Divergent binding sites on intercellular adhesion molecule-1 (ICAM-1) for variant Plasmodium falciparum isolates

Adhesion of human erythrocytes infected with the malaria parasite Plasmodium falciparum to host endothelium has been associated with severe forms of this disease. A number of endothelial receptors have been identified, and there is evidence that one of these, intercellular adhesion molecule-1 (ICAM-1), may play an important role in the pathology of cerebral malaria. Mutagenesis of domain 1 of ICAM-1, which is involved in parasite adhesion, shows that the binding sites for different parasite variants overlap to a large extent, but that there are subtle differences between them that correlate with their adhesive phenotypes. This suggests that the ability to bind to ICAM-1 has arisen from a common variant, but that subsequent changes have led to differences in binding avidity, which may affect pathogenesis. The definition of common binding determinants and the elucidation of links between ICAM-1 binding phenotype and disease will provide new leads in the design of therapeutic interventions.     

An Analysis of the Binding Characteristics of a Panel of Recently Selected ICAM-1 Binding Plasmodium falciparum Patient Isolates

The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1^Ref, ICAM-1^Kilifi, ICAM-1^S22/A, ICAM-1^L42/A and ICAM-1^L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants.     

Cytoadherence characteristics to endothelial receptors ICAM-1 and CD36 of Plasmodium falciparum populations from severe and uncomplicated malaria cases

The adhesion of infected red blood cells (IRBCs) to the cell lining of microvasculature is thought to play a central role in the pathogenesis of severe malaria. Individual IRBC can bind to more than one host receptor and parasites with multiple binding phenotypes may cause severe disease more frequently. However, as most clinical isolates are multiclonal, previous studies were hampered by the difficulty to distinguish whether a multiadherent phenotype was due to one or more parasite population(s). We have developed a tool, based on cytoadhesion assay and GeneScan genotyping technology, which enabled us to assess on fresh isolates the capacity of adherence of individual P. falciparum genotypes to human receptors expressed on CHO transfected cells. The cytoadhesion to ICAM-1 and CD36 of IRBCs from uncomplicated and severe malaria attacks was evaluated using this methodology. In this preliminary series conducted in non immune travelers, IRBCs from severe malaria appeared to adhere more frequently and/or strongly to ICAM-1 and CD36 in comparison with uncomplicated cases. In addition, a majority genotype able to strongly adhere to CD36 was found more frequently in isolates from severe malaria cases. Further investigations are needed to confirm the clinical relevance of these data.     

Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus.

The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.     

High Plasma Levels of Soluble Intercellular Adhesion Molecule (ICAM)-1 Are Associated with Cerebral Malaria

Background

Cerebral malaria (CM) is responsible for most of the malaria-related deaths in children in sub-Saharan Africa. Although, not well understood, the pathogenesis of CM involves parasite and host factors which contribute to parasite sequestration through cytoadherence to the vascular endothelium. Cytoadherence to brain microvasculature is believed to involve host endothelial receptor, CD54 or intercellular adhesion molecule (ICAM)-1, while other receptors such as CD36 are generally involved in cytoadherence of parasites in other organs. We therefore investigated the contributions of host ICAM-1 expression and levels of antibodies against ICAM-1 binding variant surface antigen (VSA) on parasites to the development of CM.

Methodology/Principal Findings

Paediatric malaria patients, 0.5 to 13 years were recruited and grouped into CM and uncomplicated malaria (UM) patients, based on well defined criteria. Standardized ELISA protocol was used to measure soluble ICAM-1 (sICAM-1) levels from acute plasma samples. Levels of IgG to CD36- or ICAM-1-binding VSA were measured by flow cytometry during acute and convalescent states. Wilcoxon sign rank-test analysis to compare groups revealed association between sICAM-1 levels and CM (p<0.0037). Median levels of antibodies to CD36-binding VSA were comparable in the two groups at the time of admission and 7 days after treatment was initiated (p>0.05). Median levels of antibodies to CD36-binding VSAs were also comparable between acute and convalescent samples within any patient group. Median levels of antibodies to ICAM-1-binding VSAs were however significantly lower at admission time than during recovery in both groups.

Conclusions/Significance

High levels of sICAM-1 were associated with CM, and the sICAM-1 levels may reflect expression levels of the membrane bound form. Anti-VSA antibody levels to ICAM-binding parasites was more strongly associated with both UM and CM than antibodies to CD36 binding parasites. Thus, increasing host sICAM-1 levels were associated with CM whilst antibodies to parasite expressing non-ICAM-1-binding VSAs were not.     

Malaria prevalence metrics in low- and middle-income countries: an assessment of precision in nationally-representative surveys

Maladaptive immune responses during cerebral malaria (CM) result in high mortality despite opportune anti-malarial chemotherapy. Rapamycin, an FDA-approved immunomodulator, protects against experimental cerebral malaria (ECM) in mice through effects on the host. However, the potential for reduced adaptive immunity with chronic use, combined with an incomplete understanding of mechanisms underlying protection, limit translational potential as an adjunctive therapy in CM. The results presented herein demonstrate that a single dose of rapamycin, provided as late as day 4 or 5 post-infection, protected mice from ECM neuropathology and death through modulation of distinct host responses to infection. Rapamycin prevented parasite cytoadherence in peripheral organs, including white adipose tissue, via reduction of CD36 expression. Rapamycin also altered the splenic immune response by reducing the number of activated T cells with migratory phenotype, while increasing local cytotoxic T cell activation. Finally, rapamycin reduced brain endothelial ICAM-1 expression concomitant with reduced brain pathology. Together, these changes potentially contributed to increased parasite elimination while reducing CD8 T cell migration to the brain. Rapamycin exerts pleotropic effects on host immunity, vascular activation and parasite sequestration that rescue mice from ECM, and thus support the potential clinical use of rapamycin as an adjunctive therapy in CM.     

Maladjusted Host Immune Responses Induce Experimental Cerebral Malaria-Like Pathology in a Murine Borrelia and Plasmodium Co-Infection Model

In the Plasmodium infected host, a balance between pro- and anti-inflammatory responses is required to clear the parasites without inducing major host pathology. Clinical reports suggest that bacterial infection in conjunction with malaria aggravates disease and raises both mortality and morbidity in these patients. In this study, we investigated the immune responses in BALB/c mice, co-infected with Plasmodium berghei NK65 parasites and the relapsing fever bacterium Borrelia duttonii. In contrast to single infections, we identified in the co-infected mice a reduction of L-Arginine levels in the serum. It indicated diminished bioavailability of NO, which argued for a dysfunctional endothelium. Consistent with this, we observed increased sequestration of CD8+ cells in the brain as well over expression of ICAM-1 and VCAM by brain endothelial cells. Co-infected mice further showed an increased inflammatory response through IL-1β and TNF-α, as well as inability to down regulate the same through IL-10. In addition we found loss of synchronicity of pro- and anti-inflammatory signals seen in dendritic cells and macrophages, as well as increased numbers of regulatory T-cells. Our study shows that a situation mimicking experimental cerebral malaria (ECM) is induced in co-infected mice due to loss of timing and control over regulatory mechanisms in antigen presenting cells.     

Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors

The Duffy binding-like (DBL) domain is a key adhesive module in Plasmodium falciparum, present in both erythrocyte invasion ligands (EBLs) and the large and diverse P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of cytoadherence receptors. DBL domains bind a variety of different host receptors, including intercellular adhesion molecule 1 (ICAM-1), a receptor interaction that may have a role in infected erythrocyte binding to cerebral blood vessels and cerebral malaria. In this study, we expressed the nearly full complement of DBLbeta-C2 domains from the IT4/25/5 (IT4) parasite isolate and showed that ICAM-1-binding domains (DBLbeta-C2(ICAM-1)) were confined to group B and group C PfEMP1 proteins and were not present in group A, suggesting that ICAM-1 selection pressure differs between PfEMP1 groups. To further dissect the molecular determinants of binding, we modelled a DBLbeta-C2(ICAM-1) domain on a solved DBL structure and created alanine substitution mutants in two DBLbeta-C2(ICAM-1) domains. This analysis indicates that the DBLbeta-C2::ICAM-1 interaction maps to the equivalent glycan binding region of EBLs, and suggests a general model for how DBL domains evolve under dual selection for host receptor binding and immune evasion.     

Experimental Cerebral Malaria Pathogenesis—Hemodynamics at the Blood Brain Barrier

Cerebral malaria claims the lives of over 600,000 African children every year. To better understand the pathogenesis of this devastating disease, we compared the cellular dynamics in the cortical microvasculature between two infection models, Plasmodium berghei ANKA (PbA) infected CBA/CaJ mice, which develop experimental cerebral malaria (ECM), and P. yoelii 17XL (PyXL) infected mice, which succumb to malarial hyperparasitemia without neurological impairment. Using a combination of intravital imaging and flow cytometry, we show that significantly more CD8+ T cells, neutrophils, and macrophages are recruited to postcapillary venules during ECM compared to hyperparasitemia. ECM correlated with ICAM-1 upregulation on macrophages, while vascular endothelia upregulated ICAM-1 during ECM and hyperparasitemia. The arrest of large numbers of leukocytes in postcapillary and larger venules caused microrheological alterations that significantly restricted the venous blood flow. Treatment with FTY720, which inhibits vascular leakage, neurological signs, and death from ECM, prevented the recruitment of a subpopulation of CD45^hi CD8+ T cells, ICAM-1+ macrophages, and neutrophils to postcapillary venules. FTY720 had no effect on the ECM-associated expression of the pattern recognition receptor CD14 in postcapillary venules suggesting that endothelial activation is insufficient to cause vascular pathology. Expression of the endothelial tight junction proteins claudin-5, occludin, and ZO-1 in the cerebral cortex and cerebellum of PbA-infected mice with ECM was unaltered compared to FTY720-treated PbA-infected mice or PyXL-infected mice with hyperparasitemia. Thus, blood brain barrier opening does not involve endothelial injury and is likely reversible, consistent with the rapid recovery of many patients with CM. We conclude that the ECM-associated recruitment of large numbers of activated leukocytes, in particular CD8+ T cells and ICAM+ macrophages, causes a severe restriction in the venous blood efflux from the brain, which exacerbates the vasogenic edema and increases the intracranial pressure. Thus, death from ECM could potentially occur as a consequence of intracranial hypertension.     

Temperature-Induced Catch-Slip to Slip Bond Transit in Plasmodium falciparum-Infected Erythrocytes

Plasmodium falciparum malaria-infected red blood cells (IRBCs), or erythrocytes, avoid splenic clearance by adhering to host endothelium. Upregulation of endothelial receptors intercellular adhesion molecule-1 (ICAM-1) and cluster of differentiation 36 (CD36) are associated with severe disease pathology. Most in vitro studies of IRBCs interacting with these molecules were conducted at room temperature. However, as IRBCs are exposed to temperature variations between 37°C (body temperature) and 41°C (febrile temperature) in the host, it is important to understand IRBC-receptor interactions at these physiologically relevant temperatures. Here, we probe IRBC interactions against ICAM-1 and CD36 at 37 and 41°C. Single bond force-clamp spectroscopy is used to determine the bond dissociation rates and hence, unravel the nature of the IRBC-receptor interaction. The association rates are also extracted from a multiple bond flow assay using a cellular stochastic model. Surprisingly, IRBC-ICAM-1 bond transits from a catch-slip bond at 37°C toward a slip bond at 41°C. Moreover, binding affinities of both IRBC-ICAM-1 and IRBC-CD36 decrease as the temperature rises from 37 to 41°C. This study highlights the significance of examining receptor-ligand interactions at physiologically relevant temperatures and reveals biophysical insight into the temperature dependence of P. falciparum malaria cytoadherent bonds.     

T cell-derived IL-10 and its impact on the regulation of host responses during malaria

Despite intense research, malaria still is the one of the most devastating diseases killing more people than any other parasitic infection. In an attempt to control the infection, the host immune system produces a potent pro-inflammatory response. However, this response is also associated with complications, such as severe anaemia, hypoglycaemia and cerebral malaria. This pronounced production of pro-inflammatory cytokines response is a common feature of malaria caused by parasites infecting humans as well as rodents and primates. A balance between pro- and anti-inflammatory responses may be fundamental to the elimination of the parasite without inducing excessive host pathology. IL-10 is a key cytokine that has been shown to have an important regulatory function in establishing this balance in malaria. Here we discuss which cells can produce IL-10 during infection, and present an overview of the evidence showing that T-cell derived IL-10 plays an important role in regulating malaria pathology. Many different subsets of T cells can produce IL-10, however, evidence is accumulating that it is effector Th1 CD4(+) T cells which provide the crucial source that down-regulates inflammatory pathology during blood-stage malaria infections.     

Cytoadherence and sequestration in Plasmodium falciparum: defining the ties that bind

Infected erythrocytes containing the more mature stages of the human malaria Plasmodium falciparum may adhere to endothelial cells and uninfected red cells. These phenomena, called sequestration and rosetting, respectively, are involved in both host pathogenesis and parasite survival. This review provides a critical summary of recent advances in the characterization of the molecules of the infected red blood cell involved in adhesion, i.e. parasite-encoded molecules (PfEMP1, MESA, rifins, stevor, clag 9, histidine-rich protein), a modified host membrane protein (band 3) and exofacial exposure of phosphatidylserine, as well as receptors on the endothelium, i.e. thrombospondin, CD36, ICAM-1 (intercellular adhesion molecule), and chondroitin sulfate.     

Intercellular adhesion molecule 1 deficiency leads to impaired recruitment of T lymphocytes and enhanced host susceptibility to infection with Trypanosoma cruzi

In this study, we investigated the involvement of Th1 cytokines in the expression of cell adhesion molecules (CAM) and recruitment of inflammatory cells to the heart of mice infected with Trypanosoma cruzi. Our results show that endogenously produced IFN-gamma is essential to induce optimal expression of VCAM-1 and ICAM-1 on the cardiac vascular endothelium of infected mice. Furthermore, the influx of inflammatory cells into the cardiac tissue was impaired in Th1 cytokine-deficient infected mice, paralleling the intensity of VCAM-1 and ICAM-1 expression on the vascular endothelium. Consistent with the importance of ICAM-1 in host resistance, ICAM-1 knockout (KO) mice were highly susceptible to T. cruzi infection, as assessed by mortality rate, parasitemia, and heart tissue parasitism. The enhanced parasitism was associated with a decrease in the numbers of CD4(+) and CD8(+) T lymphocytes in the heart tissue of ICAM-1 KO mice. Additionally, ICAM-1 KO mice mounted an unimpaired IFN-gamma response and IFN-gamma-dependent production of reactive nitrogen intermediates and parasite- specific IgG2a. Supporting the participation of ICAM-1 in cell migration during T. cruzi infection, the entrance of adoptively transferred PBL from T. cruzi-infected wild-type C57BL/6 mice into the cardiac tissue of ICAM-1 KO mice was significantly abrogated. Therefore, we favor the hypothesis that ICAM-1 plays a crucial role in T lymphocyte recruitment to the cardiac tissue and host susceptibility during T. cruzi infection.     

Lipoxin A4 and 15-Epi-Lipoxin A4 Protect against Experimental Cerebral Malaria by Inhibiting IL-12/IFN-γ in the Brain

Cerebral malaria is caused by infection with Plasmodium falciparum and can lead to severe neurological manifestations and predominantly affects sub-Saharan African children. The pathogenesis of this disease involves unbalanced over-production of pro-inflammatory cytokines. It is clear that signaling though IL-12 receptor is a critical step for development of cerebral malaria, IL-12 genetic deficiency failed to show the same effect, suggesting that there is redundancy among the soluble mediators which leads to immunopathology and death. Consequently, counter-regulatory mediators might protect the host during cerebral malaria. We have previously showed that endogenously produced lipoxins, which are anti-inflammatory mediators generated by 5-lipoxygenase (5-LO)-dependent metabolism of arachidonic acid, limit host damage in a model of mouse toxoplasmosis. We postulated here that lipoxins might also play a counter-regulatory role during cerebral malaria. To test this hypothesis, we infected 5-LO-deficient hosts with P. berghei ANKA strain, which induces a mouse model of cerebral malaria (ECM). Our results show accelerated mortality concomitant with exuberant IL-12 and IFN-γ production in the absence of 5-lipoxygenase. Moreover, in vivo administration of lipoxin to 5-LO-deficient hosts prevented early mortality and reduced the accumulation of CD8⁺IFN-γ ⁺ cells in the brain. Surprisingly, WT animals treated with lipoxin either at the time of infection or 3 days post-inoculum also showed prolonged survival and diminished brain inflammation, indicating that although protective, endogenous lipoxin production is not sufficient to optimally protect the host from brain damage in cerebral malaria. These observations establish 5-LO/LXA₄ as a host protective pathway and suggest a new therapeutic approach against human cerebral malaria (HCM). (255 words).     

Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface

The virulence of the malaria parasite Plasmodium falciparum is related to its ability to express a family of adhesive proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the infected red blood cell surface. The mechanism for the transport and delivery of these adhesins to the erythrocyte membrane is only poorly understood. In this work, we have used specific immune reagents in a flow cytometric assay to monitor the effects of serum components on the surface presentation of PfEMP1. We show that efficient presentation of the A4 and VAR2CSA variants of PfEMP1 is dependent on the presence of serum in the bathing medium during parasite maturation. Lipid-loaded albumin supports parasite growth but allows much less efficient presentation of PfEMP1 at the red blood cell surface. Analysis of the serum components reveals that lipoproteins, especially those of the low-density lipoprotein fraction, promote PfEMP1 presentation. Cytoadhesion of infected erythrocytes to the host cell receptors CD36 and ICAM-1 is also decreased in infected erythrocytes cultured in the absence of serum. The defect appears to be in the transfer of PfEMP1 from parasite-derived structures known as the Maurer's clefts to the erythrocyte membrane or in surface conformation rather than a down-regulation or switching of particular PfEMP1 variants.     

Peroxisome proliferator-activated receptor gamma-retinoid X receptor agonists increase CD36-dependent phagocytosis of Plasmodium falciparum-parasitized erythrocytes and decrease malaria-induced TNF-alpha secretion by monocytes/macrophages

Severe and fatal malaria is associated with the failure of host defenses to control parasite replication, excessive secretion of proinflammatory cytokines such as TNF-alpha, and sequestration of parasitized erythrocytes (PEs) in vital organs. The identification of CD36 as a major sequestration receptor has led to the assumption that it contributes to the pathophysiology of severe malaria and has prompted the development of antiadherence therapies to disrupt the CD36-PE interaction. This concept has been challenged by unexpected evidence that individuals deficient in CD36 are more susceptible to severe and cerebral malaria. In this study, we demonstrate that CD36 is the major receptor mediating nonopsonic phagocytosis of PEs by macrophages, a clearance mechanism of potential importance in nonimmune hosts at the greatest risk of severe malaria. CD36-mediated uptake of PEs occurs via a novel pathway that does not involve thrombospondin, the vitronectin receptor, or phosphatidylserine recognition. Furthermore, we show that proliferator-activated receptor gamma-retinoid X receptor agonists induce an increase in CD36-mediated phagocytosis and a decrease in parasite-induced TNF-alpha secretion. Specific up-regulation of monocyte/macrophage CD36 may represent a novel therapeutic strategy to prevent or treat severe malaria.     

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions.