Perillyl alcohol reduces parasite sequestration and cerebrovascular dysfunction during experimental cerebral malaria

Abstract

Cerebral malaria (CM) is a severe immunovasculopathy which presents high mortality rate (15-20%), despite the availability of artemisinin-based therapy. More effective immunomodulatory and/or antiparasitic therapies are urgently needed. Experimental Cerebral Malaria (ECM) in mice is used to elucidate aspects involved in this pathology since manifests many of the neurological features of CM. In the present study, we evaluated the potential mechanisms involved in the protection afforded by perillyl alcohol (POH) in mouse strains susceptible to CM caused by Plasmodium berghei ANKA (PbA) infection through intranasal preventive treatment. Additionally, to evaluate the interaction of POH with the cerebral endothelium using an in vitro model of human brain endothelial cells (HBEC). Pharmacokinetic approaches demonstrated constant and prolonged levels of POH in the plasma and brain after a single intranasal dose. Treatment with POH effectively prevented vascular dysfunction. Furthermore, treatment with POH reduced the endothelial cell permeability and PbA s in the brain and spleen. Finally, POH treatment decreased the accumulation of macrophages and T and B cells in the spleen and downregulated the expression of endothelial adhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain. POH is a potent monoterpene that prevents cerebrovascular dysfunction in vivo and in vitro, decreases parasite sequestration, and modulates different processes related to the activation, permeability, and integrity of the blood brain barrier (BBB), thereby preventing cerebral oedema and inflammatory infiltrates.

FIG 1

Pharmacokinetic profile of POH in the plasma and brain using GC-MS. C57BL/6 mice were treated with a single dose of 500 mg/kg of POH euthanized at predetermined times (n = 4 per group). Plasma and brains were collected, and POH was extracted from the samples and analyzed using GC-MS. (A) Pharmacokinetic profile of POH in the plasma shows that POH remained constant until 8 h after inhalation, the concentration decreased at 12 h, and it was undetected at 24 h after inhalation. (B) Pharmacokinetic profile of POH in the brain shows that the concentration decreased 30 min after inhalation, it remained constant until 12 h, and POH was detected in the brain extracts 24 h after inhalation. Data are presented as mean ± SD.

FIG 2

Effect of POH on the PbA luciferase sequestration using bioluminescence. Whole-body imaging after an injection of luciferin from a naive animal (A1), infected, untreated, and unperfused animal (A2), infected, untreated, and perfused animal (A3), and an infected, POH-treated, and perfused animal (A4). (B) Quantification of whole-body bioluminescence of perfused and nonperfused PbA-infected mice treated with POH or vehicle. (C) Quantification of bioluminescence in the brain, spleen, lungs, and liver. (D) Ex vivo imaging of the brain, spleen, liver, and lungs of untreated, perfused, and infected animals (D1, 3, 5, and 7) and perfused, infected, and POH-treated animals (D2, 4, 6, and 8). (E) Plasmodium berghei 18S RNA levels in the brain and spleen. Data are presented as mean ± standard error of the mean (SEM) (n = 5 per group). This figure is representative of data from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

POH prevents the blood-brain barrier breakdown in PbA-infected mice. C57BL/6 mice infected with PbA were treated with 500 mg kg⁻¹day⁻¹ POH or vehicle. On day 6 of infection, Evans blue solution was injected intravenously and quantified in the brain. PbA-infected mice showed increased extravasation of Evans blue in the brain, which was prevented by POH treatment (n = 8 for POH groups and n = 3 for uninfected). The results are shown as mean ± standard deviation (SD). The data are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

POH decreases endothelial dysfunction in human brain endothelial cells (HBECs). HBECs were stimulated with 50 ng/ml TNF or P. falciparum-parasitized red blood cells (at a 30:1 P. falciparum to HBEC ratio) and treated with 20 μM POH for 6 h. A range of 10 to 20 pictures of HBECs were taken for each culture using fluorescence microscopy, and representative images were presented for unstimulated cells (A1 to 2), cells stimulated with 30 mature forms of P. falciparum per cell (A3), cells stimulated with the parasite and treated with 20 μM POH (A4), cells stimulated with 50 ng/ml of TNF (A5), and cells stimulated with 50 ng/ml TNF and 20 μM POH (A6). White arrows indicate the opening of the interendothelial junctions (OIJs). (B) Data show the ratio of the area of OIJs per total area of each picture. (C) Evans blue leakage was significantly lower in the POH-treated HBECs than in TNF-stimulated and parasite. The results are shown as mean ± SD (scale bars: 50 μm). These graphs are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Regulation of ICAM-1, VCAM-1, and CD36 in the brains of PbA-infected mice by POH treatment. C57BL/6 mice were infected with PbA and treated with POH or vehicle (n = 5 per group). After 6 days of infection, the brains were removed, processed, and stored until qRT-PCR and immunochemistry analysis. (A) qRT-PCR showed an upregulation of ICAM-1, VCAM-1, and CD36 in untreated PbA-infected mice; POH treatment downregulated the expression of these three adhesion molecules. (B) Representative IHC photomicrographs displaying VCAM-1, ICAM-1, and CD36 immunostaining for all animals/groups. The IHC score (highly positive, positive, low-positive, and negative) for each animal/group (n = 3 to 5/group) is represented as a heatmap column on the right side of each representative brightfield photomicrograph. (C) The percentage of IHC score calculated from each staining is represented in graphics for all groups. Taking into consideration the IHC scores analyzed, POH-treated mice showed a lower expression for ICAM-1 and VCAM-1 than did the vehicle-treated mice. Scale bar: 50 μm.

FIG 6

Characterization of lymphoid and myeloid cell populations in the spleen of PbA-infected C57BL/6 mice treated with POH or vehicle after 6 days of infection. The spleen was collected and macerated for total splenocyte isolation. POH showed high efficacy in preventing an increase in T and B lymphocytes and macrophages compared with vehicle. The number of CD4⁺, CD8⁺, CD19⁺, and F4/80⁺ cells reduced significantly in POH-treated mice. The experiment was repeated twice, and the data represent the mean ± SD (n = 5 per group). *, P < 0.05; ***, P < 0,001.

FIG 7

Schematic representation of the mechanism of action of POH as a preventive therapy for experimental cerebral malaria. A 500 mg kg⁻¹ day⁻¹ dose of POH administered intranasally reduces the cerebrovascular inflammation, preventing leukocyte adhesion and pRBCs accumulation in the larger venules, resulting in decreased vascular congestion. Decreased congestion reduces vascular resistance, and improved blood flow may help to wash out trapped pRBCs in microvessels (black arrow). Also, BBB breakdown was prevented by treatment. In addition, molecules related to endothelial activation, such as ICAM-1, VCAM-1, and CD36, were downregulated by POH. POH treatment led to a significant reduction in T and B lymphocytes and macrophages in the spleen. Therefore, POH effect causes an improvement in neurological and clinical features, preventing ECM development.