Dietary restriction protects against experimental cerebral malaria via leptin modulation and T-cell mTORC1 suppression

Abstract

Host nutrition can affect the outcome of parasitic diseases through metabolic effects on host immunity and/or the parasite. Here we show that modulation of mouse immunometabolism through brief restriction of food intake (dietary restriction, DR) prevents neuropathology in experimental cerebral malaria (ECM). While no effects are detected on parasite growth, DR reduces parasite accumulation in peripheral tissues including the brain, and increases clearance in the spleen. Leptin, a host-derived adipokine linking appetite, energy balance and immune function, is required for ECM pathology and its levels are reduced upon DR. Recombinant leptin abrogates DR benefits, while pharmacological or genetic inhibition of leptin signalling protects against ECM. DR reduces mTORC1 activity in T cells, and this effect is abrogated upon leptin administration. Furthermore, mTORC1 inhibition with rapamycin prevents ECM pathology. Our results suggest that leptin and mTORC1 provide a novel mechanistic link between nutrition, immunometabolism and ECM pathology, with potential therapeutic implications for cerebral malaria.

Figures

FIGURE 1. Dietary restriction modulates susceptibility to ECM

Representative experiments to examine the dose-response to dietary restriction (DR) (A-E) and the kinetics of onset of DR benefits (F-J). Eight mice per group in two cages were food restricted by 10 to 50% as indicated (DR 10%-50%) relative to ad libitum (AL) fed controls for 7 days (A-D, DR D-7) or 40% restricted from the indicated day (F-I, 40% DR). All mice were infected on day 0 (black arrowhead) and monitored until day 10-12 after infection; orange arrowheads indicate onset of food restriction. (A, F)Food intake. Average daily food consumption of the indicated group expressed as grams of food eaten per total weight of animals in that cage. (B,G)Weight. Body weight was measured daily starting 7 days before the infection and expressed as a percentage of starting weight. (C, H)Peripheral parasitemia. The percentage of infected RBCs in circulation was assessed by flow cytometry on the indicated day after infection. (D, I)Survival. Kaplan-Meier curves indicative of survival of infected mice exposed to different levels of DR. (E, J)Cumulative survival. Percentage of survival 12 days after infection. Data from 2-4 independent experiments per group were pooled, with n indicating total number of mice in each group. Asterisks represent significance of the corresponding Kaplan-Meier survival curves by log rank test relative to the AL group; *p<0.05, **p<0.01, ***p<0.001. Values are means and error bars represent SEM.

FIGURE 2. Dietary restriction reduces brain parasite accumulation and immunopathology

(A) Luciferase activity from brain extracts of AL or 40% DR mice infected with transgenic luciferase-expressing P. berghei ANKA sacrificed on the indicated day. Luciferase activity indicative of parasite burden is expressed in arbitrary light units. A representative experiment with 4 animals per group is shown. Inset: Cumulative data pooled from 4 independent experiments showing luciferase activity in the brains of naïve, AL and DR mice on day 6 after infection. Asterisk indicates the significance of the difference between the indicated groups according to a Mann-Whitney test; *p<0.05. (B) Relative parasite 18S rRNA expression in brain of AL and DR mice (n=4/diet group) 6 days after infection as determined by qPCR with a p value between AL and DR groups as indicated according to a Mann-Whitney test. (C) Naïve, AL or DR mice (n=4/group) were injected with Evan's blue and the leakage of the dye into the cerebral tissue representing disruption of BBB was qualitatively (left) and quantitatively (right) assessed on day 6 after infection. A representative experiment is shown. Asterisk indicates the significance of the difference between the indicated groups according to a Mann-Whitney test; *p<0.05. (D) Relative gene expression of chemokines, chemokine receptors and inflammatory markers as indicated in brain on day 6 after infection of the indicated dietary group as measured by qPCR. Representative experiments with 4 mice/group are shown. Asterisks indicate the significance of the difference between AL and DR for a given gene according to a Mann-Whitney test; *p<0.05. (E-G) Numbers of total (E) and activated (F,G) CD4+ and CD8+ T lymphocytes with representative dot plots from brains of mice (n=4/group) of the indicated dietary groups on day 6 after infection following perfusion as determined by FACS. Asterisks indicate the significance of the difference between groups according to a Mann-Whitney test; *p<0.05. Values are means + SEM.

FIGURE 3. Dietary restriction reduces total parasite sequestration and increases parasite spleen clearance

(A)In vivo imaging of parasite luciferase expression in live mice (dorsal view, top; ventral view, bottom) of the indicated diet group on day 6 after infection. A naïve mouse (N) is shown as a control. (B, C) Parasite luciferase activity measured in extracts of lung (B) and WAT (C) over a time course after infection from a representative experiment with n=4 mice/dietary group/time point. Asterisks indicate the significance of the difference on day 6 after infection according to a Mann-Whitney test; *p<0.05. (D) Spleen weights of AL and DR mice (n=11-12/group) on day 4 and day 6 after infection. Weights of spleens harvested from naïve animals (N; n=6/group) are shown as controls. Data are cumulative results of 3 independent experiments. Asterisks indicate the significance of the difference on the indicated day according to a Mann-Whitney test; **p=0.01. (E,F) Relative parasite 18S rRNA expression as determined by qPCR (E), and parasite luciferase activity (F) in spleens of AL and DR mice (n=5/diet group) on day 6 after infection. Asterisks indicate the significance of the difference between groups according to a Mann-Whitney test; *p<0.05. (G) Percentages of splenic macrophages and representative dot plots from AL and DR mice on day 6 post-infection. (H,K) Total numbers of splenic CD4+ (H), and CD8+ (K) T cells as measured by flow cytometry on day 4 and 6 (d4, d6) after infection in spleens of naïve (N), AL and DR mice as indicated. (I,J,L,M) Total numbers of CD4+CD69+CXCR3+ (I), CD4+CD62L (J), CD8+CD69+CXCR3+ (L) and CD8+CD62L+ (M) T cells with representative dot plots from AL and DR mice measured on day 6 post-infection. A representative experiment with 5 mice per dietary group is shown. Asterisks indicate the significance of the difference on the indicated day according to a Mann-Whitney test; *p<0.05. Values are means ± SEM.

FIGURE 4. Leptin is required for pathogenesis of ECM

(A) Serum leptin levels in ad libitum fed (AL) or restricted (DR) mice (n=4/group) over a time course after infection. (B) Wild-type mice (lean or obese as indicated) or mice deficient in leptin (ob/ob) or leptin receptor (db/db), all fed ad libitum on a complete diet and infected on day 0 (n=5/group). Peripheral parasitemia. The percentage of infected RBCs in circulation was assessed by flow cytometry on the indicated day after infection. Survival. Kaplan-Meier curves indicate a significant survival advantage in mice lacking leptin or leptin receptor (log rank score p=0.0031) independent of adiposity. (C) Leptin antagonist (LA) prevented ECM in ad libitum fed WT mice. Peripheral parasitemia and survival curves for animals treated with vehicle or I mg/kg/day of leptin antagonist (LA) are shown (n=10/group; log rank score p=0.0089). Values are means ± SEM.

FIGURE 5. Leptin abrogates benefits of dietary restriction

(A) Peripheral parasitemia and survival of mice in the indicated dietary groups (n=5/group) infected on day 0 and treated with vehicle or recombinant leptin 10ug twice daily on the indicated days. Log rank scores indicated significant differences between DR and both DR/Leptin regimens (log rank score p=0.029 d1-6; p=0.002 d1-3). (B) AL, DR or DR+leptin (d1-3) mice (n=4/group) were injected with Evan's blue and the leakage of the dye into the cerebral tissue representing disruption of BBB was quantitatively assessed on day 6 after infection. A representative experiment is shown. (C-E) Numbers of total (C) and activated (D,E) CD4+ and CD8+ T lymphocytes with representative dot plots, of DR, DR+leptin and ob/ob mice (n=5/group) 6 days after infection following perfusion as determined by FACS. Asterisks indicate the significance of the difference according to a one-way ANOVA followed by Dunnett's multiple comparison testing vs. the DR group; *p<0.05. Values are means ± SEM.

FIGURE 6. Direct modulation of mTORC1 activity by leptin in T cells upon malarial infection

(A, B) Immunoblots of markers of mTORC1 signaling in spleens (A) or non-adherent splenocytes (B) of AL and DR animals on day 3 after infection. Quantitation of ratios of phosphorylated to total protein are presented below. Asterisks indicate the significance of the difference between AL and DR for the indicated protein according to a Mann-Whitney test; *p<0.05. N=4 mice/group. See Supplementary Fgure 6D and E for full blots of spleen and non-adherent splenocytes, respectively. (C-E) Analysis of S6 phosphorylation in splenocytes from naïve or infected AL, DR or DR animals treated with leptin one hour prior the experiment (DR + Lep) on day 3 post-infection; n=4-5 animals/group. (C) Percentages of CD45+ splenocytes, CD4+ (D) and CD8+ (E) T cells positive for S6 phosphorylation are shown with corresponding representative dot plots. Asterisks indicate the significant of the difference between groups according to a one-way ANOVA followed by Dunnett's multiple comparison testing vs. the AL group; *p<0.05, **p<0.01, ****p<0.0001. (F,G)In vitro assessment of mTORC1 activity. Splenocytes from AL or DR mice on day 3 of infection were isolated and incubated for one hour with or without leptin (50, 150 ng/ml as indicated) and the percentage of CD4+ (F) and CD8+ (G) T cells with phosphorylated S6 assessed by FACS. (H-K) Functional consequences of leptin signaling and mTORC1 activation early in infection. Numbers of total CD4+ (H) and CD8+ (J) and activated CD4+CD69+CXCR3+ (I) and CD8+CD69+CXCR3+ (K) from spleens of AL, DR and DR+leptin (d1-3) mice (n=5/group) on day 4 after infection as assessed by FACS. Asterisks indicate the significance of the difference between the indicated groups according to a Mann-Whitney test; *p<0.05. Values are means + SEM.

FIGURE 7. Rapamycin treatment protects against ECM

(A) Parasitemia and survival of ad libitum fed mice treated with rapamycin on days 1-3 after infection with the indicated dose (n=10/group). Vehicle-treated AL (n=5) and DR (n=10) mice served as controls. Log rank scores indicated significant survival advantage upon rapamycin treatment relative to the vehicle-treated group (p=0.005 for low dose; p=0.0008 for high dose). (B) Representative dot plots of total CD4+ and CD8+ T cells from brain of vehicle-treated AL, DR and rapamycin-treated AL (5 mg/kg) mice on day 6 after infection. Corresponding cell numbers are indicated. (C-E) Activated triple positive T cell populations CD4+CD69+LFA-1+ (C), CD8+CD69+LFA-1+ (D), and CD8+CD69+granzymeB+ (E) from brain on day 6 after infection of the indicated groups. Asterisks indicate the significant of the difference between according to a one-way ANOVA followed by Dunnett's multiple comparison testing vs. the AL group; *p<0.05, **p<0.01, ***p<0.001. Values are means ± SEM.

FIGURE 8. Model for the role of leptin and mTORC1 in pathogenesis of ECM

Sequestration of infected RBCs (iRBCs) to WAT activates leptin production by adipocytes early in infection. Increased circulating leptin activates mTORC1 in splenic T cells, leading to increased expression of chemokine receptors and effector molecules, and increasing the likelihood of trafficking to inflamed tissues such as brain later in infection. DR mitigates leptin production by WAT early in infection. Suppression of the leptin-mTORC1 axis either by DR, leptin antagonist peptide or rapamycin results in significant inhibition of effector CD4+ and CD8+ T cells migration to the brain late in infection, preserving BBB function and reducing neuropathology, while enhancing T cell function and parasite elimination in the spleen.