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. 2018 Dec 18;9:5363. doi: 10.1038/s41467-018-07768-9

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — 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 χ² 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)