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. 2024 Oct;12(19):e70077.
doi: 10.14814/phy2.70077.

Reproducible lung protective effects of a TGFβR1/ALK5 inhibitor in a bleomycin-induced and spirometry-confirmed model of IPF in male mice

Asbjørn Graver Petersen  1 , Stefanie H Korntner  1 , Jamal Bousamaki  1 , Denise Oró  1 , Alba Manresa Arraut  1 , Susanne E Pors  1 , Casper Gravesen Salinas  1 , Maja Worm Andersen  1 , Martin Rønn Madsen  1 , Yaohui Nie  2 , Jordan Butts  2 , Manuel Roqueta-Rivera  2 , Ulf Simonsen  3 , Henrik H Hansen  1 , Michael Feigh  1
Affiliations

Reproducible lung protective effects of a TGFβR1/ALK5 inhibitor in a bleomycin-induced and spirometry-confirmed model of IPF in male mice

Asbjørn Graver Petersen et al. Physiol Rep. 2024 Oct.

Abstract

This study comprehensively validated the bleomycin (BLEO) induced mouse model of IPF for utility in preclinical drug discovery. To this end, the model was rigorously evaluated for reproducible phenotype and TGFβ-directed treatment outcomes. Lung disease was profiled longitudinally in male C57BL6/JRJ mice receiving a single intratracheal instillation of BLEO (n = 10-12 per group). A TGFβR1/ALK5 inhibitor (ALK5i) was profiled in six independent studies in BLEO-IPF mice, randomized/stratified to treatment according to baseline body weight and non-invasive whole-body plethysmography. ALK5i (60 mg/kg/day) or vehicle (n = 10-16 per study) was administered orally for 21 days, starting 7 days after intratracheal BLEO installation. BLEO-IPF mice recapitulated functional, histological and biochemical hallmarks of IPF, including declining expiratory/inspiratory capacity and inflammatory and fibrotic lung injury accompanied by markedly elevated TGFβ levels in bronchoalveolar lavage fluid and lung tissue. Pulmonary transcriptome signatures of inflammation and fibrosis in BLEO-IPF mice were comparable to reported data in IPF patients. ALK5i promoted reproducible and robust therapeutic outcomes on lung functional, biochemical and histological endpoints in BLEO-IPF mice. The robust lung fibrotic disease phenotype, along with the consistent and reproducible lung protective effects of ALK5i treatment, makes the spirometry-confirmed BLEO-IPF mouse model highly applicable for profiling novel drug candidates for IPF.

Keywords: ALK5 inhibitor; TGFβ receptor; animal model; bleomycin; deep learning; histopathological scoring; idiopathic pulmonary fibrosis; spirometry; transcriptomics; translatability; whole‐body plethysmography.

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Conflict of interest statement

AGP, SHK, JB, DO, SEP, CGS, MWA, MRM, HHH and MFE are employed by Gubra; AGP, DO, CGS, MRM, HHH and MFE are shareholders in Gubra. AMA was employed by Gubra and is presently employed by IQVIA. YN, JB and MRR are employed by Enanta Pharmaceuticals; US is employed by Aarhus University, Aarhus, Denmark. No other potential conflicts of interest relevant to this article were reported.

Figures

FIGURE 1
FIGURE 1
Longitudinal study in BLEO‐IPF mice. (a) Study outline. Mice received a single intratracheal instillation of saline vehicle (n = 10) or bleomycin (BLEO‐IPF, 1.5 mg/kg) and were terminated on day 7‐42 after BLEO administration (n = 7–12 per time point). (b) Terminal body weight. (c) Lung weight. (d) Total lung hydroxyproline (HP) content. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTRL. Dunnett's test one‐factor linear model.
FIGURE 2
FIGURE 2
Progressive dynamics in pulmonary function in BLEO‐IPF mice. Respiratory physiology assessment using spirometry. (a) Forced expiratory volume in 0.1 seconds (FEV0.1). (b) Forced vital capacity (FVC). (c) Static compliance. (d) Inspiratory capacity (IC). (e) Flow‐volume curves for healthy controls (CTRL) and BLEO‐IPF mice (28 days after BLEO instillation). (f) Pressure‐volume curves for healthy controls (intratracheal instillation of saline vehicle, CTRL) and BLEO‐IPF mice (28 days after BLEO instillation). *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTRL. Dunnett's test one‐factor linear model.
FIGURE 3
FIGURE 3
Whole‐body plethysmography (WBP) in freely moving BLEO‐IPF mice. (a) Outline of the characterization study in BLEO‐IPF mice using repeated WBP assessment of respiratory function in individual mice on days 7, 14, 21, and 28 after bleomycin (BLEO) induction (see Figure S1 for all WBP data). Mice received a single intratracheal instillation of saline vehicle (CTRL, n = 10) or bleomycin (BLEO‐IPF, 2.0 mg/kg, n = 11). Terminal spirometry was performed on day 28. Correlation of baseline WBP (PenH, day 7) and terminal spirometry endpoints (day 28), including (b) forced expiratory volume in 0.1 seconds (FEV0.1), (c) forced vital capacity (FVC), (d) inspiratory capacity (IC), and (e) static compliance, respectively. Simple linear regression analysis.
FIGURE 4
FIGURE 4
Validation of automated deep learning‐based Ashcroft scoring of lung fibrosis in BLEO‐IPF mice. (a) Deep learning‐based Ashcroft scoring, using Gubra Histopathological Objective Scoring Technology (GHOST), applied to the entire left lung at 10x magnification. Representative Masson's trichome stainings used for image analysis. Heatmaps depict Ashcroft scores (score 0‐8; i.e., normal lung tissue architecture to total fibrous obliteration) in individual lung image tiles of 512 × 512 pixels. (b) Ashcroft score was computed and validated using a test set of lung samples from a total of 93 mice. There was a high concordance between manual and automated (GHOST) scoring (kappa value of 0.83).
FIGURE 5
FIGURE 5
Histological hallmarks of fibrotic lung disease in BLEO‐IPF mice. (a) GHOST‐based Ashcroft scoring on day 7‐42 after BLEO administration (n = 7–12 per group). Histopathological scoring was performed on sections stained with Masson's trichrome (MT). **p < 0.01, ***p < 0.001 vs. healthy controls (intratracheal instillation of saline vehicle, CTRL, n = 10); Dunnett's test one‐factor linear model. (b) Group‐wise distribution of Ashcroft scores. (c–g) Histomorphometric assessment of fibrosis (PSR, Col1a1, Col3), fibrogenesis (α‐SMA) and inflammation (Gal‐3) using conventional image analysis. Data were calculated as proportionate (%) area of staining. (c) PSR. (d) Collagen‐1a1 (Col1a1); (e) Collagen‐3 (Col3). (f) α‐SMA. (g) Galectin‐3 (Gal‐3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTRL). Dunnett's test one‐factor linear model. (h) Representative photomicrographs. Scale bar, 100 μm.
FIGURE 6
FIGURE 6
Progressive lung transcriptome changes in BLEO‐IPF mice validated against lung RNA sequencing data from IPF patients. (a, b) Venn diagrams depicting shared and separate differentially expressed genes (DEGs; false discovery rate <0.05) in BLEO IPF mice vs. patients with advanced IPF (Sivakumar et al., 2019). BLEO‐IPF mice (day 21 and 28 post‐administration (n = 12 per group) compared to intratracheal saline administration (n = 10). (c) Total number of DEGs in BLEO‐IPF mice (day 7‐42 post‐BLEO administration) compared to saline controls. (d) Number of DEGs shared between BLEO‐IPF mice at different time points after BLEO administration. (e) Curated list of 179 candidate genes linked to IPF pathogenesis and fibrosis, divided into three categories, that is, extracellular matrix (ECM) organization, immune system and TGFβ‐associated signaling. Color gradients indicate significantly upregulated (red color) or downregulated (blue color) genes compared to corresponding controls. White color indicates no significant change in gene expression compared to corresponding controls.
FIGURE 7
FIGURE 7
ALK5i treatment effects on body weight, lung weight and lung hydroxyproline content in BLEO‐IPF mice. TGFβR1/ALK5 inhibitor (SB525334) treatment outcomes were compared in 6 independent intervention studies in BLEO‐IPF mice. (a) Study outline. Mice received a single intratracheal instillation of saline vehicle (CTRL) or bleomycin (BLEO‐IPF, 2.0 mg/kg). BLEO‐IPF mice received vehicle (n = 10‐14 per study) or ALK5i (60 mg/kg/day, n = 10–16 per study), administered orally as bi‐daily (30 mg/kg, BID; study 1‐3) or once daily (60 mg/kg, QD; study 4–6) dosing for 21 days, starting 7 days after intratracheal BLEO instillation. Vehicle‐dosed CTRL mice served as healthy controls (n = 10 per study). BLEO‐IPF mice were randomized and stratified to treatment according to (b) PenH (primary factor, determined by whole‐body plethysmography, WBP) and (c) baseline body weight (secondary factor), measured on day 6 after bleomycin administration. (d) Terminal body weight. (e) Lung weight. (f) Total lung hydroxyproline (HP) content. Left panels: Data in individual mice according to group and study. *p < 0.05, **p < 0.01, ***p < 0.001 vs. BLEO‐IPF Vehicle, one‐way ANOVA with Dunnett's test for multiple comparisons. Right panels: Composite analysis of Alk5i treatment outcomes (group average). ***p < 0.001 vs. corresponding groups in study 1‐3, one‐way ANOVA with Dunnett's test for multiple comparisons.
FIGURE 8
FIGURE 8
ALK5i treatment effects on spirometry endpoints in BLEO‐IPF mice. Outcomes of TGFβR1/ALK5 inhibitor (SB525334) treatment were compared in 6 independent intervention studies performed in BLEO‐IPF mice. Mice received a single intratracheal instillation of saline vehicle (CTRL) or bleomycin (BLEO‐IPF, 2.0 mg/kg). Mice received vehicle (n = 10‐14 per study) or ALK5i (60 mg/kg/day, n = 10‐16 per study), administered orally as bi‐daily (30 mg/kg, BID; study 1‐3) or once daily (60 mg/kg, QD; study 4‐6) dosing for 21 days, starting 7 days after intratracheal BLEO instillation. Vehicle‐dosed CTRL mice served as healthy controls. (a) Forced expired volume over 0.1 seconds (FEV0.1). (b) Forced vital capacity (FVC). (c) Inspiratory capacity. (d) Static compliance. Left panels: Data in individual mice according to group and study. *p < 0.05, **p < 0.01, ***p < 0.001 vs. corresponding BLEO‐IPF Vehicle group, one‐way ANOVA with Dunnett's test for multiple comparisons. Right panels: Composite analysis of Alk5i treatment outcomes (group average). **p < 0.01, ***p < 0.001 vs. BLEO‐IPF Vehicle, one‐way ANOVA with Dunnett's test for multiple comparisons.
FIGURE 9
FIGURE 9
ALK5i treatment effects on lung fibrosis histological endpoints in BLEO‐IPF mice. Outcomes of TGFβR1/ALK5 inhibitor (SB525334) treatment were compared in six independent intervention studies performed in BLEO‐IPF mice. Mice received a single intratracheal instillation of saline vehicle (CTRL) or bleomycin (BLEO‐IPF, 2.0 mg/kg). Mice received vehicle (n = 10–14 per study) or ALK5i (60 mg/kg/day, n = 10–16 per study), administered orally as bi‐daily (30 mg/kg, BID; study 1–3) or once daily (60 mg/kg, QD; study 4–6) dosing for 21 days, starting 7 days after intratracheal BLEO instillation. Vehicle‐dosed CTRL mice served as healthy controls. (a, b) GHOST‐based Ashcroft fibrosis scoring. (c–e) Histomorphometric assessment of fibrosis (PSR, Col1a1, Col3) using conventional image analysis. Data were calculated as proportionate (%) area of histological staining. (c) PSR. (d) Collagen‐1a1 (Col1a1). (e) Collagen‐3 (Col3). Left panels: Data in individual mice according to group and study. **p < 0.01, ***p < 0.001 vs. vs. corresponding BLEO‐IPF Vehicle group. One‐way ANOVA with Dunnett's test for multiple comparisons. Right panels: Composite analysis of Alk5i treatment outcomes (group average), depicted as change vs. corresponding CTRL Vehicle group (for quantitative histology only). *p < 0.05, **p < 0.01, ***p < 0.001 corresponding BLEO‐IPF Vehicle group, one‐way ANOVA with Dunnett's test for multiple comparisons.
FIGURE 10
FIGURE 10
ALK5i Treatment effects on lung fibrogenesis and inflammation histological endpoints in BLEO‐IPF mice. Outcomes of TGFβR1/ALK5 inhibitor (SB525334) treatment were compared in six independent intervention studies performed in BLEO‐IPF mice. Mice received a single intratracheal instillation of saline vehicle (CTRL) or bleomycin (BLEO‐IPF, 2.0 mg/kg). Mice received vehicle (n = 10‐14 per study) or ALK5i (60 mg/kg/day, n = 10–16 per study), administered orally as bi‐daily (30 mg/kg, BID; study 1–3) or once daily (60 mg/kg, QD; study 4–6) dosing for 21 days, starting 7 days after intratracheal BLEO instillation. Vehicle‐dosed CTRL mice served as healthy controls. Histomorphometric assessment of fibrogenesis (α‐SMA) and inflammation (Gal‐3) using conventional image analysis. Data were calculated as proportionate (%) area of histological staining. (a) Alpha‐smooth muscle actin (α‐SMA). (b) Galectin‐3 (Gal‐3). Left panels: Data in individual mice according to group and study. *p < 0.05, **p < 0.01, ***p < 0.001 vs. corresponding BLEO‐IPF Vehicle group. One‐way ANOVA with Dunnett's test for multiple comparisons. Right panels: Composite analysis of Alk5i treatment outcomes (group average), depicted as change vs. corresponding CTRL Vehicle group. ***p < 0.001 vs. corresponding BLEO‐IPF Vehicle group, one‐way ANOVA with Dunnett's test for multiple comparisons.
FIGURE 11
FIGURE 11
Representative photomicrographs of lung histological stainings. Histological staining from representative ALK5i treatment study (QD dosing) in BLEO‐IPF mice. Mice receiving a single intratracheal instillation of saline vehicle served as healthy controls (CTRL Vehicle). α‐SMA, α‐smooth muscle actin; Col1a1, collagen‐1a1; Col3, collagen‐3; Gal‐3, galectin‐3; MT, Masson's trichrome; PSR, picro sirius red. Scale bar, 100 μm.
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