Background/Purpose: Repetitive alveolar epithelial injury together with dysregulated tissue repair is crucial for the transition of acute self-limiting to chronic-persisting inflammation and fibrosis in fibrosing interstitial lung disease (ILD). Yet, this aspect is not adequately mimicked in the widely used preclinical bleomycin (BLM)-induced model. We evaluated whether repetitive BLM injury leads to closer resemblance with human fibrosing ILD pathophysiology including persistent fibrosis.

Methods: C57BL/6J mice (female/male) received 4 bi-weekly intratracheal (i.t.) instillations of 0.5 U/kg, 1 U/kg, or 2 U/kg BLM (n=10 per dose/sex) to model chronic fibrosing ILD. Controls received saline i.t. (n=6 per sex). All mice were sacrificed on day 77 (i.e., 5 weeks after the final instillation), a time where resolution processes are typically evident in the single BLM injury (acute) model. Disease severity was assessed by Ashcroft score. Markers of fibrosis and inflammation were evaluated by gene expression profiling and quantification of immunolabelled lung tissue sections. Outcomes were compared to female cohorts of the acute model from a previous study, where lung tissue was collected 5 weeks after single 2 U/kg BLM (n=11) or saline (n=6) i.t. instillation.

Results: Body weight loss and recovery cycles were present after each instillation, indicating susceptibility to repetitive alveolar epithelial injury. Humane endpoints were reached in 0%, 20%, and 85% of mice that received 0.5, 1, and 2 U/kg BLM, respectively, demonstrating experimental feasibility for low and medium doses. Mean Ashcroft scores (± s.d.) for female/male mice were 2.8 (±1.3) / 4.0 (±2.0) for 0.5 U/kg, and 4.8 (±1.1) / 6.4 (±0.7) for 1 U/kg BLM, the latter being representative of human ILD. Generally, male mice displayed higher disease severity, suggesting the involvement of sex-associated factors in disease development. In contrast, mice from the acute model exhibited a score of 4.4 (±2.0). Importantly, the histological phenotype of the chronic, but not the acute model reflected typical features of human ILD, including presence of honeycomb-like lesions and a more uniformly spread interstitial fibrosis pattern. In the chronic model, we detected upregulation of fibrotic and inflammatory markers (FN1, CCL2, IL6). Additionally, cytokeratin 8 and 5, markers for a transitional alveolar epithelial cell (AEC) subtype and AEC-derived metaplastic basal cells in human ILD, respectively, were highly upregulated in the chronic, yet not the acute model. Furthermore, suppression of GLI1 and upregulation of CCSP, markers of mesenchymal stromal cells promoting metaplasia in the fibrotic and mucin-secreting club cells in human ILD, respectively, were exclusively observed in the chronic ILD setting.

Conclusion: The findings indicate that typical features of human fibrosing ILD are reflected in vivo upon repetitive, yet not upon single BLM instillation. As such, this preclinical model has great potential for investigation of mechanisms relevant to the transition of transient to persistent inflammation and fibrosis, and for testing of novel anti-fibrotic drug candidates.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/key-features-of-human-fibrosing-interstitial-lung-disease-are-captured-in-a-preclinical-mouse-model-upon-repetitive-but-not-single-intratracheal-bleomycin-dosing/