Lifespan-extending caloric restriction or mTOR inhibition impair adaptive immunity of old mice by distinct mechanisms

Abstract

Aging of the world population and a concomitant increase in age-related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age-related decline of the immune system, infectious diseases remain among the top 5-10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T-cell development in the thymus, peripheral T-cell maintenance, T-cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan.

Keywords: T cell; anti-aging; caloric restriction; cellular immunology; longevity regulation; mouse models.

Figure 1

Rapamycin and calorie restriction have different effects on thymic cellularity. Mice were placed on rapa for 2 months or on CR for life. (A) Untreated A and O mouse thymus cellularity. (B) Thymi were harvested from A and O mice from the indicated treatment groups and total thymocytes enumerated. (C) Thymus single-cell suspensions were analyzed for thymocyte development based on CD4 and CD8 expression. (D) Whole blood rapa concentration was measured in O mice being treated with the indicated doses and routes. Data represent the combination of 2 independent experiments, with n = 2–5 mice per replicate. (B) Thymic cellularity is represented as % of age-matched controls that were harvested at the same time, to control for different dosing routes. 2.5 mg kg⁻¹ and 75 μg kg⁻¹ were given as daily i.p. injections. 14 ppm was the concentration of rapa in rodent chow, as described in the experimental procedures section. Statistical differences were calculated by Student's t-tests compared to the cellularity of age-matched, control-treated mice. (C-D) Statistical differences were calculated by 1-way ANOVA with Bonferroni's post-tests. *P<0.05; **P<0.01; ***P<0.001.

Figure 2

Rapamycin and calorie restriction differently alter peripheral T-cell subset absolute counts. Mice were bled after 2 months of rapa feeding when O mice were 18 months old. (A) Total lymphocyte, total CD4, and total CD8 T-cell absolute counts in the blood. (B) Absolute counts of N (CD44^lowCD62L^hi) and regulatory (Foxp3⁺) CD4 T cells in the blood. (C) N, CM, and EM cells were identified enumerated based on CD44 and CD62L expression. Data are shown as mean ± SEM and are representative of 3 independent cohorts. Statistical differences between O groups were calculated by 1-way ANOVA with Bonferroni post-tests. Adults are shown to provide a reference point and were not included in statistical analyses. *P<0.05; **P<0.01; ***P<0.001.

Figure 3

Rapamycin and calorie restriction differently alter T-cell subset steady-state proliferation. Steady-state proliferation was evaluated by Ki-67 staining within naïve, effector memory, and central memory CD8 T-cell subsets. (A) The frequency of cells within the naïve CD4 T-cell subset or within regulatory Tregs that are Ki-67⁺. (B) The frequency of cells within the indicated CD8 T-cell subsets that were Ki-67⁺. Data are shown as mean ± SEM and are representative of 3 independent cohorts. Statistical differences between O groups were calculated by 1-way ANOVA with Bonferroni post-tests. Adults are shown only to provide a young reference point and were not included in statistical analyses. *P<0.05; **P<0.01; ***P<0.001.

Figure 4

Rapamycin and calorie restriction do not alter thymic output. Spleen and superficial lymph nodes were harvested and analyzed for TRECs and Ag-specific CD8 T-cell precursors. (A) CD8 T-cell TRECs were quantified. Data shown are pooled from 2 independent harvests, each with n = 2–3 mice per group in each replicate. (B) CD8 T-cell precursors specific for the immunodominant WVN epitope NS4b were quantified. (C) Memory phenotype within the NS4b-specific CD8 T-cell precursors was measured. (B-C) Data are shown as mean ± SEM and are combined from 3 independent harvests, each with n = 2–3 mice/group at each harvest. (A) Statistical differences between O groups were calculated by nonparametric Kruskal–Wallis test with Dunn's multiple comparison test. Adults are shown only as a reference point and were not included in statistical analyses. (B, C) Statistical differences were calculated by 1-way ANOVA with Bonferroni's post-test

Figure 5

Calorie restriction, but not rapamycin, increases WNV mortality in old mice. Mice were infected with 10³ pfu WNV s.c. and tracked 30 days after infection or sacrificed on day 10 postinfection for Ag-specific T-cell analysis. (A) Survival after WNV infection in O, O rapa, and O CR mice. (B) Total number of NS4b-specific CD8 T cells that produce IFNγ upon ex vivo peptide stimulation. (C) Total number of CD4 T cells that produce IFNγ after ex vivo peptide stimulation with a pool of CD4 epitopes as indicated in the materials and methods. (D) Adult control and CR mice were infected with 10³ pfu WNV and tracked for survival. (A) Data shown are the combination of 3 independent experiments (n = 9–12 mice/group in each experiment). Statistical differences were calculated by log-rank test. (B, C) Data are shown as mean ± SEM and are representative of 2 independent experiments each containing n = 12 mice/group. Statistical differences were calculated by nonparametric Kruskal–Wallis test with Dunn's multiple comparison test. (D) Data shown are the combination of 2 independent experiments with n = 19 mice/group total. Statistical differences were calculated by log-rank test. *P<0.05; **P<0.01; ***P<0.001.