Mutations in the CFTR gene cause CF (1). Lung disease, the source of most mortality, arises from defective host defense that predisposes airways to bacterial infection. MCT defends airways by trapping pathogens in mucus and cilia propel them out of the lung (2–4). Finding abnormal MCT in advanced CF (3) and some studies in culture (5) have led to the assumption 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, as severity of CF lung disease increased, MCT deficits increased, and in other pulmonary diseases airway inflammation is also linked to MCT defects (8). Until now, lack of an animal model that replicates human CF and lack of sensitive MCT assays prevented ascertainment 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, CF pig airways 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).

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 % of microdisks cleared CF and non-CF lungs in 10 min (Fig. 1A,1B,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 % of time moving (Fig. 1D–1F). Thus, under basal conditions, CF MCT appeared intact.

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 (Fig. S2A). However, it tended to reduce the % of microdisks that cleared CF lungs (Fig. 1A,1B, Video 1). Measuring individual microdisk behavior revealed that methacholine accelerated maximal and mean microdisk speeds in non-CF, but not CF piglets (Fig. 1D,1E, S1C–S1E). The most striking difference between genotypes was that in CF piglets, methacholine reduced the % 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 CF piglets. A limitation of this in vivo study is that piglets were supine, anesthetized and did not cough; cholinergic stimulation might have produced a different response in awake animals.

Some studies of cultured airway epithelia reported that loss of CFTR increased Na⁺ channel activity, causing excess liquid absorption from airway surfaces, decreased periciliary liquid, impaired cilia function, and thereby slowed 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, periciliary liquid depth did not differ by genotype (Fig. S2B), consistent with studies in CF pigs and humans (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-fold periciliary liquid volume (Fig. 2A, Video 2). This intervention prevents airway surface epithelia from altering the volume or composition of liquid covering airways.

Microdisks added to submerged non-CF airways rarely stopped as they progressed to the cranial and ventral edges (Fig. 2B, Video 2A,2B). In CF tracheas, some microdisks traveled like those on non-CF trachea. 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 (Video 2C).

To probe further for CF mucus abnormalities, we examined the response to methacholine because it elicited CF MCT defects in vivo and ex vivo (Fig. 1,2), 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 can activate CFTR, increase the driving force for anion secretion through 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, Video 3).

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

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, S4, Video 4). During a 15 min basal period, stationary mucus was rarely detected on non-CF airways (Fig. 4B, Video 4A). 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 strikingly 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. 4C,4E,S5, Video 4B).

Failure of mucus to detach, despite being bathed in saline of controlled composition, indicated that CF mucus was abnormal before it emerged onto the airway surface. That result pointed to a defect in 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 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. 4D,4F, Video 4C). Tracing mucus strands to their source revealed submucosal gland ducts as their origin (Video 4D). 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. Then, adding fluorescent nanospheres revealed microdisks attached to mucus (Fig. 4G, Video 4E). Similar results occurred in CF airways (Fig. 4H, Video 4F).

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 loss of CFTR and reduced anion secretion to altered mucus that has a reduced ability to break free after emerging from glands. Reduced Cl⁻ and HCO₃⁻-dependent liquid secretion (23, 24) and reduced HCO₃⁻ secretion (or acidic pH) (25–27) have both been proposed to alter CF mucus properties. Our data suggest that neither alone may be sufficient to generate abnormal CF mucus.

These plus our earlier results (28) reveal that loss of CFTR disrupts two lung defense processes, MCT and secreted antimicrobial activity. Vertebrates and invertebrates employ these two defenses at point of contact with environmental pathogens. CF does not eliminate either defense, but reduces their effectiveness. A vicious cycle caused by partial disruption of two processes may, in part, explain greater severity of lung disease in CF compared to 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. Now that newborn CF screening is universal in many countries, there is an opportunity to intervene early. These data suggest that submucosal glands and mucus tethered to them may be targets for early treatment and that MCT assays could report therapeutic efficacy.