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

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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.