Senescence intensifies bleomycin-induced injury in idiopathic pulmonary fibrosis lung epithelial cells

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease involving injury and a dysregulated repair response of an ageing epithelium. Here we investigate the contribution of senescence and telomere shortening to bleomycin-induced injury in alveolar epithelial cells (EpCs) derived from lung parenchymal tissue of patients with pulmonary fibrosis (PF), predominantly IPF, in vitro. METHODS: Injury responses of EpCs in air-liquid interface (ALI) culture were assessed by ELISA, qPCR and/or immunofluorescence at 48 and 96 h post-bleomycin (30 μg·mL⁻¹) treatment. Telomere length was evaluated by qPCR and corroborated by fluorescence in situ hybridisation. RESULTS: Bleomycin treatment increased markers of inflammation (i.e. interleukin (IL)-6 and IL-36γ) and senescence (i.e. p16 and p21) in EpCs from PF patients (PF EpCs) (p<0.05); responses markedly higher than control EpCs (Ctrl EpCs). Conversely, the effects of bleomycin on the expression of type I and type II alveolar EpC markers were supressed or downregulated in cultures of PF EpCs (when compared with Ctrl EpCs), whereas the percentage of keratin 17-positive type I and II intermediate EpCs were increased (p<0.05). Telomere length was also significantly shorter in PF than Ctrl EpCs at baseline and further reduced post-bleomycin treatment (p<0.05). Induction of telomere shortening by cell passaging augmented bleomycin-induced PF EpC injury, whereas pharmacological telomerase activation and selected senotherapeutics were protective. CONCLUSION: This study provides evidence that senescence-associated telomere shortening increases the susceptibility of IPF lung EpCs to injury. Our cell-based lung epithelial injury model has utility as a pre-clinical tool for drug discovery and the evaluation of potential senotherapeutics for IPF treatment.