PROJECT 2 ABSTRACT: Asthma is the most common chronic lung disease worldwide. Importantly, over half of the $3.1B spent in the U.S. each year on asthma health care costs derive from less than 10% of patients, those who experience severe disease. Roughly, half of patients with severe asthma exhibit eosinophilic airway inflammation driven by type 2 (T2) cytokines (IL-4, IL-5, and IL-13). T2 inhibitor drugs are highly effective in blocking exacerbations among T2 patients with eosinophil-predominant disease. However, the remaining 50% with non-T2 disease are without treatment options and have poorly understood pathobiology. This represents the greatest unmet need in asthma. We have found non-T2 asthma includes those with interferon-γ-driven T1 and IL-17-driven T17 inflammation. A central feature of these T1 and T17 endotypes is persistent airway neutrophilia, accompanied by airway obstruction and remodeling. Moreover, we find that a subgroup of T2 patients also exhibit airway neutrophilia. At National Jewish, we have integrated translational research infrastructure into our high-volume clinical asthma program, allowing for the collection of airway epithelium and biopsies, bronchoalveolar lavage, sputum cells, and blood from patients with severe asthma. These biosamples collected from well-phenotyped patients provide the opportunity to evaluate mechanisms controlling the infiltration, activation, and function of airway neutrophils in severe asthma. Here, we challenge the notion that neutrophils are a static, homogeneous population, playing a simple pathogen response role in the airway. Rather our data suggest that neutrophil subsets could be differentially recruited to, and programmed by, the airways of T2, T1, and T17 asthmatics, through their expression of different combinations of neutrophil chemoattractant genes (CXCL1/3/8, C3), survival/maturation genes (CSF3), and activation genes (IFNG, IL17). At the same time, we find that neutrophils are potent producers of cytokines (e.g. IL-1α/β, IL-6) that induce airway remodeling and epithelial dysfunction. Therefore, we hypothesize that airway recruitment and mucosal programming of neutrophils in severe asthma occur in a T2, T1, and T17 endotype-specific manner, resulting in unique neutrophil inflammatory, immunomodulatory, and proteolytic activities that drive airway epithelial remodeling. To test this hypothesis, we will perform single-cell and spatial transcriptomic sequencing of circulating and airway neutrophils from well-phenotyped severe asthma patients. These in vivo molecular data will be paired with data from ex vivo functional assays and from the modeling of neutrophil-epithelial circuits using airway organoid cultures. In Aim 1 we will determine whether T2, T1, and T17 airway mucosal niches direct differential recruitment of circulating neutrophil heterogeneity through CXCL1/3/5/8, C5, and LTB4. In Aim 2 we will determine the role of T2, T1, and T17 airway mucosal programming factors (CSF3, I...