Project Summary / Abstract Our published data reveals that semi quantitative mucus plug scores strongly associate with measures of airway type 2 inflammation and airflow obstruction in asthma. We have now developed novel approaches to move from a semi-quantitative mucus plug score to detailed quantitative mucus plug phenotyping (mucus plug number, size, and location), and this technology allows us to more deeply explore mucus plug biology in asthma. Our renewal grant application will continue to leverage CT lung images and airway biospecimen resources from the SARP-3 cohort, take advantage of an imaging team built by Dr Fahy to explore airway mucus plug phenotypes using CT lung images, and continue an ongoing collaboration between Dr Fahy’s and Dr Seibold’s labs to jointly tackle disease mechanisms in asthma. Aim 1 will explore relationships between airway mucus plugs, type 2 inflammation and airflow obstruction. Our studies of mucus plugs and type 2 inflammation will include analysis of airway mucus plugs in SARP-3 patients before and after they were started on therapeutic proteins targeting the type 2 pathway. Our studies of mucus plugs and airway location are designed to determine the number of mucus plugs and the airway sizes that are most consequential for airflow limitation. Aim 2 will determine if treatment with inhaled N-acetylcysteine (NAC) improves airflow in mucus-high asthma. NAC is mucolytic because it cleaves the excessive disulfide bridges between mucin polymers that increase airway mucus gel elasticity in asthma. Our clinical trial will directly test the hypothesis that mucus plugs cause airflow limitation in asthma and determine the influence of mucus plug phenotypes on response to mucolytic treatment. Aim 3 will determine the role of ITLN1 in the pathophysiology of airway mucus plugs in asthma. Intelectin-1 (ITLN1) is an epithelial cell protein that is upregulated by IL-13 (a type 2 cytokine), and our preliminary data show that a eQTL variant in ITLN-1 modifies risk of mucus plugging in asthma. This provided impetus for us to explore mechanisms of ITLN-1 mediated mucus pathology in bench experiments. Our aims have specific hypotheses, are supported by robust preliminary data, and are enabled by the application of state of the art imaging, computer vision, and cellular and molecular methodologies. Our research promises to advance understanding about mucus plug biology in patients with severe asthma and our data will inform drug development for mucus plugs and the potential utility of therapeutic bronchoscopy. Most importantly, our CT mucus phenotyping biomarkers will provide the field with much needed predictive and monitoring biomarker for mucus-high asthma.