The Assessment of Clinically Relevant Extracellular Matrix Markers in a Bleomycin‐Induced Mouse Model of Lung Fibrosis

Abstract

The bleomycin‐induced lung fibrosis model is routinely used to investigate efficacy of potential therapies for treating idiopathic pulmonary fibrosis (IPF). The aim here was to characterize a bleomycin model over an extended time course and correlate inflammation, lung function changes, collagen deposition and pathology with systemic markers reflecting extracellular matrix (ECM) metabolism assessed by MMP derived collagen fragments (C1M, C3M); markers associated with the diagnosis and prognosis of human idiopathic pulmonary fibrosis (IPF) (Jenkins et al, Lancet Respir Med 2015).MethodsC57BL/6J mice were anaesthetized and dosed with bleomycin (BLM) intratracheally (0.03 units/mouse). Control animals received saline. Animals were observed daily and bodyweight measured from D3. Lung function was assessed on days −1, 3, 7, 14, 21, 28, 35, 42 and 56. Animals were sacrificed on days 3, 7, 14, 21, 28, 35, 42, and 56 and serum samples taken. Markers of the formation and degradation of collagen type I (C1M) and collagen type III (C3M) were measured. Lungs from separate cohorts were lavaged for mediator and inflammatory cell analysis, then perfused and snap‐frozen for determination of hydroxyproline and protein levels. Another cohort had their lungs inflated with phosphate buffered formalin and used for histopathological analysis (including Modified Ashcroft score) and subsequent image analysis.ResultsBLM elicited a decrease in bodyweight, peaking on D7. BLM induced lung inflammation within the BALF with increases in neutrophils (peaking at D7, +135% p<0.05), lymphocytes (peaking at D14, +5240% p<0.0001) and macrophages (peaking at D21, +134%, p<0.05). Lung wet weight also increased following BLM‐administration, peaking on D14 (+44%, p<0.0001). There were observable BLM‐induced changes in lung function (peaking at D7), which were fully resolved by D21. Levels of hydroxyproline were elevated from D21 onwards (+63%, p<0.05). Pathology also increased over this time period, with a clear inflammatory response being seen at D7 and D14 and fibrotic lesions increasing in severity and prevalence from D21. The ECM degradation biomarker C1M was elevated in serum on D7 and 14 (+32%, p<0.01), with C3M increasing on D7,14 and 21 (+15–25%, all p< 0.05). These biomarkers were not observed in other models of inflammation (e.g., LPS‐induced lung inflammation).These data suggest that in this BLM‐induced lung fibrosis model, there is an early inflammatory phase, followed by a generation of fibrosis and collagen deposition. Clinically relevant biomarkers associated with diagnosis and progression of IPF can be measured in this model and may improve the predictability and translatability of such models when considering assessment of novel mechanisms and compoundsSupport or Funding InformationCharles River, Nordic Biosciences and Grunenthal