Muc5b is required for airway defence

Abstract

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Figures

Figure. 1. Muc5b is required for survival and particle clearance

a-d, Muc5b+/+, Muc5b−/−, and Muc5ac−/− olfactory glands (a), nasopharynxes (b), tracheal glands (c), and bronchial surface epithelial cells (d) stained with AB-PAS or with anti-Muc5b (c,d bottom). Solid arrowheads, mucous cells. Open arrowheads, ciliated cells. e-i, Effects of mucin expression on growth, survival, MCC, and ciliary function. j-l, Microsphere movement, transport velocity, and transport index in primary tracheal epithelial cells in vitro. Scale bars, 20 μm (a, j), 10 μm (b-d). Error bars, sem. ‘*’ significant differences (p<0.05). Numbers in parentheses, ‘n’ mice.

Figure. 2. Muc5b deficiency causes severe upper airway obstruction

a-b, Airflow and ventilation in Muc5b+/+ and Muc5b−/− mice. c, Oxygen saturation (SaO2) and rescue with 100% O2 supplementation (FiO2 1.0). d-f, Lung (RL) and lower airway (RAW) resistance and hyperresponsiveness to methacholine (MCh) in mechanically ventilated mice. g, Upper airway by-pass by tracheostomy under normoxia (FiO2 0.21). h, Upper airway obstruction in Muc5b−/− mice confirmed by micro-CT (red arrows, nasopharynxes). i,j, Hair encased in mucus-like plugs visible during necropsy. Scale bars, 1 mm. Error bars, sem. ‘*’, significant difference (p<0.05). Numbers in parentheses, ‘n’ mice.

Figure. 3. Infection is the cause of death in Muc5b−/− mice

a-c, Hyperemic blood vessels (red arrows), hair (white arrows), bacteria, and leukocytes (>95% neutrophils) in Muc5b−/− middle ears. d,e, Hair fragments and leukocytes in Muc5b−/− lungs. f,g, Chronically accumulating bacteria in lungs, leading to moribundity and septicemia in Muc5b−/− mice. h-j, Mortality, infection, and ventilation with antibiotic treatment. k, Bacterial genera, and staphylococcal species in Muc5b−/− mice. l, Survival following intranasal S. aureus inoculation (107 CFU/mouse). Scale bars, 20 μm (d,e). Error bars, sem ‘*’ statistical significance (p<0.05). Numbers in parentheses, ‘n’ mice.

Figure. 4. Muc5b maintains functioning lung macrophage populations

a-c, Temporal changes in lung lavage macrophage numbers and morphologies and in IL-23. d-g, Macrophage numbers and apoptosis, and IL-23, in spontaneously moribund and control lungs. h-j, Macrophage numbers and apoptosis, and IL-23, in young (3 mos) and old (6-7 mos) lungs 7d after intratracheal USA300 infection (108 CFU/mouse). j-l, IL-23 and macrophage MHC-II in infected Muc5bTg mice. Muc5bTg lavage USA300 DNA was >98% reduced; incidence was 25% vs. 67% (Muc5b+/+) and 100% (Muc5b−/−), χ2p=0.03. Error bars, sem. ‘*’, statistical significance (p<0.05). Representative data in k. Numbers in parentheses, ‘n’ mice.