Chronic calorie restriction attenuates experimental autoimmune encephalomyelitis

Abstract

Calorie restriction (CR) prevents many age-associated diseases and prolongs the lifespan. CR induces multiple metabolic and physiologic modifications, including anti-inflammatory, antioxidant, and neuroprotective effects that may be beneficial in multiple sclerosis (MS). The present studies sought to determine whether CR or increased calorie intake alters the course of experimental autoimmune encephalomyelitis (EAE), the leading animal model for MS. SJL and C57BL/6 mice were subjected to 40% CR beginning at 5 weeks of age. After 5 weeks of CR, EAE was induced by immunizing with proteolipid protein in SJL mice and with myelin oligodendrocyte glycoprotein in C57BL/6 mice. Clinical, histologic, and immunologic features of EAE were compared with mice fed ad libitum and to SJL mice fed a high-fat, high-calorie diet. CR ameliorated clinical EAE in both mouse strains with less severe inflammation, demyelination, and axon injury. No suppression of immune function was observed. A high-calorie diet did not alter the EAE course. CR was associated with increased plasma levels of corticosterone and adiponectin and reduced concentrations of IL-6 and leptin. The CR-induced hormonal, metabolic, and cytokine changes observed in our studies suggest a combined anti-inflammatory and neuroprotective effect. CR with adequate nutrition and careful medical monitoring should be explored as a potential treatment for MS.

Figures

Fig. 1.

CR ameliorates the clinical course of EAE in SJL and C57BL/6 mice. (A) Body weights of control mice (•), mice fed with HF diet (▪), and CR mice (○) throughout the experiment before and after immunization (values are mean±sd). The weight changes shown are representative of four experiments. The dotted vertical line indicates the day of immunization. (B) Experiment 1. Survival was increased significantly in CR mice (80%) compared with control (23%) and HF (30%) groups (P=0.02 CR vs. control by χ2). (C) Experiment 3. CR regimen reduces the clinical severity of relapsing-remitting EAE in SJL mice compared with the ad libitum diet in control mice. (D) Experiment 4. CR ameliorates the clinical course of chronic-progressive EAE in C57BL/6 mice. (C and D) Values are mean ± sem; clinical scores were calculated as the average of all animals in each group.

Fig. 2.

Spinal cord inflammation and demyelination are reduced in CR mice compared with control mice fed ad libitum. Spinal cords were obtained on Day 30 p.i. Representative SJL mice from Experiment 3 for control (A–C) and CR (D–F) groups are shown. LFB staining demonstrates extensive demyelination in the control (A and C) compared with CR (D and F). In the H&E-stained sections, inflammatory cells deeply infiltrated the CNS parenchyma in the control group (B), and they were localized mainly to the meninges in the CR group (E). Black arrows indicate infiltrating inflammatory cells; white arrow indicates demyelination. Original magnification: A and D, 4×; B and C, 20×; E and F, 16×. Original scale bars, 200 μm (A and D) or 50 μm (B, C, E, and F). (G) Inflammation and (H) demyelination quantified in a blinded manner were reduced significantly in CR (▵) compared with control mice (▪; *, P=0.03, by Mann-Whitney test; **, P<0.001, by Mann-Whitney test). Horizontal lines indicate median values.

Fig. 3.

Spinal cord axonal damage was less in CR mice compared with control mice fed ad libitum. In Experiment 3, spinal cords were taken from mice that were terminally perfused on Day 30 p.i. Representative SJL mice for control (A and B) and CR (C and D) groups are shown (clinical scores were 3.5 for the control mouse and 1.5 for CR). SMI-32, specific for dephosphorylated neurofilament H in damaged axons, is stained green. Antibodies to MBP were used to detect myelin (in red). Higher numbers of SMI-32-positive axons were detected in the spinal cord white matter of controls (A and B) compared with CR (C and D). White arrows indicate SMI-32-positive axons. Original magnification: A, 6.4×; B, 16×; C, 4.8×; and D, 12×. Original scale bars, 200 μm (A and C) or 100 μm (B and D). (E) Axonal damage quantification (n=5 in each group) shows a significant reduction in CR compared with control mice (*, P=0.005, by two-tailed t-test).

Fig. 4.

Chronic CR does not inhibit T cell priming in the EAE-induction phase. In vitro proliferation rate (A) and IFN-γ production (B) in response to PLP139–151 by cells isolated 10 days p.i. from draining lymph nodes (axillary and inguinal) did not show any significant differences amongst SJL controls, mice assigned to a HF or CR (n=2/group; all mice were Clinical Grade 0 except one in the control group that was Grade 2). Proliferation and cytokine production in response to PLP139–151 by splenocytes isolated from SJL mice 30 days p.i. was also tested (n=5/group; control mice were at clinical scores ranging from 2 to 4, two CR mice were at score 0, two were at 2, and one at score 1; C and D). Neither splenocyte proliferation in vitro nor cytokine production showed significant differences between the control and CR groups. Values are mean ± sem. These data are representative of four experiments.

Fig. 5.

CR alters plasma levels of corticosterone, leptin, and adiponectin. SJL mice were bled at different time-points during the experiment: prior to initiation of the specified diet (Baseline), after 4 weeks of the assigned diet (Pre-immunization), prior to EAE onset following immunization with PLP139–151 (Preclinical; Day 10 p.i.), and during clinical EAE (Days 16–19 or 30 p.i.). All blood draws were performed at 10 a.m. to control for circadian variations. Corticosterone (A), leptin (B), and adiponectin (C) plasma levels were not significantly different at baseline. (A) After 4 weeks of CR (Pre-immunization) corticosterone was elevated significantly compared with controls (P=0.01; by t-test with Welch’s correction). During clinical EAE, CR mice also had significantly elevated corticosterone levels over the control group (P=0.04; by t-test; n=6 for baseline and preimmunization; n=3 for preclinical; and n=8 for clinical). (B) At preimmunization, leptin levels were significantly lower in CR mice compared with the control mice (P=0.01 by t-test). During clinical EAE, leptin was significantly lower in CR than controls (P=0.02; by t-test with Welch’s correction). Within the control group and the CR group, leptin levels dropped progressively after immunization for EAE (n=7 for baseline, preimmunization, and preclinical; n=15 for clinical; at the preclinical stage, all mice were at score 0; at clinical stage, all controls and 11/15 CR mice displayed clinical EAE signs). (C) Adiponectin was significantly higher in CR than controls during clinical EAE (P=0.05; by t-test with Welch’s correction; n=6 for baseline and preimmunization; n=3 for preclinical; and n=11 for clinical; at the preclinical stage, all mice were at score 0; at clinical stage, all controls and 4/11 CR mice displayed clinical EAE signs). *, P ≤ 0.02; **, P ≤ 0.05.

Fig. 6.

IL-6 plasma levels are reduced by CR during clinical EAE. Blood samples were obtained at time of sacrifice at Day 30 p.i. from mice fed ad libitum or CR SJL mice. Serum IL-6 levels were measured by ELISA (n=5 for CR; n=9 for controls). IL-6 levels were significantly lower in the CR-treated mice compared with controls (*, P=0.02; two-tailed t-test with Welch’s correction).