Contribution of mucus concentration and secreted mucins Muc5ac and Muc5b to the pathogenesis of muco-obstructive lung disease

Abstract

Airway diseases, including cigarette smoke-induced chronic bronchitis, cystic fibrosis, and primary ciliary dyskinesia are associated with decreased mucociliary clearance (MCC). However, it is not known whether a simple reduction in MCC or concentration-dependent mucus adhesion to airway surfaces dominates disease pathogenesis or whether decreasing the concentration of secreted mucins may be therapeutic. To address these questions, Scnn1b-Tg mice, which exhibit airway mucus dehydration/adhesion, were compared and crossed with Muc5b- and Muc5ac-deficient mice. Absence of Muc5b caused a 90% reduction in MCC, whereas Scnn1b-Tg mice exhibited an ∼50% reduction. However, the degree of MCC reduction did not correlate with bronchitic airway pathology, which was observed only in Scnn1b-Tg mice. Ablation of Muc5b significantly reduced the extent of mucus plugging in Scnn1b-Tg mice. However, complete absence of Muc5b in Scnn1b-Tg mice was associated with increased airway inflammation, suggesting that Muc5b is required to maintain immune homeostasis. Loss of Muc5ac had few phenotypic consequences in Scnn1b-Tg mice. These data suggest that: (i) mucus hyperconcentration dominates over MCC reduction alone to produce bronchitic airway pathology; (ii) Muc5b is the dominant contributor to the Scnn1b-Tg phenotype; and (iii) therapies that limit mucin secretion may reduce plugging, but complete Muc5b removal from airway surfaces may be detrimental.

Figure 1. Mucus hyperconcentration/adhesion is necessary to produce bronchitic lung pathology, but Muc5b deletion ameliorates airway mucus obstruction in Scnn1b-Tg mice

(a) Representative photomicrographs of airway lumens cut in cross section proximal to the hilum from PND35 mice of the indicated genotypes, stained with H&E illustrating airway mucus obstruction and inflammatory infiltrates characteristic of bronchitic lung pathology in Scnn1b-Tg mice. (b-c) Semi-quantitative histology scores for airway inflammation (b) and air space enlargement (c) in PND35 mice from the Muc5b^−/− × Scnn1b-Tg cross in the C57:129 genetic background. n= 6-9 mice/genotype. ANOVA * p<0.05 vs. Muc5b^+/+ mice. (d) Equivalent sections as in (a) stained with AB-PAS for mucopolysaccharides, illustrating significant amelioration of mucus obstruction in Muc5b^−/−Scnn1b-Tg vs. Muc5b^+/+Scnn1b-Tg mice. Scale bar 0.1 mm. (e-f) Morphometric analysis of total (epithelial+luminal, e) and epithelial (f) airway mucus volume density (V_s) in PND35 mice (C57:129 genetic background). n= 6-9 mice/genotype. ANOVA * p<0.05 vs. Muc5b^+/+ mice, # p<0.05 vs. Muc5b^+/+Scnn1b-Tg mice. (g-h) Densitometric analysis of mucin agarose western blots of BAL from the progeny of the Muc5b^−/− × Scnn1b-Tg cross in the C57 congenic background, at PND35. Blots were probed with anti-Muc5b (g) or anti-Muc5ac (h) antibodies. n=6 mice/genotype. ANOVA * p<0.05 vs. Muc5b^+/+ mice, # p<0.05 vs. Muc5b^+/+Scnn1b-Tg mice.

Figure 2. Muc5ac deletion does not ameliorate airway mucus obstruction in Scnn1b-Tg mice

(a) Representative photomicrographs of proximal left lobe main stem bronchus from PND35 mice of the indicated genotypes, stained with AB-PAS for mucopolysaccharides, illustrating no changes in mucus obstruction in Muc5ac^−/−Scnn1b-Tg vs. Muc5ac^+/−Scnn1b-Tg mice. Scale bar 0.1 mm. (b-c) Morphometric analysis of total (epithelial+luminal, b) and epithelial (c) airway mucus volume density (V_s) in PND35 mice (C57:129 genetic background). n= 6-11 mice/genotype. ANOVA * p<0.05 vs. Muc5ac^+/+ mice. (d-e) Densitometric analysis of mucin agarose western blots of BAL from the progeny of the Muc5ac^−/− × Scnn1b-Tg cross in the C57 congenic background, at PND35. Blots were probed with anti-Muc5b (d) or anti-Muc5ac (e) antibodies. n= 6 mice/genotype. ANOVA * p<0.05 vs. Muc5ac^+/+ mice, # p<0.05 vs. Muc5ac^+/+Scnn1b-Tg mice.

Figure 3. Relative contribution of Muc5ac vs. Muc5b to neonatal survival in a model of lethal mucus obstruction, the F1 C57:FVB/NJ Scnn1b-Tg mice

Survival curves for the progeny of the Muc5b^−/−Scnn1b-Tg mice (a) or Muc5ac^−/−Scnn1b-Tg mice (b) crossed with inbred FVB/NJ mice. Muc5b heterozygosity improved overall survival of F1 C57:FVB Scnn1b-Tg mice, whereas Muc5ac heterozygosity only resulted in delayed time of death (median survival PND 9 vs. PND 5 for Muc5ac^+/−Scnn1b-Tg mice vs. Muc5ac^+/+Scnn1b-Tg mice, respectively). * p<0.05 vs. Muc^+/+ littermates, # p<0.05 vs. Muc^+/+Scnn1b-Tg littermates.

Figure 4. Mucociliary transport only partially correlates with the severity of muco-obstructive lung disease

Mucociliary clearance measurements in selected genotypes from the progeny of the Muc5b^−/− × Scnn1b-Tg cross in the C57 congenic background, at PND35. n=6-14 mice/genotype. ANOVA * p<0.05 vs. Muc5b^+/+ mice, # p<0.05 vs. Muc5b^+/+Scnn1b-Tg mice.

Figure 5. Deletion of Muc5b or Muc5ac does not affect bacterial burden or ameliorate airway inflammation in Scnn1b-Tg mice

(a,b) Quantification of colony forming units (CFU) in BAL samples from the progeny of the Muc5b^−/− × Scnn1b-Tg cross in the C57:129 genetic background, at PND5-7 (a) or PND35 (b). (Log₁₀+1)-transformed data. n= 8-24 mice/genotype (a) and n=5-8 mice/genotype (b). ANOVA * p<0.05 vs. Muc5b^+/+ mice. (c-d) BAL neutrophil counts for the progeny of the Muc5b^−/− × Scnn1b-Tg cross in the C57:129 genetic background, at PND5-7 (c) or PND35 (d) n= 7-24 mice/genotype (c) and n=11-15 mice/genotype (d). ANOVA * p<0.05 vs. Muc5b^+/+ mice.

Figure 6. Deletion of Muc5b does not alter the BAL chemokine and cytokine profile in Scnn1b-Tg mice

KC (a) and LIX (b) levels in cell-free BAL from the progeny of the Muc5b^−/− × Scnn1b-Tg cross in the C57:129 genetic background at PND35. The dotted line represents the assay lower detection limit (LOD). n= 5-8 mice/genotype. ANOVA * p<0.05 vs. Muc5b^+/+ mice.

Figure 7. Deletion of Muc5b, but not Muc5ac, worsens the incidence of bronchus-associated lymphoid tissue (BALT) in Scnn1b-Tg mice

(a) Representative micrographs of proximal left lobe main stem bronchus from PND35 mice, stained with H&E, illustrating typical histopathology for the indicated genotypes. Scale bar 0.2 mm. Airway mucus obstruction was evident in Muc5b^+/+Scnn1b-Tg mice and Muc5ac^−/−Scnn1b-Tg mice (asterisks), but it was less severe in Muc5b^−/−Scnn1b-Tg mice. However, Muc5b^−/−Scnn1b-Tg mice presented with a higher incidence of BALT (arrow and high magnification inset, scale bar 20 μm). (b) Morphometric analysis of BALT in PND35 mice (C57:129 genetic background). n= 6-9 mice/genotype. ANOVA * p<0.05 vs. Muc5b^+/+ mice. (c) BAL lymphocyte counts for the progeny of the Muc5b^−/− × Scnn1b-Tg cross in the C57:129 genetic background, at PND35. n= 11-15 mice/genotype. ANOVA * p<0.05 vs. Muc5b^+/+ mice. (d) Representative confocal images of BALT immunostained with B and T cells specific markers (B220 in red and CD3 in green, respectively), and relevant isotype negative controls (rat IgG2a,k and goat IgG, respectively). Nuclei are stained in blue (DAPI). Differential interference contrast (DIC) image is provided to illustrate the typical localization of BALT in the airway submucosal compartment.

Figure 8. Airway mucus hyperconcentration/adhesion stimulates IgA secretion

(a,b) IgA (a) and IgG₁ (b) levels in cell-free BAL from the progeny of the Muc5b^−/− × Scnn1b-Tg cross in the C57:129 genetic background at PND35. The dotted line represents the assay lower detection limit (LOD). n= 5-8 mice/genotype. ANOVA * p<0.05 vs. Muc5b^+/+ mice. (c) Immunohistochemical localization of the polymeric Ig receptor secretory component (SC) in the main stem bronchus of mice for the indicated genotypes. A serial section to the one used for Muc5b^+/+Scnn1b-Tg SC stain is shown as negative IgG control (IgG control).