Cerebral malaria - modelling interactions at the blood-brain barrier in vitro

Abstract

The blood-brain barrier (BBB) is a continuous endothelial barrier that is supported by pericytes and astrocytes and regulates the passage of solutes between the bloodstream and the brain. This structure is called the neurovascular unit and serves to protect the brain from blood-borne disease-causing agents and other risk factors. In the past decade, great strides have been made to investigate the neurovascular unit for delivery of chemotherapeutics and for understanding how pathogens can circumvent the barrier, leading to severe and, at times, fatal complications. One such complication is cerebral malaria, in which Plasmodium falciparum-infected red blood cells disrupt the barrier function of the BBB, causing severe brain swelling. Multiple in vitro models of the BBB are available to investigate the mechanisms underlying the pathogenesis of cerebral malaria and other diseases. These range from single-cell monolayer cultures to multicellular BBB organoids and highly complex cerebral organoids. Here, we review the technologies available in malaria research to investigate the interaction between P. falciparum-infected red blood cells and the BBB, and discuss the advantages and disadvantages of each model.

Keywords: In vitro models; Plasmodium falciparum; Blood–brain barrier; Malaria.

Fig. 1.

Types and functions of the endothelium. (A) Schematic of different types of endothelia found in the human body. Discontinuous endothelia are found in the liver sinusoids and bone marrow, and feature larger openings in the cells, typically 60-180 nm in diameter, which extend through the basement membrane. Fenestrated endothelia are found in the glomeruli of the kidneys and the choroid plexus in the brain (red). The fenestrations (pores) within the endothelial cells are typically 60-80 nm in diameter. Continuous endothelia form the blood–brain barrier (BBB) and are characterised by tight junctions between cells and highly selective transport of molecules. (B) The integrity of the continuous endothelium of the BBB can be compromised when P. falciparum-iRBCs bind to endothelial cell surface receptors. (1) In healthy endothelia, APC can bind to PAR1 (also known as F2R), inhibiting NF-κB and ensuring a cytoprotective state. ZO-1, claudin-5 and occludin combine to form tight junctions to help maintain barrier integrity. (2) iRBCs capable of binding to both ICAM-1 and EPCR outcompete APC, triggering a pro-inflammatory state within endothelial cells, leading to a loss of EPCR surface expression (Moxon et al., 2013). (3) iRBCs can bind to ICAM-1, which is upregulated in malaria (Tripathi et al., 2006) and (4) which clusters around bound iRBCs (Adams et al., 2021). (5) This binding to ICAM-1 and clustering inhibits the production of sphingosine-1-phosphate (S1P) whilst triggering Rho A, which induces NF-κβ. Combined, this results in a loss of the tight junctions and barrier integrity. This is exacerbated further when (6) iRBCs enter the endothelial cells, further destabilising the barrier and resulting in (7) cytokine/chemokine release and the production of microparticles. This pro-inflammatory state of the endothelial cells, coupled with increased permeability, triggers an inflammatory cascade that disrupts the neurovascular unit by loosening tight junctions, (8) causing pericyte dysfunction and retraction of astrocyte end feet. This non-functional BBB cannot adequately protect the brain. APC, activated protein C; EPCR, endothelial protein receptor C; ICAM-1, intercellular adhesion molecule 1; iRBC, infected red blood cell; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PAR1, protease-activated receptor 1; Rho A, ras homolog family protein A; S1P, sphingosine-1-phosphate; ZO-1, zonula occludens protein 1.

Fig. 2.

Summary of in vitro models of the brain and BBB. This diagram summarises the increasing complexity of the models and indicates the possible experimental approaches to interrogate the mechanisms via which P. falciparum-infected red blood cells disrupt the BBB and to identify and test potential adjuvant therapeutics. (A) Boyden chambers with mono-/co-culture systems allow for the measurement of barrier integrity in response to iRBC exposure. By activating the endothelial layer, transmigration of iRBCs/leukocytes is measurable. (B) The vasculature is under constant fluid flow and this can be mimicked using 2D or 3D microfluidic chips. Pumps generate shear stress recapitulating that found within the microvasculature. Adhesion under flow conditions can be quantified, as can the effect of antibodies against the parasite-derived PfEMP1. To assess barrier permeability, TEER and solute transport using fluorescent tracers can also be measured. Alterations of endothelial proteins such as actin (red) or the tight-junction protein ZO-1 (green) can be visualised via immunofluorescence. (C) The complexity of the BBB model can be increased by generating BBB organoids. These consist of astrocytes, pericytes and an outer layer of endothelial cells. This allows for the generation of a functional BBB and adhesion of iRBCs. Owing to the induction of swelling by specific iRBCs, which are capable of entering and migrating into the organoids, they also serve as a model for the investigation of neuroprotective agents. (D) The most complex model is the cerebral organoid. Although lacking endothelial cells/vascularisation, cerebral organoids offer a unique opportunity to investigate neurodevelopment in response to parasite exposure and the toxicity of parasite-derived products. BBB organoid and primary human brain endothelial monolayer images courtesy of Y.A., University of Copenhagen. Cerebral organoid image ©IMBA reused with permission (Lancaster et al., 2013). This image is not published under the terms of the CC-BY license of this article. For permission to reuse, please see Lancaster et al. (2013). BBB, blood–brain barrier; iRBC, infected red blood cell; TEER, transepithelial electrical resistance; ZO-1, zonula occludens protein 1.