Impaired mucus detachment disrupts mucociliary transport in a piglet model of cystic fibrosis

Abstract

Lung disease in people with cystic fibrosis (CF) is initiated by defective host defense that predisposes airways to bacterial infection. Advanced CF is characterized by a deficit in mucociliary transport (MCT), a process that traps and propels bacteria out of the lungs, but whether this deficit occurs first or is secondary to airway remodeling has been unclear. To assess MCT, we tracked movement of radiodense microdisks in airways of newborn piglets with CF. Cholinergic stimulation, which elicits mucus secretion, substantially reduced microdisk movement. Impaired MCT was not due to periciliary liquid depletion; rather, CF submucosal glands secreted mucus strands that remained tethered to gland ducts. Inhibiting anion secretion in non-CF airways replicated CF abnormalities. Thus, impaired MCT is a primary defect in CF, suggesting that submucosal glands and tethered mucus may be targets for early CF treatment.

Fig. 1. Loss of CFTR impairs MCT in vivo in newborn piglets treated with methacholine

(A) Images are reconstructed ventral-dorsal views of non-CF and CF airways under basal conditions and after 1.28 ×10⁻⁷ mol/kg, IV methacholine. Images are at beginning and end of a 10 min tracking period (Videos 1A,1B). Positions of microdisks are shown as spheres (enlarged ~40 times actual area). (B to F). Symbols indicate data from 8 non-CF and 8 CF piglets studied before and after 1.28 ×10⁻⁷ mol/kg, IV methacholine. Each data point is average behavior of individual microdisks in a piglet during a 10 min tracking run. Lines and whiskers beside individual data are mean±SEM. * indicates P<0.05, paired Student’s t-test. (B) Percentage of microdisks that cleared tracking field during 10 min tracking period. (C) Radial position of microdisks at start and end of 10 min tracking period. Data are absolute values of angles relative to ventral (0 degrees). (D,E) Maximum and mean speed of microdisks. (F) Percentage of time microdisks were moving during a tracking run. † indicates P<0.05 unpaired Student’s t-test. Analysis using linear mixed effects model with random effect for pigs yielded a similar conclusion (P=0.024). Analysis in panel F was not adjusted for multiple comparisons.

Fig. 2. Loss of CFTR increases the percentage of non-mobile microdisks on ex vivo trachea submerged in saline

(A) Schematic showing trachea removed from piglets treated with methacholine (1.28 ×10⁻⁵ mol/kg, IV), opening along the ventral surface, covering with saline, application of tantalum microdisks to the surface, and tracking of movement. Images in panel B are examples and data in panel C are averages. (B) Track of microdisks. Red circle indicates microdisk start position. Arrowhead indicates position of tracking field exit. Black circle indicates end position of microdisk that failed to clear tracking field. Red/black circle indicates disk that never moved. Dashed line indicates tracking field. Bar = 2 mm. Images are compiled from a 10 min tracking period (Videos 2A,2B). (C) Percentage of time microdisks were moving. N = 4 non-CF and 4 CF tracheas. * P<0.05 unpaired Student’s t-test.

Fig. 3. Strands of mucus emerge from submucosal gland ducts in methacholine-treated non-CF airways studied ex vivo

Submucosal gland duct openings are indicated by arrowheads. (A) Schematic of imaging procedure. All experiments were repeated at least 3 times. Non-CF trachea was removed from piglets, opened along ventral surface, pinned flat, submerged in a HCO₃⁻/CO₂ buffered Ringers, and treated with 1.28 ×10⁻⁵ mol/L methacholine. Trachea was opened along ventral surface so that cilia would propel mucus and nanospheres to lateral edges of the tracheal preparation (10). Solution bathing the trachea contained a dilute suspension of fluorescent 40 nm nanospheres. Images were obtained with a high-speed confocal microscope at the tracheal surface. Green indicates fluorescence from nanospheres (Video 3A). (B) Reconstruction of mucus (labeled with green nanospheres) emerging from submucosal gland duct onto airway surface (grey) (Video 3B). Bar = 50 μm. (C) Mucus strands grow in length from submucosal glands. In top panels, saline contained dilute suspension of green and red nanospheres, and both labeled a mucus strand. Green nanospheres were then removed from saline and mucus strand continued to elongate from gland duct as indicated by labeling with red nanospheres in bottom panels 25 min later. Bar = 50 μm. (D) Mucus strand grew from opening of submucosal gland duct and then broke free and rapidly flowed out of microscopic field (Video 3C). Time is at bottom. Grey background in first panel is reflected light image. Bar = 50 μm. (E) Mucus strand “α”, anchored at arrowhead, temporarily captures another mucus strand “β” flowing past, α stretches, and then the connection between α and β breaks at 140 sec. Immediately after the break, β leaves the field, and α snaps back to its original length (Video 3D). Grey background in first panel is reflected light image. Bar = 50 μm.

Fig. 4. Mucus strands fail to detach from submucosal glands when ex vivo CF airways are treated with methacholine or when liquid secretion is inhibited in non-CF airways

(A) Schematic of imaging procedure. Images are panoramic views of tracheal sections obtained with a time-averaging technique that visualizes static mucus labeled with fluorescent nanospheres. See Fig. S4, Video 4. Black lines on sides of images are pins holding trachea. Fluorescently-labeled mucus is shown in grey-scale. Panels B to D represent images from individual tracheas and average data and numbers of experiments are in panels E and F. (B to D) Airways were removed from methacholine-treated (1.28 ×10⁻⁵ mol/kg, IV) piglets and images were captured at end of a 15 min basal period and then 45 min after adding 1.28 ×10⁻⁵ mol/L methacholine. Bar = 1 mm. See videos 4A to 4C. (B) Non-CF trachea. Note accumulation of mucus (white) along cranial and ventral edges of tissue. Some static mucus on lower left of non-CF trachea was attached to pin at tissue edge. (C) CF trachea. (D). Non-CF airways stimulated with methacholine in HCO₃⁻-free HEPES-buffered (pH 7.4 or 6.8) saline containing 10 μM bumetanide. (E) Mucus tethered to submucosal glands on non-CF and CF airways 45 min after adding methacholine. See Methods for description of tethered mucus score. N=7 non-CF and 7 CF trachea, * P<0.05 unpaired Student’s t-test. (F) Mucus tethered to submucosal glands on non-CF trachea 45 min after adding methacholine. Trachea were bathed in HCO₃⁻/CO₂-buffered saline (N=12), HCO₃⁻/CO₂-buffered saline containing 10 μM bumetanide (N=8), HCO₃⁻-free HEPES-buffered (pH 7.4 or 6.8) saline (N=9), or HCO₃⁻-free saline containing bumetanide (N=11). * P<0.05 by one-way ANOVA and Bonferroni post-test. (G,H) Combined reflected light and fluorescence images show position of tantalum microdisks (yellow circles) and mucus; mucus often wrapped around microdisks and partly obscured them. Microdisks were applied to non-CF trachea in HCO₃⁻-free saline containing bumetanide (G) and to CF trachea (H). Subsequent addition of fluorescent nanospheres to saline revealed that all stationary microdisks were attached to mucus. These data also indicate that mucus strands formed independently of nanospheres. Arrowheads indicate submucosal gland ducts. Experiments were repeated at least 3 times. Bar = 1 mm. Video 4E,4F.