Experimental modeling of pulmonary fibrosis and combined emphysema in mice using repeated doses of bleomycin and cigarette smoke extract

Rationale

A high prevalence of smoking among idiopathic pulmonary fibrosis (IPF) patients increases the risk of emphysema. Combined pulmonary fibrosis and emphysema (CPFE) occurs predominantly in males, characterized by severe exercise-induced dyspnea, decreased diffusing capacity, and increased lung cancer risk. The exclusion of CPFE patients from clinical studies has limited understanding of its pathophysiology, treatment, and prognosis. This study aimed to close this knowledge gap by comparing CPFE with pure IPF in animal models, with the intent of developing phenotype-directed therapeutic strategies.

Methods

Eight-week-old female C57BL/6J mice received biweekly intratracheal bleomycin (BLM, 2 U/kg/100 μL) for 3 doses. For the CPFE model, additional intranasal cigarette smoke extract (CSE) was administered twice weekly. Inflammation and fibrosis were evaluated through bronchoalveolar lavage fluid (BALF), proinflammatory cytokines, fibrosis markers, and lung histology.

Results

BALF analysis showed increased cell counts in all BLM-treated groups, primarily macrophages, with elevated interleukin (IL)-6, transforming growth factor (TGF)-β1, and matrix metalloproteinases (MMP 3 and MMP 8). The BLM and CSE treated group displayed higher macrophages and lymphocytes counts than BLM alone. Lung tissue analysis revealed increased hydroxyproline, inflammation scores, Ashcroft scores, and higher α-smooth muscle actin (SMA) and collagen 1 expression in BLM-treated groups. The BLM and CSE treated group demonstrated notably higher emphysema formation as measured by mean linear intercept (MLI).

Conclusions

Through repeated BLM administrations and CSE exposure, an effective model for persistent pulmonary fibrosis and emphysema was established, enabling preclinical studies on CPFE and IPF and exploration of phenotype-directed therapeutic strategies for pulmonary fibrosis.