Reflection confocal microscopy for quantitative assessment of airway surface liquid dysregulation and pharmacological rescue in cystic fibrosis under near-physiological conditions

Proper regulation of airway surface liquid (ASL) is essential for effective mucociliary clearance (MCC) in healthy airways, and ASL depletion due to deficient cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion/fluid secretion plays an important role in the pathogenesis of mucociliary dysfunction and chronic muco-obstructive lung disease in patients with cystic fibrosis (CF). The current standard for quantitative measurements of ASL height is confocal fluorescence microscopy that has the disadvantage that it requires apical addition of volume for fluorescent staining, and hence perturbation of the ASL. Therefore, our aim was to develop a method that enables studies of ASL regulation under unperturbed conditions using reflected light by confocal microscopy of primary airway epithelial cultures grown at air-liquid interface (ALI). After apical volume addition to primary tracheal mouse cultures, confocal reflection microscopy yielded comparable ASL height as confocal fluorescence microscopy on cultures of wild-type mice, and was sensitive to detect ASL depletion on cultures of βENaC-Tg mice. Under unperturbed conditions, ASL determined by confocal reflection microscopy was significantly higher in wild-type and βENaC-Tg mice compared to values obtained by confocal fluorescence microscopy. Studies in normal and CF primary human airway epithelial cultures showed that confocal reflection microscopy was sensitive to detect effects of low temperature rescue and pharmacological modulation including improvement of CFTR function by VX-809 and VX-770 in cultures from CF patients with the F508del mutation. Our results support confocal reflection microscopy as a novel sensitive technique for quantitative studies of ASL regulation and response to therapeutic intervention under unperturbed near-physiological conditions in healthy and CF airways. ### Competing Interest Statement SYG reports grants from the Christiane Herzog Foundation, the German Cystic Fibrosis Association (Mukoviszidose), Vertex Pharmaceuticals, and a fellowship from the Berlin Institute of Health Charite Clinician Scientist Program; lecture honoraria from Chiesi and Vertex Pharmaceuticals; and advisory board participation for Chiesi and Vertex Pharmaceuticals outside the submitted work. MAM reports grants from Vertex fees form Pharmaceuticals; consulting or advisory board participation from AbbVie, Antabio, Arrowhead Pharmaceuticals, Boehringer Ingelheim, Enterprise Therapeutics, Kither Biotech, Pari, Prieris, Recode, Santhera, Splisense, and Vertex Pharmaceuticals; lecture honoraria from Vertex Pharmaceuticals; travel support from Boehringer Ingelheim and Vertex Pharmaceuticals; and a patent on the Scnn1b-transgenic mouse as an animal model for chronic obstructive pulmonary disease and cystic fibrosis outside the submitted work. No conflicts of interest, financial or otherwise, are declared by the other authors.