Airway mucins promote immunopathology in virus-exacerbated chronic obstructive pulmonary disease

Abstract

The respiratory tract surface is protected from inhaled pathogens by a secreted layer of mucus rich in mucin glycoproteins. Abnormal mucus accumulation is a cardinal feature of chronic respiratory diseases, but the relationship between mucus and pathogens during exacerbations is poorly understood. We identified elevations in airway mucin 5AC (MUC5AC) and MUC5B concentrations during spontaneous and experimentally induced chronic obstructive pulmonary disease (COPD) exacerbations. MUC5AC was more sensitive to changes in expression during exacerbation and was therefore more predictably associated with viral load, inflammation, symptom severity, decrements in lung function, and secondary bacterial infections. MUC5AC was functionally related to inflammation, as Muc5ac-deficient (Muc5ac-/-) mice had attenuated RV-induced (RV-induced) airway inflammation, and exogenous MUC5AC glycoprotein administration augmented inflammatory responses and increased the release of extracellular adenosine triphosphate (ATP) in mice and human airway epithelial cell cultures. Hydrolysis of ATP suppressed MUC5AC augmentation of RV-induced inflammation in mice. Therapeutic suppression of mucin production using an EGFR antagonist ameliorated immunopathology in a mouse COPD exacerbation model. The coordinated virus induction of MUC5AC and MUC5B expression suggests that non-Th2 mechanisms trigger mucin hypersecretion during exacerbations. Our data identified a proinflammatory role for MUC5AC during viral infection and suggest that MUC5AC inhibition may ameliorate COPD exacerbations.

Keywords: COPD; Innate immunity; Pulmonology.

Figure 1. Airway mucin expression during naturally occurring, virus-induced COPD exacerbations.

(A) A cohort of 40 patients with COPD was monitored prospectively. Sputum samples were taken during stable state (baseline), at presentation with an exacerbation associated with positive virus detection, and 2 and 6 weeks after exacerbation presentation. (B) Sputum MUC5AC and (C) MUC5B protein concentrations measured by ELISA. Baseline and peak (i.e., maximal concentrations of mucin detected during infection for each individual) concentrations of (D) MUC5AC and (E) MUC5B proteins during naturally occurring exacerbations. (F) Sputum MUC5AC and (G) MUC5B concentrations in individuals with frequent versus infrequent COPD exacerbations. (H) Sputum cell MUC5AC and (I) MUC5B mRNA expression. The red line in D and E indicates the mean concentration. In F and G, the box and whisker plots show the median (line within the box), the IQR (box), and the minimum-to-maximum values (whiskers). *P < 0.05 and **P < 0.01, by Mann-Whitney U test or Wilcoxon matched-pairs, signed-rank test.

Figure 2. Airway MUC5AC expression and correlation with immunopathology during experimental RV infection.

(A) Experimental outline. Fourteen patients with COPD and 10 healthy control volunteers underwent sampling at baseline and the indicated time points after RV-A16 infection. (B) Sputum MUC5AC concentrations in patients with COPD and healthy individuals, measured during RV-A16 infection. (C) Comparison of baseline (BL) and peak (i.e., maximal concentration detected during the infection for each individual) concentrations of MUC5AC in patients with COPD and healthy individuals. Individual data points are shown; red lines indicate the mean concentration. (D) Sputum MUC5AC to MUC5B ratio measured by mass spectrometry for 11 individuals with COPD and 10 healthy participants. (E) Correlation of peak sputum MUC5AC concentrations with inflammatory cell numbers and cytokine concentrations in sputum. Correlations between peak sputum MUC5AC levels and (F) sputum viral loads and (G) sputum neutrophil elastase. Change from baseline concentrations of (H) SLPI and (I) elafin. (J) Sputum MUC5AC concentrations in patients with COPD with positive (+ve) or negative (–ve) sputum bacterial cultures during experimental RV infection. Box and whisker plots in J show the median (line within the box), the IQR (box), and the minimum-to-maximum values (whiskers). (K) Correlation of peak sputum MUC5AC with bacterial loads as assessed by 16S qPCR. 16S qPCR was not measured for all patients because of a lack of sample availability, so only the data measured are shown. (L) Upper respiratory tract symptom scores. (M) Lower respiratory tract symptom scores. (N) PEFR change from baseline. (B and D) *P < 0.05 and **P < 0.01 (COPD vs. healthy); ^#P < 0.05 (day 3 vs. baseline in patients with COPD). (C) *P < 0.05 and **P < 0.01, by Wilcoxon matched-pairs, signed-rank test for baseline versus peak values, and Mann Whitney U test for comparison of peak values between patients with COPD and healthy individuals. (J) *P < 0.05, by Mann Whitney U test. (E) **P < 0.01 and ***P < 0.001, by Spearman’s correlation analysis of pooled data on healthy volunteers and patients with COPD. (F–I and K–N) Nonparametric Spearman’s correlation analysis was performed on pooled data on healthy volunteers and patients with COPD.

Figure 3. Muc5ac^–/– mice have attenuated inflammatory responses to RV infection.

(A) Experimental outline. Muc5ac^–/– and WT control C57BL/6 mice were i.n. infected with RV-A1 or UV-inactivated RV-A1. (B) BAL total cell count and (C) BAL neutrophil count on day 1 after infection. BAL concentrations of (D) CXCL1/KC, (E) CXCL2/MIP-2, (F) IL-1β, (G) IL-6, (H) TNF, (I) IFN-α, and (J) IFN-λ2/3 on day 1 after infection. (K) Lung tissue RV RNA copies. Box and whisker plots in K show the median (line within the box), the IQR range (box), and the minimum to maximum values (whiskers). Data in B–J are expressed as the mean ± SEM of 6 mice per treatment group and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Bonferroni’s post test.

Figure 4. Exogenous MUC5AC augments airway inflammation and bacterial loads in RV-infected mice.

(A) Experimental outline. C57BL/6 WT mice were treated i.n. with purified MUC5AC protein and additionally i.n. infected with RV-A1 or UV-inactivated RV-A1. (B) BAL total cell counts and (C) BAL neutrophil counts on day 1 after infection. BAL concentrations of (D) CXCL1/KC, (E) CXCL2/MIP-2, (F) CCL5/RANTES, (G) IL-1β, (H) IL-6, (I) TNF, (J) neutrophil elastase, and (K) SLPI on day 1 after infection. (L) Lung 16S bacterial loads on day 4 after infection. (M) IFN-α and (N) IFN-λ2/3 protein concentrations in BAL on day 1 after infection. (O) Lung tissue RV RNA copies. Box and whisker plots in O show the median (line within the box), the IQR (box), and the minimum to maximum values (whiskers). Data in B–N are expressed as the mean ± SEM of 5 mice per treatment group and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Bonferroni’s post test.

Figure 5. Neutralization of airway ATP inhibits augmentation of inflammation and bacterial loads by exogenous MUC5AC.

(A) Experimental outline. C57BL/6 WT mice were treated i.n. with purified MUC5AC protein or PBS control, infected with RV-A1 or UV-inactivated RV-A1, and additionally treated with i.n. apyrase or vehicle control (PBS). (B) BAL ATP concentrations on day 1 after infection. (C) BAL total cell counts and (D) BAL neutrophil counts on day 1 after infection. BAL concentrations of (E) CXCL1/KC, (F) CXCL2/MIP-2, (G) CCL5/RANTES, (H) TNF, (I) IL-1β, (J) neutrophil elastase, and (K) SLPI on day 1 after infection. (L) Lung 16S bacterial loads on day 4 after infection. All data are expressed as the mean ± SEM of 5 mice per treatment group and are representative of 2 independent experiments. *P < 0.05 and **P < 0.01, by 1-way ANOVA with Bonferroni’s post test.

Figure 6. EGFR inhibition inhibits MUC5AC expression and attenuates airway inflammation, bacterial loads, and airway hyperreactivity in a mouse model of virus-exacerbated COPD.

(A) Experimental outline. C57BL/6 WT mice were treated i.n. with elastase or PBS control and additionally treated i.p. with 50 mg/kg of the EGFR inhibitor AG1478, prior to challenge with RV-A1 or UV-inactivated RV-A1. (B) Lung Muc5ac mRNA expression and (C) BAL MUC5AC protein concentration. (D) Periodic acid–Schiff–stained (PAS-stained) lung sections on day 4 after infection, with scoring for PAS-positive, mucus-producing cells. Scale bars: 50 μm. Original magnification, ×400. (E) BAL total cell counts and (F) BAL neutrophil counts on day 1 after infection. (G) BAL lymphocytes on day 4 after infection. BAL concentrations of (H) CXCL1/KC, (I) CXCL2/MIP-2, (J) CCL5/RANTES, (K) IL-6, (L) IL-1β, (M) TNF, (N) GM-CSF, (O) neutrophil elastase, (P) ATP, and (Q) SLPI, on day 1 after infection. (R) Lung 16S bacterial loads on day 4 after infection. (S) Airway hyperresponsiveness (enhanced pause [Penh]) to methacholine challenge on day 1 after infection. In S, the comparison shown is for elastase-RV plus vehicle versus the elastase-RV plus AG1478 group. All data are expressed as the mean ± SEM of 5 mice per treatment group and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA (B–R) and 2-way ANOVA (S) with Bonferroni’s post test. Elas, elastase; VEH, vehicle.