Cerebral malaria is associated with differential cytoadherence to brain endothelial cells

Abstract

Sequestration of Plasmodium falciparum-infected erythrocytes (IE) within the brain microvasculature is a hallmark of cerebral malaria (CM). Using a microchannel flow adhesion assay with TNF-activated primary human microvascular endothelial cells, we demonstrate that IE isolated from Malawian paediatric CM cases showed increased binding to brain microvascular endothelial cells compared to IE from uncomplicated malaria (UM) cases. Further, UM isolates showed significantly greater adhesion to dermal than to brain microvascular endothelial cells. The major mediator of parasite adhesion is P. falciparum erythrocyte membrane protein 1, encoded by var genes. Higher levels of var gene transcripts predicted to bind host endothelial protein C receptor (EPCR) and ICAM-1 were detected in CM isolates. These data provide further evidence for differential tissue binding in severe and uncomplicated malaria syndromes, and give additional support to the hypothesis that CM pathology is based on increased cytoadherence of IE in the brain microvasculature.

Keywords: Plasmodium falciparum; PfEMP1; cerebral malaria; cytoadherence; paediatric patient isolates.

Figure 1. PfEMP1 domain structure

A schematic presentation of PfEMP1 domain structure comprising a N‐terminal head structure, 2‐6 subsequent C‐terminal domains, a transmembrane domain (TM) and an intracellular acidic terminal segment (ATS) with known receptors indicated in bold. Receptor specificity is determined by combined DBL and CIDR domains with mutually exclusive binding to EPCR and CD36 by different CIDRα domains in the head structure. Part of group A PfEMP1 and a particular subset of group B/A chimeric PfEMP1 (DC8) bind to EPCR via CIDRα1 domains, whereas group B and C PfEMP1 bind CD36 via CIDRα2‐6 domains. The atypical group E VAR2CSA PfEMP1 binds placental chondroitin sulphate A (CSA) via DBLpam1 and DBLpam2 domains. The binding phenotype of VAR 1, VAR3 and group A CIDRβ/γ/δ domains is unknown, but they do not bind EPCR or CD36. DBLβ domains can be involved in ICAM‐1 binding and are from both groups A and B. Not much is known about the other DBL domains (γ/δ/ε/ζ), but the DBLε and DBLζ domains are implicated in IgM and α₂‐macroglobulin binding.

Figure 2. Cytoadherence of IE from CM and UM cases to HBMEC and HDMEC

IE were isolated, and binding to HBMEC and HDMEC was determined under flow conditions. Number of IE bound per mm² EC surface was calculated and shown is the mean ± 95% CI for 26 CM and 33 UM cases on HBMEC and 21 CM and 35 UM cases on HDMEC on a log scale. P‐value was calculated by two‐tailed unpaired t‐test. The dotted line is 20 IE/mm², the cut‐off value for inclusion of the inhibition data.

Figure 3. Inhibition of cytoadherence of IE from CM and UM cases to HDMEC and HBMEC by αICAM‐1 and αCD36 antibody and rEPCR

1. A

   IE were isolated, and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 μg/ml αICAM‐1 or αCD36 antibody or 50 μg/ml rEPCR. Number of IE bound per mm² EC surface was determined.

2. B

   Using the same data, percentage inhibition by αICAM‐1 antibody was calculated relatively to binding in the absence of antibody.

3. C

   Using the same data, percentage inhibition by rEPCR was calculated relatively to binding in the absence of inhibitor.

4. D

   Using the same data, percentage inhibition by αCD36 antibody was calculated relatively to binding in the absence of antibody.

Data information: (A) shown is the paired analysis between the absence and presence of inhibitor, with number of cases (n) indicated. Statistical significance was determined by two‐tailed paired t‐test, and the P‐value is shown; ns is not significant. (B–D) shown are the mean ± 95% CI, and no significant differences were determined with a two‐tailed unpaired t‐test. Each assay was only conducted once for each isolate. The number of isolates tested can be seen from the dot plot.