Multiparametric senescent cell phenotyping reveals CD24 osteolineage cells as targets of senolytic therapy in the aged murine skeleton

Doolittle, Madison L.; Saul, Dominik; Kaur, Japneet; Rowsey, Jennifer L.; Vos, Stephanie J.B.; Pavelko, Kevin D.; Farr, Joshua N.; Monroe, David G.; Khosla, Sundeep

doi:10.1101/2023.01.12.523760

Cited by 6 publications

(25 citation statements)

References 121 publications

(153 reference statements)

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“…In the case of fracture, we identify these cells as OCH cells and a specific sub-set of neutrophils that express high levels of p21 and a SASP, and we also define important signaling pathways between these key cell populations. Moreover, by directly comparing the effects of genetic senescent cell clearance in p21-ATTAC vs. p16-INK-ATTAC mice, we provide further evidence that the injury-related senescent phenotype in the setting of skeletal injury (fracture, radiation (17)) is predominantly driven by p21, particularly in the early healing phase, in contrast to age-related bone loss, which appears to be principally driven by p16 (14,19,46). Finally, targeting the injury-related senescent-like populations and/or the pathways we identified may prove beneficial in accelerating fracture healing and potentially repair across multiple tissues (e.g., muscle).…”

Section: Discussionmentioning

confidence: 78%

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Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair

Saul,

Doolittle,

Rowsey

et al. 2024

Preprint

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Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells are key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings establish contextual roles of p21+ vs p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

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Section: Discussionmentioning

confidence: 78%

“…Specifically, in the context of aging, these cells appear to be principally, if not exclusively, of mesenchymal origin (14,47). Indeed, cellular senescence was originally defined by Hayflick and Moorhead for mesenchymal cells (48), and whether immune cells express the full features of classical senescent cells remains unclear.…”

Section: Discussionmentioning

confidence: 99%

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair

Saul,

Doolittle,

Rowsey

et al. 2024

Preprint

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“…Table S1 provides a list of all antibodies used in this analysis; note also that each of these antibodies have been validated for CyTOF by the Mayo Clinic CyTOF Core Laboratory, and additional validations of these antibodies in our laboratory are described in Doolittle et al . [55]. For these studies, we treated 21-month-old female mice with zoledronate or vehicle for two weeks.…”

Section: Resultsmentioning

confidence: 99%

“…Note that each of these antibodies have been validated for CyTOF by the Mayo Clinic CyTOF Core Laboratory; additional validations for the antibodies used are provided in Doolittle et al . [55].…”

Section: Methodsmentioning

confidence: 99%

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In vitroandin vivoeffects of zoledronate on senescence and senescence-associated secretory phenotype markers

Samakkarnthai

Saul

Zhang

et al. 2023

Preprint

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In addition to reducing fracture risk, zoledronate has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronate could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronate killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronate or vehicle for 8 weeks, zoledronate significantly reduced circulating SASP factors, including CCL7, IL-1β, TNFRSF1A, and TGFβ1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronate demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronate, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronate significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronate has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronate and/or other bisphosphonate derivatives for senotherapeutic efficacy.

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Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair

Saul,

Doolittle,

Rowsey

et al. 2024

Journal of Clinical Investigation

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Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21 + cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21 + cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21 + cell clearance did not alter bone loss due to aging; conversely, p16 + cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21 + neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21 + OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell–like OCH subset that was p21 + and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21 + versus p16 + senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

show abstract

Multiparametric senescent cell phenotyping reveals CD24 osteolineage cells as targets of senolytic therapy in the aged murine skeleton

Cited by 6 publications

References 121 publications

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair

In vitroandin vivoeffects of zoledronate on senescence and senescence-associated secretory phenotype markers

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair

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