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

Abstract

Proper regulation of airway surface layer (ASL) is essential for effective mucociliary clearance (MCC) in healthy airways. 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). Quantitative measurement of ASL height by confocal fluorescence microscopy following addition of fluorescent dye has contributed important insight into the (dys)regulation of ASL in health and disease. Here, we present a novel method enabling studies of ASL regulation that does not require the addition of dye by 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 near-physiological conditions that may be applicable for studies of (patho)physiology and drug screens in healthy and CF airways.

Supplementary Information: The online version contains supplementary material available at 10.1038/s41598-025-32061-3.

Keywords: Airway epithelium; Airway2 surface layer; Confocal reflection microscopy; Cystic fibrosis; βENaC-Tg mice.

Fig. 1

Principle of reflection confocal microscopy for measurements of ASL height on airway epithelial cultures. A Schematic representation of the laser beam passing through a transwell with a differentiated airway epithelial layer grown at air liquid interface and the portions of light being reflected at each interface with a change in refractive index reversing its direction of propagation. For clarity, the reflected signal is depicted separately from the laser light. FEP: fluorinated ethylene propylene. B Reflection signals obtained from normal (wild-type) murine primary tracheal epithelial cultures with the 488 nm laser by XZ-scanning and fluorescence images recorded in parallel at 488 nm for the cell layer (calcein-AM) and 561 nm for ASL (rhodamine dextran). Arrows mark the position at which the following line profiles of fluorescent intensities were taken from. Peaks of reflected light in the line profile are labeled correspondingly. In the fluorescence measurements (x) and (y) mark the basolateral and apical border of the rhodamine signal, respectively. (a) and (b) mark the borders of the calcein-AM signal representing the cell layer. For reflection measurement laser light is reflected at (1) the transition from medium to transwell, (2) the transition from transwell to cells, (3) the transition from ASL to air. Scale bar: 25 μm. C Line profiles of an XZ-scan of the recorded reflection signal and the fluorescent signal from calcein-AM and rhodamine dextran. Vertical dashed lines mark the corresponding positions in the confocal images in (B).

Fig. 2

Comparison of ASL height after apical volume challenge of primary murine airway epithelial cultures determined by reflection vs. fluorescence confocal microscopy. Primary tracheal epithelial cell cultures from wild-type mice were grown at air-liquid interface for 14 days and 20 µl of fluorescent dye (rhodamine dextran in PBS) was added to the apical compartment to label the ASL. A Representative confocal images of the reflection signal recorded with the 488 nm laser by XZ-scanning and fluorescence images recorded in parallel at 488 nm for the cell layer (calcein-AM) and 561 nm for the ASL (rhodamine dextran). Images show ASL and labeled cells immediately after volume challenge (T = 0), 2 h (T = 2) and 24 h (T = 24) thereafter. Scale bar: 25 μm. B Summary of ASL height as determined by confocal fluorescence microscopy and reflection microscopy immediately after dye addition, at 2 h, 6 h and 24 h. n = 11–16 wells per group from 4 independent isolations. Statistical analysis was performed with paired two-tailed t test.

Fig. 3

Reflection confocal microscopy detects steady state ASL depletion on primary airway cultures from βENaC-Tg mice after apical volume challenge. Primary tracheal epithelial cultures from βENaC-Tg mice and wild-type controls were grown at air-liquid interface for 14 days and numeric densities of ciliated cells, βENaC-expressing cells and regulation of steady state ASL height following an apical volume challenge (20 µl PBS) were determined. A Representative images of wild-type and βENaC-Tg cultures co-immunostained with Hoechst (blue) as nuclear stain, anti-acetylated tubulin antibody (AcTub, green) and anti-βENaC antibody (red). Scale bar: 20 μm. B Numeric cell densities of ciliated cells (AcTub-positive), βENaC-positive cells and βENaC-overexpressing cells were determined and expressed as percentages of total cells. Each data point represents a single well (technical replicate). Data are from n = 7–8 wells per group of 2 independent isolations, *P < 0.01. Statistical analysis was performed with unpaired two-tailed t test. C Representative confocal images of the reflection signal recorded with the 488 nm laser by XZ-scanning and calcein-AM staining showing the epithelial cell layer and ASL height immediately after apical volume challenge (T = 0) and 24 h (T = 24) thereafter. Scale bar: 12 μm. D: Summary of ASL height as determined by reflection confocal microscopy immediately after apical volume challenge and at 2 h, 6 h and 24 h thereafter. n = 10–16 wells per group of 4 independent isolations, *P < 0.01. Statistical analysis was performed with unpaired two-tailed t test.

Fig. 4

Reflection confocal microscopy detects ASL depletion on unperturbed primary airway cultures from βENaC-Tg mice without apical volume challenge. Primary tracheal epithelial cell cultures from βENaC-Tg and wild-type mice were grown at air-liquid interface for 14 days and ASL height was measured in unperturbed cultures without apical volume challenge. A, B Representative confocal images of the reflection signals recorded with the 488 nm laser by XZ-scanning and calcein-AM stained cell layer (A) and summary of ASL height measured every 15 min over a period of 5 h (B) on βENaC-Tg vs. wild-type airway cultures without addition of fluorescent dye from the apical side. Scale bar: 30 μm. Data are from n = 8–12 wells per group of 5 independent isolations, *P < 0.01 compared to wild-type. Statistical analysis was performed with unpaired two-tailed t test. C Comparison of steady state ASL height determined at 24 h after the first measurement by reflection confocal microscopy in unperturbed airway cultures vs. fluorescence confocal microscopy after apical addition of rhodamine dextran (conditions as in Fig. 3). Each data point represents a single well (technical replicate) from primary mouse airway epithelial cell cultures. Data are from n = 10–20 technical replicates per isolation, with 4–5 independent isolations per group, *P < 0.01 compared to wild-type; ^#P < 0.001 compared to measurements with volume addition. Statistical analysis was performed with Wilcoxon signed rank test corrected for multiple comparisons.

Fig. 5

Reflection confocal microscopy detects ASL dysregulation and response to pharmacological modulation of ion transport in non-CF and CF primary airway epithelial cultures. Primary airway epithelial cultures from non-CF controls and CF patients with at least one F508del mutation (Table 1) were grown at air-liquid interface for 14 days and effects of pharmacological modulation of airway ion transport on ASL height was measured by reflection confocal microscopy without apical addition of fluorescent dye. A Summary of acute effects of inhibition of ENaC by benzamil (100 µM), cAMP-dependent activation with IBMX (100 µM) and forskolin (1µM), and inhibition of transepithelial chloride transport by bumetanide (100 µM) on ASL height on non-CF and CF primary airway epithelial cultures determined by confocal reflection microscopy during sequential addition of compounds to the basolateral compartment each added at 45-minute intervals. Each data point represents a single well (technical replicate). To facilitate intra-well comparison of pharmacological responses, data points acquired from the same well are represented using identical colors. Data are from n = 8–15 technical replicates from 4–6 individuals per group, *P < 0.01 compared to non-CF cultures. For statistical analysis linear mixed-effects models were applied, followed by Tukey’s post-hoc test for pairwise comparisons. B, C Effects of low temperature (27 °C) and treatment with the CFTR modulator combination VX-809/VX-770 (lumacaftor/ivacaftor) on ASL height on F508del-expressing CF primary airway epithelial cultures determined by reflection confocal microscopy. B Representative confocal images of the reflection signals recorded with the 488 nm laser by XZ-scanning showing steady state ASL height and the calcein-AM stained cell layer. Scale bar: 30 μm. C Summary of ASL height in untreated non-CF vs. CF cultures and effects of incubation at 27 °C and treatment with VX-809 and VX-770. Each data point represents a single well (technical replicate). Data are from n = 10–16 technical replicates per individual, with 5–7 individuals per group, *P < 0.001 compared to non-CF cultures; ^#P < 0.01 compared to untreated CF cultures. Statistical analysis was performed with unpaired two-tailed t test corrected for multiple comparisons.