Excess mucus viscosity and airway dehydration impact COPD airway clearance

Abstract

The mechanisms by which cigarette smoking impairs airway mucus clearance are not well understood. We recently established a ferret model of cigarette smoke-induced chronic obstructive pulmonary disease (COPD) exhibiting chronic bronchitis. We investigated the effects of cigarette smoke on mucociliary transport (MCT).Adult ferrets were exposed to cigarette smoke for 6 months, with in vivo mucociliary clearance measured by technetium-labelled DTPA retention. Excised tracheae were imaged with micro-optical coherence tomography. Mucus changes in primary human airway epithelial cells and ex vivo ferret airways were assessed by histology and particle tracking microrheology. Linear mixed models for repeated measures identified key determinants of MCT.Compared to air controls, cigarette smoke-exposed ferrets exhibited mucus hypersecretion, delayed mucociliary clearance (-89.0%, p<0.01) and impaired tracheal MCT (-29.4%, p<0.05). Cholinergic stimulus augmented airway surface liquid (ASL) depth (5.8±0.3 to 7.3±0.6 µm, p<0.0001) and restored MCT (6.8±0.8 to 12.9±1.2 mm·min^-1, p<0.0001). Mixed model analysis controlling for covariates indicated smoking exposure, mucus hydration (ASL) and ciliary beat frequency were important predictors of MCT. Ferret mucus was hyperviscous following smoke exposure in vivo or in vitro, and contributed to diminished MCT. Primary cells from smokers with and without COPD recapitulated these findings, which persisted despite the absence of continued smoke exposure.Cigarette smoke impairs MCT by inducing airway dehydration and increased mucus viscosity, and can be partially abrogated by cholinergic secretion of fluid secretion. These data elucidate the detrimental effects of cigarette smoke exposure on mucus clearance and suggest additional avenues for therapeutic intervention.

Figure 1.. In vivo mucociliary clearance is impaired in a ferret model of COPD.

(A) Representative images depicting percent clearance of DTPA-conjugated Tc⁹⁹ from the lungs over time in ferrets exposed to room air or nose-only cigarette smoke for 6 months. Red = areas of maximal clearance (~2%/min). Dark blue/black = regions of minimal clearance (0%/min). (B) Quantification of percent clearance over time. (C) Area under the curve (AUC) for percent retention after 60 min. (D) Percentage of total Tc-DTPA remaining after 60 min of gamma imaging. n = 8-12 animals per group, *P < 0.05, **P < 0.01, ***P < 0.001 compared to air control, using two-way ANOVA.

Figure 2.. A ferret model of COPD exhibits abnormal local airway epithelial anatomy and function.

(A) Representative μOCT stills of trachea excised from air- or smoke-exposed ferrets. Red line = airway surface liquid (ASL). Yellow line = periciliary layer (PCL). Epi = epithelium. Mu = mucus. (B) Representative re-slices of μOCT videos for mucociliary transport (MCT) rate quantification. Yellow arrows represent mucus transport. (C) ASL, (D) PCL, (E) ciliary beat frequency (CBF), and (F) MCT rate were quantified for each ferret. n = 27 air control and 28 smoke-exposed, *P < 0.05 compared to air control, as assessed by unpaired Mann-Whitney. Data are presented as box-and-whisker (median ± quartiles, using Tukey’s method).

Figure 3.. Cholinergic stimulation rescues mucus transport in COPD ferret trachea.

A-D: ASL (A), PCL (B), CBF (C), and MCT (D) were quantified for each animal. Data is plotted as pre- and post-stimulation pairs by individual animal. n = 27 air control and 28 smoke-exposed ferrets, *P < 0.05, ***P < 0.001, ****P < 0.0001 compared to baseline and based on paired Wilcoxon tests.

Figure 4.. Smoke-exposed ferrets exhibit a trend toward increased mucus viscosity.

(A) Representative H&E and AB/PAS staining of tracheal sections and lung tissue from air control and smoke-exposed ferrets. Black arrows depict submucosal glands. B-E: Particle tracking microrheology (PTM) was used to measure ferret tracheal mucus viscosity. (B) Representative tracings of the Brownian motion of individual 500-nm particles moving through mucus collected from air- or smoke-exposed ferrets. MSD (C) and corresponding effective viscosities over a range of frequencies (D) and at 0.6 Hz (E) for each group. (F) Mucus percent solid content was calculated from measured wet and dry weights. n = 12-15 animals per group, *P < 0.05 compared to air control, using unpaired Student’s t-test.

Figure 5.. Histopathology of normal control, healthy smoker, and COPD human airway tissue.

Representative H&E and AB/PAS staining of bronchial sections obtained from non-smoker, healthy smoker, and COPD donors. g = submucosal glands.

Figure 6.. Healthy smoker and COPD HBE cells perpetuate mucus abnormalities.

(A) Representative tracings of the Brownian motion of individual 1-μm particles moving through normal control, healthy smoker, and COPD mucus. B-C: PTM was used to measure mean-squared displacement (MSD) of particles over time (B) within mucus secreted by HBE cells (normal, healthy smoker, COPD), from which effective viscosity (C) was calculated. (D) Comparison of effective viscosity of each group at 0.6 Hz. (E) Mucus percent solids content by weight was calculated for each donor group. (F) The relationship between mucus solid content and effective viscosity for each donor. n = 1 normal, 3 healthy smoker, 3 COPD donors (with 2-4 samples per donor for PTM, and 6 samples per donor for percent solids), *P<0.05, **P<0.01, ****P<0.0001 compared to COPD using two-way ANOVA (panel B), compared to healthy smoker unless otherwise denoted with two-way ANOVA (panel C), or compared to COPD unless otherwise denoted by one-way ANOVA (panels D-E).