Persistent, Progressive Pulmonary Fibrosis and Epithelial Remodeling in Mice

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease with underlying mechanisms that have been primarily investigated in mice after intratracheal instillation of a single dose of bleomycin. However, the model has significant limitations, including transient fibrosis that spontaneously resolves and its failure to fully recapitulate the epithelial remodeling in the lungs of patients with IPF. Thus, there remains an unmet need for a preclinical model with features that more closely resemble the human disease. Repetitive intratracheal instillation of bleomycin has previously been shown to recapitulate some of these features, but the instillation procedure is complex, and the long-term consequences on epithelial remodeling and fibrosis persistence and progression remain poorly understood. Here, we developed a simplified repetitive bleomycin instillation strategy consisting of three bi-weekly instillations that leads to persistent and progressive pulmonary fibrosis. Lung histology demonstrates increased collagen deposition, fibroblast accumulation, loss of type I and type II alveolar epithelial cells within fibrotic areas, bronchiolization of the lung parenchyma with CCSP⁺ cells, remodeling of the distal lung into cysts reminiscent of simple honeycombing, and accumulation of hyperplastic transitional KRT8⁺ epithelial cells. Micro-computed tomographic imaging demonstrated significant traction bronchiectasis and subpleural fibrosis. Thus, the simplified repetitive bleomycin instillation strategy leads to progressive fibrosis and recapitulates the histological and radiographic characteristics of IPF. Compared with the single bleomycin instillation model, we suggest that the simplified repetitive instillation model may be better suited to address mechanistic questions about IPF pathogenesis and preclinical studies of antifibrotic drug candidates.

Keywords: IPF; KRT8+ epithelial cells; bleomycin; bronchiolization; progressive pulmonary fibrosis.

Figure 1.

Fibrosis development 2 weeks after repetitive bleomycin instillation. (A) Schematic of three-dose and eight-dose instillation and harvest strategy. Black arrows indicate bleomycin doses. Red arrows indicate harvest times. (B) Hydroxyproline concentrations measured in saline- and bleomycin-instilled mice. (C) Static compliance in saline- and bleomycin-instilled mice. (D) Quantification of BAL cells after bleomycin instillation. (E) BAL differential cell counts from saline-instilled and (F) bleomycin-instilled mice. (G) Mouse lung sections stained with Mason’s trichrome and (H) anti–α-SMA (α-smooth muscle actin) antibody after three and eight bleomycin instillations. n = 4–5. Scale bars: G and H, 40 μm and 20 μm. ***P < 0.001. Bleo = bleomycin; ns = not significant; SMA = smooth muscle actin.

Figure 2.

Fibrosis is persistent after three instillations of bleomycin. (A) Static compliance in saline- and bleomycin-instilled mice. (B) Hydroxyproline concentrations measured in saline- and bleomycin-instilled mice. (C) Quantification of TGF-β in the BAL. (D) Quantification of BAL cells identifying macrophages, lymphocytes, and neutrophils from saline- and (E) bleomycin-instilled mice. (F) Quantification of lineage-negative fibroblasts (CD31⁻, CD45⁻, and CD326⁻) from lung digest. (G–J ) Lung histology after a three-dose regime of saline (G) or bleomycin (H–J) at 6, 12, and 24 weeks after the last instillation. Top panel, trichrome images (collagen blue); middle panel, Picrosirius Red (PSR) images (collagen red); bottom panel, identification of α-SMA–positive cells (green) and collagen-1 staining (red). (K) Whole-lung Aperio scans 24 weeks after saline or bleomycin after PSR staining. (L) Three-dimensional (3D) reconstruction of collagen in the lungs 24 weeks after saline or bleomycin. One hundred serial sections/images were combined for each lung to generate the reconstruction, which is quantified in N. (M) Ex vivo microCT imaging analysis and 3D reconstruction of saline- and bleomycin-instilled lungs at 24 weeks. Quantification of the (O) total lung tissue volume and (P) Hounsfield Units. n = 4 saline; n = 6 bleomycin. Scale bars, 200 μm, 100 μm, and 40 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with saline control and ^# P < 0.05 between 16 and 24 weeks bleomycin. MicroCT = micro–computed tomographic; TGF = transforming growth factor.

Figure 3.

Presence of aberrant epithelial cell remodeling after three instillations of bleomycin. (A) Identification of type I alveolar epithelial cells (purple), type II alveolar epithelial cells (AEC2s) (green), and CCSP (club cell secretory protein)⁺ cells (red) in saline-instilled or bleomycin-instilled mice at 6, 12 and 24 weeks after the last instillation. Magnified inserts show individual AEC2s (white arrowheads) and cells with dual positive staining for pro-SPC and CCSP (bronchoalveolar stem cells) in areas of bronchiolization (yellow arrowheads). (B) Identification of CCSP⁺ (purple) and KRT8 (cytokeratin 8)⁺ (green) transitional cells and KRT5⁺ cells (red). Magnified inserts show CCSP⁺/KRT8⁺/KRT5⁻ cells (yellow arrowheads) and CCSP⁻/KRT8⁺/KRT5⁻ cells (yellow circles). (C) Identification KRT8+ (green) transitional cells in relation to KRT5⁺ (red) and pro-SPC⁺ (purple) cells. Magnified inserts show that most KRT8⁺ cells are pro-SPC⁻ but some are KRT8⁺/pro-SPC⁺ (white circles) and some are KRT8⁺/KRT5⁺ (white arrowheads). Scale bars, 40 μm. SPC = surfactant protein C.