Plasmodium falciparum binding interactions with human brain endothelial cells

Abstract

Cerebral malaria is the most severe form of malaria and mostly affects children under 5 years causing impaired consciousness, coma and neurological disorders, with life-threatening consequences in affected individuals. A pathological feature of the disease is the sequestration of mature Plasmodium falciparum infected erythrocytes (IEs) in the microvasculature of the brain. P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) expressed on the surface of IEs is thought to enable the parasites bind to human brain endothelial cells (HBEC) to avoid splenic clearance. An in vitro model of cytoadherence in cerebral malaria has been developed using a human brain endothelial cell line called HBEC-5i, which enables the study of IEs binding to HBEC. Previous work based on three laboratory parasite lines showed that HBEC-binding IEs express a specific subset of the diverse PfEMP1 family which contain sets of cysteine-rich domains called domain cassettes (DC) 8 and 13. Parasites from children diagnosed with cerebral malaria have also been independently shown to express the DC8 and DC13 PfEMP1 types. The adhesion of IEs to HBEC is suggested to occur by binding of a domain of the DC8 and DC13 PfEMP1 variants called CIDRα1, to Endothelial Protein C Receptor (EPCR) on HBEC. However, investigations of the effect of parasite and host environmental factors on adhesion to HBEC are lacking, and further studies are needed to confirm the association of these DC8 and DC13 PfEMP1 to HBEC-binding and the role of EPCR in mediating the cytoadhesion. Therefore, the aim of this thesis was to examine the hypothesis that HBEC-binding would be affected by changes in environmental conditions, and that all IEs that bind to HBEC would express group A-like PfEMP1 containing DC8 and DC13, for binding to EPCR on HBEC. In this study, the effect of pH, parasitaemia, gas, temperature, and serum on cytoadhesion, were investigated using four HBEC-binding parasite lines. Adhesion of IEs to HBEC was found to be pH and parasitaemia -dependent with optimal binding at pH 7.3 and IE adhesion positively correlated with parasitaemia. There was no significant effect of increase in temperature to 39°C and no significant difference between hypoxic and normoxic conditions on adhesion in all parasite lines. Human serum, however, abolished binding of the DC8-expressing parasite line but had minimal effects on adhesion of the DC13-expressing parasite lines. Two Kenyan isolates recently adapted to culture were selected for binding to HBEC and were found to also predominantly express group A-like PfEMP1 including a DC8 PfEMP1 variant and PfEMP1 (s) that contained DBLα1.2 domains. Attempts were made to localise the binding domain within the DC8 and DC13 PfEMP1 variants using recombinant proteins and antibodies. However, the CIDRα1 domain appeared to mediate adhesion of the DC8-expressing parasite line but had no effect on adhesion of the DC13-expressing parasite lines. Only antibodies to the N-terminal domain, known as NTS.DBLα, significantly inhibited binding of all the parasites lines. The role of EPCR and other receptor molecules on endothelial cells including ICAM-1, CD36, CSA, PECAM-1, HABP-1 and heparin, in mediating adhesion to HBEC was also investigated. Using EPCR recombinant protein, monoclonal and polyclonal antibodies, and EPCR-siRNA knockdown in binding assays, EPCR was shown to be involved in adhesion of only the DC8-expressing parasite line and did not affect adhesion of the DC13-expressing parasite lines to HBEC. Binding of DC8-expressing parasite line to HBEC was also inhibited by soluble recombinant PECAM-1. There was no significant adhesion of both types of parasite lines to the other receptor molecules, although minimal binding to HABP-1 was observed. This study expands current knowledge on the parasite binding interactions with HBEC by elucidating some of the environmental factors that affect the binding properties, and gives the optimal conditions for the in vitro model of HBEC-adhesion in cerebral malaria. Findings presented here confirm the association of expression of group A-like PfEMP1 to HBEC-binding and shows that the EPCR-CIDRα1 interaction does not mediate adhesion of all DC8 and DC13- expressing parasite lines to HBEC. Additional receptors, other than EPCR, are therefore required for HBEC-binding in cerebral malaria. The ability of normal human serum to abolish binding of the DC8-expressing parasite line also raises the question of whether IE binding to EPCR is physiologically relevant and suggest that the DC8- expressing parasites associated with cerebral malaria may contribute to the disease in a mechanism other than binding to brain endothelial cells