Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs

Abstract

The respiratory tract is normally kept essentially free of bacteria by cilia-mediated mucus transport, but in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), bacteria and mucus accumulates instead. To address the mechanisms behind the mucus accumulation, the proteome of bronchoalveolar lavages from COPD patients and mucus collected in an elastase-induced mouse model of COPD was analyzed, revealing similarities with each other and with the protein content in colonic mucus. Moreover, stratified laminated sheets of mucus were observed in airways from patients with CF and COPD and in elastase-exposed mice. On the other hand, the mucus accumulation in the elastase model was reduced in Muc5b-KO mice. While mucus plugs were removed from airways by washing with hypertonic saline in the elastase model, mucus remained adherent to epithelial cells. Bacteria were trapped on this mucus, whereas, in non-elastase-treated mice, bacteria were found on the epithelial cells. We propose that the adherence of mucus to epithelial cells observed in CF, COPD, and the elastase-induced mouse model of COPD separates bacteria from the surface cells and, thus, protects the respiratory epithelium.

Keywords: COPD; Glycobiology; Mouse models; Pulmonology.

Figure 1. Altered proteome in BALF from never-smokers, asymptomatic smokers, and chronic obstructive pulmonary disease (COPD) patients.

(A) Number of proteins identified in BALF in each sample group, never-smokers (n = 5), smokers (n = 12), and COPD (n = 42); data presented as medians ± IQR. (B) Principal component analysis. (C) Absolute quantification with isotopically labeled peptides by mass spectrometry, n = 5–42; data presented as medians ±IQR, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, Kruskal-Wallis and Dunn’s multiple comparisons test. (D) Ratio of MUC5AC to MUC5B calculated from absolute quantification data in C, n = 5–42 patients/group; data presented as medians ± IQR, **P ≤ 0.01, Kruskal-Wallis and Dunn’s multiple comparisons test. AGR2, anterior gradient protein 2; DMBT1, deleted in malignant brain tumors 1; FCGBP, Fc fragment of IgG binding protein; PSCA, prostate stem cell antigen; TFF3, trefoil factor 3; TGM2, transglutaminase-2; ZG16B, zymogen granule protein 16 B.

Figure 2. Mice exposed to elastase (PPE) show goblet cell hyperplasia/metaplasia and mucus accumulation in the airways.

(A) Alcian blue/Periodic acid-Schiff–stained (AB/PAS-stained) tissue section of mice airways exposed i.n. to saline (vehicle). (B) AB/PAS-stained tissue section of mice airways exposed i.n. to elastase (PPE), showing a marked increase in AB/PAS-positive cells. Representative images of 4–5 animals/group. Scale bar: 100 μm (left panels) or 33 μm (right panels). (C and D) High- and low-magnification images of lungs from mice exposed to saline or elastase (PPE). Scale bar: 100 μm. (E) Paraffin sections stained with H&E revealed few immune cells around intrapulmonary airways and intact alveoli from saline-instilled mice. Mild perivascular and peribronchiolar lymphocytic infiltration and damaged alveoli were detected in PPE-challenged mice. Scale bar: 100 μm. (F) Airway obstruction presented by measuring the percentage of airway luminal area containing AB/PAS-stained material in 1 entire lung section per animal; n = 4–9 animals/group, median ± IQR, saline vs. PPE (**P = 0.001), inactivated PPE vs. PPE (*P = 0.01), Kruskal-Wallis test with Dunn’s multiple comparisons test. (G) Differential white blood cell counts in BALF of vehicle- and PPE-exposed mice; n = 9–17 animals/group, data presented as median ± IQR, neutrophils (***P = 0.0008), eosinophils (****P < 0.0001), macrophages (***P = 0.0005), and lymphocytes (****P < 0.0001), Mann-Whitney U test.

Figure 3. Most abundant proteins (excluding albumin) detected by mass spectrometry in airway mucus plugs obtained from mice exposed to PPE and changes induced in the epithelial cell and BALF proteome in vivo.

BALF, bronchoalveolar lavage fluid; ID, identification; LOD, limit of detection; SN, supernatant. Color code: red (most abundant) to yellow (least abundant) as found in the plugs.

Figure 4. Elastase (PPE) administration alters BALF proteome.

(A and B) Major proteins in BALF supernatants obtained from lungs lavaged with PBS in animals exposed to saline (A) and elastase (PPE) (B) analyzed by mass spectrometry proteomics. (C) Absolute amounts of Muc5ac and Muc5b in BALF supernatant from saline- and PPE-exposed mice analyzed by mass spectrometry. The detection limit for Muc5ac was set to 0.01 fmol/μl, n = 6 animals/group in A–C; data presented as median ± IQR, **P = 0.002, ***P = 0.0002; Mann-Whitney U test. (D) Isolation of mucus plugs from BALF in a PPE-exposed mouse after lavage with an Alcian blue solution. Blue-stained mucus plugs (arrowhead) were obtained and transferred to a dry tube (right). (E) The most abundant proteins in mucus plugs detected by proteomics. (F) Absolute quantification of Muc5ac and Muc5b protein amounts in mucus plugs by mass spectrometry; data presented as median ± IQR, n = 12 animals in E and F, **P = 0.0011, Mann-Whitney U test.

Figure 5. Airway epithelial cell proteome changes after elastase (PPE) administration.

(A) H&E-stained section of bronchi before (left) and after epithelial cell isolation (right). Scale bars: (left) 200 μm, (right) 50 μm. (B) Heatmap of the most changed proteins in the airway epithelial cell proteome after PPE exposure. (C and D) The most abundant proteins in airway epithelial cells detected by label-free proteomics after saline (C) and PPE (D) instillation. (E) Label-free quantification (LFQ) of the most changed proteins after PPE exposure, n = 7 pools/group with 3 mice/pool, data presented as median ± IQR, *P = 0.017, **P = 0.006, ***P = 0.0006, Mann-Whitney U test. The detection limit for Muc5ac was set to 1 × 10⁶. (F) Absolute quantification of Muc5ac and Muc5b with isotopically labeled peptides by mass spectrometry; n = 8 pools/group with 3 mice/pool, data presented as median ± IQR, **P = 0.001, Mann-Whitney U test. Detection limit for Muc5ac was set to 1 fmol. (G) Ratio of Muc5ac to Muc5b in isolated mucus plugs, BALF supernatants (BALF SN), and epithelial cells, n = 6–12 samples/group. Data presented as median ± IQR, *P = 0.04, **P = 0.0016, Kruskal-Wallis and Dunn’s multiple comparisons test. Chi3l4, chitinase-3-like protein 4; Clca1, chloride channel accessory 1; Retnla, resistin-like α; Chi3l3, chitinase-3-like protein 3; Ltf, lactotransferrin; C3, complement C3; Agr2, anterior gradient protein 2 homolog; Cp, ceruloplasmin; Bpifb1, BPI fold-containing family B member 1; Chi3l1, chitinase-3-like protein 1; Scin, adseverin; Fcgbp, Fcgbp protein; Pla2g4c, Pla2g4c protein; St3gal4, β-galactoside α-2,3-sialyltransferase 4; Tff2, trefoil factor 2; Pglyrp1, peptidoglycan recognition protein 1; Reg3g, regenerating islet-derived protein 3-γ; Qsox1, sulfhydryl oxidase 1; Chia, acidic mammalian chitinase; Chad, chondroadherin; Fer1l6, Fer-1-like 6; Fn1, fibronectin; Cbr2, carbonyl reductase [NADPH] 2; Aldh1a1, retinal dehydrogenase 1; Sec14l3, SEC14-like 3; Hist1h2bj, histone H2B; Scgb1a1, uteroglobin.

Figure 6. Airway mucus accumulated after elastase administration in mice has a stratified appearance.

(A) Lower (left) and higher magnification (right) images of an AB/PAS-stained paraffin section showing bronchial walls lined by mucus with lamellar appearance in a mouse exposed to PPE. Representative of 9 animals. Scale bars: left, 200 μm; right, 50 μm. In this and the other pictures, the white line marks the mucus layer. (B) A paraffin section from a mouse exposed to PPE stained with specific antibodies against Muc5b (green) and Muc5ac (red), in addition to nuclear stain (blue), shows that this stratified structure consisted Muc5b and Muc5ac. Representative of 3 animals. Scale bar: 10 μm. (C) The stratified appearance of mucus has been observed in the distal colon, where the mucus is organized to separate the epithelium from bacterial contact in humans as well as mice. Representative image showing mouse distal colon in an AB/PAS-stained paraffin section. Scale bar: 50 μm. In airway sections from patients with (D) COPD and (E) cystic fibrosis stained with AB/PAS, stratified mucus can be observed covering the epithelium. Scale bars: 50 μm. In mice as well as humans, mucus does not accumulate in the airways in (F) control subjects. Section from patients with (G) COPD and (H) cystic fibrosis stained with antibodies against MUC5B (green) and MUC5AC (red), in addition to nuclear stain (blue), shows a stratified structure of MUC5B and MUC5AC in the mucus. The MUC5B cell surface staining in F could be due to cross-reactivity. Scale bars: 20 μm.

Figure 7. Accumulated airway elastase–exposed mucus is anchored in the goblet cells and separate bacteria from the epithelial cells.

(A) Immunostaining of a paraffin section with specific antibodies against Muc5b (green) and Muc5ac (red), in addition to nuclear stain (blue). Both Muc5b and Muc5ac seem to be attached inside goblet cells. Representative of 4 mice. Scale bar: 20 μm. (B) Immunostaining of a paraffin section from a PPE-exposed mouse with specific antibodies against Muc5b (green) and tubulin (red) to visualize cilia, in addition to nuclear stain (blue). The cilia are not compressed by the accumulated mucus. Representative of 3 mice. Scale bar: 20 μm. (C) PPE-exposed mice were instilled with P. aeruginosa, and the lungs isolated, fixed in Carnoy, and immunostained for Muc5ac (red), bacteria (white), and nuclei (blue). (D) P. aeruginosa were instilled in saline-treated mice (control) and stained as in C. Representative of 6 PPE-exposed and 3 saline-exposed mice.

Figure 8. Accumulated airway elastase (PPE) mucus is attached to the epithelium and can be removed by hypertonic saline.

(A) Representative image of an airway from a mouse exposed to PPE and lavaged with PBS (BAL; 2 × 1 min). Paraffin section stained with AB/PAS. Representative of 8 animals. Scale bar:200 μm. Note the lighter AB/PAS staining and the mucus still covering the epithelial surface. (B) Percentage of airway obstruction measured as airway luminal area containing AB/PAS-stained material in an entire lung section per animal. n = 8–9 animals/group, 695–729 airway sections/group. BAL was performed by washing 2 × 1 min with PBS. Results presented as median ± IQR. (C) Graph showing the large increase in percentage of airway surface covered by AB/PAS-stained material in mice exposed to PPE compared with inactivated PPE. The inactivated PPE gives no increase in surface area covered by mucus, corroborating that the enzymatic activity of PPE-induces the mucus accumulation. In addition, two 1-min washes with PBS did not affect the percentage of epithelium covered by mucus after PPE exposure; n = 4–9 animals/group, median ± IQR, **P = 0.009, Kruskal-Wallis and Dunn’s multiple comparisons test. (D) Airway obstruction after BAL (2 × 1 minute) in PPE-treated Muc5ac^–/– and Muc5b^–/– as compared with WT, n = 8-9, **P = 0.011, Kruskal-Wallis and Dunn’s multiple comparisons test. (E) Percentage of airway surface covered by AB/PAS-stained material after BAL (2 × 1 minute) in PPE-exposed Muc5ac^–/– and Muc5b^–/– mice as compared with WT; n = 8–9, ****P < 0.0001, Kruskal-Wallis and Dunn’s multiple comparisons test. (F) Airway obstruction was reduced after 2 × 20 min treatment with 7% saline, compared with 2 × 20 min wash with PBS measured in AB/PAS-stained paraffin sections; n = 9–12 animals, **P = 0.002, Kruskal-Wallis and Dunn’s multiple comparisons test. (G) The percent of the epithelium covered by mucus was not affected by 2 × 20 min 7% saline treatment measured in AB/PAS-stained paraffin sections; n = 9–13 animals, Kruskal-Wallis and Dunn’s multiple comparisons test. (H) Transport of Alcian blue–stained mucus in naive and PPE-exposed mice; n = 6–9, *P = 0.02, Mann-Whitney U test.