Patients and sampling
Patients of all ages were enrolled in three Gabonese regional hospitals: Franceville, Koulamoutou and Lastourville between October 2014 and February 2015 (Fig. 1). Blood samples were collected in EDTA tubes and malaria diagnosis was based on microscopic examination of thick and thin smears, and the clinical status of each patient was determined according to the WHO classification [22].
Plasmodium falciparum maturation
Parasitized red blood cells were selected for their viability, because pRBC from patients having self-medicated (and who rarely inform their physician) do not grow well in culture. Samples with parasite loads of at least 5000 parasites per microliter were washed 4 to 5 times in RPMI 1640 medium to remove white cells. pRBC were immediately grown at 37 °C for 24 to 48 h (until parasites matured into schizonts) in RPMI 1640 with L-glutamine (Sigma) supplemented with 8.3 g/l HEPES, 2.1 g/l sodium bicarbonate, 0.05 g/l hypoxanthine, 0.1 mg/ml gentamicin, 1 mg/ml fungizone, 2 g/l D-glucose and 0.4% Albumax II (InVitrogen, Cergy Pontoise, France), with 5% hematocrit as described [23].
Mature pRBC were enriched by gelatin flotation as previously reported [24] and counted under a microscope. Samples with more than 6% parasitaemia were diluted with non-parasitized red blood cells until to obtain 6% parasitaemia and hematocrit was adjusted at 2%. Also, all the pRBC preparations were free of white blood cell and reticulocytes. All experiments were performed during the first cycle of parasite development and isolates were confirmed to be Mycoplasma-free by PCR (data not shown).
Human endothelial cell culture
HLEC culture medium
HLEC were obtained from Professor Frederick Gay (INSERM UMRS 945). These cells predominantly express the CD36 receptor, in addition to other receptors such as Willebrand factor, ICAM1, VCAM1, CD31, E/P-selectin and chondroitin sulfate A [6].
HLEC were cultured in a pulmonary culture medium (PCm) composed of M199 medium (Lonza) supplemented with 10% inactivated FBS (Life Technologies), 5 μg/ml endothelial cell growth supplement (Sigma-Aldrich), 50 U/ml streptomycin-penicillin (Life Technologies), and 0.25 μg/ml fungizone (Lonza). Cells were grown to confluence at 37 °C with 5% CO2 and were used at the 10th passage. The cell lines were tested for Mycoplasma with the MycoAlert Detection Kit (Lonza) (data not shown).
hCMEC/D3 culture medium
The hCMEC/D3 were provided by Professor Pierre Olivier Couraud (INSERM, U1016, Paris, France). This immortalized line of human brain endothelial cells was produced from freshly isolated adult cells. It maintains in stable manner (at least 80 doublings) a differentiated phenotype similar to that of cerebral endothelial cells and forms homogeneous and stable confluent monolayers. hCMEC/D3 express many endothelial markers, including CD31, ICAM1, vWF and VE-cadherin, and blood-brain-barrier (BBB) markers such as proteins associated with tight junctions (ZO-1, JAM-A, claudin-5) [18].
The hCMEC/D3 were cultured in a cerebral culture medium (CCm) composed of EBM-2 basal medium (Lonza) supplemented with 5% inactivated FBS (Life technologies), 1% penicillin-streptomycin (Life Technologies), 0.25 μg/ml fungizone (Lonza), 1.4 μM hydrocortisone (Sigma-Aldrich), 5 μg/ml ascorbic acid (Sigma-Aldrich), 1/100 chemically defined lipid concentrate (Life Technologies), 10 mM HEPES and 1 ng/ml basic fibroblast growth factor (Merck Millipore). The cells were used at the 25th passage. The hCMEC/D3 were grown to confluence at 37 °C with 5% CO2 and cell lines were tested for Mycoplasma with the MycoAlert Detection Kit (Lonza) (data not shown).
Cytoadherence assay
Parasitized red blood cells cytoadherence to HLEC and hCMEC/D3 were determined using microscopy as described elsewhere [25]. Briefly, each cell line was grown to confluence (approximately 8.4 × 104 HLEC and 7 × 104 hCMEC/D3) in its respective culture medium in 8-well Lab-Tek™ chamber slides. Confluent HLEC and hCMEC/D3 were then used directly in adhesion assays, or fixed for one hour at 37 °C with 2% paraformaldehyde and kept at 4 °C in 400 μl of phosphate buffered saline (PBS) for a maximum of 2 months [26]. A suspension of 300 μl of mature pRBC at 6% parasitaemia and 2% haematocrit (approximately 4 × 106 schizonts for 63 × 106 uninfected RBC) in complete RPMI culture medium without bicarbonate was added to each well containing confluent HLEC and hCMEC/D3 monolayers from Labtek in duplicate, and incubated for 1 h at 37 °C. Also, cytoadherence was performed using 3D7 strain at the same conditions as positive control.
After 1 h of incubation, unfixed pRBC were removed gently by washing 5 times with PBS and the Labtek was fixed with 2% glutaraldehyde for 20 min at room temperature, rinsed with PBS and stained with 30% Giemsa. The number of adherent pRBCs was determined microscopically, and expressed as the number of pRBC adhering to 700 cells. Cytoadherence was considered positive if the number of adhering pRBC was greater than to 35 per 700 cells (more than 5% of adhering pRBC).
Apoptosis assay
HLEC and hCMEC/D3 were grown respectively in PCm and CCm until 80–90% confluence in both 24-well (about 3 × 105 cells for HLEC and 2.55 × 105 cells for hCMEC/D3) and 6-well Transwell plates (about 106 cells for HLEC and 9 × 105 cells for hCMEC/D3) with polyester 0.4 μm pore-size filters (Costar, Corning Incorporated). The 24-well plates were used for apoptosis assay in contact condition whereas the 6 well plates (Transwell) were used for apoptosis assay in non-contact experiment.
Contact and non-contact co-culture
Co-cultures of P. falciparum field isolates with hCMEC/D3 or HLEC were carried out in parallel using two modified media that lacked bicarbonate [11, 21]. Briefly, for hCMEC/D3, the modified medium contained RPMI 1640 + L-glutamine +25 mM HEPES (Gibco) supplemented with 5% inactivated FBS, 1.4 μM hydrocortisone, 5 μg/ml ascorbic acid, 1/100 chemically defined lipid concentrate and 1 ng/ml of basic fibroblast growth factor and was called MCCm. For HLEC, it contained RPMI 1640 + L-glutamine +25 mM HEPES (Gibco) supplemented with 10% of inactivated FBS and 5 μg/ml of endothelial cell growth supplement and was called MPCm.
For contact co-culture experiment, a suspension of 133 μl of mature pRBC (isolates and positive control 3D7 strain) at 6% parasitaemia and 1% haematocrit (approximately 5.31 × 105 schizonts for 9 × 106 uninfected red blood cells (RBC)) in both complete MPCm and complete MCCm has been co-culture respectively with HLEC and hCMEC/D3 separately, in quadruplicate using the 24-well plates.
In non-contact coculture experiment, a suspension of 500 μl of mature pRBC (isolates and positive control 3D7 strain) at 6% parasitaemia and 1% haematocrit (approximately 2 × 106 schizonts for 35 × 106 uninfected RBC) in both complete MPCm and complete MCCm has been coculture respectively with HLEC and hCMEC/D3 separately, in quadruplicate using the 6 well plates Transwell. Each apoptosis assay condition was performed using a suspension of uninfected RBC at 1% haematocrit for negative controls (approximately 9.5 × 106 RBC). Each coculture was then incubated for 24 h at 37 °C with 5% CO2.
Apoptosis detection
Apoptosis was measured with an enzyme immunoassay (Cell Death Detection ELISA plus, Roche Diagnostics) as recommended by the manufacturer. This ELISA detects cytoplasmic nucleosomes released from apoptotic cells and has an estimated detection limit of 625 apoptotic cells. Both HLEC and hCMEC cell line was incubated with 3D7 stain as positive control and uninfected RBC as negative controls. After 24 h of incubation, the 24-well plates were washed 5 times with RPMI 1640, and twice with PBS to remove unbound pRBC, 3D7 stain and RBC. In the mean time, the upper compartment of the 6-well Transwell plates containing pRBC, 3D7 stain and RBC was removed, the supernatant was discarded and the cells were also washed twice with PBS. Two hundred (200) μl and 500 μl of lysis buffer provided with the ELISA kit were added to the 24 and 6-well plates respectively to permeabilize HLEC and hCMEC/D3. The plates were then centrifuged at 1000 rpm for 10 min, and 20 μl of supernatant per well was used in the ELISA test. Optical density (OD) was read with a Stat FaxH 3200 (Fisher Bioblock Scientific, Illkirch France) at 405 nm, with a reference filter of 492 nm. The mean OD of pRBC-activated HLEC and hCMEC/D3 to RBC-activated HLEC and hCMEC/D3 and mean OD of 3D7-activated HLEC and hCMEC/D3 to RBC-activated HLEC and hCMEC/D3 was calculated, using a positivity cut-off of 3, as recommended [6, 14].
Statistical analysis
Categorical data were analyzed with Fisher’s test. McNemar’s test was used to compare paired proportions. The non parametric Mann-Whitney U test was used for non-normally distributed quantitative data. Significance was set at p < 0.05.