Journal: Nature Communications

Article Title: Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice

doi: 10.1038/s41467-018-07768-9

Figure Lengend Snippet: Muc5b overexpression in the distal lung is associated with greater fibrogenesis following bleomycin. a , b In situ hybridization of human lung specimens from control and IPF subjects with MUC5B variant rs35705950. Arrowheads depict cells co-expressing SFTPC (red) and MUC5B (blue) in control bronchioles and IPF bronchiolar structures a and in control type 2 alveolar epithelia and IPF type 2 alveolar structures b . c Transgenic mice expressing Muc5b under control of the mouse Scgb1a1 and the human SFTPC promoters demonstrate Muc5b protein in conducting airways and in type 2 alveolar epithelia. d , e After repeated doses of bleomycin (2.5 U/kg, IT on day 0, 1.5 U/kg on days 14 and 28), SFTPC-Muc5b Tg demonstrated worse survival d and tissue injury e relative to wild type (+) littermates. Survival data in d were evaluated by χ 2 statistic using 17 Muc5b −/− , 16 Muc5b +/+ , 15 Scgb1a1- Muc5b Tg , 23 Scgb1a1-Muc5b +(wt) , 26 SFTPC- Muc5b Tg , and 25 SFTPC- Muc5b +(wt) , mice. f – i Scgb1a1- Muc5b Tg , Muc5b −/− , and control mice were treated IT with bleomycin as in d . To induce similar levels of fibrosis while limiting survivor bias, SFTPC-Muc5b Tg and controls received 2.0 U/kg bleomycin on day 0, followed by 1.0 U/kg on days 14 and 28. HP content increased in Scgb1a1-Muc5b Tg and SFTPC-Muc5b Tg but decreased in Muc5b −/− mice compared to wild type (+) controls for each strain. g – i Fibrillar collagen (magenta in g ) was assessed in peripheral lung tissues by confocal/multi-photon fluorescence microscopy with SHG. h , i Fibrillar collagen volumes following bleomycin were increased per mouse h and showed heterogeneous distributions i in Tg mice compared to controls. Data in i were analyzed by t- test ( n = 105 Scgb1a1-Muc5b Tg and 105 Scgb1a1-Muc5b +(wt) images, and n = 120 SFTPC-Muc5b Tg and 120 Scgb1a1-Muc5b +(wt) littermate images. Scale bars, 10 μm a , b , c , 250 μm e , and 100 μm g . In f and h , data are means ± sem, numbers in italics indicate n animals used per experiment, and p -values indicate differences determined by ANOVA with Holm-Sidak’s multiple comparisons test. In i , * indicates statistical significance ( p < 10 −5 )

Article Snippet: For immunohistochemistry, tissues were heated in citrate buffer for antigen retrieval (20 min boiling), and tissues were then incubated overnight in goat primary antisera against Muc5b (1:1000, Everest Biotech, Upper Heyford, UK) or with rabbit primary anti-mouse Muc5b antisera (1:20,000) .

Techniques: Over Expression, In Situ Hybridization, Variant Assay, Expressing, Transgenic Assay, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice

doi: 10.1038/s41467-018-07768-9

Figure Lengend Snippet: Muc5b overexpression in SFTPC-Muc5bTg mice is associated with impaired mucociliary transport. a Representative μOCT images of excised SFTPC-Muc5bTg mice trachea in comparison to trachea of wild type (+) littermate controls. b – e Quantitative metrics from image analysis reveal increased mucus layer depth b without significant alteration of periciliary layer (PCL) depth c in Tg mice compared to + controls. Functional analysis demonstrated reduced ciliary beat frequency d and diminished mucociliary transport rates e in Muc5b-overexpressing mice. Data in b – e are means ± sem and were analyzed by Mann–Whitney test

Article Snippet: For immunohistochemistry, tissues were heated in citrate buffer for antigen retrieval (20 min boiling), and tissues were then incubated overnight in goat primary antisera against Muc5b (1:1000, Everest Biotech, Upper Heyford, UK) or with rabbit primary anti-mouse Muc5b antisera (1:20,000) .

Techniques: Over Expression, Functional Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice

doi: 10.1038/s41467-018-07768-9

Figure Lengend Snippet: P-2119 effectively cleaves mucus in vitro and in vivo, enhancing the acute clearance of inflammatory cells. a , b P-2119 hydrolyzed DTNB disulfide bonds more quickly than n-acetylcysteine (NAC) a , and at pH 6 P-2119 cleaved more bonds than NAC b . c In human saliva, P-2119 reduced MUC5B in salivary mucus at lower concentrations than NAC. d – g In concentrated normal human bronchial epithelial cell culture mucus (5% solids), P-2119 reduced MUC5B at a potency similar to that seen in saliva in c . Reduction of MUC5B by P-2119 dose dependently lowered mucus viscosity as demonstrated by enhanced mean square displacement (MSD) of fluorescent microspheres e , and as shown by improved macrorheological complex viscosity f that was strongly correlated with reduced molecular mass g . Data in e represent 900 technical and three biological replicates; data in f represent three technical and two biological replicates. Cyan symbols, vehicle; magenta symbols, P-2119. h – j In vivo effects of aerosolized P-2119 (68–135 mM for 60 min). h Wild-type mice challenged with LPS (20 μg, IT) 48 h prior to P-2119 aerosol. P-2119 decreased Muc5b mass detected by immunoblot of lung lavage fluid over a 120 min period. i SFTPC-Muc5b Tg mice were challenged with bleomycin (2.5 U/kg, IT) 7 d prior to P-2119 treatment (Tx). P-2119 caused Muc5b reduction detected by immunoblot 120 min post initiation of P-2119 aerosol. j The effect of P-2119-induced mucolysis on MCC was assessed by quantifying the acute elimination of leukocytes in lung lavage fluid obtained from bleomycin treated SFTPC-Muc5b Tg mice ( n = 9 vehicle and 12 P-2119 treated) and wild type (+) controls ( n = 12 vehicle and 13 P-2119 treated). Total cells were significantly lower in P-2119 treated mice compared to vehicle treated animals, reflecting decreases in all leukocyte subtypes in bleomycin-injured lungs. Data in a , e – g , and j are means ± sem. Data in a were analyzed between P-2119 and NAC treated groups using t -tests. Data in e – g were analyzed statistically on biological replicates: ANOVA of results between 0 mM vehicle and 0.1–10 mM P-2119 treatment groups e , f and linear regression of complex viscosity vs mass g

Article Snippet: For immunohistochemistry, tissues were heated in citrate buffer for antigen retrieval (20 min boiling), and tissues were then incubated overnight in goat primary antisera against Muc5b (1:1000, Everest Biotech, Upper Heyford, UK) or with rabbit primary anti-mouse Muc5b antisera (1:20,000) .

Techniques: In Vitro, In Vivo, Cell Culture, Western Blot

Journal: Nature Communications

Article Title: Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice

doi: 10.1038/s41467-018-07768-9

Figure Lengend Snippet: P-2119 treatment results in reduced collagen deposition in the setting of excess Muc5b. a SFTPC-Muc5bTg mice were subjected to bleomycin challenge with a 2 wk P-2119 intervention protocol, with daily P-2119 treatments (Tx) or saline vehicle (Veh) exposures starting 7 or 56 d post bleomycin challenge and ending 24 h prior to tissue harvest. b – h Injury and fibrosis induced in bleomycin-treated mice exposed to saline vehicle were decreased in P-2119 treated animals. Compared to vehicle treated animals, P-2119 treated mice had significantly fewer leukocytes in lung lavage b , less hydroxyproline in lung homogenates at 10 wks c , and decreased peripheral lung fibrillar collagen d – h . In b – d , data are means ± sem, numbers in italics indicate n animals used per experiment, and p -values indicate differences determined by t -test with Welch’s correction for unequal variances. In e and f , * indicates statistical significance by t -test ( p < 0.00001). Histograms in e are from 15 Veh mice (225 images) and from 12 Tx mice (165 images); histograms in f are from 9 Veh mice (180 images) and from 6 Tx mice (120 images). Images in g and h show greater amounts of fibrillar collagen (red) in Veh vs. Tx groups. Scale bars, 100 μm g , h

Article Snippet: For immunohistochemistry, tissues were heated in citrate buffer for antigen retrieval (20 min boiling), and tissues were then incubated overnight in goat primary antisera against Muc5b (1:1000, Everest Biotech, Upper Heyford, UK) or with rabbit primary anti-mouse Muc5b antisera (1:20,000) .

Techniques: