Electrolyte transport properties in distal small airways from cystic fibrosis pigs with implications for host defense

Abstract

While pathological and clinical data suggest that small airways are involved in early cystic fibrosis (CF) lung disease development, little is known about how the lack of cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to disease pathogenesis in these small airways. Large and small airway epithelia are exposed to different airflow velocities, temperatures, humidity, and CO2 concentrations. The cellular composition of these two regions is different, and small airways lack submucosal glands. To better understand the ion transport properties and impacts of lack of CFTR function on host defense function in small airways, we adapted a novel protocol to isolate small airway epithelial cells from CF and non-CF pigs and established an organotypic culture model. Compared with non-CF large airways, non-CF small airway epithelia cultures had higher Cl(-) and bicarbonate (HCO3 (-)) short-circuit currents and higher airway surface liquid (ASL) pH under 5% CO2 conditions. CF small airway epithelia were characterized by minimal Cl(-) and HCO3 (-) transport and decreased ASL pH, and had impaired bacterial killing compared with non-CF small airways. In addition, CF small airway epithelia had a higher ASL viscosity than non-CF small airways. Thus, the activity of CFTR is higher in the small airways, where it plays a role in alkalinization of ASL, enhancement of antimicrobial activity, and lowering of mucus viscosity. These data provide insight to explain why the small airways are a susceptible site for the bacterial colonization.

Keywords: cystic fibrosis transmembrane conductance regulator.

Fig. 1.

Large and small airways have different cellular compositions. A: image of intact airway tree dissected from the left lung of a newborn non-cystic fibrosis (CF) pig. The red square indicates where large airway epithelia was isolated, and the red circle indicates where small airway epithelia was isolated and used for expansion. B and C: hematoxylin and eosin (H&E) staining of large (B) and distal small (C) airways. In B, large airways consist of airway epithelial cells, submucosal glands (SMG), airway smooth muscle (ASM), and cartilage. Scale bar = 500 μm. In C, small airways lack complete cartilage ring support and SMG. Scale bar = 100 μm. D: Alcian blue staining of an intact right lung airway tree dissected from a newborn piglet. Scale bar = 1 cm. E: H&E staining of large cartilaginous airway. Scale bar = 2 mm. F: H&E staining demonstrates that only isolated cartilage plate is present in small airways. Scale bar = 1 mm.

Fig. 2.

Expanded small airway cells form differentiated epithelia when cultured at the air-liquid interface. A and B: expanded large and small airway cells cultured at the air-liquid interface form tight junctions [zonula occludens (ZO)-1, red] and develop cilia (acetylated α-tubulin, green). Nuclei (DAPI, blue), Scale bar = 20 μm. C and D: goblet cells are present in expanded large and small airway cells cultured at the air-liquid interface. Goblet cells (Muc5AC, green), tight junctions (ZO-1, red), and nuclei (DAPI, blue) are shown. Scale bar = 20 μm. E–H: cellular composition of the native large and small airways, which are mostly covered by ciliated cells (acetylated α-tubulin, green in E and F) with fewer goblet cells in small vs. large airway tissue (Muc5AC, green in G and H). Phalloidin was used to stain F-actin in red. DAPI staining for nuclei is in blue. Area surrounded by dotted box is enlarged in insets. Scale bar = 100 μm in large image and 20 μm in insets.

Fig. 3.

Gene expression profile between expanded large and small airway epithelia is different. A: top five significantly upregulated and downregulated genes in expanded small vs. large airway epithelia from non-CF pigs. N = 3 non-CF piglets. Three Transwell inserts of expanded epithelia from each pig were pooled to isolate RNA. ANOVA was used, and the P value was listed. B: validation of surfactant protein D [SFTPD (SP-D)] mRNA changes in native large and small airway tissue by qRT-PCR. Data were normalized to ZO-1. N = 6 non-CF piglets. *P = 0.0498; Ratio-paired t-test. C and D: immunostaining of SP-D in native large and small airway tissue. The white arrowhead in D indicates the SP-D-positive surface epithelia in small airway but not in large airways. Scale bar = 60 μm in C and 40 μm in D.

Fig. 4.

Non-CF small airway cells have increased cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl⁻ current. A and B: summarized short-circuit current (I_sc) data of expanded large and small airway epithelial cells from non-CF (A) and CF (B) pigs. C: summary data of amiloride-sensitive ΔI_sc (ΔI_scAmil) in expanded large and small airway epithelia from non-CF and CF pigs. D: summary data of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive ΔI_sc (ΔI_scDIDS) in expanded large and small airway epithelia from non-CF and CF pigs. E: summary data of forskolin (F) + 3-isobutyl-1-methylxanthine (I)-stimulated ΔI_sc (ΔI_scF&I) in expanded large and small airway epithelia from non-CF and CF pigs. N = 11 non-CF piglets and 11 CF piglets. One Transwell insert of expanded epithelia from each pig was used. *P = 0.002 vs. non-CF large airway epithelia, Wilcoxon signed-rank test. F: summary data of GlyH-101 (a CFTR inhibitor)-sensitive ΔI_sc (ΔI_scGlyH) in expanded large and small airway epithelia from non-CF and CF pigs. N = 11 non-CF piglets and 11 CF piglets. One Transwell insert of expanded epithelia from each pig was used. *P = 0.0029 vs. non-CF large airway epithelia, Wilcoxon signed-rank test.

Fig. 5.

CFTR-mediated bicarbonate (HCO₃⁻) transport and airway surface liquid (ASL) pH are higher in non-CF small vs. large airway cells. A and B: representative I_sc traces of expanded large and small airway epithelial cells from non-CF (A) and CF (B) pigs. C: summary data of ΔI_scF&I in expanded large and small airway epithelia from non-CF and CF pigs. N = 7 non-CF piglets and 7 CF piglets. One Transwell insert of expanded epithelia from each pig was used. *P = 0.0313 vs. non-CF large airway epithelia, Wilcoxon signed-rank test. D: summary data of ΔI_scGlyH in expanded large and small airway epithelia from non-CF and CF pigs. N = 7 non-CF piglets and 7 CF piglets. One Transwell insert of expanded epithelia from each pig was used. *P = 0.0313 vs. non-CF large airway epithelia, Wilcoxon signed-rank test. E: calibration curve for ASL pH measurement. x-Axis indicates the ratio of fluorescence reading of SNARF at 580 vs. 640 nm at different pH values; n = 5, error bars are hidden by symbols. F: ASL pH in non-CF small airway epithelia is higher than that in non-CF large airway epithelia. ASL pH is similar in CF large and small airway epithelia. Epithelia were exposed to 5% CO₂ under stimulated conditions with cAMP-elevating agents (F&I). N = 7 non-CF piglets and 7 CF piglets. One Transwell insert of expanded epithelia from each pig was used. *P = 0.0469 vs. non-CF large airway epithelia, Wilcoxon signed-rank test.

Fig. 6.

CF small airway epithelia have higher ASL viscosity and lower bacterial killing ability compared with non-CF small airway epithelia. A: ASL viscosity as determined by fluorescence recovery after photobleaching of FITC-dextran in expanded small airway epithelia from non-CF and CF pigs. N = 6 non-CF piglets and 6 CF piglets. One Transwell insert of expanded epithelia from each pig was used. *P = 0.0043 vs. non-CF large airway epithelia, Wilcoxon rank-sum test. B: antimicrobial activity of expanded small airway epithelia from non-CF and CF pigs. Data are presented as the relative luminescence (RLU) of Staphylococcus aureus (Xen-29) as a percentage of control. N = 6 non-CF piglets and 6 CF piglets. One Transwell insert of expanded epithelia from each pig was used. *P = 0.0043 vs. non-CF large airway epithelia, Wilcoxon rank-sum test. C: antimicrobial activity of expanded small airway epithelia from non-CF and CF pigs as evaluated by S. aureus-coated grid assay. Data are presented as the percentage of live bacteria. N = 5 non-CF piglets and 5 CF piglets. One Transwell insert of expanded epithelia from each pig was used. *P = 0.0159 vs. non-CF small airway epithelia, Wilcoxon rank-sum test.