An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis
Jin Young Lee, Nabora S. Reyes, Supriya Ravishankar, Minqi Zhou, Maria Krasilnikov, Christian Ringler, Grace Pohan, Chris Wilson, Kenny Kean-Hooi Ang, Paul J. Wolters, Tatsuya Tsukui, Dean Sheppard, Michelle R. Arkin, Tien Peng
Jin Young Lee, Nabora S. Reyes, Supriya Ravishankar, Minqi Zhou, Maria Krasilnikov, Christian Ringler, Grace Pohan, Chris Wilson, Kenny Kean-Hooi Ang, Paul J. Wolters, Tatsuya Tsukui, Dean Sheppard, Michelle R. Arkin, Tien Peng
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Research Article Aging Pulmonology

An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis

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.

Authors

Jin Young Lee, Nabora S. Reyes, Supriya Ravishankar, Minqi Zhou, Maria Krasilnikov, Christian Ringler, Grace Pohan, Chris Wilson, Kenny Kean-Hooi Ang, Paul J. Wolters, Tatsuya Tsukui, Dean Sheppard, Michelle R. Arkin, Tien Peng

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Figure 5

XL888 deletes p16Ink4a+ fibroblasts and attenuates fibrotic remodeling in vivo.

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XL888 deletes p16Ink4a+ fibroblasts and attenuates fibrotic remodeling i...
(A) Schematic outline of animal experiments to validate in vivo efficacy of candidate senolytics. (B and C) Flow cytometry analysis of GFP+ fibroblasts (% of fibroblasts that are GFP+) in bleomycin-injured lungs of vehicle or XL888 delivered INKBRITE animals (n = 11–12 biological replicates, experiment repeated twice). (D and E) Immunofluorescence analysis (D) and quantification (E) of GFP+ cells among ACTA2+ fibroblasts in the lungs of vehicle or XL888-treated INKBRITE mice (n = 4 biological replicates, experiment repeated twice). Scale bars: 100 μm. (F) Representative images (left) and quantification of Masson’s trichrome staining of lung sections from indicated group of mice after bleomycin injury (n = 4 biological replicates). Scale bars: 1,000 μm. (G) Quantitative analysis of collagen in lung homogenates from vehicle or XL888 treated animals injured with bleomycin (n = 19–20 biological replicates, experiment repeated twice). Data are represented as mean ± SD. *P < 0.05; **P < 0.01; 2-tailed Student’s t test (C); or 1-tailed Student’s t test (E–G).