Disulfide disruption reverses mucus dysfunction in allergic airway disease

Abstract

Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma.

Fig. 1. Polymeric mucins in asthmatic airways are targets for disulfide disruption.

a Alcian blue/periodic acid-Schiff (AB-PAS) stained tissues from the lungs of patients (n = 2) who died during asthma exacerbations demonstrate mucin glycoproteins in large and small airways. Scale bars, 500 μm and 100 μm in insets (trachea), 200 μm (bronchus and bronchiole). b MUC5AC and MUC5B assemble via amino (N-) and carboxyl (C-) terminal disulfide bonds that are sensitive to reducing agents. c A mucus plug in a fatal asthma airway was examined in consecutive sections stained with AB-PAS (purple), or labeled with biotinylated-maleimide (brown) after incubation at 37 °C for 10 min with saline vehicle (Veh) or tris(2-carboxyethyl)phosphine (TCEP, 10 mM). Scale bars, 250 μm; image representative of airways from n = 2 patients. d–f. Expectorated sputum (n = 3 patients) was treated with TCEP (0.1–10 mM, 37 °C, 30 min), separated by electrophoresis (1% SDS/agarose, non-reducing), and detected by immunoblot for MUC5AC and MUC5B (d). Note that full reduction causes epitope loss for both anti-mucin antibodies resulting in decreased signal intensities at higher TCEP concentrations in d. Diffusion of 2-µm carboxylated micro-particles was evaluated in mucus samples aspirated from fatal asthma bronchi (e, f). Compared to vehicle controls (Veh, cyan, n = 1303), particle mean square displacement (MSD) increased significantly after TCEP (magenta, n = 1425). Bars in e are means ± sem of summarized linear-scale values, with individual points (open circles). Data in f are log distributions of particles with curves showing Gaussian non-linear fits (r² = 0.99 for Veh and TCEP). Significance was determined by two-tailed Mann–Whitney U-tests with p-value and “*” denoting significance from Veh in f. Inverted arrows in f indicate locations of median values. Source data are provided as a Source Data file.

Fig. 2. Mucolytic treatment improves mucus function in allergic mouse airways.

a Mucus was probed in tracheal preparations ex vivo using 100 nm muco-inert particles (MIPs). b, c In Aspergillus oryzae extract (AOE) challenged mice receiving 500 mM aerosol TCEP treatment (Tx, magenta, n = 13 biological replicates), MIP diffusion measured as mean square displacement (MSD) increased significantly compared to vehicle (Veh, cyan, n = 14 biological replicates) and non-AOE exposed animals (Healthy, gray, n = 7 biological replicates). Cumulative distribution data in b show the distributions of MSD values (means ± sem). Scatter plot data in c show individual median MSD values per animal. d–f Mucociliary clearance was tested in AOE challenged mice treated by nose-only aerosol with TCEP in a concentration dependent manner or Veh, followed by immediate lung lavage. Lectin blot analysis using Ulex europaeus agglutinin I (UEA-1) (α1,2-fucose) in d shows disruption of mucin polymers in TCEP-treated animals (Tx, 500 mM, magenta) compared to vehicle (cyan). Image shows four samples per group. Intensities of high molecular weight (polymerized) and low molecular weight (depolymerized) mucins were evaluated using Image Studio software (e). Numbers of total eosinophils (f) recovered in lung lavage decreased significantly in Tx (magenta, n = 8 biological replicates) vs. Veh (cyan circles, n = 6 biological replicates) exposed animals. Lines and error bars in c–f are means ± sem. ‘*’ denotes significance using a cut-off of 0.05 determined by unpaired two-tailed t-tests using a two-stage step-up method at a 5% false discovery rate from Veh in b, by two-tailed Mann–Whitney U-test in c, and by non-parametric one-way ANOVA in e, f. Source data are provided as a Source Data file.

Fig. 3. Mucolytic treatment reverses allergic airway hyperreactivity.

Dose response curves to methacholine (MCh, 4–64 μg kg⁻¹ min⁻¹, i.v.) were generated in AOE challenged allergic wild type (WT) mice (magenta, n = 7 biological replicates) treated with TCEP between MCh doses (100 mM, inverted yellow triangles). For comparison, saline challenged non-allergic WT mice (gray, n = 6 biological replicates), AOE challenged allergic WT mice (cyan, n = 12 biological replicates), and AOE challenged Muc5ac^−/− mice (black, n = 4 biological replicates) were treated with nebulized vehicle (Veh) during i.v. MCh dose response tests. Values are means ± sem. For each mouse, dose response curves were fitted by log-linear best-fit regression analysis, and slopes of regression lines were analyzed by one-way ANOVA. “*”, p < 0.05 using Dunnett’s test for multiple comparisons relative to AOE-challenged Veh-treated WT mice (p-values are shown). Total lung resistance (R_L in a, b), conducting airway resistance (R_AW in c, d), tissue resistance (G_TI in e, f) and tissue elastance (H_TI in g, h) are shown. Source data are provided as a Source Data file.

Fig. 4. Mucolytic treatment disrupts mucus plugging.

a AB-PAS stained lungs obtained after methacholine dose response tests show mucin glycoproteins obstructing airspaces in vehicle (Veh, n = 8 biological replicates) treated mice that are disrupted by mucolytic treatment (Tx, n = 7 biological replicates). Scale bars, 500 μm (low power) and 25 μm (high power). b–j Calculated mucus volumes (b, e, h), mean fractional occlusion (c, f, i), and heterogeneous plugging (d, g, j) were significantly decreased in AOE-challenged mice receiving mucolytic (Tx, magenta, n = 7 biological replicates) compared to controls (Veh, cyan, n = 8 biological replicates). Mucolytic treatment significantly reduced obstruction across all airways (b–d), and this effect was most prevalent in bronchi (e–g). Although total mucus volume in bronchioles was low (h), occasional mucus aggregates were obstructive and are sensitive to TCEP treatment (i, j). Data in b, c, e, f, h, and i are means ± sem on scatter plots, with p-values shown and significance (*) indicating p < 0.05 by two-tailed Mann–Whitney U-test. Cumulative frequency distributions in d, g, and h show percentages of airways demonstrating occlusion, with circles and error bars identifying means ± sem, and “*” demonstrating significance by t-test using a two-stage step-up method at a 5% false discovery rate. Source data are provided as a Source Data file.