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

Cystic fibrosis (CF) is a hereditary disease caused by mutations in CFTR, a gene encoding an ion channel [cystic fibrosis transmembrane conductance regulator (CFTR)] that transports chloride and bicarbonate across epithelial cell membranes (1). Lung disease, the source of most mortality in people with CF, arises from defective host defense that predisposes airways to bacterial infection. Mucociliary transport (MCT) defends airways by trapping pathogens in mucus, and cilia propel them out of the lung (2–4). The observation that MCT is abnormal in advanced CF (3) and several in vitro studies (5) have led to the proposal that impaired MCT causes CF lung disease. Yet, in vivo data suggest that CF MCT defects may be secondary to inflammation and airway remodeling. For example, in some studies CF adults showed no MCT abnormality, and CF children exhibited no MCT defects (3, 6, 7). Moreover, MCT deficits increased as the severity of CF lung disease increased, and in other pulmonary diseases, airway inflammation is also linked to MCT defects (8). Until now, lack of both sensitive MCT assays and an animal model that replicates human CF prevented determination of whether MCT is impaired from the outset and thus contributes to pathogenesis or is a secondary defect.

A porcine CF model provides an opportunity to test MCT in vivo at the disease’s origin (9). At birth, the airways of pigs with CF lack infection and inflammation but spontaneously develop hallmark CF features including infection, inflammation, mucus accumulation, and obstruction. To assay MCT in vivo with good spatial and temporal resolution, we developed an x-ray computed tomography–based assay to track movement of 350-μm diameter tantalum microdisks (10) (see supplementary materials and methods).

Most current MCT assays measure disappearance of radioactivity (clearance) after inhalation of radiolabeled particles (3, 6, 7). After insufflation into newborn piglet airways, a similar percentage of microdisks cleared CF and non-CF lungs in 10 min (Fig. 1, A and B, and fig. S1) (10). As microdisks were swept toward the larynx, they migrated to the ventral tracheal surface in both genotypes (Fig. 1C), consistent with our finding that ciliary orientation drives microdisks ventrally (10). Both genotypes propelled microdisks with similar maximal and mean speeds, and microdisks spent a similar percentage of time moving (Fig. 1, D to F). Thus, under basal conditions, CF MCT appeared intact.

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Airway insults can elicit a cholinergic reflex, a protective response that increases cilia beating and generates copious submucosal gland mucus secretion (4, 11, 12). The cholinergic agonist methacholine raised cilia beat frequency in CF and non-CF lungs (fig. S2A). However, it tended to reduce the percentage of microdisks that cleared CF lungs (Fig. 1, A and B, and movie S1). Measurements of individual microdisk behavior revealed that methacholine accelerated maximal and mean microdisk speeds in piglets without CF, but not those with the disease (Fig. 1, D and E, and fig. S1, C to E). The most marked difference between genotypes was that in piglets with CF, methacholine reduced the percentage of time individual microdisks were in motion by ~40% (Fig. 1F). Thus, rather than enhancing MCT and airway defense, a cholinergic agonist impeded MCT in piglets with CF. One possible limitation of this in vivo study is that the piglets were supine, anesthetized, and did not cough; cholinergic stimulation might have produced a different response in awake animals.

Several studies of cultured airway epithelia reported that loss of the CFTR increased Na⁺ channel activity, causing excess liquid absorption from airway surfaces; decreased periciliary liquid; impaired cilia function; and, thus, a reduction in MCT (5). However, culture models do not recapitulate the complexity of MCT because mucus cannot enter or escape cultures and the model lacks submucosal glands, which are estimated to produce ~95% of mucus in large airways (4). After infusing methacholine, we found that periciliary liquid depth did not differ by genotype (fig. S2B), consistent with previous studies in pigs and humans with CF (13, 14). However, mucus overlying cilia was often dislodged during processing, and we could not assess potential effects of reduced volume secretion by CF submucosal glands (15, 16). Together, the results suggested that MCT might be disrupted by a CF abnormality in glands rather than surface epithelia. To test this, we flooded tracheas from methacholine-treated piglets with saline in a volume >1000 times the periciliary liquid volume (Fig. 2A and movie S2). This intervention prevents airway surface epithelia from altering the volume or composition of liquid covering airways.

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Microdisks added to submerged non-CF airways rarely stopped as they progressed to the cranial and ventral edges (Fig. 2B and movie S2, A and B). In CF tracheas, some microdisks traveled like those on non-CF tracheas. However, CF decreased the time that microdisks were in motion by ~50% (Fig. 2C), in good agreement with in vivo data (Fig. 1F). These results further excluded periciliary liquid depletion as the cause of impaired MCT. Moreover, on some occasions, we could see and remove CF mucus strands with attached microdisks (movie S2C).

To probe further for CF mucus abnormalities, we examined the response to methacholine because it elicited CF MCT defects in vivo and ex vivo (Figs. 1 and 2) and it stimulates submucosal gland secretion, consisting of liquid and proteins including mucins, which confer key structural properties to mucus (11, 12). Cholinergic stimulation can also activate the CFTR, increase the driving force for anion secretion through the CFTR, and thereby enhance liquid secretion (17, 18). To visualize mucus in real time, we covered excised tracheas with saline that contained a dilute suspension of fluorescent nanospheres (40-nm diameter) (Fig. 3A and movie S3).

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When nanospheres attached to mucus, their increased local density highlighted strands and globules of mucus flowing over or linked to airways (movie S3A). Methacholine stimulated production of mucus strands that emanated from submucosal gland ducts and sometimes extended hundreds of micrometers from their anchor point (Fig. 3B and movie S3B). Strands grew in length from gland ducts (Fig. 3C). As mucus strands grew, they broke free and were carried up the airway (Fig. 3D and movie S3C). Mucus strands flowing over the surface sometimes attached to and stretched anchored strands. If the two strands separated, the strand attached to a submucosal gland often snapped back like a rubber band (Fig. 3E and movie S3D), indicating elasticity and tenacity, important properties of mucus. Reflected light studies (movie S3E), as well as scanning electron microscopy and immunocytochemistry studies (fig. S3, A and B), also revealed mucus strands emerging from submucosal gland ducts.

Because our preliminary observations suggested more mucus attached to CF airways, we developed a time-averaging procedure to preferentially visualize stationary mucus and provide panoramic views of entire tracheal segments (Fig. 4A, fig. S4, and movie S4). During a 15-min basal period, stationary mucus was rarely detected on non-CF airways (Fig. 4B and movie S4A). After adding methacholine, nanosphere-labeled mucus was mobile and therefore not visualized; it only became apparent after accumulation on ventral and cranial edges of airway segments. Under basal conditions, the surface of CF trachea revealed only small amounts of stationary mucus. However, methacholine stimulation generated a markedly different appearance, with many immobile mucus strands and globules failing to detach from submucosal gland ducts (or attaching to anchored strands) and not flowing across the airway (Fig. 4, C and E, fig. S5, and movie S4B).

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Failure of mucus to detach, despite being bathed in saline of controlled composition, indicates that CF mucus was abnormal before it emerged onto the airway surface. This result points to a defect in the submucosal glands; they express abundant CFTR (19, 20) and conduct Cl^– and HCO₃^– (21) that contribute to liquid secretion (11, 17, 22, 23). To test if the loss of anion transport might be responsible for altered mucus behavior in CF, we bathed non-CF trachea in HCO₃^–-free saline or saline containing bumetanide, which inhibits basolateral Cl^– entry into epithelia (23). Neither alone caused a failure of mucus detachment (fig. S6). However, when combined, they reproduced the CF phenotype of tethered mucus strands and globules (Fig. 4, D and F, and movie S4C). Tracing mucus strands to their source revealed submucosal gland ducts as their origin (movie S4D). Because ion transport by epithelia lining airways would not alter the composition of liquid covering the surface, these changes can be attributed to reduced anion secretion into submucosal gland lumens. The results are also consistent with previous findings that inhibiting both Cl^– and HCO₃^– secretion altered viscoelastic properties of submucosal gland secretions (4, 24).

To explore whether mucus impeded movement of the microdisks we used in vivo (Fig. 1), we applied them to non-CF epithelia in HCO₃^–-free saline plus bumetanide and found that they failed to move across the surface. Adding fluorescent nanospheres revealed microdisks attached to mucus (Fig. 4G and movie S4E). We obtained similar results in airways of piglets with CF (Fig. 4H and movie S4F).

Our findings identify impaired mucus detachment from CF submucosal glands as a primary defect that disrupts MCT. Na⁺ hyperabsorption by airway surface epithelia and periciliary liquid depletion (5) do not explain the data. These in vivo and ex vivo results link the loss of CFTR and reduced anion secretion to altered mucus that has a reduced ability to break free after emerging from glands. Both reduced Cl^–- and HCO₃^–-dependent liquid secretion (23, 24) and reduced HCO₃^– secretion (or acidic pH) (25–27) have been proposed to alter CF mucus properties. Our data suggest that neither alone may be sufficient to generate abnormal CF mucus.

In combination with our earlier results (28), these findings reveal that the loss of CFTR disrupts two lung defense processes: MCT and secreted antimicrobial activity. Vertebrates and invertebrates employ these two defenses at the point of contact with environmental pathogens. CF does not eliminate either defense, but it does reduce their effectiveness. A vicious cycle caused by partial disruption of two processes may partly explain greater severity of lung disease in CF compared with primary ciliary dyskinesia, which obliterates MCT (29), because compromising one defense may accentuate the other defect. For example, mucus that fails to detach would impair MCT and provide a nidus for bacteria to grow under conditions that promote resistance to host defenses already weakened by CF (28, 30). Conversely, reduced antibacterial activity could precipitate infection that triggers submucosal gland secretion, and defective mucus detachment would impair MCT. Inflammation resulting from both defects would evoke submucosal gland hypertrophy, further increasing the amount of static mucus. Because newborns are universally screened for CF in many countries, an opportunity for early intervention exists. Our data suggest that submucosal glands and the mucus tethered to them may be targets for early treatment and that MCT assays could report therapeutic efficacy.