Perforin deficiency accelerates senescence with age

The prevalence of senescent cells in tissues increases with chronological age10,11. While senescent cells are subjected to immune cell cytotoxicity, it is not clear whether age-related impaired cell cytotoxicity could account for their accumulation. To examine this possibility, we set an in vivo experiment for assessment of systemic cytotoxicity of CD8+ T cells in young and old mice. The systemic cytotoxicity of CD8+ T cells in vivo was reduced more then 3-fold (P < 0.01) in aged mice compared to the young ones (Supplementary Figure 1). Age-related decline in cell cytotoxicity was shown to be a consequence of reduced release and binding of perforin at the immunological synapse24. To determine whether immune cell cytotoxicity plays a role in regulation of tissue burden of senescent cells throughout aging, we used Prf1−/− mice, in which immune surveillance of senescent cells is impaired22. We established cohorts of Prf1−/− and control WT mice, both on the background of C57BL/6, and examined selected organs including livers, pancreas, lungs, and skin in 2, 12, and 24-month old mice (defined hereafter as “young”, “adult”, and “old”, respectively). To assess time-dependent accumulation of senescent cells in those tissues, we first assayed them for senescence-associated-β-galactosidase (SA-β-Gal) activity, an assay commonly used to identify senescent cells in tissues and in culture10. We observed an increase in the number of SA-β-Gal + cells with age in all tissues examined. Increase was more pronounced in the Prf1−/− mice (Fig. 1a, b, Supplementary Figure 2a). Quantitative analysis of these cells in WT mice indicated that they comprise around 10% of the examined tissues by the time these mice reach 24 months of age. At the same age in Prf1−/− mice those cells comprised up to 43% of the total cells, demonstrating a significant (P < 0.005) increase of 2- to 4-fold (depending on the tissue) compared to WT mice (Fig. 1b). These finding indicate that tissues of old Prf1−/− mice extensively accumulate SA-β-Gal + cells.

Fig. 1 Old Prf1−/− mice accumulate more senescent cells then old WT mice. Cohorts of Prf1−/− and wild type (WT) C57BL/6 female mice at the age of 2, 12, and 24 months were sacrificed and their livers, pancreas, lungs, and skin were examined for the presence of senescent cells. a SA-β-Gal activity representative frozen sections of livers from 24-months-old mice. Scale bar, 100 µm. b Quantification of cells with marked SA-β-Gal activity, based on Nuclear Fast Red counterstaining, in liver, pancreas, bronchial epithelia, and skin epidermis. (n = 5 mice per group). c Percentages of cells stained positively for p16 in the liver. Values are means ± SEM, (n = 5 mice per group). d Representative images of IHC staining for p16 on liver frozen section. Scale bar, 100 µm. e Real-time qPCR analysis for expression of p16 in livers. Values are means ± SEM, (n = 5 mice per group). f Representative image of SA-β-Gal activity combined with IHC staining for p16 on liver frozen section from 24 old Prf1−/− mice. Scale bar, 100 µm. g Scheme of examination of senescence by ImageStreamX. h Representative ImageStreamX images of CD45− negative cells derived from indicated organs that were stained for HMGB1 and SA-β-Gal. Scale bar, 50 µm. i Quantification of CD45−/SA-β-Gal + /HMGB1− population in each organ, as analyzed in (h). (n = 5 mice per group). j Percentages of cells stained positively for γH2AX, p15, p53, p53BP1, and DcR2 in livers from old mice. (n = 5 mice per group). k Immunofluorescence staining for p65 (pink) in SA-β-Gal + cells (indicated by arrows in black/white photos) in livers from old mice. Scale bar, 50 µm. l Percentages of nuclei positive for p65 in livers. For all graphs, values are means ± SEM, (n = 5 mice per group). Student’s t-test was used for all comparisons between Prf1−/− and WT female mice (*P < 0.05, **P < 0.01, ***P < 0.001) Full size image

The most established molecular marker of senescence and a key component of the senescence program is p16 (Cdkn2a)25. Accumulation of senescent p16-positive cells shortens mouse lifespan (Baker et al, 2016). We studied the correlation of SA-β-Gal activity in the liver with the pattern of p16 expression. The dynamics of p16 expression mimicked the levels of SA-β-Gal + cells (Fig. 1c–e). The expression of p16 assessed by immunohistochemistry (Fig. 1c, d) or by quantitative RT-PCR (Fig. 1e) increased with age in WT mice, while Prf1−/− old mice had a significant increase in p16 expression compared to WT of the same age. Moreover, expression of p16 overlapped substantially with SA-β-Gal activity in the livers of old Prf1−/− mice, mostly in non-hepatocytes cells (Fig. 1f). Therefore, both p16-positive and SA-β-Gal-positive cells accumulate more extensively in the liver of Prf1−/− mice compared to WT mice.

To achieve a more reliable quantification of senescent cells in tissues, we applied a method based on a combination of SA-β-Gal and molecular markers of senescence on a single-cell level26. One such marker is loss of the nuclear high-mobility group box 1 protein (HMGB1)27. We therefore studied the prevalence of SA-β-Gal + /CD45−/HMGB1− cells as a cell population representative of tissue-resident senescent cells by the quantitative single-cell based method and visualized by the ImageStreamX apparatus which combines flow cytometry and microscopy (Fig. 1g). After validating the presence of the SA-β-Gal + populations in the liver, pancreas, and lung (Supplementary Figure 2b), we analyzed the nuclear HMGB1 staining in CD45−/SA-β-Gal + cells. While nuclear HMGB1 is ubiquitously expressed in the tissues examined, most of CD45−/SA-β-Gal + cells were found to be negative for nuclear HMGB1 staining (Fig. 1h, Supplementary Figure 2c). The presence of the SA-β-Gal + /CD45−/HMGB1− cells was increased in an age-dependent manner in both groups with a significant (P < 0.05), 2- to 6-fold increase in old Prf1−/− mice compared to old WT mice (Fig. 1i). We also examined the expression of an additional set of senescence markers, previously used to identify senescent cells28, comprised of γH2AX foci, p15, p53, p53BP1 foci, and DcR2, in the tissues. A marked increase in expression all of those proteins was observed in old Prf1−/− mice compared to the old WT mice and it was overlapping with SA-β-Gal staining of the consecutive sections (Supplementary Figure 2d). Quantitative analysis of each of these markers showed a significant (P < 0.0001) increase in positive cells in the old Prf1−/− mice (Fig. 1j). Apparently, expression of a variety of senescence markers increases in old Prf1−/− mice.

Due to their pro-inflammatory nature, accumulation of senescent cells in tissues can potentially drive age-related chronic inflammation29,30,31. NF-κB pathway is one of the main regulators of the pro-inflammatory profile of senescent cells2,32,33,34. The p65 subunit of NF-κB (also known as a RelA) translocates to the nucleus to drive transcriptional regulation. Co-staining of liver sections with p65 and SA-β-Gal demonstrated that SA-β-Gal + cells in the livers of old Prf1−/− mice display nuclear p65 frequencies that are 10-fold higher than in the livers of old WT mice, reflecting activation of the NF-κB pathway in these cells in Prf1−/− mice (Fig. 1k). Quantification of nuclear p65 + cells in liver sections showed age-dependent increase, which was further elevated in old Prf1−/− mice compared to the old WT mice (Fig. 1l). Taken together, our findings indicate that Prf1−/− leads to increased accumulation of senescent cells in tissues with age, as identified by multiple markers representing different characteristics of the senescence phenotype.

Perforin deficiency drives age-dependent chronic inflammation

Chronic inflammation is one of the characteristics of aging that are associated with the presence of senescent cells3,16,34. We evaluated the expression of known SASP components in the liver tissue of young, adult, and old WT and Prf1−/− mice by qPCR analysis (Fig. 2a). The expression of Il-6, RANTES, and JE was significantly increased in old Prf1−/− mice compared to old WT mice, while other cytokines show a similar tendency. Together with the increase in the cytokine expression, the number of tissue-infiltrating immune cells gradually increased with a similar kinetic to accumulation of senescent cells (Fig. 2b). This immune infiltration was further escalated in old Prf1−/− mice compared to WT (P < 0.05), in a manner that was largely represented by infiltration of T and NK cell subsets (Fig. 2b). Histological analysis verified the existence of accumulating immune cells in different sites of Prf1−/− mice (Supplementary Figure 3a). Examination of the liver cryosections from old Prf1−/− mice revealed multiple white dots that were barely noticeable in the livers from old WT mice (Fig. 2c, left panel). These structures appeared to be occupied by CD45+ cells and were therefore suspected to be immune foci. On examining these foci for the presence of different subsets of immune cells, we found that they were dominated by CD3+ cells and NK1.1+ cells (Fig. 2c, right and Fig. 2d). The robust infiltration observed in livers of the Prf1−/− mice was accompanied by an increase of 1.5 fold in their liver weight compared to the WT (Supplementary Figure 3b, c).

Fig. 2 Prf1−/− mice develop chronic systemic and local inflammation. a Real-time qPCR analysis for expression of p16 and SASP components in livers from 2, 12, and 24-months-old Prf1−/− and WT female mice. (n = 5 mice per group). b Flow-cytometric quantification of total immune cells (CD45+), T cells (CD45+/CD3+/NK1.1−), NKT cells (CD45+/CD3+/NK1.1+), and NK cells (CD45+/CD3-/NK1.1+) in livers, pancreas, and lungs of 2, 12, and 24-months-old Prf1−/− and WT mice. (n ≥ 5 mice per group). c Immune foci observed (indicated in squares, left panel) in liver cryosections from old Prf1−/− and WT mice. The immune foci were analyzed on sections stained immunofluorescently for CD45, Gr-1, B220, CD3, and NK1.1. Scale bar, 100 µm. d Numbers of the different immune subsets in livers from old Prf1−/− mice and WT mice, based on the immunofluorescence staining presented in (c). e Array of serum cytokine levels in 2, 12, and 24-months-old Prf1−/− female mice relative to WT female mice at the corresponding age. Colors represent increase (red) or decrease (blue) in the average intensity of 3 pooled samples. Values are shown in log2 according to the legend panel. f White blood cell counts of 2, 12, and 24-months-old Prf1−/− and WT female mice. (n = 5 mice per group). g Representative photos of spleens from 2, 12, and 24-months-old Prf1−/− and WT female mice. h Spleen weights of 2, 12, and 24-months-old Prf1−/− and WT female mice. (n = 7 mice per group). For all graphs, values are means ± SEM. Student’s t-test was used for all comparisons between Prf1−/− and WT mice. (*P < 0.05, **P < 0.01, ***P < 0.001) Full size image

To determine whether the increased accumulation of senescent cells and immune cells in tissues of Prf1−/− mice are accompanied by systemic inflammation, we used a cytokine array to compare their serum cytokine levels with those of WT mice at the age of 2, 12, and 24 months. Interestingly, at the age of 2 months there was no difference between Prf1−/− and WT mice, and at the age of 12 months only slight elevations were detected in the Prf1−/− mice. Nevertheless, in 24-months-old Prf1−/− mice we found a strong upregulation of pro-inflammatory factors (such as RANTES, TNF-α, IP-10, and MIG), in addition to an upregulation of some anti-inflammatory factors (such as IL-10 and IL-1RA) (Fig. 2e, Supplementary Figure 3d). Furthermore, the white blood counts (WBCs) of the old Prf1−/− mice were more then 2-fold higher than those of the old WT mice (P = 0.014, Fig. 2f). In addition to a raised WBC, chronic inflammation usually leads to an increase in spleen size. The spleens of old Prf1−/− mice were larger, both in size and in weight, than in their WT counterparts (Fig. 2g, h, Supplementary Figure 3e). In order to better characterize the inflammatory situation in the Prf1−/− mice throughout aging, we looked for the presence of inflammatory cells in some of their internal organs. Flow-cytometry analysis of liver, pancreas, and lung of young Prf1−/− mice did not show differences in their immune composition at young age comparing to the WT (Supplementary Figure 3f). Our observations thus suggest that accumulation of senescent cells in tissues of Prf1−/− mice is accompanied by an increased inflammatory response, which might facilitate the establishment of age-related chronic inflammation.

Perforin deficiency promotes age-related disorders and death

The establishment of chronic inflammation, also known as “inflammaging”, is a major risk factor for both morbidity and mortality in elderly people16,35. Owing to the increase in general inflammation in old Prf1−/− mice, we set to monitor physiological integrity of organs in these mice compared to WT mice. We examined different organs by pathological analysis of hematoxylin and eosin (H&E)-stained sections. Our analysis demonstrated an acceleration of age-dependent general impairment of tissues in the Prf1−/− mice compared to the WT mice (Supplementary Figure 4a). One manifestation of tissue impairment is the abnormal accumulation of extracellular matrix in tissues, commonly called fibrosis. In old Prf1−/− mice, fibrotic areas in the liver, pancreas, skin, and kidney were extensively formed, and were increased more than 2-fold compared to old WT controls (P < 0.01) (Fig. 3a, Supplementary Figure 4a). Additionally, in the kidney of Prf1−/− mice, periodic acid−Schiff (PAS) staining revealed enhanced formation of glomerular sclerosis compared to WT controls (P < 0.01, Fig. 3b, c, Supplementary Figure 4b). In the skin of old Prf1−/− mice, loss of the subcutaneous fat layer and a moderate attrition of hair follicles have resulted in a significantly reduced skin thickness, compared to the old WT mice (P = 0.005, Fig. 3d, e). Skin abnormalities in old Prf1−/− mice also resulted in significant reduction in hair density (P = 0.004), and a 20-fold increase in the prevalence of gray hair (Fig. 3f, g). Due to these phenotypes, old Prf1−/− mice appeared visibly older than their WT counterparts (Fig. 3h). To investigate whether the described tissue impairments are affecting the physiological condition of old Prf1−/− mice, we examined their blood for markers widely used to assess functions of different organs. Blood tests indicated a significant increase in general markers of tissue damage in internal organs, including the liver (ALP, AST, and ALT), pancreas (amylase), lungs (LDH), and muscles (CPK) (Fig. 3i). Analysis of markers in the blood also showed that urea levels in the blood of old Prf1−/− mice were significantly higher than in old WT (P = 0.016; Fig. 3i). This might be a consequence of a reduction in kidney’s filtration capacity and glomerular sclerosis (Fig. 3b, c), frequently observed in both humans and mice of advanced age36,37. Apparently, therefore, accumulation of senescent cells in perforin-deficient mice is associated with impaired tissue structure and function of multiple organs.

Fig. 3 Prf1−/− mice exhibit reduced fitness, higher rates of age-related disorders and a shorter lifespan. Cohorts of Prf1−/− and WT male mice were followed for survival and analyzed at the age of 2, 12, and 24 months. a Percentages of fibrotic areas in indicated tissues based on the Sirius red-staining in Supplementary Figure 4a. (n ≥ 5 mice per group). b PAS staining of glomeruli from kidneys of 24-months-old Prf1−/− and WT mice. Scale bar, 25 µm. c Percentages of sclerotic glomeruli, based on PAS-stained kidney sections. (n = 5 mice per group). d H&E-stained sections of lower-back skin. Scale bar, 200 µm. e Quantification of skin thickness of old mice. (n ≥ 6 mice per group). f Hair density on the lower back of old male mice. (n = 4 mice per group). g Numbers of gray-hair follicles per square centimeter of skin on the lower back of old mice. (n ≥ 6 mice per group). h Representative photos of the old mice with considerable differences in skin and in hair between the two genotypes. i Levels of common markers of damage in the sera of old mice. (n = 3 mice per group). j Average activity levels based on voluntary exercise of old mice. (n ≥ 4 mice per group). k Grip strength analysis based on a hang-wire test in old mice. Values indicate time that a mouse managed to hold on the wire. (n ≥ 4 mice per group). l Weight curves of Prf1−/− and WT male mice. Values are means, dashed lines represent ± SEM (n ≥ 20 mice per group). m µCT-3D images of old mice. The arrow indicates dorsal kyphosis. Kyphotic index, (n = 3 mice per group). n The prevalence of seminal gland (shown) gangrene in old Prf1−/− and WT male mice. o Kaplan–Meier curves for Prf1−/− and WT mice. Chi square Gehan–Breslow-Wilcoxon test was used for statistical analysis. For all graphs, values are means ± SEM. Student’s t-test was used for all other comparisons between Prf1−/− mice and WT mice. (*P < 0.05, **P < 0.01, ***P < 0.001) Full size image

The progressive decline in cellular function that occurs during aging ultimately affects the fitness on the organism level38. For example, old mice, like old humans, tend to be less active than young ones. To evaluate the overall fitness of Prf1−/− mice we tested their voluntary exercise, as well as their muscle strength and coordination. We found that old Prf1−/− mice showed a reduction of more than 2-fold in voluntary exercise (P < 0.05; Fig. 3j, Supplementary Figure 5a), and their grip strength and coordination in a “hang-wire” test were significantly decreased, compared to old WT mice (P = 0.044; Fig. 3k). Mammals also tend to lose weight during late stages of life39. Old Prf1−/− mice also suffered from progressive weight loss (Fig. 3l), which was comprised of both muscle loss and fat loss (Supplementary Figure 5b). Another aspect of organism-level fitness reduction is kyphosis, an exaggerated rounding of the spine. Old Prf1−/− mice exhibited a higher incidence of age-related kyphosis than old WT mice (P < 0.05; Fig. 3m). An age-related pathology reported to be common in C57BL/6 males is enlargement of the seminal glands39,40. Interestingly, many of the old Prf1−/− males in our study had enlarged seminal glands (in 70% of Prf1−/− mice compared to 10% in WT), which eventually resulted in gangrene (Fig. 3n). These glands were enriched with SA-β-Gal + cells in the Prf1−/− mice, and the thickness of their fibrotic layer was 3-fold higher in these mice than in the old WT (Supplementary Figure 5c, d). Therefore, our finding showed Prf1−/− mice develop multiple physiological disorders usually associated with older age, which could lead to shorten lifespan. We monitored the natural lifespan of cohorts of Prf1−/− and WT mice from both genders. The median survival of Prf1−/− mice was shorter (616 days) than that of WT mice (780 days), owing to the higher rate of early deaths, starting from the age of 14 months (Fig. 3o). Notably, the decreased median lifespan of Prf1−/− mice was evident in both males and females (Fig. 3o). Overall, perforin deficiency is associated with accumulation of senescent cells, increase in total body inflammation, impaired function of multiple organs, and reduced survival.

ABT-737 alleviates age-related phenotype of Prf1 −/− mice

Clearance of p16Ink4a-positive senescent cells in mice extends their health-span36,41. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-73742. Senescent cell viability is dependent on the expression of anti-apoptotic proteins from the BCL-2 family42,43,44,45,46. Accordingly, treatment with their specific inhibitors, such as ABT-737 or ABT-263, skews senescent cells toward apoptosis both in vitro and in vivo42,43,44. We administrated ABT-737 (25 mg/kg) or a vehicle solution to 18-months-old Prf1−/− mice, for two consecutive days monthly for a period of two months (Fig. 4a). Two weeks after the first ABT-737 injection, voluntary activity of ABT-737 treated mice increased compared to control mice (Fig. 4b). While the activity of the control group decreased over the two months as expected from aged mice, the ABT-737-treated group increased their activity 2-fold at the end of the first month and this increase was retained till the end of the experiment (P = 0.026; Fig. 4c). At the end of the two-month period, the amount of senescent cells was reduced in the ABT-737 treated mice as evaluated by SA-β-Gal staining (Fig. 4d) and by quantitative analysis of SA-β-Gal + /CD45-/HMGB1− cells in different tissues (Fig. 4e). To examine whether reduction on senescent cell burden was accompanied by a reduction of the hyper-inflammatory profile of Prf1−/− mice, we tested their serum by cytokine array. Interestingly, the top five proteins that were reduced by ABT-737 administration (Fig. 4f, Supplementary Figure 6a) include some of the most elevated proteins in the serum of old Prf1−/− mice compared to WT mice (Fig. 2e). Moreover, both WBCs counts and spleen weight were significantly reduced in ABT-737 treated group (P = 0.017 and P = 0.039, respectively, Fig. 4g, h). In line with this effect, ABT-737 treatment led to reduced immune infiltration into tissues (Fig. 4i), and reduced fibrotic area (Fig. 4j, Supplementary Figure 6b). In order to evaluate the effect of ABT-737 on the tissues by an independent approach, we systematically characterized the mRNA expression profile of ABT-737-treated old Prf1−/− mice and vehicle-treated old Prf1−/− mice using genome wide RNA sequencing (RNA-seq). Differential expression analysis and subsequent functional (Gene Ontology) analysis on kidney, liver, lung, and skin indicates a general repression of immune response, cytokine production, and endocytosis (P = 1.4e−92; 1.2e−25 and 2.3e−13, respectively; Fig. 4k, Supplementary Data 1). The analysis also revealed several upregulated processes including fatty acid metabolism, cell morphogenesis, and development (P = 6.6e−8; 3e−5 and 3.1e−4, respectively; Fig. 4k, Supplementary Data 1). Encouraged by significant downregulation in cytokine production, we evaluated ABT-737 effects on a more confined SASP signature47 in these tissues. Out of 56 established SASP factors, we detected 40 factors in at least one of the tissues, while different tissues express from 20 to 34 of the factors (Fig. 4l). In all four tissues examined, affected genes were enriched for SASP factors (hypergeometric test P < 0.001). To assess whether ABT-737 treatment drives gene expression levels towards those of young mice, we performed RNA-seq on the same tissues from 3-month-old Prf1−/− mice. We found that expression levels of genes affected by ABT-737 were driven towards the levels in young mice (Fig. 4l, Supplementary Figure 6c). ABT-737 treatment positioned overall SASP expression levels of old Prf1−/− mice closer to those of young Prf1−/− mice in kidney, liver, and skin (P < 0.01, P < 0.05, P < 0.001, respectively; Fig. 4l).

Fig. 4 Treatment with ABT-737 counteract accelerated aging process of Prf1−/− male mice. a Starting from 18 months, ABT-737 or vehicle were administered to Prf1−/− mice at the beginning of each month, the mice were analyzed at the age of 20 months. b Change in distance run on a voluntary exercise test. Values are means and dashed lines represent ± SEM for each curve (n = 5 mice per group). c Average voluntary exercise levels during the last 3 days of the two months (n = 5 mice per group). d SA-β-Gal activity in indicated organs. Scale bar, 100 µm. (n = 5 mice per group). e CD45−/SA-β-Gal + /HMGB1− population in indicated organs (n ≥ 4 mice per group). f Relative cytokine levels in the serum for top five altered cytokines. Bars represent average intensity of 3 pooled samples. g WBC counts. (n = 5 mice per group). h Spleen weights. (n = 3 mice per group). i Flow-cytometry quantification of total immune cells (CD45+), T cells (CD45+/CD3+/NK1.1-), NKT cells (CD45+/CD3+/NK1.1+), and NK cells (CD45+/CD3-/NK1.1+) in indicated organs (n ≥ 4 mice per group). j Fibrotic areas in indicated organs based on the Sirius Red-stained sections in Supplementary Figure 6b. (n = 5 mice per group). Student’s t-test was used for all comparisons between Prf1−/− mice and WT mice. k Differentially expressed genes in indicated organs. Notable enriched GO terms are highlighted (n ≥ 3 mice per group). l SASP genes score, presented as expression mean log ratio of old ABT-737 and Vehicle treated over young Prf1−/− mice (Supplementary Figure 6c). The bottom panel depicts number of detected SASP genes. (n ≥ 3 mice per group). m Upper panel: Euler diagram of differentially expressed genes from all analyzed tissues. Lower panel: Log ratio of gene expression for 20-months-old ABT-737 (y axis) and Vehicle (x axis)-treated over young Prf1−/− mice. Dashed line represents linear regression fitted to the data; p value computed against the null hypothesis that tilt = 1; (n ≥ 3 mice per group). For all graphs, values are means ± SEM. Student’s t-test was used for all other comparisons. (*P < 0.05, **P < 0.01, ***P < 0.001) Full size image

Effects observed in SASP factors point to the possibility of global shift of the transcriptional profile to a younger state following the ABT-737 treatment. Indeed, 74% of genes affected by ABT-737 treatment were also affected by aging (Fig. 4m, upper panel). This significantly larger-than-random (Hypergeometric test, P < 2e−16) overlap suggests an interaction between ABT-737 treatment and age-related transcriptional changes. Importantly, ABT-737 effect counteracts the effect of aging on the vast majority of these genes, literally rejuvenating the expression profile of the mice. This effect becomes clear when the data are presented by regression of gene expression log fold-change old ABT-737/Young on old Vehicle/Young (Fig. 4m, lower panel, Supplementary Data 2) for all age-affected genes. The linear regression line (dashed line, Fig. 4m, lower panel, Supplementary Data 2) has a tilt < 1, indicated that the expression profile of old ABT-737 treated mice is more similar to young compared to old vehicle-treated mice (P < 5e−5). This result is further supported by correlation analysis, showing that log(old Vehicle/Young) anti-correlates with log(ABT-737/Vehicle) (Spearman correlation test ρ = −0.63, −0.46, −0.59, and −0.25 for kidney, liver, lung, and skin respectively, P < 2e−16). Overall, our results show that the senolytic drug ABT-737 eliminates senescent cells and counteracts age-related phenotype caused by Prf1 gene knockout in old mice.

ABT-737 extends the lifespan of progeroid mice

In some cases of accelerated aging, senescent cell formation can be enhanced by a repetitive intrinsic stress; that appears to occur in Hutchinson–Gilford progeria syndrome (HGPS), where accumulation of the progerin protein causes defects of the nuclear lamina and frequent damage to DNA48,49. LMNA+/G609G mouse harbors a point mutation in the LMNA gene, which is identical to the HGPS causing mutation50. This HGPS mouse model demonstrates an extensive accumulation of senescent cells (Supplementary Figure 7a, b)50. To inquire the possibility that pharmacological elimination of senescent cells could also increase the lifespan of progeroid mice, we have tested the effect of the senolytic drug ABT-737 on LMNA+/G609G mice. LMNA+/G609G mice were treated with ABT-737 for two consecutive days monthly from the age of 7 months and their tissues were analyzed at 11 months (Fig. 5a). We quantified the SA-β-Gal + cells present in the livers, pancreas, lungs, and skin of LMNA+/G609G mice. ABT-737 treatment resulted in a substantial decrease in SA-β-Gal + cells in all of the tested tissues (Fig. 5b), with an efficacy of elimination ranging from 50% (liver) to 70% (lungs) (Fig. 5c). Elimination of senescent cells was further validated by an overall reduction in molecular markers of senescence (γH2AX, p15, p53, and p53BP1) (Fig. 5d, Supplementary Figure 7c). Therefore, treatment with ABT-737 resulted in considerable decrease in the numbers of senescent cells in tissues of LMNA+/G609G mice.

Fig. 5 Treatment with ABT-737 increases median lifespan of progeroid mice. Starting the age of 7 months ABT-737 or DMSO-based vehicle solution was administered to LMNA+/G609G mice. a Scheme of drug administration to LMNA+/G609G mice. b Representative images depicting SA-β-Gal activity in frozen sections of livers, pancreas, lungs, and skin from ABT-737-treated and vehicle-treated LMNA+/G609G female mice at the age of 11 months. Scale bar, 100 µm. c Percentage of cells with SA-β-Gal activity in liver, pancreas, lungs, and skin from ABT-737-treated and vehicle-treated LMNA+/G609G female mice. (n = 5 mice per group). d Percentages of cells stained positively for γH2AX, p15, p53, p53BP1, and DcR2 in ABT-737-treated and vehicle-treated LMNA+/G609G female mice at the age of 11 months. (n = 5 mice per group). e Representative images of immunofluorescence staining for p65 (arrows) in liver sections from ABT-737-treated and vehicle-treated LMNA+/G609G female mice at the age of 11 months. Scale bar, 50 µm. f Percentage of p65 + nuclei in livers from ABT-737-treated and vehicle-treated LMNA+/G609G female mice at the age of 11 months. (n = 5 mice per group). g Serum cytokine levels in ABT-737-treated LMNA+/G609G female mice relative to age-matched vehicle -treated LMNA+/G609G female mice. Bars represent the average intensity of 3 pooled samples (log2). Values are shown for cytokines that were decreased by more than 2-fold. h Kaplan–Meier survival curves of ABT-737-treated and vehicle-treated LMNA+/G609G mice. Data is shown for males (n = 10), females (n = 6), and both genders combined (n = 16). Chi square Gehan–Breslow–Wilcoxon Test was used for statistical analysis. i Weight curves of ABT-737-treated and vehicle-treated LMNA+/G609G mice. (n = 16 mice per group). For all graphs, values are means ± SEM. Student’s t-test was used for all comparisons between the two groups. (*P < 0.05, **P < 0.01, ***P < 0.001) Full size image

The accelerated aging phenotype in LMNA+/G609G mice has been previously linked to an NF-κB-mediated systemic inflammatory response50, similarly observed in Prf1−/− mice. To determine whether the reduced tissue burden of senescent cells following ABT-737 treatment is associated with reduced inflammatory profile in old LMNA+/G609G mice, we evaluated the activation levels of NF-κB in the liver. The number of cells that are positive for nuclear p65 was more than two-fold decreased in the ABT-737 treated mice comparing to the control mice (P = 0.019; Fig. 5e, f). To examine the systemic effect of ABT-737 treatment in these mice we assayed cytokines in their serum using a cytokine array (Fig. 5g, Supplementary Figure 7d). Interestingly, the amounts of the eight cytokines that had been among the most strongly upregulated in old Prf1−/− mice were reduced upon ABT-737 treatment in LMNA+/G609G mice (Fig. 5g, Fig. 2a). We next determined the lifespan of LMNA+/G609G mice treated with ABT-737 or with a vehicle. Strikingly, ABT-737-treatment resulted in a significant increase in median survival (377 vs. 353 days, P = 0.0007), compared with vehicle-treated mice (Fig. 5h). ABT-737 effect on lifespan was observed in both male and female LMNA+/G609G mice (Fig. 5h). The observed increase in median survival in the ABT-737-treated mice was accompanied by a delayed decrease in weight loss during their late stages of life (Fig. 5i). Our data thus suggest that the treatment with ABT-737 eliminates senescent cells, reduces the levels of circulating cytokines, and extend the median lifespan in LMNA+/G609G mice.

Perforin deficiency shortens the lifespan of progeroid mice

The involvement of immune cell cytotoxicity in the regulation of the presence of senescent cells and in accelerated aging in progeroid mice is largely unknown. By crossing Prf1−/− mice with LMNA+/G609G mice, we generated LMNA+/G609G/Prf1−/− mice, LMNA+/G609G/Prf1+/− mice, and LMNA+/G609G/Prf1+/+ mice, and followed their survival. In a similar manner to Prf1−/− mice, LMNA+/G609G/Prf1−/− mice exhibited reduced median survival compared to control LMNA+/G609G/Prf1+/+ mice, suggesting that immune cell cytotoxicity acts to restrain accelerated aging in progeroid mice (Fig. 6a). Intrigued by this finding, and by the finding that in LMNA+/G609G mice ABT-737 treatment reduced the presence of cytokines upregulated in old Prf1−/− mice, we asked whether clearance of senescent cells could counteract accelerated aging which stems from a combination of progerin accumulation and impaired immune cell cytotoxicity. For this purpose, we administered ABT-737 or vehicle to LMNA+/G609G/Prf1−/− female mice two consecutive days monthly, since the age of 7-month until the age of 11 months (Fig. 6b). In line with the accumulation of senescent cells in Prf1−/− mice, selected tissues from LMNA+/G609G/Prf1−/− mice showed a more than 2-fold increase in the numbers of SA-β-Gal + cells when compared to LMNA+/G609G control mice, (Fig. 6c, d). As expected, vehicle-treated LMNA+/G609G/Prf1−/− mice were not different from the untreated mice of the same age. Importantly, ABT-737 treatment in LMNA+/G609G/Prf1−/− mice caused a significant decrease in the numbers of SA-β-Gal + cells, bringing them close to the levels of these cells in the LMNA+/G609G control mice (Fig. 6c, d). Accumulation of senescent cells in tissues of LMNA+/G609G/Prf1−/− mice could intensify the systemic inflammatory response that is driving the accelerated aging phenotype of LMNAG609G mice50. Indeed, staining of livers from LMNA+/G609G/Prf1−/− mice showed an increase in the numbers of cells that were stained positively for nuclear p65 (Fig. 6e, f). Relative to LMNA+/G609G/Prf1+/+ mice, the LMNA+/G609G/Prf1−/− mice had an increased WBC count (Fig. 6g) and enlarged spleen (Fig. 6h). Administration of ABT-737 was not only able to alleviate all of those aspects of inflammation (Fig. 6e–h), but also reconcile compromised integrity and tissue fibrosis that are associated with high inflammatory load in the tissues (Fig. 6i). Lastly, by carrying out the same ABT-737 administration protocol while monitoring survival, we found that ABT-737 treatment extended the median lifespan in both male and female LMNA+/G609G/Prf1−/− mice (Fig. 6j). Our data thus indicate that Prf1 deficiency in progeroid mice further escalates their aging process by promoting the early establishment of chronic inflammation. Pharmacological clearance of senescent cells by ABT-737, on the other hand, is able to halt deleterious consequences of impaired cell cytotoxicity and increase survival in this model.