B29-09 Oxidative Imbalance Disrupts Mucus Transport

Rationale Mucociliary transport removes airborne irritants through coordinated ciliary beating and mucus flow. Mucus is a gel-forming polymer whose viscoelastic properties are essential to its transport function. These characteristics emerge as mucins unfold, hydrate and undergo crosslinking in the airway environment. This host defense mechanism plays an important role in the development of several airway diseases, including chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis and cystic fibrosis (CF). We propose that CFTR dysfunction creates a pro-oxidant state promoting excessive disulfide crosslinking that impairs mucus clearance and transport. Methods We used human CF airway epithelia from transplant donors and newborn piglet epithelia with the G551D mutation. We measured viscoelastic and transport characteristics of an oxidation-sensitive synthetic hydrogel (thiol-hyaluronic acid). To preserve the mucus layer, we used differential dynamic microscopy and measured, in situ, the scattering function of microspheres embedded in the mucus layer. In addition, we measured reactive oxygen species, viscosity with FRAP, free thiol content, and mitochondrial respiration. Results Human CF airway cultures significantly altered thiol-HA biophysical properties—storage modulus G’: CF 30.7 ± 17.1 Pa vs. non-CF 1.7 ± 1.0 Pa; loss modulus G”: CF 30.9 ± 14.8 Pa vs. non-CF 5.0 ± 2.1 Pa. These changes were independent of pH or polymer concentration. Pig airway cultures with G551D mutation showed similar alterations—G’: G551D 29.4 ± 8.0 Pa vs. WT 1.4 ± 0.4 Pa; G”: G551D 14.6 ± 6.9 Pa vs. WT 2.1 ± 0.5 Pa; transport speed: G551D 45.0 ± 4.4 μm/s vs. WT 91.5 ± 20.6 μm /s. These changes were accompanied by elevated hydrogen peroxide levels and decreased free thiol availability. Ivacaftor treatment partially reversed these effects—G’: G551D with Ivacaftor 1.2 ± 0.2 Pa; G”: 1.6 ± 0.4 Pa; speed: 85.2 ± 6.5 μm/s. Seahorse analysis revealed mitochondrial dysfunction in CF with elevated basal respiration (G551D 3.5 ± 1.1 vs. WT 0.7 ± 0.7 pmol/min), proton leak (G551D 1.1 ± 0.4 vs. WT 0.2 ± 0.2 pmol/min), and non-mitochondrial oxygen consumption (G551D 0.6 ± 0.2 vs. WT 0.1 ± 0.1 pmol/min). Ivacaftor partially normalized these parameters. Conclusion Optimal disulfide crosslinking is essential for mucus transport. In CF, even without infection, a pro-oxidant environment creates unfavorable mucus viscoelastic properties, worsened by inflammation and partially reversed by CFTR modulator therapy. These findings suggest early antioxidant interventions may restore optimal mucus crosslinking in CF patients, complementing CFTR modulators by maintaining oxidative balance necessary for effective mucociliary transport. This abstract is funded by: NIH