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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)1,2. However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases1. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus1,3. Genetic variants are linked to diverse lung diseases4,5,6, 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 normally7. 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 sputum1,8. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%9,10,11. 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.

Figure 1: Muc5b is required for survival and particle clearance.
Figure 2: Muc5b deficiency causes severe upper airway obstruction.
Figure 3: Infection is the cause of death in Muc5b−/− mice.
Figure 4: Muc5b maintains functioning lung macrophage populations.

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Acknowledgements

We thank F. Ttofali, D. Harper, D. Raclawska, V. Mdoe, C. Ramsey and J. Parker-Thornburg for their assistance. We also thank K. Naff and the MD Anderson Cancer Center Department of Veterinary Medicine and Surgery for support in animal care. This work was supported by the National Institutes of Health Grants R01 HL080396 (C.M.E.); R01 AA008769 (J.H.S.), R01 HL109517 (W.J.J.); R01 HL114381 (P.M.H.); R01 HL097000 (B.F.D.), P01 HL108808, P01 HL110873, P50 HL107168, P30 DK065988 (R.C.B.), Medical Research Council Grant G1000450 (D.J.T.) and Cystic Fibrosis Foundation Grants 06IO (C.M.E.) and RDP R026-CR11 (R.C.B.). Additional support was provided by National Institutes of Health Cancer Center Support Grants CA016672 for the MD Anderson Cancer Center and CA046934 for the University of Colorado transgenic mouse facilities; by CA016086 for the UNC Biomedical Research Imaging Center Small Animal Imaging Facility, and for the UNC Michael Hooker Microscopy Facility funded by an anonymous private donor.

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Authors

Contributions

M.G.R., A.L.-B. and A.A.F designed and performed survival, histological, particle clearance, inflammation, and infectious agent identification experiments, performed and collected data for S. aureus infection, macrophage and neutrophil identification experiments, and cytokine analyses. A.M.W., R.C.K., C.E.H. and D.A.S. generated and assisted in studies in Scgb1a1-Muc5b mice. M.M.M., R.M.B, I.R., A.S.B., M.G.B., W.K.O., K.A.T., S.C.F., A.C.-G., J.M.D. and C.A.L. performed infectious pneumonia and infectious agent identification experiments. S.N.A., L.K.B., A.S.S. and Y.M.P. constructed and generated Muc5b knockout mice. S.E.E., M.M.D.G., S.J.M. and M.J.T. assisted in the design and performance of inflammation studies. B.R.G., R.A., H.K.-Q. and M.R.B. assisted in the design and performance of hypoxemia studies. J.H.S., S.M.D. and B.R.G. assisted in the design and performance of tracheal and nasal mucociliary function studies. D.J.T. and K.R. provided purified MUC5B protein. W.J.J. and L.B. designed and performed macrophage activation and apoptosis assays. I.V.Y, P.M.H., P.G.W., C.W.D., R.C.B and B.F.D. assisted in the analysis and interpretation of data, and C.W.D. provided Muc5b antisera. C.M.E. designed the study, analysed data, and wrote the manuscript.

Corresponding author

Correspondence to Christopher M. Evans.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation and characterization of Muc5ac- and Muc5b-mutant mice.

a, Muc5b-knockout mice were generated by flanking exon 1 with loxP sites, selecting embryonic stem cells for neomycin resistance, and crossing allele-positive mice with germline active FLP and Cre deleter strains. b, Muc5b transgenic mice were generated by inserting a full-length Muc5b genomic DNA (gDNA) transgene driven by the airway specific rat Scgb1a1 promoter. c, Muc5b transgene expression throughout the conducting airways (top part, bronchi; bottom part, bronchioles). Note the lack of Muc5ac induction in Scgb1a1-Muc5b mouse airways (top right). d, Immunoblot analysis of wild-type (WT) and Scgb1a1-Muc5b mouse lung homogenates. e, Pulmonary MCC in Scgb1a1-Muc5b mice. f, Loss of Muc5ac and retention of Muc5b in ovalbumin sensitized and challenged Muc5ac−/− airways. Tg, transgenic.

Extended Data Figure 2 Pathways for the clearance of bacteria and particles from the lower airways.

Inhaled agents such as bacteria (yellow) impact upon epithelium-lined surfaces in conducting airways or deposit in alveoli. Bacteria trapped in surface and glandular mucous secretions (green) are eliminated directly by MCC (1) or are killed and then eliminated by MCC (2). Bacteria that evade these mechanisms or deposit more deeply in the lungs are detected and phagocytosed by dendritic cells (DCs) and macrophages (3; present in the airways and alveoli, but shown only in the latter here for clarity). DCs and macrophages secrete IL-23 and facilitate innate anti-bacterial immune responses through innate myeloid- and lymphoid-cell (4), and monocyte and granulocyte recruitment (5). Recruited inflammatory cells accumulate in the lungs and kill bacteria through phagocytic, exocytic and extracellular trap-mediated mechanisms (6). To resolve inflammation, recruited leukocytes undergo apoptosis and are phagocytosed by macrophages, which then repress IL-23 production (7), undergo apoptosis (8) and are eliminated by MCC. In the studies presented here, we demonstrate that these defences are critically dependent upon the expression of Muc5b.

Extended Data Figure 3 Increased Muc5ac production in Muc5b−/− mice.

a, Steady-state Muc5ac messenger RNA levels in Muc5b+/+ and Muc5b−/− mice (age 12 weeks, n = 3 per group). *P = 0.019, one-tailed t-test. b, c, AB-PAS and Muc5ac staining (arrows) in Muc5b+/+ and Muc5b−/− mice. Note the increased PAS (pink) and lack of AB (blue or purple) positivity in Muc5b−/− airway goblet cells.

Extended Data Figure 4 Otitis media and upper airway congestion in Muc5b−/− mice.

a, b, Micro-CT reveals dark and radiopaque middle-ear cavities (red arrows) in Muc5b+/+ and Muc5b−/− mice, respectively. c, Mass of accumulated mucus plugs removed from nasal cavities at the indicated time points. Plug mass was compared to age using Pearson’s product–moment correlation (ρ). d, Hair in the middle ear of a Muc5b−/− mouse. The ventral side of the tympanic bulla was removed to visualize middle-ear content. White arrowhead identifies encased hair fragments. Et, Eustachian tube; tm, tympanic membrane.

Extended Data Figure 5 Further characterization of bacteria in Muc5b−/− mouse lungs.

a–e, Bacteria grown from lungs under microaerophilic conditions. a, b, Total growth in 12-week-old Muc5b−/− and Muc5b+/+ mice, and in moribund and control mice. ce, Bacterial genera, staphylococcal species and S. aureus incidence. f–h, Streptococcal group and S. acidominimus growth in mouse lungs. f, Streptococci identified by taxonomic classifications were assessed for abundance (g) and prevalence (h). Symbols identify samples from the lungs of individual Muc5b+/+ (blue) and Muc5b−/− (red) mice at 12-week-old (open circles) and moribund or cage-mate control cohorts (filled circles). Data were analysed by t-tests in a, b, ANOVA in c, d, g, and using chi-squared tests in e, h. P < 0.05 was considered significant. Total numbers of mice in each group are listed as denominators below e and h. ND, none detected; NS, not significant.

Extended Data Figure 6 Systemic polymicrobial infection in Muc5b−/− mice.

a–e, Bacteria from spleen homogenates obtained from moribund Muc5b−/− mice and Muc5b+/+ controls were identified by 16SrRNA gene sequencing. a, Genera in spleens from Muc5b−/− mice (red circles). Staphylococcus was identified in the spleen of a single Muc5b+/+ control (blue circle), but its incidence was 20% compared to 80% in Muc5b−/− mice (b). c, Sequence identification of staphylococci present. d, e, Prevalence and abundance of streptococci. Data were analysed by ANOVA in a, c, e, and using chi-squared tests in b, d. P < 0.05 was considered significant. Total numbers of mice in each group are listed as denominators below b and d.

Extended Data Figure 7 Effects of airway mucins on infection and growth of S. aureus.

a–e, USA300 infection (107 CFU intranasally) in Muc5b+/+ and Muc5b−/− mice. a, Changes in body weights during the first week in Muc5b+/+ animals (blue circles, n = 7), Muc5b−/− animals that recovered fully from infection (open red circles, n = 4), and Muc5b−/− animals that did not survive to 21 days (filled red circles, n = 6). b–e, On day 21 (surviving mice), or on the day in which a >15% loss of body weight was discovered (non-surviving mice), lungs and spleens were cultured. One non-survivor was found to have died in its cage and not cultured. Incidence of positive cultures (b) and numbers of CFUs (c) in infected Muc5b+/+ and Muc5b−/− lungs. d, e, Spleen-culture incidences and numbers of CFUs. Error bars, s.e.m. in a, c, e. Data were analysed using Student’s t-test in a (day 7) and c, and Fisher’s exact test in b and d. Because all values for Muc5b+/+ mice are zeroes in e, Mann–Whitney and Wilcoxon ranked sums tests were used. For all statistical analyses, P < 0.05 was considered significant. f, USA300 growth in medium containing MUC5AC or MUC5B (2–250 µg ml−1). Purified mucin stocks were solubilized in urea. Separate growth curves were thus also measured in serially diluted urea at matched concentrations (1.2–150 mM). Each line represents mean growth at a single concentration of mucin or urea over time. Error bars, s.d. from triplicate experiments.

Extended Data Figure 8 Effects of Muc5b expression on pulmonary inflammation.

a, Lung lavage leukocytes from Muc5b+/+ (blue, n = 15, 23, 7), Scgb1a1-Muc5b (green, n = 11, 18, 5), and Muc5b−/− (red, n = 12, 5, 5) mice in the steady state at ages 3, 6 and 12 months and moribund Muc5b−/− animals (red; n = 8, various ages) and Muc5b+/+ cagemates (blue, n = 6). *P < 0.05 by ANOVA (timecourse studies) and by Student’s t-test (moribund Muc5b−/− versus Muc5b+/+ cage mates). b, Three-month-old Muc5b+/+ (blue, n = 7), Scgb1a1-Muc5b (green, n = 3) and Muc5b−/− (red, n = 3) mice were challenged intratracheally with 5 × 107 CFU USA300. Inflammation was assessed 24 h post infection. No significant differences in macrophages or neutrophils were seen among groups. Error bars, s.e.m. c, Young (3 months of age) and ageing (6–7 months of age) Muc5b+/+ (blue), Scgb1a1-Muc5b (green) and Muc5b−/− (red) mice were exposed to 2–5 × 107CFU of live or heat-killed Staphylococcus USA300, or to saline (SAL) as indicated. Lung lavage leukocytes were assessed 7 days post challenge. Ageing Muc5b−/− mice died before lavage sampling. Infected Muc5ac−/− mice showed no differences in inflammation compared to Muc5b+/+ or C57BL/6J (not shown). Error bars, s.e.m. Numbers in brackets, n mice. *P < 0.05 across genotypes by ANOVA and within genotypes by Student’s t-test. Tg, Scgb1a1-Muc5b transgenic.

Extended Data Figure 9 Effects of Muc5b expression on pulmonary macrophages.

a, Microsphere phagocytosis in Muc5b+/+ and Muc5b−/− mouse lung macrophages. Data are percentages of cells containing microspheres per mouse. Error bars, s.e.m. *P < 0.05 between groups as determined by Student’s t-test. b, Muc5b was not detected in lung lavage macrophages from allergic wild-type mice. Data are representative of five mice. Scale bar, 5 μm. c, Accumulation of efferocytotic macrophages in Muc5b−/− lung tissues. Neutrophils in lung tissues from Muc5b+/+ and Muc5b−/− mice were immunolabelled and stained with DAB (brown). Asterisk in lower right panel identifies whole neutrophils contained within pulmonary macrophages. Arrows identify areas in c shown at high magnification. Scale bar, 50 μm in low-magnification and 15 μm in high-magnification images.

Extended Data Table 1 Cytokines and chemokines in mouse lung lavage fluid

Supplementary information

Mucociliary transport in Muc5b+/+ mTEC cultures

Microsphere transport measured by video microscopy. Time-lapse video speed is 300x. (MPG 503 kb)

Mucociliary transport in Muc5b-/- mTEC cultures

Microsphere transport measured by video microscopy. Time-lapse video speed is 300x. (MPG 503 kb)

Labored breathing pattern of a moribund Muc5b-/- mouse before tracheostomy

An isoflurane anesthetized Muc5b-/- mouse displays deep and extended ventilatory patterns. (AVI 9397 kb)

Quiet tidal breathing pattern of a moribund Muc5b-/- mouse immediately after tracheostomy

Ventilation following tracheostomy is shallow and rapid. Note isoflurane administration through the tracheal cannula. (AVI 11064 kb)

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Roy, M., Livraghi-Butrico, A., Fletcher, A. et al. Muc5b is required for airway defence. Nature 505, 412–416 (2014). https://doi.org/10.1038/nature12807

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