的细胞将用于基于FACS的全基因组CRISPR敲除和活化文库筛选以鉴定基因候选物。将通过标准分子生物学技术确定最相关候选物的作用机制和细胞类型特异性。 目的2定位III型干扰素信号通路调控ISG表达的蛋白质。在IFN-L2处理后，将通过基于FACS的全基因组CRISPR敲除和激活文库筛选在氖绿色标记的OAS-2蛋白报告细胞系中鉴定候选基因。在对所选候选物进行验证之后，将研究IFN信号通路中调节的确切步骤和所涉及的潜在机制。 意义：III型IFN在屏障组织中对入侵病原体的宿主免疫中发挥核心作用。对调节IFN-L通路的细胞蛋白质的全面了解将使我们能够理解这些组织中宿主细胞和病原体之间复杂的相互作用，并揭示病原体与该通路相互作用和破坏该通路的新方式。在IFN-L通路中识别新的可药物靶点也将增强我们在感染和自身免疫性疾病期间调节它的能力。

Rationale: Interferons (IFNs) are cytokines that play an essential role in protection against fungal, bacterial and viral pathogens. They also play an active role in antitumor response, mucosal immunity and lipid metabolism. Expression of interferons are tightly regulated in cells. My long-term objective is to understand the role of cellular proteins in regulation of induction and signaling of various IFNs in cells. Based on evolutionary relatedness, interferons are grouped into three classes namely type I, type II and type III. Among these, the recently identified group is type III IFNs which include IFN lambda 1-4 (IFN-L1-4). Despite being a major component of host barrier immune response, a systematic screening for cellular proteins modulating type III IFN induction and signaling has not been done yet. We hypothesise that several cellular proteins and pathways may play a critical role in regulating the interferon system. In this discovery grant we propose to comprehensively map the cellular proteins that positively or negatively regulate the induction and signaling of IFN-L using genome-wide CRISPR knockout and activation library screens and TurboID-based proximity labeling interactome screen. Aim 1 will characterize cellular proteins regulating the induction of IFN-L3 following activation of two PRRs, RIG-I and TLR-3 by their cognate ligand, double stranded RNA. Cells expressing neon green fluorescent protein tagged IFN-L3 gene in genomic context, generated by CRISPR-gene tagging will be used in a FACS-based genome wide CRISPR knock-out and activation library screen to identify gene candidates. The mechanism of action and cell type specificity of the most relevant candidates will be determined by standard molecular biology techniques. Aim 2 will map the proteins modulating ISG expression by type III IFN signaling. The gene candidates will be identified by a FACS-based genome-wide CRISPR knock-out and activation library screen in neon green tagged OAS-2 protein reporter cell line following IFN-L2 treatment. Following verification of selected candidates, the exact steps in the IFN signaling pathway regulated and the potential mechanism involved will be studied. Significance: Type III IFNs play a central role in host immunity against invading pathogens in barrier tissues. A comprehensive understanding of the cellular proteins modulating the IFN-L pathway will enable us to understand the complex interaction between host cells and pathogens in these tissues, and to uncover novel ways in which pathogens interact with and undermine this pathway. Identifying novel druggable targets in IFN-L pathway will also augment our ability to regulate it during infections and autoimmune diseases.