In vitro model of brain endothelial cell barrier reveals alterations induced by Plasmodium blood stage factors

Abstract

Cerebral malaria (CM) is a severe neurological condition caused by Plasmodium falciparum. Disruption of the brain-blood barrier (BBB) is a key pathological event leading to brain edema and vascular leakage in both humans and in the mouse model of CM. Interactions of brain endothelial cells with infected red blood cells (iRBCs) and with circulating inflammatory mediators and immune cells contribute to BBB dysfunction in CM. Adjunctive therapies for CM aim at preserving the BBB to prevent neurologic deficits. Experimental animal and cellular models are essential to develop new therapeutic strategies. However, in mice, the disease develops rapidly, which offers a very narrow time window for testing the therapeutic potential of drugs acting in the BBB. Here, we establish a brain endothelial cell barrier whose disturbance can be monitored by several parameters. Using this system, we found that incubation with iRBCs and with extracellular particles (EPs) released by iRBCs changes endothelial cell morphology, decreases the tight junction protein zonula occludens-1 (ZO-1), increases the gene expression of the intercellular adhesion molecule 1 (ICAM-1), and induces a significant reduction in transendothelial electrical resistance (TEER) with increased permeability. We propose this in vitro experimental setup as a straightforward tool to investigate molecular interactions and pathways causing endothelial barrier dysfunction and to test compounds that may target BBB and be effective against CM. A pre-selection of the effective compounds that strengthen the resistance of the brain endothelial cell barrier to Plasmodium-induced blood factors in vitro may increase the likelihood of their efficacy in preclinical disease mouse models of CM and in subsequent clinical trials with patients.

Keywords: BBB; Brain endothelium; Cerebral malaria; Plasmodium.

Fig. 1

Establishment of a brain endothelial cell barrier in vitro. a Schematic diagram of the experimental setup consisting of brain endothelial cells (BECs) isolated from mouse brains and cultured on cell culture inserts with a polyethylene terephthalate (PET) membrane of 5 μm porous to form an endothelial cell barrier. The formation of this endothelial barrier was monitored by transendothelial electrical resistance (TEER) measurements with a voltmeter. b Barrier formation in six independent wells (#1–6) showed similar kinetics and was established by day 20 after cell seeding. c Immunofluorescence analysis of endothelial cells showing F-actin or ZO-1 staining (green). Nuclei were stained with DRAQ5 (blue)

Fig. 2 iRBCs compromise the brain endothelial cell function. a Transendothelial electrical resistance (TEER) was measured before (t=0) and after adding non-infected red blood cells (RBCs), infected red blood cells (iRBCs), or culture medium (CTR) to cultures with established endothelial cell barrier. The permeability to Lucifer yellow (LY) was assessed after 24 h of incubation. b TEER was measured 2, 4, 6, and 24 h after incubation with non-infected RBC, iRBCs, or culture medium (mean±s.d., n=3; two-way ANOVA, **P<0.01; **** P<0.0001). c Diffusion across the barrier was assessed by detecting LY in the lower chamber 2 h after adding it to the upper chamber of cultures exposed to RBCs, iRBCs, or CTRL for 24 h (mean±s.d., n=3; one-way ANOVA, *P<0.05). Data are representative of three independent experiments

Fig. 3

iRBCs alter cell morphology and expression of tight junction components and adhesion molecules genes in brain endothelial cells. a As described in Fig. 1c (F-actin and ZO-1), immunofluorescence staining revealed alterations in cell morphology in the distribution of the tight junction protein ZO-1 in cultures incubated with infected red blood cells (iRBCs) for 24 h. b Relative quantification of Cldn5 and Icam1 mRNA in cultures of endothelial cells barrier exposed to non-infected (RBCs), iRBCs, or culture medium (CTRL) for 24 h (mean±s.d., n=3; one-way ANOVA, **P<0.01)

Fig. 4

Blood stage-derived EPs decrease TEER values. a Extracellular particle (EP) preparations from the supernatant of infected red blood cell (iRBC) cultures were detected by flow cytometry using side scatter height from the blue-violet laser, SSC (BV)-H, and forward scatter height, FSC-H. b The EP preparation contained GFP⁺ particles corresponding to P. berghei-GFP merozoites that do not stain for TER119 as well as TER119^+/Annexin V⁺ particles that contain Pba-GFP-derived products. c The effect of EPs in the endothelial barrier was evaluated by transendothelial electrical resistance (TEER) measurements and d permeability assay to Lucifer yellow (LY) after 24 h of incubation with red blood cells (RBCs) (5×10⁶), iRBCs (5×10⁶, 111 μg/ml), EPs (183 μg/ml), or TNF (10 ng/ml) (mean±s.d., n=3; one-way ANOVA, *P<0.05 and ****P<0.001). Data are representative of three independent experiments