Highly Calibrated Relationship Between Bleomycin Concentrations and Facets of the Active Phase Fibrosis in Classical Mouse Bleomycin Model

The mouse bleomycin model is useful in pre-clinical IPF research to understand pathophysiological mechanisms and pharmacological interventions. In the present study, we systematically investigated the effects of bleomycin at a 60-fold dose range on experimental features of lung fibrosis in the mouse bleomycin model. We analyzed the effect of intratracheal (i.t.) dosing of 0.05-3 U/kg bleomycin on disease phenotypes, including weight loss, morbidity and mortality, pulmonary inflammation, lung collagen content, various BALF biomarkers, and histology in a 14-day mouse model when the animals are in the active phase of fibrosis. In mice, challenge with 1-2 U/kg bleomycin doses induced significant and saturated responses on fibrotic endpoints, confirmed by collagen content, BALF biomarker levels, and marked weight loss compared to the normal control (NC). We observed 100% mortality in 3 U/kg of bleomycin-treated mice. In contrast, 0.05-0.5 U/kg bleomycin doses induced a dose-dependent fibrotic phenotype. The mice challenged with doses of 0.25-0.5 U/kg bleomycin showed optimum body weight loss, a significant increase in pulmonary inflammation, and the fibrotic phenotype compared to NC. Furthermore, we showed 0.25-0.5 U/kg bleomycin increases expression levels of (pro-) fibrotic cytokines, which are the mediators involved in the activation of myofibroblast during fibrogenesis (TGF-β1, IL-13, IL-6, WISP-1, VEGF), angiogenesis (VEGF), matrix remodeling (TIMP-1), and non-invasive lung function biomarker (CRP) compared to NC. A modified Ashcroft scale quantified that the fibrotic changes in the lungs were significantly higher in the lung of mice dosed at 0.25-0.5 U/kg > 0.1 U/kg bleomycin and non-significant in mice lung dosed at 0.05 U/kg bleomycin compared to NC. We demonstrated that the changes due to 0.25-0.5 U/kg i.t. bleomycin on protein biomarkers are enough to drive robust and detectable fibrotic pathology without mortality. The 0.1 U/kg has a moderate phenotype, and 0.05 U/kg had no detectable phenotype. The Goodness of Fit (