Project Summary / Abstract Type 2 inflammation is initiated at the airway epithelium through the release of master cytokines such as IL-33 that drive type 2 cytokine production, eosinophilia, and mucus pathology. Tyope 2 inflammation becomes persistent when homeostatic mechanisms that normally contain it fail causing persistent disease. We find that lung imaging (computed tomography) frequently reveals mucus plugging in asthmatic airways and that the plugs are highly eosinophilic and persist for many years. These findings lead us to propose that airway injury leads to reprogramming of the epithelium to cause focal areas of type 2 inflammation and mucus plugging (“type 2 airway niches”). We have three Aims to characterize the biology of type 2 niches in asthma with an emphasis on reprogramming of immune cells and epithelial cells and on IL-13 driven mechanisms of mucus plug formation. AIM 1 will characterize the subtypes of immune cell, their receptor expression, and their niche specific gene expression. We will use mass cytometry (CyTOF) to enumerate type 2 cytokine producing cells and their receptor expression repertoire. AIM 2 will character epithelial cells in the niche using bulk and single cells sequencing and also methods to uncover niche-specific epigenetic changes in these cells with a focus on genes that regulate type 2 cytokines (IL-33, TSLP, IL25, IL1β). ATAC-seq and whole genome methylation studies will be included to characterize epigenetic changes in epithelial cells from plugged and non-plugged airways. AIM 3 will explore how cross-talk between epithelial cells and eosinophils results in mucus plug formation in the type 2 airway niche. Emphasis in this aim will be placed on IL-13 regulated pathways that caused epithelial cells to upregulate transport of redox-relevant halides such as thiocyanate and to increase section of mucin-like molecules such as FcγBP. To achieve its three aims, Project 3 will interact closely with projects 1 and 2, and it will take advantage of all cores, especially the resources of the human subjects core and the analytic capabilities of Core C. Our project will advance knowledge of the type 2 niche in ways that could point to novel treatment strategies to switch off type 2 inflammation and fundamentally modify asthma.