Genes, other than Muc5b, play a role in bleomycin-induced lung fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is an incurable genetic disease that affects 5 million people worldwide. The gain-of-function MUC5B promoter variant rs35705950 is the dominant genetic risk factor for IPF, yet has a low penetrance. This raises the possibility that other genes and transcripts affect the penetrance of MUC5B. Previously, we have shown that the concentration of Muc5b in bronchoalveolar epithelia is directly associated with the extent and persistence of bleomycin-induced lung fibrosis in mice. In this study, we investigated whether bleomycin-induced lung injury is Muc5b dependent in genetically divergent strains of mice. Specifically, mice from the eight Diversity Outbred (DO) founders were phenotyped for Muc5b expression and lung fibrosis 3 wk after intratracheal bleomycin administration. Although we identified strains with low Muc5b expression and minimal lung fibrosis (CAST/EiJ and PWK/PhJ) and strains with high Muc5b expression and extensive lung fibrosis (NZO/H1LtJ and WSB/EiJ), there also were strains that did not demonstrate a clear relationship between Muc5b expression and lung fibrosis (129S1/SvlmJ, NOD/ShiLtJ, and C57BL/6J, A/J). Hierarchical clustering suggests that other factors may work in concert with or potentially independent of Muc5b to promote bleomycin-induced lung injury and fibrosis. This study suggests that these strains and their recombinant inbred crosses may prove helpful in identifying the genes and transcripts that interact with Muc5b and cause lung fibrosis.

Keywords: IPF; Muc5b; diversity outbred mice; lung fibrosis.

Figure 1.

Mouse family tree and single nucleotide polymorphisms (SNPs) in the Muc5b promoter region. A: the eight strains tested are distantly related. [Adapted from Petkov et al. (12) with permission from Cold Spring Harbor Laboratory Press under Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) license; https://creativecommons.org/licenses/by-nc/4.0/.] B: SNPs map 4 kb upstream of Muc5b gene region for six inbred mouse lines.

Figure 2.

Comparisons of phenotypes after bleomycin-induced lung injury. Three weeks after a single challenge with intratracheal bleomycin (2.5 U/kg), mice showed considerable variation at baseline and injury. A: representative images of hematoxylin and eosin (H&E) stain across the eight strains of mice show differences in severity and localization of injury. The progressive amount of injured tissue (condensed, darker, with infiltrating immune cells) from PWK/PhJ (almost no injury) to A/J (about 50% of the injured tissue) is shown. Bar is 1 mm. Injury scoring (B); macrophages counts in bronchoalveolar lavage fluid (BALF) (C and D) and hydroxyproline (HP) measurements for total lung collagen (E and F). n = 8–13 mice were analyzed after bleomycin-induced injury and compared with n = 5–7 saline treated-control mice from each strain.

Figure 3.

Second-harmonic generation (SHG) imaging of fibrillar collagen is a more reliable measure of parenchymal fibrosis. Fibrillar collagen was assessed in peripheral lung tissues by confocal/multi-photon fluorescence microscopy with SHG. A: representative images showing the localization of fibrillar collagen (red) around the airways and in the parenchyma following bleomycin exposure. Bar, 500 µm. Fibrillar collagen amount (B) was calculated per mouse following exposure to bleomycin or vehicle. C: fibrillar collagen accumulation correlates with the percent affected area in injured lung. n = 8–13 mice were analyzed after bleomycin-induced injury and compared with n = 5–7 saline treated-control mice from each strain.

Figure 4.

Muc5b gene expression and protein secretion do not fully correlate with other measures of injury and fibrosis. Muc5b protein in bronchoalveolar lavage fluid (BALF) (A and C, Supplemental Fig. S5, top row) and whole lung gene expression (B and D, Supplemental Fig. S5, bottom row) vary at baseline and after injury among mouse strains and their correlation with percent affected area in injured lung (F and G, correspondingly). Hierarchical clustering of mouse strains based on %changes in Muc5b gene expression, protein, and measures of lung fibrosis (E). n = 8–13 mice were analyzed after bleomycin-induced injury and compared with n = 5–7 saline treated-control mice from each strain.

Figure 5.

Airways Muc5b expression. A: representative images for Muc5b (purple) staining in the proximal airways for 8 strains of mice. Tissues were counterstained with nuclear staining (DAPI, blue). Bar, 100 µm. Quantification for Muc5b mean intensity in proximal (B and F, Supplemental Fig. S6, top row) and distal (C and G, Supplemental Fig. S7, top row) airways, and number of Muc5b expressing cells in proximal (D and H, Supplemental Fig. S6, bottom row) and distal (E and I, Supplemental Fig. S7, bottom row) airways. n = 4–5 mice were analyzed after bleomycin-induced injury and compared with n = 4–5 saline treated-control mice from each strain.

Figure 6.

Muc5b expressing honeycomb-like cysts. A: representative images for Muc5b immunohistochemistry (IHC) staining in eight mouse strains (lower magnification; top row) and regions with cyst-like structures shown at higher magnification (middle row). Immunofluorescent images for representative cyst-like regions with Muc5b (purple), proSPC (yellow), keratin5 (green), and SGH (red) signals in all analyzed mouse strains (bottom row). Bar, 60 µm. Quantification for the number of cyst-like structures (B), area % taken by cyst-like structures (C), number of cyst-like structures in fields of view (FOV; D), and average cyst-like structures size (E). n = 8–13 mice were analyzed after bleomycin-induced injury and compared with n = 5–7 saline treated-control mice from each strain.