ABSTRACT
The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.
Subject(s)
Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p16 , Fibroblasts , Idiopathic Pulmonary Fibrosis , Animals , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Mice , Humans , Fibroblasts/metabolism , Fibroblasts/pathology , Fibroblasts/drug effects , Cellular Senescence/drug effects , Idiopathic Pulmonary Fibrosis/pathology , Idiopathic Pulmonary Fibrosis/metabolism , Idiopathic Pulmonary Fibrosis/drug therapy , Idiopathic Pulmonary Fibrosis/genetics , Senotherapeutics/pharmacology , Male , Lung/pathology , Lung/metabolism , Female , HSP90 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/geneticsABSTRACT
We engineered an ultrasensitive reporter of p16INK4a, a biomarker of cellular senescence. Our reporter detected p16INK4a-expressing fibroblasts with certain senescent characteristics that appeared shortly after birth in the basement membrane adjacent to epithelial stem cells in the lung. Furthermore, these p16INK4a+ fibroblasts had enhanced capacity to sense tissue inflammation and respond through their increased secretory capacity to promote epithelial regeneration. In addition, p16INK4a expression was required in fibroblasts to enhance epithelial regeneration. This study highlights a role for p16INK4a+ fibroblasts as tissue-resident sentinels in the stem cell niche that monitor barrier integrity and rapidly respond to inflammation to promote tissue regeneration.
