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.
Fahy, J. V. & Dickey, B. F. Airway mucus function and dysfunction. N. Engl. J. Med. 363, 2233–2247 (2010)
Button, B. et al. A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 337, 937–941 (2012)
Young, H. W. et al. Central role of Muc5ac expression in mucous metaplasia and its regulation by conserved 5′ elements. Am. J. Respir. Cell Mol. Biol. 37, 273–290 (2007)
The Collaborative Study on the Genetics of Asthma. A genome-wide search for asthma susceptibility loci in ethnically diverse populations. Nature Genet. 15, 389–392 (1997)
Seibold, M. A. et al. A common MUC5B promoter polymorphism and pulmonary fibrosis. N. Engl. J. Med. 364, 1503–1512 (2011)
Kamio, K. et al. Promoter analysis and aberrant expression of the MUC5B gene in diffuse panbronchiolitis. Am. J. Respir. Crit. Care Med. 171, 949–957 (2005)
Janssen, W. J. et al. Fas determines differential fates of resident and recruited macrophages during resolution of acute lung injury. Am. J. Respir. Crit. Care Med. 184, 547–560 (2011)
Thornton, D. J., Rousseau, K. & McGuckin, M. A. Structure and function of the polymeric mucins in airways mucus. Annu. Rev. Physiol. 70, 459–486 (2008)
Woodruff, P. G. et al. T-helper type 2-driven inflammation defines major subphenotypes of asthma. Am. J. Respir. Crit. Care Med. 180, 388–395 (2009)
Innes, A. L. et al. Epithelial mucin stores are increased in the large airways of smokers with airflow obstruction. Chest 130, 1102–1108 (2006)
Ordoñez, C. L. et al. Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am. J. Respir. Crit. Care Med. 163, 517–523 (2001)
Kawakubo, M. et al. Natural antibiotic function of a human gastric mucin against helicobacter pylori infection. Science 305, 1003–1006 (2004)
Johansson, M. E. et al. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc. Natl Acad. Sci. USA 105, 15064–15069 (2008)
Van der Sluis, M. et al. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology 131, 117–129 (2006)
Velcich, A. et al. Colorectal cancer in mice genetically deficient in the mucin Muc2. Science 295, 1726–1729 (2002)
Hasnain, S. Z. et al. Muc5ac: a critical component mediating the rejection of enteric nematodes. J. Exp. Med. 208, 893–900 (2011)
Preciado, D. et al. Muc5b is the predominant mucin glycoprotein in chronic otitis media fluid. Pediatr. Res. 68, 231–236 (2010)
Labandeira-Rey, M. et al. Staphylococcus aureus panton-valentine leukocidin causes necrotizing pneumonia. Science 315, 1130–1133 (2007)
Stark, M. A. et al. Phagocytosis of apoptotic neutrophils regulates granulopoiesis via IL-23 and IL-17. Immunity 22, 285–294 (2005)
Patnode, M. L. et al. Galactose-6-o-sulfotransferases are not required for the generation of siglec-F ligands in leukocytes or lung tissue. J. Biol. Chem. 288, 26533–26545 (2013)
Moser, C. et al. β-defensin 1 contributes to pulmonary innate immunity in mice. Infect. Immun. 70, 3068–3072 (2002)
Manitz, M. P. et al. Loss of S100a9 (Mrp14) results in reduced interleukin-8-induced CD11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro. Mol. Cell. Biol. 23, 1034–1043 (2003)
Flo, T. H. et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432, 917–921 (2004)
Ward, P. P., Mendoza-Meneses, M., Cunningham, G. A. & Conneely, O. M. Iron status in mice carrying a targeted disruption of lactoferrin. Mol. Cell. Biol. 23, 178–185 (2003)
O'Riordan, T. G., Zwang, J. & Smaldone, G. C. Mucociliary clearance in adult asthma. Am. Rev. Respir. Dis. 146, 598–603 (1992)
Goodman, R. M., Yergin, B. M., Landa, J. F., Golivanux, M. H. & Sackner, M. A. Relationship of smoking history and pulmonary function tests to tracheal mucous velocity in nonsmokers, young smokers, ex-smokers, and patients with chronic bronchitis. Am. Rev. Respir. Dis. 117, 205–214 (1978)
Rowe, S. M., Miller, S. & Sorscher, E. J. Cystic fibrosis. N. Engl. J. Med. 352, 1992–2001 (2005)
Talbot, T. R. et al. Asthma as a risk factor for invasive pneumococcal disease. N. Engl. J. Med. 352, 2082–2090 (2005)
Lee, T. A., Weaver, F. M. & Weiss, K. B. Impact of pneumococcal vaccination on pneumonia rates in patients with COPD and asthma. J. Gen. Intern. Med. 22, 62–67 (2007)
Hendley, J. O. Clinical practice. Otitis media. N. Engl. J. Med. 347, 1169–1174 (2002)
Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl Acad. Sci. USA 97, 5978–5983 (2000)
Zhu, Y. et al. Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways. J. Physiol. (Lond.) 586, 1977–1992 (2008)
Roy, M. G. et al. Mucin production during prenatal and postnatal murine lung development. Am. J. Respir. Cell Mol. Biol. 44, 755–760 (2011)
Sisson, J. H., Stoner, J. A., Ammons, B. A. & Wyatt, T. A. All-digital image capture and whole-field analysis of ciliary beat frequency. J. Microsc. 211, 103–111 (2003)
Voronina, V. A. et al. Inactivation of chibby affects function of motile airway cilia. J. Cell Biol. 185, 225–233 (2009)
Coote, K., Nicholls, A., Atherton, H. C., Sugar, R. & Danahay, H. Mucociliary clearance is enhanced in rat models of cigarette smoke and lipopolysaccharide-induced lung disease. Exp. Lung Res. 30, 59–71 (2004)
You, Y., Richer, E. J., Huang, T. & Brody, S. L. Growth and differentiation of mouse tracheal epithelial cells: Selection of a proliferative population. Am. J. Physiol. Lung Cell. Mol. Physiol. 283, L1315–L1321 (2002)
Dillard, P., Wetsel, R. A. & Drouin, S. M. Complement c3a regulates muc5ac expression by airway clara cells independently of th2 responses. Am. J. Respir. Crit. Care Med. 175, 1250–1258 (2007)
Matsui, H. et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95, 1005–1015 (1998)
Okada, S. F. et al. Voltage-dependent anion channel-1 (vdac-1) contributes to atp release and cell volume regulation in murine cells. J. Gen. Physiol. 124, 513–526 (2004)
Weinberg, E. D. Iron withholding: a defense against infection and neoplasia. Physiol. Rev. 64, 65–102 (1984)
Weinberg, E. D. Nutritional immunity. Host's attempt to withold iron from microbial invaders. J. Am. Med. Assoc. 231, 39–41 (1975)
Weinberg, E. D. Iron and susceptibility to infectious disease. Science 184, 952–956 (1974)
Evans, S. E. et al. Stimulated innate resistance of lung epithelium protects mice broadly against bacteria and fungi. Am. J. Respir. Cell Mol. Biol. 42, 40–50 (2010)
DeSantis, T. Z. et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72, 5069–5072 (2006)
Drancourt, M. et al. 16s ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J. Clin. Microbiol. 38, 3623–3630 (2000)
Moghaddam, S. J. et al. Haemophilus influenzae lysate induces aspects of the chronic obstructive pulmonary disease phenotype. Am. J. Respir. Cell Mol. Biol. 38, 629–638 (2008)
Steinkamp, J. A., Wilson, J. S., Saunders, G. C. & Stewart, C. C. Phagocytosis: flow cytometric quantitation with fluorescent microspheres. Science 215, 64–66 (1982)
Lozupone, C. et al. Widespread colonization of the lung by Tropheryma whipplei in HIV infection. Am. J. Respir. Crit. Care Med. 187, 1110–1117 (2013)
Moghaddam, S. J. et al. Promotion of lung carcinogenesis by chronic obstructive pulmonary disease-like airway inflammation in a k-ras-induced mouse model. Am. J. Respir. Cell Mol. Biol. 40, 443–453 (2009)
Evans, C. M. et al. Mucin is produced by clara cells in the proximal airways of antigen-challenged mice. Am. J. Respir. Cell Mol. Biol. 31, 382–394 (2004)
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.
The authors declare no competing financial interests.
Extended data figures and tables
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.
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.
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.
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.
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. c–e, 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.
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.
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.
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.
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.
Microsphere transport measured by video microscopy. Time-lapse video speed is 300x. (MPG 503 kb)
Microsphere transport measured by video microscopy. Time-lapse video speed is 300x. (MPG 503 kb)
An isoflurane anesthetized Muc5b-/- mouse displays deep and extended ventilatory patterns. (AVI 9397 kb)
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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