Airway Mucus Hyperconcentration in Non-Cystic Fibrosis Bronchiectasis

Abstract

Rationale: Non-cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized.Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity.Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured.Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV₁ and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non-cystic fibrosis bronchiectasis mucus concentration by 5%.Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.

Keywords: gene expression; induced sputum; mucin.

Figure 1.

The biochemical properties of induced sputum from individuals with non–cystic fibrosis bronchiectasis (NCFB) and healthy control subjects. (A) Mucus percent solids, (B) total mucin concentrations, and (C) extracellular DNA concentration were each significantly higher in individuals with NCFB (gray circles) than in healthy control subjects (white circles). (D) Total mucin (Spearman rho [95% confidence interval] = 0.81 [0.73–0.88], P < 0.001) and (E) extracellular DNA concentrations (Spearman rho [95% confidence interval] = 0.72 [0.60–0.84], P < 0.001) correlated significantly with mucus percent solids (NCFB: gray circles; control subjects: white circles). (A–C) Individual data points, median, and interquartile ranges are shown; Mann-Whitney nonparametric test was used. n = 99 subjects with NCFB for all panels; n = 15 healthy control subjects; ****P < 0.0001. wt = weight.

Figure 2.

Mucus percent solids and mucin concentration in induced and spontaneously expectorated sputum from individuals with non–cystic fibrosis bronchiectasis from BLESS (Bronchiectasis and Low-Dose Erythromycin Study) (n = 99) and the University of North Carolina at Chapel Hill (UNC) Hospitals (n = 15). There were no differences in the (A) mucus percent solids or (B) total mucin concentration in induced sputum samples from individuals with bronchiectasis from the BLESS study and induced sputum samples from individuals with bronchiectasis at UNC Hospitals. Hypertonic saline–induced sputum samples had significantly lower mucus percent solids and total mucin concentrations than spontaneously expectorated sputum samples from the same individuals with bronchiectasis recruited from UNC Hospitals. Statistics: bars represent median and interquartile range; Mann-Whitney nonparametric test was used; *P < 0.05. wt = weight.

Figure 3.

Mucin glycoproteins in induced sputum from individuals with non–cystic fibrosis bronchiectasis and healthy control subjects. (A) Quantification of MUC5B (mucin 5B) (Ai) and MUC5AC (Aii) by internal standard labeled mass spectrometry and comparisons of MUC2 to MUC5AC and MUC5B by peptide intensity (Aiii). (B) Representative blots after agarose gel electrophoresis of denatured and reduced proteins extracted from sputum showing staining of all mucins with periodic acid–Schiff (PAS) staining and immunoblotting for MUC5AC and MUC5B (LCI = low-charge isoform; HCI = high-charge isoform). Note that the band pattern of the combined MUC5AC/MUC5B overlay (bottom panel) matches exactly with that in the PAS blot (top panel) for each sample. (C) Quantification of HCI and LCI of MUC5B from agarose gel electrophoresis blots. Statistics: box-and-whisker plots display median, interquartile ranges, and range; Mann-Whitney nonparametric test was used; ****P < 0.0001. LFQ = label-free quantification; tot = total.

Figure 4.

The biophysical properties of induced sputum in individuals with non–cystic fibrosis bronchiectasis (NCFB). (A) Osmotic pressure, (B) viscous moduli, (C) elastic moduli, and (D) complex viscosity of induced sputum were higher in individuals with NCFB (gray circles) than in healthy control subjects (white circles). The (E) osmotic pressure (Spearman rho [95% confidence interval] = 0.71 [0.59–0.82], P < 0.001) and (F) complex viscosity (0.49 [0.32–0.66], P < 0.001) of induced sputum correlated significantly with mucus percent solids (NCFB: gray circles; control subjects: white circles). Dashed line/PCL = periciliary layer osmotic pressure. (A–D) Individual data points, median, and interquartile ranges are shown; Mann-Whitney nonparametric test was used. n = 99; **P < 0.01 and ****P < 0.0001. G = viscous modulus; wt = weight.

Figure 5.

Association between mucus percent solids and clinical outcomes in BLESS (Bronchiectasis and Low-Dose Erythromycin Study) subjects with non–cystic fibrosis bronchiectasis (NCFB). (A) The high-resolution chest computed tomography bronchiectasis extent score significantly correlated with FEV₁ (Spearman rho [r] [95% confidence interval (CI)] = 0.40 [−0.61 to −0.19], P < 0.001). (B) FEV₁ significantly correlated inversely with mucus percent solids (r [95% CI] = −0.22 [−0.43 to −0.01], P = 0.03), and (C) mucus concentration correlated positively with high-resolution chest computed tomography extent scores (r [95% CI] = 0.41 [0.19–0.63], P < 0.001). Mucus percent solids was significantly associated with the concentration of (D) IL-8 (r [95% CI] = 0.48 [0.32–0.64], P < 0.001) and (E) IL-1β (r [95% CI] = 0.40 [0.22–0.56], P < 0.001) in sputum. (F) Ability of mucus concentration (percent solids) to diagnose NCFB. Area under the receiver operating characteristic (ROC) curve = 0.9603. Area under the ROC curve for total mucins in NCFB versus control cohorts = 0.949 (see Figure E5). wt = weight.