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, 8 (7), 1294-315

Aging of Mice Is Associated With p16(Ink4a)- And β-Galactosidase-Positive Macrophage Accumulation That Can Be Induced in Young Mice by Senescent Cells


Aging of Mice Is Associated With p16(Ink4a)- And β-Galactosidase-Positive Macrophage Accumulation That Can Be Induced in Young Mice by Senescent Cells

Brandon M Hall et al. Aging (Albany NY).


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-gal(pH6)), 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-gal(pH6)-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-gal(pH6)-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-gal(pH6)-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-gal(pH6)-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Keywords: chronological aging; clodronate; inflammaging; inflammation; p16INK4a; senescence-associated beta-galactosidase; senescence-associated secretory phenotype.

Conflict of interest statement

statement A.P., O.B.C. and A.V.G. are co-founders and shareholders of Everon Biosciences.


Figure 1
Figure 1. SC implantation in vivo
(A-B) Bioluminescent signal accumulation in a cohort (n=5 mice) of chronologically aged mice harboring a hemizygous p16(Ink4a) knock-in of luciferase (p16LUC mice; p16Ink4a/Luc). (A) Whole body luminescence (total flux; p/s) for individual mice are depicted. (B) Serial bioluminescence imaging of chronologically aged mice. Color scale indicates signal intensity (same thresholds across all time points). (C-D) A model of SC implantation into SCID mice. NDF cells harboring a secreted GLuc reporter construct (NDF-GLuc) were implanted intraperitoneally into SCID mice as microcarrier bead cultures that were, prior to injection, cultured in low serum (0.2% FBS) for induction of quiescence (Qui NDF) or irradiated at 20 Gy for induction of senescence (Sen NDF). Alternatively, irradiated NDFs were coated in protective alginate gel (Sen NDF + Alg). Kinetics of NDF-GLuc survival was monitored via measurement of GLuc activity in mouse plasma collected at regular intervals over 28 days. The amount of GLuc activity remaining in the blood over time is expressed as a percentage of activity in plasma 24 hours after cell inoculation. Values depicted are means ± SEM for each group (n = 4-6 mice/group). Differences between all groups are statistically significant after day 7 (p≤0.001). (D) Microphotographs of empty alginate beads (no cells) and alginate beads containing embedded irradiation-induced senescent NDFs (bright field images). After embedding senescent NDF cells and before implantation into mice, viability of embedded cells was assessed by labeling live cells with Calcein AM (green) and dead cells with propidium iodide (PI; red), followed by fluorescent microscopy. Senescent NDFs in alginate beads were also assessed for β-galpH6 staining. Representative images are shown (magnification 100x). Successful embedding of cells was indicated by >90% viability (< 10% PI-positive cells).
Figure 2
Figure 2. Accumulation of β-galpH6- and p16(Ink4a)-positive immunocytes in response to SC implantation
(A) Images of whole alginate beads ex vivo (either empty or containing SCs) following retrieval from peritoneal cavity of p16LUC mice. The dense encapsulating cell layers surrounding the alginate beads were visualized by phase contrast light microscopy (top panel) or by fluorescent microscopy of samples stained with a DNA dye kit (CyQUANT™ Direct) for visualization of nuclei within live cells (middle panel). β-galpH6 staining reveals activity in cells encapsulating SC-embedded alginate beads (magnification 100x). (B) Tissue sections (15-μm) of cryopreserved SC-embedded alginate beads were stained with Geimsa for visualization of histology via light microscopy at 100x and 400x magnification (top left and right panels, respectively), for F4/80 immunofluorescence (green) for visualization of macrophages, showing specific staining of this outer membrane-localized protein (bottom left panel; 400X magnification), and for β-galpH6 activity via X-Gal substrate with nuclear fast red counterstain (400X magnification). Alginate gel containing SCs is indicated (Alg). (C) Bioluminescent in vivo imaging of p16LUC mice following i.p. inoculation of empty alginate bead (Empty) or alginate-embedded SCs (Sen). Representative serial images acquired two days before bead injection (baseline), and days 5 and 12 after injection, depict increased luminescent signal in mice bearing SCs. The colored scale depicts relative luminescent signal intensity of minimum and maximum thresholds, displayed in terms of radiance. Red arrow indicates injection site wound from alginate bead implantation. (D) The amount of bioluminescence on day 12 post-SC injection is expressed as the total flux (p/s) from the abdomen, expressed as the fold increase in signal compared to baseline measurements. (E) Analysis of the cell composition of peritoneal lavage from mice bearing SCs collected 2-3 weeks post-inoculation, as analyzed by flow cytometry on live cells immunostained for surface markers. The percent contribution to major cell types is depicted: macrophages (Mac), B lymphocytes (B cells; B), eosinophils (Eos) and remaining cell populations (Rest). This analysis depicts a representative experiment (E-G) in which these 4 cell populations were isolated via FACS and assayed for luciferase activity (F) and β-gal activity (G), normalized to cell number. The gating scheme used for FACS is presented in Supplemental Figure S1. Values depicted are means ± SEM of fold induction for each group (n = 3-6 mice/group).
Figure 3
Figure 3. Pharmacological clearance of macrophages in vivo depletes luciferase and β-galpH6 activity from p16LUC mice bearing SCs
(A) Representative serial images depicting in vivo bioluminescence from p16LUC mice acquired 12 days after inoculation of empty beads (Empty) or alginate-embedded SCs (before treatment) and one week later (after treatment; 18-20 days post-inoculation) after two i.p. administrations of liposomes containing PBS control (Veh) or clodronate (Clod). Colored scale depicts relative luminescent signal intensity of minimum and maximum thresholds, displayed in terms of radiance. (B) The amount of luminescence (total flux; p/s) from the abdomen after treatment is expressed as the fold difference compared to the signal measured before treatment for each group. (C) Total yield of cells recovered from peritoneal lavage from naïve mice, of liposomal vehicle-treated mice bearing empty beads (Em/Veh), or of liposomal vehicle- or clodronate-treated mice bearing SCs (Sen/Veh and Sen/Clod, respectively). (D) The amount of macrophages present in peritoneal lavage of treated mice bearing SCs is expressed as the percentage of F4/80-positive cells present within the population of live CD45-positive cells, as assessed via flow cytometry on immunostained cells. Cell lysates of whole lavage after treatment were assayed for luciferase activity (E) and β-galpH6 activity (F), normalized to cell number. Values depicted are means +/− SEM (n = 3-7 mice/group). ns = not statistically significant, p>0.05; n.d. = not detectable, values depicted indicate detection limit (defined as 2-fold above background reading) per cell number analyzed.
Figure 4
Figure 4. Clodronate treatment depletes p16(Ink4a)-positive and β-galpH6-positive cells from chronologically aged p16LUC mice
(A) Bioluminescent baseline readings from the abdomen of young (13 weeks) versus old (90 weeks) p16LUC mice (n=5 and 17 mice/group, respectively). Geometric mean is depicted on graph. (B-C) Old mice were randomized among 3 groups based on bioluminescence from the abdomen (n=5-6 per group): treatment with PBS, vehicle liposomes in PBS (Veh), or liposomal clodronate (Clod). Bioluminescence of the abdomens was measured after two clodronate treatments (i.p., three days prior and i.v., one days prior to luminescent measurement). (B) Representative serial images of p16LUC mice depicting luminescence (in radiance) before and after treatment regimen. Colored scale depicts relative luminescent signal intensity of minimum and maximum thresholds, displayed in terms of radiance. (C) The amount of luminescent signal (total flux; p/s) from the abdomen of treated p16LUC mice is expressed as the fold difference compared to measurement before treatment. Geometric mean is depicted on graph. (D) Inguinal and visceral (perigonadal) depots of white adipose tissue (iWAT and vWAT, respectively) were collected from vehicle and clodronate liposome treated 90-week old p16LUC mice and stained for β-galpH6 activity. Representative photographic images are presented. (E) Representative light microscopy images (magnification, 200x) of β-galpH6-stained visceral adipose tissue counterstained with nuclear fast red. Cells residing between adipocytes (indicated by the presence of nuclear stain) are β-galpH6-negative (white arrow) or -positive (black arrow). These cells are altogether absent from large regions in clodronate-treated mice (as depicted). (F) Representative images of β-galpH6-stained cultures of mouse adipose-derived mesenchymal stromal cells (mAdMSC) from p16LUC mice at early passage (p1 cultures) or 10 days after 20Gy gamma-irradiation (Senescent). SCs stain positive for β-galpH6 and are enlarged and morphologically distinct from early passage. (G) Phase contrast light microscopy images of senescent mAdMSCs following overnight (20 hr) with 50 μg/mL clodronate liposomes (Clod), or similar dilution (1:100) of vehicle liposomes (Veh) or PBS (non-treated), indicating no observable cell death or effects on these cells.
Figure 5
Figure 5. Schematic of hypothetical model of in vivo accumulation of p16(Ink4a)/β-galpH6-positive cells in naturally aged organisms
In young mammals (top panel), the secretion of SASP by p16(Ink4a)/β-galpH6-positive SCs facilitates the attraction of innate immune components necessary for efficient targeting and destruction of SCs. SC secretions activate recruited macrophages, inducing a p16(Ink4a)/β-galpH6-positive phenotype in them. After the successful eradication of SCs, inflammatory factors subside and tissue homeostasis resumes. This resolution results in the loss of p16(Ink4a)/β-galpH6-positive cells from the tissue, as macrophages with this phenotype are cleared or discharge their activated state. However, in old animals (bottom panel), impairments in innate immunity result in the inability to efficiently recognize or destroy SCs. This results in establishment of chronic, inflammation induced by products of secretion of SCs and SC-associated macrophages (SAM). Accumulation of SAMs can be a manifestation of unresolved innate immune response leading to chronic sterile systemic inflammation typical for aged organisms.

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