Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells

Abstract

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-galpH6), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-galpH6-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-galpH6-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-galpH6-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-galpH6-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Senescence in Health and Disease

“…Beads that elaborate SASP-components attract macrophages, which show some features of senescence (e.g. SA-β-gal and p16 INK4a expression) (Hall et al, 2016). It is likely that some bone marrow-derived cells found in nascent neoplasms or after UV injury are also p16 INK4a –expressing macrophages (Burd et al, 2013; Sorrentino et al, 2014a), and these cells may accumulate with aging.…”

Discovery of piperlongumine as a potential novel lead for the development of senolytic agents

“…Studies have shown that the genetic clearance of SCs extends the lifespan of mice and delays the onset of several age-associated diseases in both progeroid and naturally-aged mice [12–15]. It has also been shown that rapamycin and metformin increase lifespan in mice and marmoset monkeys, by suppressing the induction of senescence [16–20].…”

Senescence associated macrophages and “macroph-aging”: are they pieces of the same puzzle?

“…However, when Hall and co-authors analyzed the adipose tissue of old mice for SCs before and after clodronate treatment, they found a large reduction in β-gal+ cells. In addition, most β-gal+ cells expressed F4/80, a well-known and widely used marker of murine macrophage populations, thus reinforcing the notion that most SCs accumulating in fat during aging are a macrophage subset, rather than pre-adipocytes [4]. …”

“…Now, Hall and co-workers have demonstrated that SCs can spread senescence to immune cells [4]. By implanting into the peritoneal cavity of SCID mice senescent fibroblasts capable of releasing a small bioluminescent protein, the authors showed that acquired immunity is not mandatory for SC clearance.…”

“…A considerable fraction of these cells were cleared by liposomal clodronate, which kills cells with phagocytic activity. These cells were defined senescence associated macrophages (SAMs) [4]. The possibility that clodronate treatment affected SC types other than macrophages cannot be totally excluded, since fibroblasts and endothelial cells also have a certain phagocytic activity and become senescent both in vivo and during in vitro replicative senescence [2].…”