PROJECT SUMMARY/ABSTRACT The gastrointestinal tract is composed of a single layer of epithelial cells that is in equipoise with immune cells and a vast number of microorganisms. Inappropriate responses to these microorganisms, either through genetic predisposition, altered immune or epithelial responses, or yet to be defined environmental influences, are postulated to lead to inflammatory bowel diseases (IBD). The immune signals that recognize and respond to bacterial and viral components of the microbiome remain incompletely understood. Interferons (IFNs) play a major role in antiviral immune defense in the intestinal epithelium, and are also important in regulating proliferation, differentiation, survival and effector functions of immune and non-immune cells. There are three classes of IFNs: type I IFNs (IFNα, β, and others), type II IFN (IFN) and type III IFNs, or IFNλs. To date, most studies investigating the use of IFNs on IBD have focused on type I IFNs and were not found to be effective. Dr. Ivan Zanoni reported that IFNλ decreases oxidative stress and intestinal damage in a murine model of colitis and that exogenous IFNλ can suppress intestinal inflammation. Importantly, we identified two unrelated patients with infantile-onset IBD with rare and functionally deleterious mutations in IFNλ2 and IFNλ3. Of note, each patient’s disease improved significantly with age. We have preliminary data that IFNλ2 and IFNλ3 may be more important in the first months of life than FNλ1, and data illustrating more severe murine colitis in Ifnλ3-/- mice as compared to wild type mice. Taken together, we hypothesize that INFλs are essential modulators of mucosal homeostasis, prevent development of IBD, and hold therapeutic potential. Current therapeutics available for the management of IBD fail to treat a large number of patients. This work will provide a better understanding of the role of IFNλ in mucosal homeostasis, and may provide the groundwork to implement novel strategies to treat IBD by manipulating IFNλ signaling. Unraveling the role of IFNλ in maintaining mucosal homeostasis will be achieved through the following aims: (1) Establishing the developmental expression of IFNλs, related cytokines and receptors in humans at different ages using bulk and single cell RNA sequencing technologies (2) Determining the role of IFNλ signaling in predisposition to development of colitis in vivo using various murine models of colitis; (3) Characterizing the functional consequences of patient-encoded IFNλ variants in vitro using T84 cells as well as human control and patient-derived intestinal organoids. The K08 award is instrumental in enabling Dr. Ouahed’s acquisition of the necessary structured training to become proficient in critical skills: RNAseq analysis, murine models of colitis and immune analyses, and generation/manipulation of intestinal organoids and epithelial analyses, complimented with didactic coursework, assisting her path to independence as a succe...