Abstract
Introduction
Pulmonary fibrosis is an age-related, progressive disease that can develop following thoracic radiation therapy. Radiation-induced lung fibrosis is associated with chronic inflammation and impaired tissue repair. An increasingly recognized driver of pulmonary fibrosis is cellular senescence, which is characterized by stable cell-cycle arrest and a potent pro-inflammatory 'senescence-associated secretory phenotype,' or 'SASP'. Senescent cells accumulate with age and repeated injury, and are recognized as a critical link between aging and age-related diseases, including pulmonary fibrosis. Previously, the euchromatin mark H4K16ac was reported to promote profibrotic gene expression in lung fibrosis. Here, we will establish clinically relevant, repeated low-dose radiation-induced senescence model in ex vivo 3D organoid and 2D cellular models to determine whether radiation-induced senescence is mediated by H4K16ac.
Methods
IMR90 human lung fibroblasts were exposed to repeated low-dose radiation (1 or 2Gy every 2–3 days for 10 days) to model fractionated radiation exposure. Senescence was assessed by growth arrest, SA-βgal staining, apoptosis resistance, and p16 and p21 expression. Inflammatory gene expression was evaluated by mRNA profiling. To extend these findings to a 3D lung tissue environment, we established a mouse precision-cut lung slice (PCLS) model and evaluated radiation-induced changes by mRNA profiling and immunofluorescence. The active histone mark, H4K16ac, levels are examined in the 3D model by immunofluorescence, and the occupancy of H4K16ac at SASP-associated genes in lung fibroblasts was examined in 2D model following radiation by chromatin immunoprecipitation (ChIP) assays.
Results
Repeated low-dose radiation induced a senescent phenotype in IMR90 fibroblasts, with upregulation of inflammatory SASP-associated genes. In our PCLS model, radiation increased mRNA markers of senescence, inflammation, and fibrosis, along with increased p21 and H4K16ac by immunofluorescence. H4K16ac is enriched in lung fibroblasts following radiation and at promoters of key inflammatory SASP genes (IL-1B, IL-6). These findings demonstrate that repeated low-dose radiation induces senescence, inflammatory, and fibrotic changes in lung tissue and supports a potential role for H4K16ac in this response. Ongoing studies aim to identify other SASP-related genes that are linked to H4K16Ac in radiation-induced senescence.
Conclusion
We have established complementary fibroblast and PCLS models for investigating senescence induced by fractionated radiation exposure. The euchromatin mark H4K16Ac is enriched in lung tissue following irradiation and mediates the up-regulation of key SASP-related inflammatory genes. Ongoing studies to define the role of H4K16ac in radiation induced SASP-related gene transcription. Establishing these mechanisms may identify pathways that contribute to persistent lung injury and provide potential targets for limiting radiation-induced pulmonary fibrosis.