Rapamycin improves healthspan but not inflammaging in nfκb1-/- mice

Abstract

Increased activation of the major pro-inflammatory NF-κB pathway leads to numerous age-related diseases, including chronic liver disease (CLD). Rapamycin, an inhibitor of mTOR, extends lifespan and healthspan, potentially via suppression of inflammaging, a process which is partially dependent on NF-κB signalling. However, it is unknown if rapamycin has beneficial effects in the context of compromised NF-κB signalling, such as that which occurs in several age-related chronic diseases. In this study, we investigated whether rapamycin could ameliorate age-associated phenotypes in a mouse model of genetically enhanced NF-κB activity (nfκb1^-/- ) characterized by low-grade chronic inflammation, accelerated aging and CLD. We found that, despite showing no beneficial effects in lifespan and inflammaging, rapamycin reduced frailty and improved long-term memory, neuromuscular coordination and tissue architecture. Importantly, markers of cellular senescence, a known driver of age-related pathology, were alleviated in rapamycin-fed animals. Our results indicate that, in conditions of genetically enhanced NF-κB, rapamycin delays aging phenotypes and improves healthspan uncoupled from its role as a suppressor of inflammation.

Keywords: SASP; aging; inflammaging; mTOR; rapamycin; senescence.

Figure 1

Rapamycin prevents age‐related frailty in nf‐κb1 ^−/− mice without impacting on lifespan. (a) Kaplan–Meier survival curves of nf‐κb1 ^−/− mice fed with a control (n = 44) or rapamycin‐supplemented diet (n = 24), from 4 to 5 months old until death; (b) percentage of body weight change from baseline (start of diet) in control or rapamycin‐fed nf‐κb1 ^−/− mice at 9.5 months and in moribund animals (n = 7–8 per group); (c) Clinical Frailty Index at 15 and 18 months of age in nf‐κb1 ^−/− with or without rapamycin diet (n = 8–11 per group); (d) representative images of nf‐κb1 ^−/− mice with or without rapamycin feeding at 18 months of age; (e) Barnes maze test in nf‐κb1 ^−/− mice with or without rapamycin diet (n = 9 per group); (f) neuromuscular coordination measured as % number of successful attempts (in purple) to remain on a straight rod for 60 s (n = 4–5 per group; 9.5 months old); (g) forelimb grip strength measured as number of trials required to remain hanging for total of 90 s (% success in purple (n = 5–9 per group; 18 months old); (h) linear regression of mean forelimb grip time (seconds). All data are mean ± SEM. *p < 005, **p < 0.01, ***p < 0.001 (One‐way ANOVA)

Figure 2

Rapamycin ameliorates several aging‐associated histological parameters in nf‐κb1 ^−/− mice. (a) Representative images of H&E staining of lung tissue sections from nf‐κb1 ^−/− mice fed a control or rapamycin‐supplemented diet with quantifications of mean linear intercept (MLI) at 9.5 months of age; (b) representative images of H&E staining of skin sections from 9.5‐month‐old nf‐κb1 ^−/− mice fed with control or rapamycin‐supplemented diet plus quantifications of mean epidermal thickness; (c) representative images of heart sections labelled with wheat germ agglutinin WGA from 9.5‐month‐old nf‐κb1 ^−/− mice fed with control or rapamycin‐supplemented diet plus quantifications of cross‐sectional cardiomyocyte area. All data are mean ± SEM (n = 4–8 mice per group). *p < 005, **p < 0.01, ***p < 0.001 (One‐way ANOVA)

Figure 3

Rapamycin does not reduce inflammation in nf‐κb1 ^−/− mice. (a) Serum cytokine array from wild‐type (n = 5) or nfκb1 ^−/− mice (n = 6) fed a control (−) or rapamycin‐supplemented (+) diet; (b) spleen weight, represented as percentage of total body weight in wild‐type or nfκb1 ^−/− mice fed control or rapamycin‐supplemented diet at 9.5 months of age; (c) IL‐6 mRNA levels in whole lung, or liver (f), tissue from wild‐type or nfκb1 ^−/− mice fed control or rapamycin‐supplemented diet, normalized to 18S; (d) ChIP analysis of RELA enrichment at the IL‐6 promoter in whole lung, or liver (g), from nfκb1 ^−/− mice fed a control or rapamycin‐supplemented diet; (e) and (h) representative images of CD68 immunohistochemical staining in lung or liver tissue sections, respectively, from wild‐type or nf‐κb1 ^−/− mice fed with control or rapamycin‐supplemented diet plus quantifications of CD68+ cells/field. Data represent group mean ± SEM (n = 3–7 mice per group). *p < 005, **p < 0.01, ***p < 0.001 (One‐way ANOVA)

Figure 4

Rapamycin reduces markers associated with cellular senescence and mitochondrial dysfunction in vivo and in vitro. (a) Representative images of immunofluorescence in situ hybridization (immuno‐FISH) staining in lung airway epithelium, or hepatocytes in liver sections (c) of nfκb1 ^−/− mice fed control or rapamycin‐supplemented diet at 9.5 months of age. Images are maximum intensity projections of at least 50 planes with areas of colocalization in single Z planes shown on the right. Graphs represent quantification of γH2A.X and telomere signals in selected regions of interest (dotted lines); (b) frequencies of telomere‐associated foci (TAF) in small airway epithelial cells, or hepatocytes (d), from 9.5‐month‐old wild‐type and nfκb1 ^−/− mice fed a control or rapamycin‐supplemented diet; (e) p16 and p21 mRNA levels in whole lung tissue from wild‐type or nfκb1 ^−/− mice (9.5 months old) fed control or rapamycin‐supplemented diet, normalized to 18S; (f) mouse adult fibroblasts (MAFs) isolated from the ears of wt or nfκb1 ^−/− mice were induced to senescence by 10 Gy irradiation and cultured in the presence or absence of rapamycin at 3% O₂; (g) representative western blot showing phospho‐S6 (pS6), S6 and p21 expression in senescent MAFs from wild‐type and nfκb1 ^−/− mice treated with rapamycin (+) or DMSO (−) with quantifications on the right (h); (i) representative images of senescence‐associated β‐galactosidase staining in MAFs from wild‐type or nfκb1 ^−/− mice treated with rapamycin (+) or DMSO (−) with quantifications of percentage positive cells shown on the right; (j) heatmap depicting fold change in indicated cytokines in medium of proliferating and senescent MAFs from wild‐type and nfκb1 ^−/− mice treated with or without rapamycin measured using a Multiplexing LASER Bead Assay; (k) mitochondrial ROS (MitoSOX) levels in proliferating and senescent MAFs from wild‐type and nfκb1 ^−/− mice treated with or without rapamycin and hydrogen peroxide generation in isolated mitochondria from the livers of wild‐type and nfκb1 ^−/− mice (9.5 months old) fed a control or rapamycin‐supplemented diet. Data are mean ± SEM (n = 3–7 mice per group for all analyses). *p < 005, **p < 0.01, ***p < 0.001 (One‐way ANOVA)