Pathogenesis of pediatric cerebral malaria: A series of unfortunate events
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Despite reductions in incidence and mortality, over 400,000 lives are lost to Plasmodium falciparum malaria annually. Roughly 70% of deaths occur in sub-Saharan African children, a population particularly at risk for severe malaria, including cerebral malaria (CM). The clinical case definition of CM is imperfect, as autopsy studies indicate 25% of children presenting with WHO-defined CM have an alternative, non-malaria cause of death. Imprecise diagnosis has impeded the understanding of CM pathophysiology; however, the application of advanced technologies, including ophthalmological funduscopy and MRI, have improved the clinical characterization of children presenting with CM and identified those most at risk of fatality. Funduscopic examination to identify malarial retinopathy has provided unprecedented utility in accurately identifying children with histological CM. Brain swelling is a key process in pediatric CM, and parasitized red blood cell (pRBC) sequestration in the brain microvasculature is a hallmark of CM and thought to be essential for swelling. Sequestration is mediated by erythrocyte membrane protein 1 (PfEMP 1). PfEMP 1 proteins encode binding domains conferring unique host receptor adhesion properties (i.e. CD36, endothelial protein C receptor (EPCR)). Using transcript profiling, machine learning approaches, functional assays, and sequencing, we investigated the link between malarial retinopathy (Ret+CM), brain swelling, and PfEMP1 variants. Our data indicate that EPCR-binding parasites dominate in children with Ret+CM and/or severe swelling, and specific EPCR-binding isolates disrupt EPCR's protective effects (i.e. barrier permeability), demonstrating utility of therapeutics that restore EPCR protective pathways in children with CM. Prior to sequestration, parasites evade opsonization and clearance by the host immune system. Transfer experiments, in concert with the acquisition of non-sterilizing immunity, argue a key role for antibodies in pediatric disease. We used a protein microarray to compare the antibody profiles of children with uncomplicated malaria (UM) to those with Ret+CM to understand why certain children develop severe disease and others do not. Our findings indicate that the breadth and magnitude of P. falciparum and PfEMP 1 antibody – and degree of prior malaria exposure – is similar between groups despite differences in case severity. Responses to antigens previously deemed as `protective' were no different between groups, suggesting that certain P. falciparum antibodies are important for protection from symptomatic but not severe disease. We identified low platelet count as a key feature distinguishing Ret+CM and brain swelling from UM and the only feature distinguishing CM cases with vs. without swelling. Thrombocytopenia is associated with disease severity and fatality, and autopsies of children with histologically confirmed CM revealed abundant platelet sequestration in the brain microvasculature. Platelets may bind to activated cerebral endothelium but may also play a direct role in disease processes. Platelet-mediated pathology in pediatric CM – thrombocytopenia, thrombosis, and microvascular endothelial activation – mirrors that observed in heparin-induced thrombocytopenia (HIT), an adverse reaction observed upon administration of heparin. HIT is caused by anti-platelet factor-polyanion antibodies that bind and activate platelets via Fcy receptors. Activated platelets release factors/microparticles that establish and perpetuate a feedback loop of thrombin production, endothelial activation, and coagulation dysfunction. We assayed the plasma of children with UM and CM for HIT platelet factor and antibodies using standard immunology and molecular biology methods. Our work identified HIT antibodies in pediatric malaria patients for the first time. Confirmatory studies are underway to test whether there is a role for antibody-mediated thrombocytopenia in CM. This work highlights parasite and host factors important for CM pathogenesis, which is characterized by abundant parasite sequestration, disruption of biological pathways important for cytoprotection and microvascular integrity, and the establishment of a platelet-endothelial activation feedback loop that amplifies disease pathology. Our work improves the current understanding of CM disease pathophysiology and highlights avenues for future therapeutic developments for pediatric CM.