Pathological mucus and impaired mucus clearance in cystic fibrosis patients result from increased concentration, not altered pH

Abstract

Cystic fibrosis (CF) is a recessive genetic disease that is characterised by airway mucus plugging and reduced mucus clearance. There are currently alternative hypotheses that attempt to describe the abnormally viscous and elastic mucus that is a hallmark of CF airways disease, including: 1) loss of CF transmembrane regulator (CFTR)-dependent airway surface volume (water) secretion, producing mucus hyperconcentration-dependent increased viscosity, and 2) impaired bicarbonate secretion by CFTR, producing acidification of airway surfaces and increased mucus viscosity.A series of experiments was conducted to determine the contributions of mucus concentration versus pH to the rheological properties of airway mucus across length scales from the nanoscopic to macroscopic.For length scales greater than the nanoscopic, i.e. those relevant to mucociliary clearance, the effect of mucus concentration dominated over the effect of airway acidification.Mucus hydration and chemical reduction of disulfide bonds that connect mucin monomers are more promising therapeutic approaches than alkalisation.

Figure 1:

Relevant biophysical size scales in mucus, showing mucin polymers (black lines) with an average correlation length, or mesh size, of 300 to 500 nm. 1-μm diameter beads (Blue) are larger than the correlation length. 250-nm beads (red) are sufficiently near to the mesh size that their diffusive motion reflects both the polymeric mucins network as well as the small proteins and solvent that permeate the intra-mucin space. The 5 nm probes (Purple) that are typically used in FRAP assays report the rheological properties of the solvent / small protein component of mucus. B: Frap recovery time ratio of mucus ratioed to saline recovery time for HBE mucus and CF sputum at various concentrations ranging from 3 to 17% solids. C: Recovery times for HBE mucus (no significance, p = 0.6). D: Recovery times for whole, raw sputum at 5 and 15% CO2, resulting in a change in pH of 7.2 to 6.6 (no significances, p = 0.19). E: Recovery times for filtered, mucin-less solvent component of sputum at the same CO₂’s.

Figure 2:

Microbead Rheology: A: PGM (20mg/ml (Blue) and 50mg/ml (Red)) MSD curves at pH 6, 7, and 8. B: BSM (20mg/ml (Blue) and 50mg/ml (Brown)) MSD curves at pH 6, 7, and 8. C: HBE mucus 2% solids (Blue), and 4% solids (Red) MSD curves at pH 6, 7, 8. D: CF HBE mucus 2% solids (Blue) and 4% solids (Red). In each case, the results of two-way ANOVA showed significant dependence of MSD on concentration (p << 0.01) and no significance on pH (P > 0.05).

Figure 3:

Macroscopic Rheology of two concentrations of mucus at pH 6,7, and 8. A: Complex viscosity, (η*), of 20 (white) and 50 mg/ml (grey) PGM. B: Complex viscosity of 20 (white) and 50 mg/ml (grey) BSM. C: Complex viscosity of 2.0% (white), and 4% (grey) normal HBE mucus. D: Complex viscosity of 2.0% (white), and 4% (grey) CF HBE mucus. Two-way ANOVA showed that the complex viscosities of PGM and normal and CF HBE were significantly dependent on concentration (p = 0.031 and p = 0.038), while BSM was of borderline significance (p = 0.05). In no case was there a significant correlation between pH and complex viscosity (p = 0.46 for PGM, p = 0.77 for BSM, and p = 0.83 for HBE). For reference, the viscosity of water is ~ 0.001 Pa·s.

Figure 4:

Effect of pH on MCT, osmotic pressure, and PCL height A: Effect of pH on mucus osmotic pressure (●) and PCL height (△) of 6.5% solids mucus on HBE culture. Note, the PCL height is normally ~ 7μm with a normal 2% solids (100 Pa) mucus layer apposed to it. B: Example trace of the effects of acidification (5% CO₂, pH 7.2, to 15% CO₂, pH 6.6) compared to changes in mucus concentration (by dehumidification) on MCT rate during a single experiment. C: Summary of the MCT rates comparing changes in concentration, 2.8% solids (△) vs. 12.6% solids (●) vs. pH (6.6, 7.2, and 7.9).

Figure 5:

CF sputum targets and candidate muco-corrective therapeutic strategies. A: Complex viscosity vs. % solids for CF sputum exhibited a significant relationship (n = 11, p = 0.006, r² = 0.58). B: Complex viscosity (η*) vs. pH for CF sputum samples exhibited no significant correlation (p = 0.42). C: Complex viscosity of pooled, homogenized CF sputum at 5.2 (grey) and 2.6% (white) solids, pH 6, 7, and 8. Two-way ANOVA indicated that complex viscosity was correlated to concentration (p = 0.013), but not pH (p = 0.10). D: Reduction of normal HBE mucus by DTT. The reduction of mucus by 10 mM DTT was found to be significantly different than control (p = 0.027). E: Reduction of 5% pooled CF sputum shows a similar reduction when treated with 10mM DTT (p = 0.008). For reference, the viscosity of water is ~ 0.001 Pa·s.