Samuel C. Wassmer

Platelets Reorient Plasmodium falciparum–Infected Erythrocyte Cytoadhesion to Activated Endothelial Cells

Severe malaria is characterized by the sequestration of Plasmodium falciparum-infected erythrocytes (IEs). Because platelets can affect tumor necrosis factor (TNF)-activated endothelial cells (ECs), we investigated their role in the sequestration of IEs, using IEs that were selected because they can adhere to endothelial CD36 (IE(CD36)), a P. falciparum receptor that is expressed on platelets. The results of coincubation studies indicated that platelets can induce IE(CD36) binding to CD36-deficient brain microvascular ECs. This induced cytoadhesion resisted physiological shear stress, was increased by EC stimulation with TNF, and was abolished by anti-CD36 monoclonal antibody. Immunofluorescence and scanning electron microscopy results showed that platelets serve as a bridge between IEs and the surface of ECs and may therefore provide receptors for adhesion to microvascular beds that otherwise lack adhesion receptors. This novel mechanism of cytoadhesion may reorient the sequestration of different parasite phenotypes and play an important role in the pathogenesis of severe malaria.

Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children.

Cerebral malaria (CM) is a major cause of mortality in African children and the mechanisms underlying its development, namely how malaria-infected erythrocytes (IEs) cause disease and why the brain is preferentially affected, remain unclear. Brain microhemorrhages in CM suggest a clotting disorder, but whether this phenomenon is important in pathogenesis is debated. We hypothesized that localized cerebral microvascular thrombosis in CM is caused by a decreased expression of the anticoagulant and protective receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR) and that low constitutive expression of these regulatory molecules in the brain make it particularly vulnerable. Autopsies from Malawian children with CM showed cerebral fibrin clots and loss of EPCR, colocalized with sequestered IEs. Using a novel assay to examine endothelial phenotype ex vivo using subcutaneous microvessels, we demonstrated that loss of EPCR and TM at sites of IE cytoadherence is detectible in nonfatal CM. In contrast, although clotting factor activation was seen in the blood of CM patients, this was compensated and did not disseminate. Because of the pleiotropic nature of EPCR and TM, these data implicate disruption of the endothelial protective properties at vulnerable sites and particularly in the brain, linking coagulation and inflammation with IE sequestration.

Platelets Potentiate Brain Endothelial Alterations Induced by Plasmodium falciparum

ABSTRACT Brain lesions of cerebral malaria (CM) are characterized by a sequestration of Plasmodium falciparum-parasitized red blood cells (PRBC) and platelets within brain microvessels, as well as by blood-brain barrier (BBB) disruption. In the present study, we evaluated the possibility that PRBC and platelets induce functional alterations in brain endothelium. In a human brain endothelial cell line, named HBEC-5i, exhibiting most of the features demanded for a pathophysiological study of BBB, tumor necrosis factor (TNF) or lymphotoxin α (LT-α) reduced transendothelial electrical resistance (TEER), enhanced the permeability to 70-kDa dextran, and increased the release of microparticles, a recently described indicator of disease severity in CM patients. In vitro cocultures showed that platelets or PRBC can have a direct cytotoxic effect on activated, but not on resting, HBEC-5i cells. Platelet binding was required, as platelet supernatant had no effect. Furthermore, platelets potentiated the cytotoxicity of PRBC for TNF- or LT-α-activated HBEC-5i cells when they were added prior to these cells on the endothelial monolayers. This effect was not observed when platelets were added after PRBC. Both permeability and TEER were strongly affected, and the apoptosis rate of HBEC-5i cells was dramatically increased. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM.

Pathophysiology of Cerebral Malaria

Abstract: Cerebral malaria (CM), one of the most serious complications of Plasmodium falciparum infection, is characterized by the sequestration of infected erythrocytes (IEs) in cerebral microvascular beds. The precise mechanisms involved in the onset of neuropathology remain unknown, but parasite sequestration in the brain, metabolic disturbances, and host immune responses all play a role. Studies in a murine model of CM showed a potential role for host cells, especially platelets, in the pathogenesis of CM. Indeed, urokinase plasminogen activator receptor (uPAR; CD87) deficiency attenuates the severity of CM, most likely by its important role in platelet kinetics and trapping. These results led us to evaluate whether platelets have a role in the human disease. By immunostaining of brain samples from Malawian patients, we determined that the surface of platelet accumulation and the proportion of vessels filled with platelets were significantly higher in patients who died of CM than in those who died of other causes. We then investigated the role of platelets in IE cytoadhesion in vitro, using CD36‐binding IE (IECD36) and CD36‐deficient (CD36DEF) brain microvascular endothelial cells (ECs). Coincubation studies indicated that platelets can induce strong IECD36 binding to CD36DEF ECs and, conversely, can hide constitutively expressed falciparum receptors such as chondroitin sulfate A. Thus, platelets may provide an adhesion receptor to microvascular beds originally devoid of it. This novel mechanism of cytoadhesion may reorient the sequestration of different parasite phenotypes and play an important role in the pathogenesis of severe malaria.

Inhibition of endothelial activation: a new way to treat cerebral malaria?

Background Malaria is still a major public health problem, partly because the pathogenesis of its major complication, cerebral malaria (CM), remains incompletely understood. However tumor necrosis factor (TNF) is thought to play a key role in the development of this neurological syndrome, as well as lymphotoxin α (LT). Methods and Findings Using an in vitro model of CM based on human brain–derived endothelial cells (HBEC-5i), we demonstrate the anti-inflammatory effect of LMP-420, a 2-NH2-6-Cl-9-[(5-dihydroxyboryl)-pentyl] purine that is a transcriptional inhibitor of TNF. When added before or concomitantly to TNF, LMP-420 inhibits endothelial cell (EC) activation, i.e., the up-regulation of both ICAM-1 and VCAM-1 on HBEC-5i surfaces. Subsequently, LMP-420 abolishes the cytoadherence of ICAM-1-specific Plasmodium falciparum–parasitized red blood cells on these EC. Identical but weaker effects are observed when LMP-420 is added with LT. LMP-420 also causes a dramatic reduction of HBEC-5i vesiculation induced by TNF or LT stimulation, as assessed by microparticle release. Conclusion These data provide evidence for a strong in vitro anti-inflammatory effect of LMP-420 and suggest that targeting host cell pathogenic mechanisms might provide a new therapeutic approach to improving the outcome of CM patients.

Platelet-induced clumping of Plasmodium falciparum-infected erythrocytes from Malawian patients with cerebral malaria - possible modulation in vivo by thrombocytopenia

Platelets may play a role in the pathogenesis of human cerebral malaria (CM), and they have been shown to induce clumping of Plasmodium falciparum-parasitized red blood cells (PRBCs) in vitro. Both thrombocytopenia and platelet-induced PRBC clumping are associated with severe malaria and, especially, with CM. In the present study, we investigated the occurrence of the clumping phenomenon in patients with CM by isolating and coincubating their plasma and PRBCs ex vivo. Malawian children with CM all had low platelet counts, with the degree of thrombocytopenia directly proportional to the density of parasitemia. Plasma samples obtained from these patients subsequently induced weak PRBC clumping. When the assays were repeated, with the plasma platelet concentrations adjusted to within the physiological range considered to be normal, massive clumping occurred. The results of this study suggest that thrombocytopenia may, through reduction of platelet-mediated clumping of PRBCs, provide a protective mechanism for the host during CM.

Vascular endothelial cells cultured from patients with cerebral or uncomplicated malaria exhibit differential reactivity to TNF

Plasmodium falciparum malaria is a major cause of morbidity and mortality in African children, and factors that determine the development of uncomplicated (UM) versus cerebral malaria (CM) are not fully understood. We studied the ex vivo responsiveness of microvascular endothelial cells to pro‐inflammatory stimulation and compared the findings between CM and UM patients. In patients with fatal disease we compared the properties of vascular endothelial cells cultured from brain tissue to those cultured from subcutaneous tissue, and found them to be very similar. We then isolated, purified and cultured primary endothelial cells from aspirated subcutaneous tissue of patients with CM (ECCM) or UM (ECUM) and confirmed the identity of the cells before analysis. Upon TNF stimulation in vitro, ECCM displayed a significantly higher capacity to upregulate ICAM‐1, VCAM‐1 and CD61 and to produce IL‐6 and MCP‐1 but not RANTES compared with ECUM. The shedding of endothelial microparticles, a recently described parameter of severity in CM, and the cellular level of activated caspase‐3 were both significantly greater in ECCM than in ECUM. These data suggest that inter‐individual differences in the endothelial inflammatory response to TNF may be an additional factor influencing the clinical course of malaria.

Investigating the Pathogenesis of Severe Malaria: A Multidisciplinary and Cross-Geographical Approach

More than a century after the discovery of Plasmodium spp. parasites, the pathogenesis of severe malaria is still not well understood. The majority of malaria cases are caused by Plasmodium falciparum and Plasmodium vivax, which differ in virulence, red blood cell tropism, cytoadhesion of infected erythrocytes, and dormant liver hypnozoite stages. Cerebral malaria coma is one of the most severe manifestations of P. falciparum infection. Insights into its complex pathophysiology are emerging through a combination of autopsy, neuroimaging, parasite binding, and endothelial characterizations. Nevertheless, important questions remain regarding why some patients develop life-threatening conditions while the majority of P. falciparum-infected individuals do not, and why clinical presentations differ between children and adults. For P. vivax, there is renewed recognition of severe malaria, but an understanding of the factors influencing disease severity is limited and remains an important research topic. Shedding light on the underlying disease mechanisms will be necessary to implement effective diagnostic tools for identifying and classifying severe malaria syndromes and developing new therapeutic approaches for severe disease. This review highlights progress and outstanding questions in severe malaria pathophysiology and summarizes key areas of pathogenesis research within the International Centers of Excellence for Malaria Research program.

Platelets alter gene expression profile in human brain endothelial cells in an in vitro model of cerebral malaria.

Platelet adhesion to the brain microvasculature has been associated with cerebral malaria (CM) in humans, suggesting that platelets play a role in the pathogenesis of this syndrome. In vitro co-cultures have shown that platelets can act as a bridge between Plasmodium falciparum-infected red blood cells (pRBC) and human brain microvascular endothelial cells (HBEC) and potentiate HBEC apoptosis. Using cDNA microarray technology, we analyzed transcriptional changes of HBEC in response to platelets in the presence or the absence of tumor necrosis factor (TNF) and pRBC, which have been reported to alter gene expression in endothelial cells. Using a rigorous statistical approach with multiple test corrections, we showed a significant effect of platelets on gene expression in HBEC. We also detected a strong effect of TNF, whereas there was no transcriptional change induced specifically by pRBC. Nevertheless, a global ANOVA and a two-way ANOVA suggested that pRBC acted in interaction with platelets and TNF to alter gene expression in HBEC. The expression of selected genes was validated by RT-qPCR. The analysis of gene functional annotation indicated that platelets induce the expression of genes involved in inflammation and apoptosis, such as genes involved in chemokine-, TREM1-, cytokine-, IL10-, TGFβ-, death-receptor-, and apoptosis-signaling. Overall, our results support the hypothesis that platelets play a pathogenic role in CM.

Fatal Pediatric Cerebral Malaria Is Associated with Intravascular Monocytes and Platelets That Are Increased with HIV Coinfection

ABSTRACT  Cerebral malaria (CM) is a major contributor to malaria deaths, but its pathophysiology is not well understood. While sequestration of parasitized erythrocytes is thought to be critical, the roles of inflammation and coagulation are controversial. In a large series of Malawian children hospitalized with CM, HIV coinfection was more prevalent than in pediatric population estimates (15% versus 2%, P < 0.0001, chi-square test), with higher mortality than that seen in HIV-uninfected children (23% versus 17%, P = 0.0178, chi-square test). HIV-infected (HIV+) children with autopsy-confirmed CM were older than HIV-uninfected children (median age, 99 months versus 32 months, P = 0.0007, Mann-Whitney U test) and appeared to lack severe immunosuppression. Because HIV infection is associated with dysregulated inflammation and platelet activation, we performed immunohistochemistry analysis for monocytes, platelets, and neutrophils in brain tissue from HIV+ and HIV-uninfected children with fatal CM. Children with autopsy-confirmed CM had significantly (>9 times) more accumulations of intravascular monocytes and platelets, but not neutrophils, than did children with nonmalarial causes of coma. The monocyte and platelet accumulations were significantly (>2-fold) greater in HIV+ children than in HIV-uninfected children with autopsy-confirmed CM. Our findings indicate that HIV is a risk factor for CM and for death from CM, independent of traditional measures of HIV disease severity. Brain histopathology supports the hypotheses that inflammation and coagulation contribute to the pathogenesis of pediatric CM and that immune dysregulation in HIV+ children exacerbates the pathological features associated with CM. IMPORTANCE There are nearly 1 million malaria deaths yearly, primarily in sub-Saharan African children. Cerebral malaria (CM), marked by coma and sequestered malaria parasites in brain blood vessels, causes half of these deaths, although the mechanisms causing coma and death are uncertain. Sub-Saharan Africa has a high HIV prevalence, with 3 million HIV-infected (HIV+) children, but the effects of HIV on CM pathogenesis and mortality are unknown. In a study of pediatric CM in Malawi, HIV prevalence was high and CM-attributed mortality was higher in HIV+ than in HIV-uninfected children. Brain pathology in children with fatal CM was notable not only for sequestered malaria parasites but also for intravascular accumulations of monocytes and platelets that were more severe in HIV+ children. Our findings raise the possibility that HIV+ children at risk for malaria may benefit from targeted malaria prophylaxis and that adjunctive treatments targeting inflammation and/or coagulation may improve CM outcomes. There are nearly 1 million malaria deaths yearly, primarily in sub-Saharan African children. Cerebral malaria (CM), marked by coma and sequestered malaria parasites in brain blood vessels, causes half of these deaths, although the mechanisms causing coma and death are uncertain. Sub-Saharan Africa has a high HIV prevalence, with 3 million HIV-infected (HIV+) children, but the effects of HIV on CM pathogenesis and mortality are unknown. In a study of pediatric CM in Malawi, HIV prevalence was high and CM-attributed mortality was higher in HIV+ than in HIV-uninfected children. Brain pathology in children with fatal CM was notable not only for sequestered malaria parasites but also for intravascular accumulations of monocytes and platelets that were more severe in HIV+ children. Our findings raise the possibility that HIV+ children at risk for malaria may benefit from targeted malaria prophylaxis and that adjunctive treatments targeting inflammation and/or coagulation may improve CM outcomes.