All animal experiments at UAB were conducted in accordance with UAB IACUC approved protocols. Experiments were conducted using two different rat strains. SD-CFTR^tm1sage rats (Horizon Discovery, St. Louis, MO), either the CFTR^−/− (termed KO) or their littermate controls, CFTR^+/+ (termed WT), were bred and genotyped as previously described (Tuggle et al., 2014). In brief, heterozygote (CFTR^+/−) male and female were paired to generate WT and KO pups. Some experiments used the humanized-G551D-CFTR strain (Birket et al., 2020), either homozygous G551D (termed hG551D) or their littermate controls without the G5521D insert (termed WT). This rat strain was bred and genotyped as previously described. Heterozygote (CFTR^hG551D/−) male and female were paired to generate WT and hG551D pups, as above. All animals were bred and housed in standard cages maintained on a 12 h light/dark cycle with ad libitum access to food and water, in temperatures ranging from 71°F–75°F. KO and hG551D, along with WT controls, were maintained on a standard rodent diet with supplemental DietGel 76A (Clear H20, Westbrook, ME, United States) and 50% Go-LYTLEY (Braintree Laboratories, Inc., Braintree, MA, United States) added to the water from weaning, as a means to reduce mortality from gastrointestinal obstruction. WT and KO rats were assayed at 1, 3, or 6 months of age, while the hG551D studies were conducted at 6 months of age. Groups were split as evenly as possible between males and females.

Rats were euthanized via intraperitoneal injection of 500 μL pentobarbital sodium (390 mg/ml). Rats were exsanguinated, the thoracic cavity exposed, and intubated via the lower trachea. 5 ml of sterile, cold PBS pushed into the lungs and recollected into a separate sterile syringe using a two-way stopcock. Bronchoalveolar lavage fluid (BALF) was centrifuged at 1200 rpm for 7 min, and the supernatant transferred for cytokine and mucin analysis. IL-1β was analyzed by ELISA (Abcam, Cambrige, MA, United States).

Muc5b and Muc5ac were detected on nitrocellulose membranes using modified dot blot methods (Thornton et al., 1996) and antibodies selective for each mucin (Muc5b ab121025, lot# GR3383105-1; Muc5ac ab24071, lot# GR3321372-2) Abcam, Cambridge, MA, United States), as previously performed (Henderson et al., 2022). Signal was detected with HRP-secondary antibodies and the SuperSignal West Femto Maximum Sensitivity Substrate (Thermofisher Scientific, Grand Island, NY, United States). Blots were detected for chemiluminescence and analyzed by densitometry using ImageJ software. Samples were normalized to age-matched WT control average values.

Lung tissue harvested from WT, KO, and hG551D rats was homogenized, followed by Rneasy (Qiagen, Venlo, Netherlands) extraction and purification, following the manufacturer instructions. RNA was converted to cDNA and amplified using the Taqman RNA-to-CT 1-Step Kit (ThermoFisher Scientific, Waltham, MA, United States), according to instructions. Taqman probes for Muc5b (Rn01502008_m1), and Muc5ac (Rn01451252_m1), were compared to rat Gapdh as a housekeeping gene, and normalized to WT. Amplifications were conducted using a ProFlex PCR System (Applied Biosciences, ThermoFisher Scientific, Waltham, MA, United States). Data were analyzed to present ddCt.

Tracheae and lungs were immersion fixed in 10% phosphate-buffered formalin and embedded in paraffin blocks for sectioning, as previously performed (Tuggle et al., 2014). Sections were stained with hematoxylin and eosin (H&E) or alcian blue Periodic acid Schiffs (ABPAS).

Tracheae were excised from WT and KO rats, at baseline or treated with methacholine as below. Tracheae were opened along the dorsal side and placed on gauze soaked in F12 media. 5 μL of 10% Alcian Blue dissolved in PBS was pipetted into the lumen, evenly distributed along the length. Tracheae were imaged followed by three successive washes with 20 μL of PBS and imaged again. Using ImageJ, the overall density of the Alcian Blue compared to the surface area of the trachea before washing was used to calculate the % area covered. The surface area of the trachea after the wash steps was calculated to indicate the % of Alcian Blue retained.

Functional microanatomic measurements of ex vivo tissue were performed using micro-Optical Coherence Tomography (μOCT), a high-speed, high-resolution microscopic reflectance imaging modality, as previously described (Liu et al., 2013; Chu et al., 2016). Trachea were excised and immediately placed on gauze soaked in F12 media, such that the apical surface of the trachea remained media-free, and incubated under physiologic conditions (37°C, 5% CO₂, 100% humidity; using live-cell imaging incubation systems, Carl Zeiss, Oberkochen, Germany). Trachea were allowed to equilibrate for 30 min before imaging. Mucociliary transport (MCT) rate was determined using time elapsed and distance traveled of native particulates in the mucus over multiple frames. For each trachea, images were acquired at standard distances along the ventral surface with the optical beam scanned along the longitudinal axis.

Particle-tracking microrheological techniques were used to measure viscosity of mucus on rat tracheae in situ. Tracheae were treated with 0.1% benzalkonium chloride (Acros Organics, New Jersey, United States) 1 h at 37°C to stop ciliary beating. Polystyrene (PS) beads were used as previously described (Chu et al., 2016). Baseline μOCT images were acquired, tracheae were incubated for 30 min at 37°C on media with 100 μM acetylcholine, and then μOCT images were acquired. Images were analyzed using ImageJ and the SpotTracker plugin (http://bigwww.epfl.ch/sage/soft/spottracker/SpotTrackerX2D_.jar). Resulting particle tracks were analyzed with custom Matlab procedures to compute mean squared displacement (MSD) while subtracting spurious bulk motion common to all tracks. Dynamic viscosity was derived from MSD by application of the generalized Stokes-Einstein relation.

WT and KO rats at 6 months of age received methacholine (MilliporeSigma, St. Louis, MO, United States) administration to stimulate mucus release in the airways (Fischer et al., 2019). Methacholine was reconstituted in sterile normal saline and administered at a dose of 10 μg/kg/day via subcutaneous injection. Rats received methacholine daily for 7 days before sacrifice for tissue collection. hG551D rats received administration of ivacaftor, obtained from Selleckchem (Houston, TX, United States), and suspended in methylcellulose. Rats were dosed for 14 days with at 30 mg/kg/day or 3% methylcellulose vehicle by oral gavage.

Increased mucin concentrations of airway secretions in the CF lung have been long documented (Button et al., 2012; Button et al., 2016), but it is not clear whether this observation is because the CF epithelium is producing more mucin protein or because the mucin that is secreted is not cleared via mucociliary transport (MCT). Our lab has previously shown that tracheal MCT decreases as the rats age but has not until now measured the content of the specific mucins in airway secretions, or determined if increased mucin production correlates with decreasing MCT. To determine this, we measured Muc5b and Muc5ac secretions in the airways, via bronchoalveolar lavage fluid (BALF), as well as concentrations of the cytokine IL-1β, which has been shown to increase mucin production as part of a positive feedback signaling loop (Chen et al., 2019). Importantly, the rats that were included in this study were not infected (Tuggle et al., 2014), thus allowing us to assess mucin production at baseline. Compared to the 1 month WT control, the amount of Muc5b did not change as the WT littermates aged, while the amount of Muc5b present in the CFTR-KO airways increased significantly by 3 months of age, remaining high in the KO rats at 6 months of age as well (Figure 1A). Similarly, the amount of Muc5ac in the airways did not change as the WT rats aged, while the CFTR-KO had significantly higher Muc5ac by 6 months of age (Figure 1B). Correspondingly, concentrations of the cytokine IL-1β in the BALF increased in the CFTR-KO by 3 and 6 months of age, significantly higher than the amount of IL-1β in the BALF of the WT littermates (Figure 1C), even considering that IL-1β increases in the WT over age (with no statistical difference between 1 and 3 months, and the 6 months WT increased over 1 month, with a p value < 0.0001).

We also extracted mRNA from the lungs of WT and KO rats at 1, 3, and 6 months of age, to assess for gene production of Muc5b and Muc5ac. Again, compared to the 1 month WT rats, 3 and 6 months WT rats had a slightly higher, though not statistically significant, increase in Muc5b (Figure 1D) and Muc5ac (Figure 1E) gene expression. Compared to the WT, the CFTR-KO gene expression of each mucin was again slightly increased but not significantly different. This suggests that the excess mucin in the airways of CFTR-KO rats at 3 and 6 months of age is due to accumulation, rather than overproduction.

In previous studies, we have found that the KO rats have higher numbers and size of goblet cells in the large airways as they age, compared to WT (Birket et al., 2018). While gene expression of Muc5b and Muc5ac was no different between the two genotypes, at any age, we wondered if the epithelial layer of the CFTR-KO had more mucins in storage, prepared for release into the airway in the event of an infection or in response to an airway irritant. To determine if this was the case, we treated WT and KO rats at 6 months of age with systemic methacholine, to cause release of any mucus granules into the airway. After 7 days of treatment, we assessed the lungs and BALF of the WT and KO rats for the same metrics. We found again that, compared to WT rats that had received methacholine, KO rats had higher amounts of Muc5b (Figure 2A) and Muc5ac (Figure 2B). Correspondingly, there was more IL-1β in the KO rat BALF as well (Figure 2C). However, comparing the change in each mucin in the airway over what was measured in the baseline 6 month old rats revealed some important physiology. WT rats had slightly less Muc5b after methacholine compared to baseline (Figure 2D) and no change in Muc5ac (Figure 2E). This suggests that methacholine resulted in overall increased mucin clearance from the airways, rather than increasing the concentration in the airway. In contrast, compared to the baseline rats, KO rats treated with methacholine had increased Muc5b (Figure 2D) and increased Muc5ac (Figure 2E), indicating the KO rats have increased mucin stores that are not able to be cleared via the normal mucociliary clearance process. This was confirmed on histological examination. WT rats at baseline had very few mucus-positive stained cells, by AB-PAS and no detectable mucus in the airway lumen (Figure 2F). Methacholine administration resulted in an increase in mucus-positive stained cells in the epithelial layer but did not cause mucus to accumulate in the airways. KO rats at baseline had both increased numbers of mucus-positive stained cells, by AB-PAS, and some mucus strands present on the surface of the epithelium (Figure 2F, black arrows) compared to the WT. However, the KO rats treated with methacholine had dramatically increased numbers of mucus-positive stained cells as well as mucus detected along the surface of the epithelium. The accumulations on histological examination suggest that there is adherence of the mucin to the epithelial layer in the KO rat only.

In order to determine if the mucus in the KO rat is more adherent to the epithelia, we used a modified version of retention measurement, adapted from previous methods conducted on piglet tracheae (Ermund et al., 2017a). In this experiment, tracheae were excised from WT and KO rats at 6 months of age and incubated at physiologic conditions. Alcian Blue, dissolved in PBS, was applied to the apical surface of the tracheae, incubated to allow binding to the mucus on the airway surface, and imaged. Repeat washing steps removed mucus that was not adherent to the airway epithelium. Comparing images from before and after the wash steps allowed us to calculate a percent of Alcian Blue retained. Representative images are shown from the WT and KO rats at baseline, both before (Figure 3A) and after (Figure 3B) washing, as well as the rats treated with methacholine, both before (Figure 3C) and after (Figure 3D) washing. In the WT trachea, very little mucus is retained after the wash steps, in both baseline (Figure 3E) and methacholine (Figure 3F) conditions. However, there was no difference in the mucus present on the KO rat tracheae after the wash steps at baseline (Figure 3E) or in the rats treated with methacholine (Figure 3F). As expected, trachea from KO rats treated with methacholine had more mucus present compared to the baseline conditions. These data indicate that the mucus secreted on the surface of the KO rat airway is adherent to the epithelium.

Because older KO rats had increased amounts of both Muc5b and Muc5ac, an increase that coincided with changes to mucus transportability, we wanted to know if one mucin was predominantly associated with the previously observed decrease in MCT rates or increase in mucus effective viscosity (Birket et al., 2018). To determine this, we calculated the ratio of Muc5b:Muc5ac in each BALF sample, including all three ages of WT and KO rats, and plotted the results against the previously observed viscosity and transport measurements. When compared to effective viscosity, there was a moderate correlation between the Muc5b:Muc5ac ratio and effective viscosity in samples from WT rats (r² = 0.4112, Figure 4A), although the trendline was not significantly different than zero (p = 0.244). However, in the KO rat samples, the trendline was highly correlated (r ² = 0.7845, Figure 4A) and the slope was significantly non-zero (p < 0.05). These data indicate that in the KO, the more Muc5b compared to Muc5ac in the mucus sample, the higher the viscosity. Interestingly, there was no relationship between the Muc5b:Muc5ac ratio and MCT rates in either WT or KO rat samples (Figure 4B), indicating that transportability is not dependent upon the amount of single mucin.

Previous studies have determined that highly effective modulator therapies (HEMT), such as ivacaftor, can increase mucociliary clearance in patients with targeted mutations (Donaldson et al., 2018). We have similarly shown that ivacaftor administration to rats with a humanized-G551D version of the CFTR protein (hG551D rats) have normalization of MCT rates and effective viscosity of airway mucus (Birket et al., 2020), returning to WT values. We wanted to know if these corrections were due to a decrease in mucins present in the airway secretions or due to a reduction in mucus adhesion to the epithelial surface. To determine this, we treated 6 month old hG551D rats with either ivacaftor or methylcellulose vehicle for 14 days, and then identified the content of Muc5b and Muc5ac. Compared to age-matched WT rats, hG551D rats had more Muc5b detected in the BALF (p < 0.0001, Figure 5A). Treatment of the hG551D rats with ivacaftor normalized the Muc5b content to WT levels. Similarly, hG551D rats treated with vehicle alone had significantly more Muc5ac in the BALF (p < 0.001, Figure 5B), which was also decreased when the rats were treated with ivacaftor. Additionally, vehicle treated hG551D rats had no difference between the before and after wash mucus staining in the retention test. However, treatment with ivacaftor resulted in reduced mucus adhesion (Figure 5C), with percent retention values after wash reduced, similar to the WT (p < 0.05 for each).