Stiffness-dependent alveolar type II cell senescence in idiopathic pulmonary fibrosis

IPF is characterized by the fibrotic response involving abnormally activated ATII cells. A stiffened matrix exerts extrinsic microenvironment-derived forces, leading to mechanical stress and ATII cell dysfunction. However, these mechanisms remain largely unknown. ATII cells were isolated from control organ donors and the explanted lungs of patients with IPF. DNA damage was assessed by the comet assay and western blotting. ATII cell senescence was determined by β-galactosidase staining. TERRA transcripts, the long noncoding RNA telomeric repeat-containing RNA, and R-loops, which can impact genome integrity, were analyzed by qPCR. H3K27me3 and H3K9me3 levels were assessed by immunofluorescence. ATII cells were cultured on PDMS hydrogel to study mechanical stiffness. The impact of YAP/TAZ inhibition on control ATII cells cultured on a 50 kPa PDMS hydrogel was also evaluated. A significant increase in γH2AX expression and impairment of NHEJ were detected in ATII cells in IPF. The data obtained indicate defective DDR and decreased DNA damage repair capacity in ATII cells in patients with this disease, which can contribute to senescence, replication stress, transcription replication conflicts, and genomic instability. Moreover, the increased levels of H3K27me3 and H3K9me3 in ATII cells in IPF suggests repressed transcriptional activity. Nuclear lamina discontinuity was identified by immunofluorescence using emerin staining. Mechanotransduction signaling influenced epigenetic regulation in ATII cells in response to mechanical stiffness. This study provides new mechanistic insights into the function of ATII cells and the integrity of the alveolar epithelium under highly stressed conditions in IPF. The data underscores the importance of mechanical stiffness on ATII cell dysfunction and senescence.