Date Published: August 2, 2017
Publisher: Impact Journals LLC
Author(s): Brandon M. Hall, Vitaly Balan, Anatoli S. Gleiberman, Evguenia Strom, Peter Krasnov, Lauren P. Virtuoso, Elena Rydkina, Slavoljub Vujcic, Karina Balan, Ilya I. Gitlin, Katerina I. Leonova, Camila R. Consiglio, Sandra O. Gollnick, Olga B. Chernova, Andrei V. Gudkov.
Constitutive p16Ink4a expression, along with senescence-associated β-galactosidase (SAβG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16Ink4a-positive cell killing to the eradication of accumulated SCs. However, detection of p16Ink4a/SAβG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16Ink4a and SAβG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16Ink4a/SAβG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16Ink4a-positive cells may not be solely attributed to SCs but also to non-senescent p16Ink4a/SAβG-positive macrophages.
Senescence is a cellular phenotype, initially described in cell culture, that is acquired in a variety of normal and tumor-derived cells following activation of the p53/p21Cip1/Waf1-, Rb/p16Ink4a-dependent DNA damage response and characterized by irreversible proliferation arrest coupled with a constitutive pro-inflammatory secretory phenotype (SASP) [1–3]. Accumulation of senescent cells (SCs) in mammalian tissues with age and the proinflammatory activity resulting from SASP has been proposed as a major factor responsible for aging-associated chronic systemic sterile inflammation (“inflammaging”, see [4–9]). Recently, this hypothesis received strong support from a series of reports that described attempts to selectively eradicate SCs in mice using genetic and pharmacological approaches [10–16]. In most models, a decrease in the number of cells with SC markers was associated with positive physiological outcomes that were interpreted as indications of rejuvenation. However, the accurate interpretation of these studies requires the reliable identification of SCs in vivo, and thus, depends on a level of exclusivity of SC markers that is currently lacking .
The accumulation of p16Ink4a-positive cells is observed in aged mice, and their eradication has been linked to certain improvements in the health state of older animals consistent with rejuvenation [10,11]. Even though p16Ink4a-positive cells in vivo have been assumed to be senescent, little evidence exists to directly support this assumption. Our previous work identifying macrophage subtypes that co-express markers conventionally assigned to SCs (p16Ink4a/SAβG)  has prompted additional interpretations of previously published experimental data regarding the role of p16Ink4a-positive cells in aging and age-related diseases, as recently articulated by Bennett and Clarke (2017)  and Kirkland and Tchkonia (2017) . As such, defining the exact nature of p16Ink4a-positive cells is crucial for proper development of therapeutics for the prevention and treatment of aging and age-related diseases. Today, the field of aging is focused on the development of senolytic compounds that are isolated for their ability to selectively kill SCs generated in vitro [16,56,57]. If these cells are different from p16Ink4a-positive cells accumulating in vivo with age, this could misdirect both academic studies of senescence as a phenomenon, as well as practical efforts to develop anti-aging therapeutics. These considerations motivated our present work, which was aimed at defining the nature of p16Ink4a-positive cells found in mouse tissues in vivo and their relation to the phenomenon of cellular senescence.