Senescent lung fibroblasts attenuate alveolar epithelial cell proliferation and migration in IPF

Introduction/Aim Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterised by excessive accumulation of fibroblast (Fbs) and collagen in lung parenchyma. Our laboratory has shown that, in comparison to age-matched controls, lung Fbs from IPF patients in vitro exhibit increased characteristics of senescence including growth arrest and an activated secretome. We hypothesised that senescent Fbs disrupt alveolar epithelial regeneration following injury to perpetuate fibrosis in IPF. The aim of this study was to examine the effect of senescent lung Fbs on the growth and wound repair responses of alveolar epithelial cells. Methods Primary Fb cultures were established from lung tissue of patients with IPF (IPF-Fbs) and age-matched controls (Ctrl-Fbs). Ctrl-Fbs were treated with H202 (150 μM, 2h) followed by recovery for 72 h, to induce senescence. Senescence was detected by increased senescence-associated β-galactosidase activity and p16/p21 expression. IPF-Fbs or senescence-induced Ctrl-Fbs were co-cultured with A549 alveolar epithelial cells maintained in transwell inserts in the presence of 5% foetal bovine serum. After 48 h in co-culture, A549 cells were harvested for cell counting and cell-cycle analysis, or migration was analysed using a ‘scratch-wound' assay. Furthermore, the effects of conditioned media (CM) from Fbs on A549 cell growth and wound repair were evaluated. Results A549 cell proliferation was attenuated when either co-cultured with or grown in CM from senescence-induced Ctrl-Fbs, as compared to non-senescent Ctrl-Fbs (P