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.
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. SC implantation
A-B) Bioluminescent signal accumulation in a cohort (n=5 mice) of chronologically aged mice harboring a hemizygous p16(Ink4a) knock-in of luciferase (p16 LUC mice; p16 Ink4a/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-gal pH6 staining. Representative images are shown (magnification 100x). Successful embedding of cells was indicated by >90% viability (< 10% PI-positive cells).
Figure 2. Accumulation of β-gal
pH6- 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 p16 LUC 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-gal pH6 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-gal pH6 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 p16 LUC 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. Pharmacological clearance of macrophages
in vivo depletes luciferase and β-gal pH6 activity from p16 LUC mice bearing SCs
A) Representative serial images depicting in vivo bioluminescence from p16 LUC 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-gal pH6 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. Clodronate treatment depletes p16(Ink4a)-positive and β-gal
pH6-positive cells from chronologically aged p16 LUC mice
A) Bioluminescent baseline readings from the abdomen of young (13 weeks) versus old (90 weeks) p16 LUC 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 p16 LUC 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 p16 LUC 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 p16 LUC mice and stained for β-gal pH6 activity. Representative photographic images are presented. ( E) Representative light microscopy images (magnification, 200x) of β-gal pH6-stained visceral adipose tissue counterstained with nuclear fast red. Cells residing between adipocytes (indicated by the presence of nuclear stain) are β-gal pH6-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-gal pH6-stained cultures of mouse adipose-derived mesenchymal stromal cells (mAdMSC) from p16 LUC mice at early passage (p1 cultures) or 10 days after 20Gy gamma-irradiation (Senescent). SCs stain positive for β-gal pH6 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. Schematic of hypothetical model of
in vivo accumulation of p16(Ink4a)/β-gal pH6-positive cells in naturally aged organisms
In young mammals (top panel), the secretion of SASP by p16(Ink4a)/β-gal
pH6-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-gal pH6-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-gal pH6-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.
p16(Ink4a) and Senescence-Associated β-Galactosidase Can Be Induced in Macrophages as Part of a Reversible Response to Physiological Stimuli
BM Hall et al.
Aging (Albany NY) 9 (8), 1867-1884.
p16 expression, along with senescence-associated β-galactosidase (SAβG), are commonly accepted biomarkers of senescent cells (SCs). Re …
Cells Exhibiting Strong
p16 Promoter Activation in Vivo Display Features of Senescence
JY Liu et al.
Proc Natl Acad Sci U S A 116 (7), 2603-2611.
The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This …
Murine Mesenchymal Cells That Express Elevated Levels of the CDK Inhibitor p16(Ink4a) in Vivo Are Not Necessarily Senescent
D Frescas et al.
Cell Cycle 16 (16), 1526-1533.
Age-related health decline has been attributed to the accumulation of senescent cells recognized in vivo by p16(Ink4a) expression. The pharmacological elimination of p16( …
Methods to Detect Biomarkers of Cellular Senescence: The Senescence-Associated Beta-Galactosidase Assay
K Itahana et al.
Methods Mol Biol 371, 21-31.
Most normal human cells undergo cellular senescence after accruing a fixed number of cell divisions, or are challenged by a variety of potentially oncogenic stimuli, in c …
The Molecular Balancing Act of p16(INK4a) in Cancer and Aging
KM LaPak et al.
Mol Cancer Res 12 (2), 167-83.
p16(INK4a), located on chromosome 9p21.3, is lost among a cluster of neighboring tumor suppressor genes. Although it is classically known for its capacity to inhibit cycl …
PubMed Central articles
lncRNA-Triggered Macrophage Inflammaging Deteriorates Age-Related Diseases
L Nie et al.
Mediators Inflamm 2019, 4260309.
Aging and age-related diseases (ARDs) share basic mechanisms largely involving inflammation. A chronic, low-grade, subclinical inflammation called inflammaging occurs dur …
Bifidobacterium lactis BB-12 Attenuates Macrophage Aging Induced by D-Galactose and Promotes M2 Macrophage Polarization
DY Zhang et al.
J Immunol Res 2019, 4657928.
Immunosenescence comprises a set of dynamic changes occurring in innate and adaptive immune systems, and macrophage aging plays an important role in innate and adaptive i …
miR-140 Attenuates the Progression of Early-Stage Osteoarthritis by Retarding Chondrocyte Senescence
HB Si et al.
Mol Ther Nucleic Acids 19, 15-30.
Osteoarthritis (OA) is a major cause of joint pain and disability, and chondrocyte senescence is a key pathological process in OA and may be a target of new therapeutics. …
Hyperglycemia-induced Inflamm-Aging Accelerates Gingival Senescence via NLRC4 Phosphorylation
P Zhang et al.
J Biol Chem 294 (49), 18807-18819.
Inflamm-aging was recently affiliated with the progression of diabetic complications. Local cellular senescence together with senescence-associated secretory phenotype (S …
Signaling Pathways Regulating Hematopoietic Stem Cell and Progenitor Aging
AK Singh et al.
Curr Stem Cell Rep 4 (2), 166-181.
Here we review our current understanding of the signalling pathways that are differentially activated or repressed during HSC/P aging, focusing on the oxidative, metaboli …
Cannizzo ES, Clement CC, Sahu R, Follo C, Santambrogio L. Oxidative stress, inflamm-aging and immunosenescence. J Proteomics. 2011;74:2313–23.
Larbi A, Franceschi C, Mazzatti D, Solana R, Wikby A, Pawelec G. Aging of the immune system as a prognostic factor for human longevity. Physiology (Bethesda) 2008;23:64–74.
Sagiv A, Krizhanovsky V. Immunosurveillance of senescent cells: The bright side of the senescence program. Biogerontology. 2013;14:617–28.
Campisi J, Andersen JK, Kapahi P, Melov S. Cellular senescence: a link between cancer and age-related degenerative disease? Semin Cancer Biol. 2011;21:354–59.
Chung HY, Cesari M, Anton S, Marzetti E, Giovannini S, Seo AY, Carter C Y BP LC. Molecular Inflammation: Underpinnings of Aging and Age- related Diseases. Ageing Res Rev. 2009;8:18–30.
Research Support, Non-U.S. Gov't
Cellular Senescence / physiology
Cyclin-Dependent Kinase Inhibitor p16 / metabolism
Macrophages / metabolism
beta-Galactosidase / metabolism
Cyclin-Dependent Kinase Inhibitor p16
LinkOut - more resources
Full Text Sources Other Literature Sources Medical