A CRISPR based organoid screen for mediators of epithelial-myeloid interactions - Project Summary Protective immunity in mucosal organs requires tightly regulated communication between epithelial cells and immune cells. The airways are particularly complex, with multiple airway epithelial cell (AEC) and immune cell populations, which must coordinate to defend against pathogens via the regulation of airway inflammation. However, inappropriate immune activation in the airway can result in diseases such as asthma. Indeed, inhibition of AEC-derived immune mediators is beneficial in asthma, demonstrating the therapeutic potential for targeting epithelial-immune cell crosstalk. Tissue macrophages are important for organ homeostasis with roles in antigen surveillance and presentation as well as tissue repair. Recently, a novel macrophage population that is located within the epithelial cell layer of the conducting airways has been described in both mice and humans. These intraepithelial airway macrophages (IAMs) have been shown to be important for normal regeneration of the airway following injury, and for antigen surveillance via stimulating local memory T cells and transfering antigens to adjacent dendritic cells to induce T cell responses in lymphoid tissue. These data suggest that IAMs are important modulators of airway inflammation, injury, and repair, yet little is known about their specific role in lung disease. Identification of the mechanisms that maintain airway mucosal immune homeostasis has broad implications for understanding lung immunity and may help identify therapeutic targets for inflammatory airway diseases. The objective of this proposal is to define novel mechanisms of communication between AEC and IAMs by developing an innovative high-throughput airway organoid screen. Our preliminary studies demonstrate that: (1) IAMs are necessary for allergic airway inflammation, (2) interactions between AECs and IAMs drive allergic airway inflammation, (3) establishment of a novel AEC-IAM organoid co-culture model, (4) the ability to genetically modify AEC via lentiviral delivered guide RNAs and CRISPR, and (5) the ability to sort and analyze AEC-IAM organoids for novel mediators of IAM homeostasis and function. We hypothesize that a novel AEC- IAM organoid system will allow high-throughput screening for novel mediators of AEC-immune cell interactions. Investigation of this hypothesis will define novel mechanisms of AEC-airway immune cell crosstalk with relevance for inflammatory diseases of the airway. The specific aims are: 1) To establish a CRISPR-based airway organoid platform for screening mechanisms involved in AEC-IAM interactions, and 2) To utilize our CRISPR-based airway organoid platform to identify genes important for IAM maintenance in the airway epithelium. In this project, we will test and validate methods and then execute a high-throughput pool-based CRISPR-Cas9 screen for mechanisms of AEC-IAM communication utilizing our novel airway organoid model with both murine and human cells. Our results will not only establish a novel screening method for AEC-IAM interactions but could also be extended to screen for genes involved in other airway functions.
