Senolytic-Resistant Senescent Cells Have a Distinct SASP Profile and Functional Impact: The Path to Developing Senosensitizers

Abstract

The senescent cell (SC) fate is linked to aging, multiple disorders and diseases, and physical dysfunction. Senolytics, agents that selectively eliminate 30%-70% of SCs, act by transiently disabling the senescent cell antiapoptotic pathways (SCAPs), which defend those SCs that are proapoptotic and pro-inflammatory from their own senescence-associated secretory phenotype (SASP). Consistent with this, a JAK/STAT inhibitor, Ruxolitinib, which attenuates the pro-inflammatory SASP of senescent human preadipocytes, caused them to become "senolytic-resistant". Administering senolytics to obese mice selectively decreased the abundance of the subset of SCs that is pro-inflammatory. In cell cultures, the 30%-70% of human senescent preadipocytes or human umbilical vein endothelial cells (HUVECs) that are senolytic-resistant (to Dasatinib or Quercetin, respectively) had increased p16^INK4a, p21^CIP1, senescence-associated β-galactosidase (SAβgal), γH2AX, and proliferative arrest similarly to the total SC population (comprising senolytic-sensitive plus-resistant SCs). However, the SASP of senolytic-resistant SCs entailed less pro-inflammatory/apoptotic factor production, induced less inflammation in non-senescent cells, and was equivalent or richer in growth/fibrotic factors. Senolytic-resistant SCs released less mitochondrial DNA (mtDNA) and more highly expressed the anti-inflammatory immune evasion signal, glycoprotein non-melanoma-B (GPNMB). Transplanting senolytic-resistant SCs intraperitoneally into younger mice caused less physical dysfunction than transplanting the total SC population. Because Ruxolitinib attenuates SC release of proapoptotic SASP factors, while pathogen-associated molecular pattern factors (PAMPs) can amplify the release of these factors rapidly (acting as "senosensitizers"), senolytic-resistant and senolytic-sensitive SCs appear to be interconvertible.

Keywords: cellular senescence; senescent cell subtypes; senolytics; senosensitizers.

FIGURE 1

The SASP impacts extent of SC clearance by senolytics. (A) TUNEL‐positive nuclei as a percent of total cells and (B) representative images of surviving (crystal violet⁺) senescent preadipocytes and quantification relative to vehicle‐treated cells after exposure to vehicle or Ruxolitinib (1 μM), which attenuates the pro‐inflammatory SASP, for 3 days followed by Dasatinib 800 nM for 24 h. (C) TUNEL‐positive nuclei as a percent of total cells and (D) survival of SCs pre‐treated with 10 μg/mL LPS for 3 days followed by treatment with Dasatinib 800 nM for 24 h. Quantification of images (N = 5) is shown on the right. Data are shown as means +/− SEM; 1‐way ANOVA; and post hoc comparisons with by Tukey's HSD multiple comparison (A, C), and are expressed as a function of vehicle‐treated cells; means +/− SEM; paired, 2‐tailed Student's t‐tests.

FIGURE 2

Senolytics target a pro‐inflammatory subset of senescent preadipocytes in obese mice. (A) CyTOF experimental scheme. (B) Representative t‐SNE plots of FlowSOM clusters of cells from obese vehicle‐ or senolytic‐treated mice. (C) Heatmap of senescence marker expression within FlowSOM clusters (N = 5). (D) Heatmap comparing expression of pro‐inflammatory SASP factors among preadipocyte clusters in vehicle‐treated obese mice (N = 5). (E) Percent of cells cleared in the indicated clusters in senolytic‐ vs. vehicle‐treated mice (N = 5). Means ± SEM; unpaired two‐tailed Mann–Whitney tests.

FIGURE 3

Cellular senescence markers are similar in the human preadipocyte senolytic‐resistant versus total SC preadipocyte populations. (A) Cells were analyzed for proliferative arrest by BrdU staining. See quantification of BrdU positive cells in Figure S4A. (B) Gene expression of the indicated senescence markers (N = 9) in total senescent preadipocyte populations vs. senolytic‐resistant senescent preadipocytes. Data are expressed as a function of non‐senescent control cells. Means ± SEM; unpaired, 1‐way ANOVA; post hoc comparisons by Tukey's HSD multiple comparison test. (C) Total and senolytic‐resistant preadipocyte SC populations were immunostained for γ‐H2AX, p16^INK4a, and p21^CIP1; representative images of N = 3 subjects are shown. See quantification of p16^INK4a, and p21^CIP1 expression in Figure S4B. (D) Quantification of γ‐H2AX expression. Means ± SEM; unpaired 2‐tailed Student's t‐tests. (E) SAβgal (pH 6) in the total and senolytic‐resistant senescent preadipocyte populations. See quantification of SAβgal intensity in Figure S4C. Analogous findings in human senescent endothelial populations are in Figure S4D–F.

FIGURE 4

Senolytic‐resistant human senescent preadipocyte SASP profiles are distinct from the total SC population. (A) Heat map of differentially expressed genes (DEGs). (B) Volcano plot of the DEGs indicating key genes. (C, D) Gene ontology analysis of biological functions of DEGs in the total versus senolytic‐resistant SC populations. (E) SASP factor expression (rt‐PCR) in senescent preadipocytes. Data are expressed as a function of vehicle‐treated non‐senescent cells. Means ± SEM; paired, 2‐tailed Student's t‐tests.

FIGURE 5

Effects of the senolytic‐sensitive vs. total SC populations on induction of inflammation and secretion of mt‐DNA. (A) Experimental scheme. (B) Non‐senescent preadipocytes were treated with conditioned media (CM) from resistant vs. total SC populations for 24 h, and inflammatory factors were analyzed by rt‐PCR. Means ± SEM; paired, two‐tailed Student's t‐tests. (C) Secreted mt‐DNA by indicated cell types. Means ± SEM; paired, 1‐way ANOVA; post hoc pairwise comparison by Tukey's HSD multiple comparison test.

FIGURE 6

SC subtypes transplanted into younger mice differ in impact on physical function. (A) Experimental scheme. (B) Grip strength and (C) wire hanging endurance (sec × BW g) in 2‐month‐old male SCID‐Beige mice 1 month after being transplanted with 1 × 10⁶ senolytic‐resistant or total senescent human preadipocyte population cells by intraperitoneal injection (N = 15). Baseline grip strength was similar in each group of mice before transplantation (Figure S9). Data are shown as means ± SEM with individual values; unpaired two‐tailed Student's t‐tests.