Acidic pH increases airway surface liquid viscosity in cystic fibrosis

Abstract

Cystic fibrosis (CF) disrupts respiratory host defenses, allowing bacterial infection, inflammation, and mucus accumulation to progressively destroy the lungs. Our previous studies revealed that mucus with abnormal behavior impaired mucociliary transport in newborn CF piglets prior to the onset of secondary manifestations. To further investigate mucus abnormalities, here we studied airway surface liquid (ASL) collected from newborn piglets and ASL on cultured airway epithelia. Fluorescence recovery after photobleaching revealed that the viscosity of CF ASL was increased relative to that of non-CF ASL. CF ASL had a reduced pH, which was necessary and sufficient for genotype-dependent viscosity differences. The increased viscosity of CF ASL was not explained by pH-independent changes in HCO3- concentration, altered glycosylation, additional pH-induced disulfide bond formation, increased percentage of nonvolatile material, or increased sulfation. Treating acidic ASL with hypertonic saline or heparin largely reversed the increased viscosity, suggesting that acidic pH influences mucin electrostatic interactions. These findings link loss of cystic fibrosis transmembrane conductance regulator-dependent alkalinization to abnormal CF ASL. In addition, we found that increasing Ca2+ concentrations elevated ASL viscosity, in part, independently of pH. The results suggest that increasing pH, reducing Ca2+ concentration, and/or altering electrostatic interactions in ASL might benefit early CF.

Figure 1. CF does not have major changes in airway mucin or ASL glycan composition.

(A) MUC5AC and MUC5B mRNA in trachea and primary epithelial cultures (data points are from individual pigs on the left and from cultures from individual pigs on the right; bars indicate mean ± SEM). Values are relative to MUC5AC in non-CF trachea or MUC5AC in non-CF epithelial cultures. n = 3–4, each from a different piglet. (B and C) Immunocytochemistry of non-CF and CF (B) trachea and (C) primary airway epithelial cultures. B shows MUC5AC (green), MUC5B (red), DAPI (nucleus, blue), and actin (gray). C shows MUC5AC (green), MUC5B (green), β-catenin (red), and DAPI (blue). Scale bars: 10 μm. (D) Western blot of MUC5B (lanes 2 and 3) and MUC5AC (lanes 4 and 5) in ASL isolated from non-CF and CF tracheas. Lane 1 shows high-molecular-weight standards. The arrowhead indicates migration of mucin. Similar data were obtained in 5 to 6 other experiments. (E) O-glycan structures released from ASL of methacholine-stimulated newborn pigs determined by MALDI-TOF mass spectrometry. Area of peak normalized to the protein concentration is shown. Corresponding proposed O-glycan structures are indicated below the mass. n = 3 non-CF and 3 CF ASL samples, each from a different pig; littermate controls were used.

Figure 2. CF ASL has an increased viscosity.

τ_ASL/τ_saline was measured in ASL from newborn CF and non-CF piglets or pig and human cultured epithelia. Each data point is from a different pig or human donor (mean ± SEM). (A) ASL removed from methacholine-stimulated newborn non-CF and CF littermates. n = 6 per genotype. (B) ASL on unperturbed, well-differentiated primary airway epithelial cultures. n = 21 per genotype (see also Supplemental Figure 3). (C) ASL on differentiated primary airway epithelial cultures from non-CF and CF humans (see also Supplemental Figure 4 for ASL pH of human epithelia). n = 7 per genotype. *P < 0.05 by unpaired Student’s t test. The dashed lines indicate the viscosity of saline.

Figure 3. CF ASL has an increased percentage of nonvolatile material and an increased sulfate content.

(A) Percentage of nonvolatile material in ASL collected from newborn piglets under basal conditions (n = 17 non-CF and n = 12 CF) and after administering methacholine (n = 16 non-CF and n = 6 CF) (2.5 mg/kg, i.v.). (B) ASL depth in unperturbed cultures of airway epithelia determined by z-scanning confocal microscopy. n = 11 per genotype. (C) τ_mucin/τ_saline of bovine salivary mucin (n = 6) and porcine gastric mucin (n = 4) exposed to 21% O₂ or 95% O₂ (oxidized condition). The dashed horizontal lines indicate the viscosity of saline. (D) Sulfate content in ASL collected from airway epithelia cultured from newborn CF and non-CF piglets. n = 6 per genotype. Data were normalized to the amount of protein. *P < 0.05 by unpaired Student’s t test. In A, B, and D, each data point is from a different pig, and error bars represent mean ± SEM. In C, data represent mean ± SEM and error bars are hidden by symbols in left graph.

Figure 4. Stimulating epithelial HCO₃^– secretion reduces ASL viscosity.

(A) ASL pH, τ_ASL/τ_saline, and ASL depth measured in non-CF and CF primary airway epithelial cultures before and after stimulation with 10 μM forskolin and 100 μM IBMX for 2 hours (F&I). Forskolin and IBMX were added to the basolateral medium. Each data point indicates epithelia from a different animal. (B) NaHCO₃ or NaCl (3 μl, 150 mM) was added to the apical surface of non-CF (blue) and CF (red) cultured airway epithelia, and τ_ASL/τ_saline and ASL pH were measured 5 minutes later (mean ± SEM). n = 6 non-CF and 4 CF, each from different piglets. The dashed horizontal lines indicate the viscosity of saline. *P < 0.05 by unpaired Student’s t test.

Figure 5. Increasing ASL pH reduces ASL viscosity.

(A) 21 mM NaHCO₃ (3 μl, 5% CO₂) or 68 mM NaHCO₃ (3 μl, 15% CO₂) was added to the apical surface of non-CF cultured airway epithelia; HCO₃^– concentration and CO₂ were balanced to achieve the same pH. τ_ASL/τ_saline and ASL pH were measured 5 minutes later. n = 9 epithelia per genotype, each from a different pig. (B) 24 mM NaHCO₃ (3 μl with 5% or 15% CO₂) was applied to the apical surface of non-CF cultured airway epithelia. Five minutes later, τ_ASL/τ_saline and ASL pH were measured. n = 6 epithelia per genotype, each from a different pig. (C) Non-CF cultured airway epithelia were exposed to 5% or 15% CO₂ in a humidified chamber at 37°C. τ_ASL/τ_saline and ASL pH were measured 5 minutes later. n = 6 epithelia per genotype, each from a different pig. (D) To eliminate HCO₃^–/CO₂, HEPES buffer (3 μl, 20 mM in saline) at a pH of 6.8 or 7.8 was applied to the apical surface of non-CF cultured airway epithelia. τ_ASL/τ_saline and ASL pH were measured 5 minutes later (see also Supplemental Figure 5, which indicates that changes in viscosity were not due to HEPES per se). n = 6 epithelia per genotype, each from a different pig. (E) Methacholine-stimulated ASL was collected from newborn non-CF (blue) and CF (red) pigs after methacholine stimulation and immediately assayed for τ_ASL/τ_saline and pH in a humidified chamber containing either 5% or 15% CO₂. The line is a linear regression. n = 5 pigs per genotype; littermate controls were used. The dashed horizontal lines indicate the viscosity of saline. *P < 0.05 by unpaired Student’s t test. Mean ± SEM.

Figure 6. An acidic pH does not increase ASL viscosity by inducing disulfide bond formation.

(A) Effect of tris(2-carboxyethyl)phosphine (TCEP; 10 mM, 3 μl in PBS for 1 hour) on τ_ASL/τ_saline of cultured non-CF and CF airway epithelia. n = 6 epithelia per condition, each from a different pig. (B) Effect of tris(2-carboxyethyl)phosphine on τ_ASL/τ_saline of cultured non-CF airway epithelia exposed to 5% or 15% CO₂. n = 5 epithelia per condition, each from a different pig. (C) Effect of IAA (25 mM, 3 μl in PBS for 15 minutes) on τ_ASL/τ_saline of cultured non-CF airway epithelia exposed to 5% or 15% CO₂ (see also Supplemental Figure 6A for ASL pH.). n = 6 epithelia per condition, each from a different pig. (D) Effect of 25 mM IAA on the τ_ASL/τ_saline response to an oxidizing environment (95% O₂ for 15 minutes) (see also Supplemental Figure 6B for ASL pH). n = 6–7 epithelia per condition, each from a different pig. The dashed horizontal lines indicate the viscosity of saline. *P < 0.05 by unpaired Student’s t test. Mean ± SEM.

Figure 7. pH affects ASL viscosity by altering ionic interactions.

(A) Effect of heparin (3 μl of 1 mg/ml in PBS for 2 hours) on τ_ASL/τ_saline of non-CF and CF cultured airway epithelia exposed to 5% or 15% CO₂ (see Supplemental Figure 7 for ASL pH). n = 5–6 per condition, each from a different pig. (B) Effect of adding 0.9% or 7% NaCl (4 μl added to 10 μl ASL for 30 minutes) on τ_ASL/τ_saline. ASL was collected from non-CF and CF piglets that were stimulated with methacholine. ASL was exposed to 5% or 15% CO₂ (see Supplemental Figure 8 for ASL pH). n = 6 per condition, each from a different pig. The dashed horizontal lines indicate the viscosity of saline. *P < 0.05 by unpaired Student’s t test. Mean ± SEM.

Figure 8. An increased Ca²⁺ concentration increases ASL viscosity.

(A) Effect of Ca²⁺ concentration on τ_ASL/τ_saline at varying ASL pH values. ASL (10 μl) collected from newborn non-CF piglets was studied after addition of 20 mM EGTA, 10 mM NaCl, 10 mM CaCl₂, or 100 mM CaCl₂ in 4 μl saline containing 20 mM HEPES at a pH of 7.35 or 6.8. The final Ca²⁺ concentrations were calculated based on volume of addition, ASL volume, Ca²⁺ in added solution, and an ASL Ca²⁺ concentration of 3 mM. n = 7 for 20 mM EGTA, 10 mM NaCl, and 10 mM CaCl₂ and n = 4 for 100 mM CaCl₂; n refers to the number of pigs. (B) Effect on τ_ASL/τ_saline of adding 4 μl of saline (20 mM HEPES, pH 7.35) containing 10 mM CaCl₂, 10 mM MgCl₂, 10 mM ZnCl₂, or 30 mM NaCl to 10 μl ASL for 30 minutes. ASL was collected from newborn non-CF pigs (see Supplemental Figure 10 for ASL pH). n = 6–7 per condition, each from a different pig. (C) Effect of 7% NaCl on Ca²⁺-induced increase in ASL viscosity. ASL was collected from newborn non-CF piglets and studied after addition of 20 mM EGTA (0 mM Ca²⁺) or 100 mM CaCl₂ (30 mM Ca²⁺ calculated as described for A). Additions were in 4 μl of 0.9% or 7% NaCl containing 20 mM HEPES at pH 6.8 (see Supplemental Figure 11 for pH values). n = 6 per condition, each from a different pig. The dashed horizontal lines indicate the viscosity of saline. *P < 0.05 by 1-way ANOVA. Mean ± SEM.