PROJECT SUMMARY/ABSTRACT Cystic fibrosis (CF) remains a life-threatening disorder caused by inherited mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In CF airways, impaired mucus clearance set up an environment prone to chronic bacterial infections, prominent neutrophilic inflammation, remodeling, and progressive loss of lung function. Early interventions that enhance mucus clearance and prevent mucus accumulation could dramatically change the outcome of CF lung disease. The lack of an animal model and a sensitive mucus clearance prevented understanding of early CF lung disease pathogenesis. Two novel milestones made this possible: 1) CF pig, a model that develops the hallmarks of human CF airway disease. 2) CT-based MCT, a sensitive computed-tomography (CT) based mucus clearance assay with high spatial and temporal resolution. We found that in CF pigs, MCT is impaired after cholinergic stimulation. We identified failed detachment of mucus strands from CF submucosal gland (SMG) ducts impaired mucus clearance. Hypertonic saline (HS) is widely used to improve mucus clearance. However, its use is limited by unwanted side-effects and mixed efficacy in CF. A better understanding of the mechanism of HS on mucus clearance will advance development of therapeutic strategies. HS is supposed to enhance mucus clearance in several putative mechanisms; by increasing ASL height, by stimulating SMG secretions, by disrupting ionic interactions, or by altering ciliary beat frequency. Our novel mucus clearance assay will provide a framework to investigate these mechanisms. The overarching goal of this project is to understand the mechanisms of mucus transport in the airways. The project addresses two main questions. First, how does HS increase mucus clearance under basal condition? By blocking the cholinergic response with atropine and examining the effects in vivo and ex vivo will elucidate the mechanism. Investigating in CF will suggest whether HS have unintended potentially adverse effects and whether these adverse effects could be blocked by atropine. Second, does HS alone or in combination with a reducing agent (TCEP) enhance mucus clearance in CF after cholinergic stimulation? Here, in contrast to the prior question, submucosal gland secretion will be stimulated prior to delivering HS. Answers will indicate whether HS increases mucus clearance by disrupting the ionic interactions that hold mucus together, or by increasing ASL height. Combining HS with a reducing agent that can disrupt mucus disulfide interactions, will suggest whether the effect is additive or not. This research will allow us to better understand the mechanism of HS effect on mucus clearance. Whether HS might work through stimulation of submucosal glands or osmotically increasing ASL height should make inroads to develop new therapies.