An ex vivo model of cellular senescence and inflammaging in precision-cut lung slices

Abstract

Cellular senescence and an increased pro-inflammatory milieu, known as inflammaging, contribute to the development of chronic lung diseases like idiopathic pulmonary fibrosis. Precision-cut lung slices (PCLS) preserve lung cellular diversity and architecture and have provided insights into pulmonary fibrosis&nbsp;pathobiology but we lack a model to study aging-related mechanisms. Therefore, we aimed to establish an <i>ex vivo</i> model of lung inflammaging based on PCLS. Mouse PCLS were analyzed by single-cell sequencing and <i>ex vivo</i> aging was induced by irradiating (15-120Gy) mouse and human PCLS. Viability was evaluated by WST-1 assay, markers of senescence and inflammaging were assessed by RT-qPCR, flow cytometry, and immunofluorescence (IF). Inflammatory mediators were detected by ELISA. We found that PCLS remained viable at least for 7 days post-irradiation (dpir). Single-cell sequencing analysis confirmed the preservation of all compartments including alveolar epithelial cells as well as adaptive and innate immune cells during 7 days of culture. Irradiated PCLS (IR-PCLS) had an increased DNA damage response and cellular senescence as evident by the nuclear accumulation of γH2Ax and increased β-galactosidase activity. Expression of the cell cycle inhibitors, p21 and p16, and secretion of Interleukin 6, an inflammatory mediator, were increased 7 dpir. IF staining and flow cytometry analysis showed an enrichment of senescent cells in the epithelial compartment in IR-PCLS.&nbsp;In conclusion, we present a novel <i>ex vivo</i> model of cellular senescence and inflammaging. This model provides a platform to study age-related lung diseases&nbsp;and future work will focus on understanding senescence-mediated fibrotic changes.