A single rapamycin dose protects against late-stage experimental cerebral malaria via modulation of host immunity, endothelial activation and parasite sequestration

Wild-type female C57BL/6J mice 8–10 weeks of age were purchased from Jackson Labs (Bar Harbor, ME). Animals were housed 4–5 per cage and kept under standard laboratory conditions and allowed free access to water and food.

All animal experiments were performed either with the approval of the Animal Research Ethics Committee of the Canton Bern, Switzerland and the University of Bern Animal Care and Use Committee, or the Harvard Medical Area Animal Care and Use Committee according to the PHS Policy on Humane Care and Use of Laboratory Animals by Awardee Institutions and NIH Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training.

Mice were fed a purified diet (D124570B, Research Diets, New Brunswick, NJ) with 72% calories from carbohydrate (sucrose, maltodextrin, corn starch), 18% calories from protein (casein) and 10% from fat (soybean oil, lard) supplemented with 15 mg of PABA per 100 g of food. Powder diets were mixed with 1 L/kg diet of hot water containing 2% agar; the cooled mixture was given daily to mice either on an ad libitum basis.

Cryopreserved transgenic P. berghei ANKA parasites expressing luciferase/GFP under a constitutive promoter [12] were thawed and passaged once in vivo before being used to infect experimental mice with 0.5 million parasitized RBCs/mouse by tail vein injection. Parasites from peripheral blood were stained with SYTO16 and the percentage of infected cells was calculated by flow cytometry [13]. Individual mouse weights and food intake per cage were calculated daily.

Infected mice were sacrificed on the day 6 after infection and perfused with cold PBS intracardially. The organs were harvested, weighed and homogenized in an equal volume/mg tissue of luciferase activity assay buffer (Invitrogen). Equal volumes were mixed with luciferin substrate and measured in a 96 well luminometer (Biotek Synergy 2).

On day 6 after infection, mice were injected intravenously with 200 μL of PBS-2% Evans blue, sacrificed 1 h later, and perfused intracardially with cold PBS. Brains were harvested, photographed, weighed and placed in 2 mL 100% formamide (Merck) for 48 h at 37 °C. Absorbance of the formamide supernatant was measured at 620 nm. Evans blue concentration was calculated from a standard curve and results expressed as microgram of Evans blue/gram of brain tissue.

Intra-vital and ex vivo imaging were performed in 5–6 week-old female C57BL/6 mice infected with P. berghei ANKA expressing mCherry under the Hsp70 promoter, and Firefly luciferase under the ef1a promoter [14]. A mixture of anaesthetics comprising 125 mg/kg ketamine (Ketasol, Graeub) and 12.5 mg/kg xylazine (Xylason, Graeub), was prepared and diluted in 1 × PBS (1:2:5). Mice were injected intraperitoneally with 100 mL per 20 g of body weight, of the mixed anaesthetics. Following anaesthesia, mice were injected intravenously with 200 µL of 70 kDa fluorescin isothiocyanate (FITC)-conjugated dextran (Sigma Aldrich, St. Louis, MO) diluted in 1 × PBS, at a final concentration of 10 mg/mL. Mice were imaged immediately after FITC-Dextan injection each day post-infection, in a Leica SP8-STED confocal microscope, using a HC PL APAO CS2 63X 1.4NA oil immersion objective. In the LeicaSP8-STED microscope, a white laser was used, and wavelengths were defined for the spectra corresponding to FITC and mCherry. Experiments were performed in three mice at each day post-infection. The images were assembled and processed using Fiji imaging software.

Brains and spleens were harvested at the indicated days after infection following perfusion with cold PBS. Spleen single-cell suspensions were prepared by homogenization through a 70 µm cell strainer (BD Falcon). Brains were digested with 50 U/mL of type II and IV collagenase (Gibco, Invitrogen) in RPMI, and mononuclear cells isolated on a 33% Percoll density gradient (GE Healthcare). After lysing red blood cells and determining the total cell concentration, cells were incubated with an FcR blocker (Miltenyi Biotech), labelled for the extracellular antigens CD3, CD8, CD4 (BD Pharmingen), CD69, LFA-1, CD62L, CXCR3, CD25 (all from Biolegend), fixed with 4% paraformaldehyde (PFA) and stained for the intracellular markers Granzyme B (Biolegend) and Foxp3 (eBioscience) using the Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer’s instructions. Single fluorochrome-labelled cells were used to compensate for spectral overlap. Isotype-matched fluorescence minus one controls were used to set gate cutoffs. Acquisition was performed using a BD Fortessa and data analysed using FlowJo (Tree Star Inc.).

Mice were perfused intracardially with PBS on the indicated day after infection. For morphometric analyses, brains were fixed in 4% paraformaldehyde in PBS, embedded in paraffin, cut and H&E stained using standard procedures. For immunofluorescence microscopy, brains were flash frozen in liquid nitrogen and stored at − 80 °C until use. Sections were cut with a cryotome, fixed in ice-cold 4% PFA, permeabilized in 0.25% Triton X-100, washed in PBS and blocked in 10% normal goat serum diluted in TBS-T (TBS buffer containing 0.2% Tween 20). After blocking, sections were incubated with primary rabbit anti CD31 and rat anti-ICAM-1 (both from Abcam, Cambridge, MA) overnight at 4 °C. After washing with PBS, sections were labelled with Alexa 488 goat and rabbit IgG and Alexa 568 goat anti rat IgG for 2 h at room temperature. After extensive washing, slides were mounted in anti-fade mounting media with DAPI (Vector Labs, Burlingame, CA). Images were taken with a Zeiss Axio observed fluorescence microscope with an APOTOME attachment with a 20 ×/0.8 M27 plan-apochromat objective and an AxioCam monochromatic MRm digital camera. Slices stained only with secondary antibodies were used to set parameters. For quantitation, all images in both groups were taken using the same acquisition parameter. Image processing analysis was performed using the using the AxioVision 4.7.1 software as well as Fiji imaging software.

Total RNA was extracted from frozen tissue with RNA Bee (Qiagen). Hexamer-primed complementary DNA (cDNA) was synthesized with Verso cDNA kit (Thermo Scientific) according to manufacturer’s instructions. Quantitative real-time PCR was performed with a MyIQ (Bio-Rad) using SYBR Green. Relative expression was calculated with the ΔΔC_t method. cDNA expression of each sample was standardized to RPLA13. Each sample was tested in duplicate at least twice. Primer sequences used: CCL-5, fwd: 5′-GCAAGTGCTCCAATCTTGCA-3′; rev: 5′-CTTCTCTGGGTTGGCACACA-3′. CXCL-9, fwd: 5′-GCCATGAAGTCCGCTGTTCT-3′; rev: 5′-GGGTTCCTCGAACTCCACACT-3′. CXCL-10, fwd: 5′-GACGGTCCGCTGCAACTG-3′; rev: 5′-GCTTCCCTATGGCCCTCATT-3′. ICAM-1, fwd: 5′-GCCTCCGGACTTTCGATCTT-3′; rev: 5′-GTCAGGGGTGTCGAGCTTTG-3′. Pb18S, fwd: 5′-AAGCATTAAATAAAGCGAATACATCCTTAC-3′; rev: 5′-GGAGATTGGTTTTGACGTTTATGTG-3′. P-Selectin, fwd: 5′ CCCTGGCAACAGCCTTCAG-3′; rev: 5′-GGGTCCTCAAAATCGTCATCC-3′. VCAM, fwr: 5′-AGTTGGGGATTCGGTTGTTCT-3′; rev: 5′-CCCCTCATTCCTTACCACCC-3′.

Snap frozen tissues were homogenized in 1 mL of lysis buffer consisting of 10% glycerol, 1% NP-40, 1 mM MgCl₂, 150 mM NaCl, 50 mM β-glycerophosphate, 20 mM Tris–HCl pH 7.4 supplemented with a protease inhibitor and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Rockford, IL) with an IKA T10 Ultra-Turrax (Wilmington, NC) tissue disperser. Homogenized samples were centrifuged for 20 min at 10,000g, and the protein concentration determined from extracts using the Pierce BCA protein assay kit (Thermo Scientific). 40 μg of protein extract was resolved by SDS-PAGE, transferred to Immobilon-P polyvinylidene difluoride (PDVF) membranes (Millipore), blocked in 5% skim milk diluted in TBST (10 mM Tris–HCl pH 7.4 plus 0.1% Tween 20), washed in TBST, and incubated overnight at 4 °C with primary antibody (CD36 and tubulin, both from Abcam, Cambridge MA) 1:1000 diluted in antibody buffer (5% BSA, 0.1% sodium azide in TBST). After washing in TBST, membranes were incubated 90 min at room temperature with goat anti-mouse or goat anti-rabbit IgG conjugated with horseradish peroxidase (Dako, Carpinteria, CA) diluted 1:5000 in 5% skim milk. After extensive washing with TBST, blots were developed using the Super Signal West Femto chemiluminescent substrate kit (Pierce, Thermo Scientific).

A stock solution of rapamycin (LC Laboratories, Woburn MA) dissolved in 100% ethanol was further diluted in 5% Tween/5% PEG-400 vehicle solution and was administered i.p. to infected mice at 5 or 25 mg/kg/day on day 4, 5 or 6 of infection; vehicle solution was used as a control.

Data are expressed as mean ± SEM unless indicated otherwise. Statistical analyses were performed in GraphPad Prism with Mann–Whitney, one-way analysis of variance (ANOVA) or Kaplan–Meier survival tests as indicated.

Previous studies have demonstrated that early rapamycin treatment on days 1–3 post-infection with P. berghei ANKA, before onset of symptoms, protects mice against ECM pathology and mortality [11]. Here, the efficacy of a single injection of vehicle or rapamycin at different doses (5 or 25 mg/kg), provided on day 4 post-infection when symptoms including weight loss and reduced food intake indicative of sickness behaviour emerge (Fig. 1a), was tested. At this time point, an increase in circulating pro-inflammatory cytokines likely causative of this anorectic response, including IFN-γ IL-6 and TNFα, was observed (Fig. 1b). Also on day 4, increased brain vascular leakage was observed by intra-vital confocal microscopy, evidence of initial blood brain barrier dysfunction (BBB) (Fig. 1c). Vascular leakage remained elevated on days 5 and 6 post infection (Fig. 1c).

Clinically, both the high and low doses of rapamycin resulted in complete abrogation of neurological symptoms, and protected against the high mortality associated with this model (Fig. 1d). Although chronic mTORC1 inhibition can interfere with host immune clearance of parasites [10] or inhibit parasite growth directly [15], a single rapamycin dose did neither, as peripheral parasitaemia levels, compared to vehicle treated controls, were not significantly affected (Fig. 1d). Interestingly, infection-induced anorexia was transiently but significantly abrogated on the day after rapamycin treatment, but only at the highest dose (Fig. 1b). The efficacy of treatment with rapamycin at a later time point (days 5 and 6), when brain vascular dysfunction is more pronounced and better established, was next tested. As shown in (Fig. 1e), mice treated with a single high dose (25 mg/kg) of rapamycin as late as day 5 post infection were also protected from neuropathology and death. However, treatment at day 6 only conferred a non-significant 25% survival rate (Fig. 1e). Nonetheless, neither day 5 nor day 6 rapamycin treatment affected the course of peripheral parasitaemia (Fig. 1e). Taken together, the results show that a single dose of rapamycin, provided as late as day 5 of infection, after the emergence of sickness behaviour and vascular leakage, protected mice from ECM. In order to establish the mechanism(s) involved in the protection afforded by rapamycin, subsequent experiments were performed using a dosing strategy consisting of one injection of 5 mg/kg on day 4 post-infection, which maximizes protection from ECM after sickness behavior and vascular leakage associated with ECM have emerged.

Cytoadherence, or sequestration of parasitized red blood cells (RBSs) to endothelial cells in peripheral organs including white adipose tissue (WAT) and lungs constitutes a mechanism by which mature Plasmodium parasites avoid circulation through and clearance by the spleen [4]. To monitor sequestration, a P. berghei ANKA strain constitutively expressing a GFP-luciferase transgene was employed [12], and used to measure luminescence in extracts of perfused tissues on day 6 post-infection. The results demonstrated that a single rapamycin treatment on day 4 post infection significantly reduced sequestration in lung (Fig. 2a) and WAT, including both perigonadal and subcutaneous depots, relative to vehicle-treated controls (Fig. 2b).

The potential mechanisms by which rapamycin reduced peripheral sequestration were next explored. In mice and humans, the fatty acid receptor CD36 is a major host determinant of sequestration in peripheral tissues including lung and WAT (but not brain) [16‐19]. Interestingly, rapamycin treatment resulted in reduced CD36 protein expression in WAT but not lungs on day 6 post-infection (Fig. 2c, d). These results suggest the existence of CD36 dependent and independent mechanisms of peripheral parasite sequestration, selectively and differentially regulated in different tissues by mTORC1 activity.

Because sequestration of mature parasites in lungs and WAT represents a strategy to avoid clearance of parasitized erythrocytes in spleen, reduced sequestration (Fig. 2) without a significant increase in circulating parasitized RBCs (Fig. 1) could potentially be explained by increased splenic clearance. Parasite-derived luciferase activity detected in the spleens from mice treated with rapamycin on day 4, tended to be reduced by day 6 compared to vehicle-treated controls per mg of tissue, although this difference did not reach statistical significance (Fig. 3a). However, as splenic clearance may also destroy parasite luciferase activity, it is not a reliable measure of clearance activity.

Host immune cell-based measures of splenic activity were next explored. Treatment with rapamycin on day 4 translated into an increase in total splenocytes, as well as an increase in total lymphocytes, including both CD4+ and CD8+ T cells, by day 6 post-infection (Fig. 3a). Amongst CD8+ T cells, the proportion displaying the activated phenotype, characterized by CD69, LFA-1 and CXCR3 expression, was significantly reduced following rapamycin treatment (Fig. 3b). However, amongst the population of activated CD8+, CD69+, LFA1+ T cells, the proportion expressing granzyme was increased upon rapamycin treatment, consistent with more robust effector function, possibly leading to enhanced clearance (Fig. 3c). Lastly, the proportion of naïve CD62L+ CD8+ T cells, characterized of reduced migratory capacity, significantly increased following rapamycin treatment (Fig. 3d). Together, these data suggest that a single dose of rapamycin on day 4 of infection alters splenic T cell differentiation, resulting in an overall decrease in T cell activation and migratory capacity, but an increase in effector function.

The simultaneous presence of parasites and activated CD8+ T cells in brains of infected mice is required for BBB disruption and ECM neuropathology [4]. Therefore, the effect of day 4 rapamycin treatment on T cell accumulation in brains of infected mice by day 6 post-infection was investigated. Quantitation of leukocytes isolated from brains of infected animals after perfusion on day 6 revealed significantly fewer total leukocytes present in the rapamycin group (Fig. 4a), including reduced accumulation of CD4+ and CD8+ T cells (Fig. 4b). Moreover, these fewer CD4+ and CD8+ T cells displayed reduced expression of activation markers such as CD69 and LFA1 (Fig. 4c, d), consistent with data in spleen showing that mTORC1 inhibition of T cells on day 4 prevented T cell activation and trafficking to the brain later in infection.

Vascular endothelial cell activation and dysfunction is another feature of CM that correlates with pathology [20]. mTORC1 plays a key role in controlling the expression of endothelial cell adhesion molecules such as ICAM-1, VCAM-1 and P-selectin [21‐23]. Analysis of ICAM-1 expression in brain sections on day 6 post-infection revealed rapamycin-dependent reduction in the intensity of endothelial ICAM-1 expression (Fig. 5a) as well as the total endothelial area expressing this adhesion molecule (CD31/ICAM-1 vascular co-localization, Fig. 5b). Interestingly, except for VCAM-1, rapamycin did not reduce mRNA expression of ICAM-1 or other chemokines crucial for T cells migration into the brain (Fig. 5c). These results are consistent with effects of rapamycin on neurovascular activation on the post-transcriptional level.

ECM neuropathology results from accumulation of infected RBCs in the brain, together with activated CD8+ T cells, which promotes destruction of the BBB [4, 24]. Having demonstrated vascular leakage starting as early as day 4 post-infection, the effect of a single dose of rapamycin at this time point on brain pathology was evaluated. Rapamycin treatment on day 4 significantly reduced the accumulation of parasites in brains on day 6 post-infection (Fig. 6a). This result was obtained by quantitating luminescence from parasites and confirmed by measurement of the parasite-specific Pb18s gene from brains using real-time PCR (Fig. 6a).

Reduce parasite brain sequestration correlated with preserved BBB integrity, as evidenced by reduced vascular leakage of injected Evan’s blue dye in rapamycin-treated mice (Fig. 6b). Furthermore, histological analyses revealed significantly fewer brain haemorrhages in the rapamycin-treated group compared to controls (Fig. 6c). However, no differences in the size of the haemorrhages were observed (Fig. 6c).