A significant fraction of asthma patients face challenges in managing their symptoms with available therapies. Uncovering new drug targets holds the promise of alleviating their suffering and improving their quality of life. Anticholinergics, which bind and inhibit muscarinic acetylcholine receptors, have shown recent success in clinical trials. Animal research suggests these drugs may mitigate the lung inflammation and remodeling observed in asthma, although the underlying mechanisms are unclear. These drugs bind M1 and M3 muscarinic receptor subtypes in the airway epithelium, where each receptor’s role in asthmatic allergen uptake, inflammation, and remodeling is unknown. We identified secretory cell associated antigen passages (SAPs) in the airway epithelium, which uptake asthmatic allergens into the epithelium and directly associate with underlying antigen-presenting cells. Acetylcholine signaling increases the fraction of airway epithelial cells internalizing allergens into SAPs, but it is not known whether M1 or M3 receptors mediate this response. We hypothesize that M1 and M3 differentially regulate SAP formation to modulate antigen provision. In mouse models of asthma, anticholinergic drugs that inhibit M1 and M3 reduce type 2 inflammation and goblet cell metaplasia, hallmark pathologies of the disease. However, genetic deletion of the M1 or M3 receptor causes only the reduced inflammatory or metaplastic phenotype, respectively. We therefore hypothesize that epithelial acetylcholine signaling through the M1 and M3 receptors separately regulates type 2 inflammation and goblet cell metaplasia during the asthmatic response. To test our hypotheses, we will use mouse genetics to delete M1 or M3 receptors. By using confocal and two-photon microscopy to visualize fluorescently labeled allergens in airway epithelial cultures and tracheal explants, we will determine which receptor mediates the stimulation of SAP formation by acetylcholine. Further, we will generate conditional knockout mice with established promoters to excise the M1 and M3 receptors specifically in airway epithelial cell types. We will determine the consequences for allergic response using the house dust mite (HDM)-induced asthma mouse model. We will measure the expression of type 2 inflammatory markers using flow cytometry, qPCR, and ELISA and will detect goblet cell metaplasia by immunofluorescence imaging. The training plan outlined will enable me to acquire advanced skills in murine genetics, airway epithelial culture and physiology, two-photon microscopy, and immunology. It will also further enhance my abilities in scientific communication. Obtaining these skills is crucial for my development into an interdisciplinary scientist employing quantitative and biomedical techniques to study lung disease. The Rajagopal laboratory and the greater MGH community provide an inimitable environment in which to learn these technical skills and to receive the professional training I need t...