Elastic mucus strands impair mucociliary clearance in cystic fibrosis pigs

Abstract

SignificanceIn many lung diseases, increased amounts of and/or abnormal mucus impair mucociliary clearance, a key defense against inhaled and aspirated material. Submucosal glands lining cartilaginous airways secrete mucus strands that are pulled by cilia until they break free from the duct and sweep upward toward the larynx, carrying particulates. In cystic fibrosis (CF) pigs, progressive clearance of insufflated microdisks was repeatedly interrupted as microdisks abruptly recoiled. Aerosolizing a reducing agent to break disulfide bonds linking mucins ruptured mucus strands, freeing them from submucosal gland ducts and allowing cilia to propel them up the airways. These findings highlight the abnormally increased elasticity of CF mucus and suggest that agents that break disulfide bonds might have value in lung diseases with increased mucus.

Keywords: cystic fibrosis; epithelia; lung; mucus; submucosal gland.

Fig. 1.

Aerosolized saline induces abrupt retrograde movement of microdisks in CF airways. Maximal intensity projection of a dynamic CT scan from a CF pig in coronal reconstruction, showing microdisk deposition and movement in the airways. (A) Basal conditions. Black circles indicate a cluster of nonmoving microdisks; red circles indicate a cluster of moving microdisks. (B) After IV methacholine and intratracheal saline. Black circles indicates a cluster of nonmoving microdisks; red and blue circles indicate clusters of moving microdisks that are transported ∼2 cm from their original location in ∼2.5 min before they abruptly (∼8.5 s) recoiled backward in a single frame (asterisk). Real time in minutes and frame number are indicated for each panel. Images were inverted to enhance contrast between microdisks and airway tree shadow. (Scale bar = 1 cm.).

Fig. 2.

Aerosolized saline increases microdisk movement, but recoil transport impairs their clearance from CF airways. (A–C) Percentage of microdisks cleared, stratified by experimental condition. Each animal is depicted with a unique color. Lines with 0% clearance are nudged for visualization purposes. Conditions include (A) basal unstimulated (mean = 33 ± 7%, n = 22), (B) simultaneous IV methacholine and inhaled saline (mean = 42 ± 10%, n = 14), and (C) simultaneous IV methacholine and inhaled TCEP (mean = 61 ± 10%, n = 8). Dots and whiskers are mean ± SEM. (D–F) Vertical position of microdisks as a function of time in representative experiments. (D) Under basal conditions, most microdisks move slowly, and some are stationary. (E) After simultaneous treatment with IV methacholine and inhaled saline, microdisk velocity was increased compared to basal conditions. Discontinuous traces indicate times when microdisks abruptly moved backward (Movie S2). (F) After IV methacholine and inhaled TCEP, microdisks advanced through the airway without apparent recoil reversals. (G and H) Average speed of moving microdisks. Each set of data points and connecting lines is from a different pig. Lines and error bars represent mean ± SEM. (I and J) Percentage of time microdisks spend in motion. Each set of data points and connecting lines is from a different pig. Some data points overlap. Lines and error bars represent mean ± SEM. (K) Percentage of pigs with observed recoil movements. Bars represent the percentage of pigs observed to have recoil. Number of experiments per condition are given in the bars. (L) Mean percentage of microdisks cleared from the field. Lines represent mean and vertical lines represent SEM for basal (black), IV methacholine and inhaled saline (blue), or IV methacholine and TCEP (red). Mch: Methacholine; S: Saline; *P < 0.05 and **P < 0.01 by paired t test.

Fig. 3.

TCEP largely eliminates recoil transport of microdisks and allows their clearance from CF airways. CF pigs were treated with methacholine (McH) to increase SMG secretion, trapping Tantalum microdisks. After 6 min, the pigs were treated with either inhaled saline or inhaled TCEP in saline. (A and B) Data show the percentage of microdisks cleared as a function of time. Lines represent individual experiments performed in the presence of saline (A) or TCEP/saline (B). (C and D) Data show vertical positions of microdisks as they are transported in two representative experiments in CF pigs. Each microdisk is given a unique color. Dotted line is the limit of the tracking field at the level of the larynx. (E) Percentage of experiments with observation of recoil movements. (F) Mean percentage of microdisks cleared from the field. Lines represent mean and vertical lines represent SEM for inhaled saline (blue) or TCEP in saline (red). Mch: Methacholine; gray bar represents 1 min pause in image acquisition to administer intratracheal intervention; **P < 0.01 by paired t test.

Fig. 4.

TCEP breaks mucus strands in ex vivo CF airway. (A) Sequential confocal microscopy images (60 ms/frame) depict a mucus strand from a CF pig, labeled with red fluorescent nanospheres. Ciliary transport directs free particles toward the top of the image (arrowheads). Arrows show the position of the distal portion of a mucus strand. The distal end of the strand spontaneously retracts. See also Movie S3. (B) Confocal microscopy images show stationary mucus strands before and after treatment with TCEP from a representative experiment. (C) Number of stationary strands in the microscopy field before and after treatment with TCEP. Mch: methacholine; open circles: before treatment with TCEP; filled circles: after treatment with TCEP; *P < 0.05 by paired t test.