Adipose tissue parasite sequestration drives leptin production in mice and correlates with human cerebral malaria

Abstract

Circulating levels of the adipokine leptin are linked to neuropathology in experimental cerebral malaria (ECM), but its source and regulation mechanism remain unknown. Here, we show that sequestration of infected red blood cells (iRBCs) in white adipose tissue (WAT) microvasculature increased local vascular permeability and leptin production. Mice infected with parasite strains that fail to sequester in WAT displayed reduced leptin production and protection from ECM. WAT sequestration and leptin induction were lost in CD36KO mice; however, ECM susceptibility revealed sexual dimorphism. Adipocyte leptin was regulated by the mechanistic target of rapamycin complex 1 (mTORC1) and blocked by rapamycin. In humans, although Plasmodium falciparum infection did not increase circulating leptin levels, iRBC sequestration, tissue leptin production, and mTORC1 activity were positively correlated with CM in pediatric postmortem WAT. These data identify WAT sequestration as a trigger for leptin production with potential implications for pathogenesis of malaria infection, prognosis, and treatment.

Fig. 1. PbA iRBC sequestration in WAT correlates with leptin production in the ECM model.

(A) Time course of luciferase-expressing PbA parasites in live C57BL6 mice (BL6-PbA) using in vivo imaging from days 1 to 6 after infection. (B) Quantitation of sequestration of luciferase-expressing transgenic PbA parasites in the indicated tissue from infected C57BL/6 mice versus naïve controls (n = 3 per group) measured ex vivo in organ homogenates after perfusion on day 6 after infection; Mann-Whitney test between groups within a given organ/tissue. (C) Sequestration kinetics of luciferase-expressing PbA parasites in Pg and ScWAT homogenates harvested after perfusion on the indicated day after infection (n = 3 per time point; Kruskal-Wallis test with Dunn’s multiple comparisons test on the indicated day versus day 0). (D) Sequestration of iRBCs in WAT microvasculature of PbA-infected mice on day 6 after infection by fluorescence microscopy with mCherry-expressing PbA (red to visualize intact parasites) in UBC promoter–driven GFP C57BL/6 mice (ubiquitous green expression to visualize host cells) in PgWAT (left) or H&E in ScWAT in WT C57BL/6 mice (right). (E) RBCs harboring mature parasites accumulate rapidly in WAT. Sequestration was measured ex vivo in PgWAT homogenates harvested following perfusion 3 hours after intravenous injection of saline vehicle (saline) or 5 × 10⁶ iRBCs; n = 4 mice per group; Mann-Whitney test. (F and G) Representative images of leptin expression and quantitation of fluorescence intensity in ScWAT (F) and PgWAT (G) of PbA-infected C57BL/6 on days 0, 4, 5, and 6 after infection; n = 10 microscopic fields per mouse tissue with two mice per group; Kruskal-Wallis test with Dunn’s multiple comparisons test against day 0. (H) Representative images of FITC-labeled dextran (70 kDa) in WAT 2 hours after intravenous injection into mice on the indicated day after infection; quantitation of dextran Fire LUTs (lookup tables) outside of vessel relative to day 0 at right; Kruskal-Wallis test with Dunn’s multiple comparisons test against day 0. All data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. MFI, mean fluorescence intensity.

Fig. 2. Parasite sequestration in WAT is required for systemic leptin production and mortality.

(A) Percent peripheral parasitemia over time grouped by experiment with corresponding controls as indicated, including SBP1-deficient versus parental WT PbA into WT female BL6 (n = 10 per group), SMAC-deficient versus parental WT PbA into WT female BL6 (n = 5 per group), PbK173 versus WT PbA into WT female BL6 (n = 5 to 6 per group), and WT PbA into male or female WT (n = 13 to 16 per group) versus male (M) or female (F) CD36KO (n = 16 to 17 per group) BL6 mice. (B) Peripheral parasitemia on day 6 (above) or day 5 (below) expressed as a percent of the corresponding sex-matched control. (C to E) Day 6 organ-specific sequestration (C), brain parasite accumulation (D), and serum leptin (E) expressed as a percentage of the control for that particular experiment as indicated in (A) and grouped by parasite (top row) or host (bottom row) mutant/strain. Statistics in (B) to (E) were performed using unpaired t tests with Welch’s correction or Mann-Whitney tests between mutants/strains and their corresponding host/strain control. (F) Kaplan-Meier survival curves of mice from the indicated experimental groups infected on day 0, including the number in each group and the log-rank statistic relative to sex-matched BL6-PbA controls. (G) Evan’s blue–stained brains of the indicated sex/genotype on day 6 after infection; white numbers indicate quantification of Evan’s blue after extraction in micrograms per gram of tissue. (H and I) Circulating leptin levels of the indicated sex/genotype presented as absolute values before (day 0, green triangles) and on day 6 after infection (red triangles), with dotted lines representing individual animals (H) or as differences between circulating leptin on day 6 and day 0 (I); one-way analysis of variance (ANOVA) with Sidak’s multiple comparisons tests as indicated. All data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant; d0, day 0; d6, day 6.

Fig. 3. WAT leptin production is controlled by mTORC1 activity.

(A) Representative images of ^S235/236phospho-S6 expression and quantitation of fluorescence intensity in PgWAT of female PbA-infected C57BL/6 on days 0, 4, 5, and 6 after infection; 5 to 10 fields were imaged in at least three sections from two different animals in each group; **P < 0.01 and ****P < 0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test relative to day 0. (B) Immunoblots of total and ^T389phospho-S6K in PgWAT obtained from C57BL/6 female mice that were uninfected (naïve) or from the indicated mouse-parasite experimental group on day 6 after infection or PbA-infected and injected with rapamycin (Rap; 1 mg/kg) 2 days earlier, with quantitation below; Mann-Whitney test as indicated. (C) Immunoblots of total and ^T389phospho S6K in PgWAT from male CD36KO and WT littermates infected with PbA with quantitation below; Mann-Whitney test. (D) Serum leptin levels on day 6 after infection (left, Mann-Whitney test), survival curves (middle, log-rank test P = 0.0031), and peripheral parasitemia (right) of female BL6-PbA mice treated with vehicle or rapamycin (1 mg/kg) on day 4 after infection; n = 5 per group. (E) Leptin and adiponectin gene expression and protein secretion into the media in primary adipocyte cultures infected with adenovirus expressing shRNAs targeting the mTORC1 inhibitor TSC2 (shTsc2) or control scrambled RNA (shScr) at the indicated multiplicity of infection (MOI). Fibroblasts were used as negative controls. NI, not infected; ND, not detected. Kruskal-Wallis test with Dunn’s multiple comparisons test versus NI. All data are expressed as mean ± SD. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 4. Positive correlation between ScWAT parasite sequestration, leptin expression, and mTORC1 activation in human CM.

(A and B) Human plasma leptin levels measured by ELISA in the following groups: (A) CM cases from Cambodia and Senegal, with uninfected controls (Mal neg) from Senegal or France; (B) CM cases from Uganda and Malawi, as well as SMA, uncomplicated malaria (Mal pos) and uninfected controls from Uganda (Mal neg); Kruskal-Wallis test versus uninfected controls. (C) Representative differential interference contrast (DIC) image with 4′,6-diamidino-2-phenylindole (DAPI) counterstain of ScWAT from a fatal case of CM. DAPI stains endothelial and adipocyte nuclei as well as P. falciparum iRBCs sequestered in the microvasculature; dark hemozoin pigment (arrows) marks iRBCs. Right: Quantification of iRBC sequestration in ScWAT sections from fatal CM (n = 24) and P. falciparum–infected non-CM cases (n = 22). Each dot represents the median of the number of iRBCs per 10 high-power fields (HPF); Mann-Whitney test. (D) Representative thermal-scale images (magnification, ×10) and quantification of relative intensity of leptin protein levels in ScWAT from postmortem P. falciparum–infected non-CM (n = 11) and CM (n = 15) cases relative to uninfected controls (n = 6); Kruskal-Wallis test with Dunn’s multiple comparisons tests as indicated. (E) Representative thermal-scale images (magnification, ×20) and quantification of relative intensity of ^S235/236phospho-S6 protein levels in ScWAT from uninfected (n = 6), P. falciparum–infected non-CM (n = 11), and CM (n = 15) cases; Mann-Whitney test. (F) Representative confocal microscopy images of ScWAT from P. falciparum–infected non-CM and CM cases showing leptin (red), ^S234/235pS6 (green), parasite [DAPI/DIC and hemozoin (black)], and colocalization (composite). All data are expressed as mean ± SD; *P < 0.05 and ****P < 0.0001.

Fig. 5. Model for the role of WAT iRBC sequestration in the neuropathology of CM.

(A to C) CD36-dependent adhesion of infected RBCs to WAT endothelium (A) results in local vascular permeability potentially allowing contact between parasite material and adipocytes (B) and subsequent mTORC1 activation in these cells (C). (D) mTORC1 activation triggers the local expression and production of adipocyte-derived leptin. Circulating leptin increases upon infection and is required for full penetrance of neuropathology and mortality in the ECM model. Rapamycin treatment suppresses mTORC1 activation in WAT, reduces circulating leptin, and protects against neuropathology. In humans, although P. falciparum infection does not increase circulating leptin levels, iRBC sequestration, tissue leptin expression, and mTORC1 activity are positively correlated with CM in ScWAT.