Insights into the Cytoadherence Phenomenon of Plasmodium vivax: The Putative Role of Phosphatidylserine

Plasmodium vivax is the most geographically widespread and the dominant human malaria parasite in most countries outside of sub-Saharan Africa and, although it was classically recognized to cause benign infection, severe cases and deaths caused by P. vivax have remarkably been reported. In contrast to Plasmodium falciparum, which well-known ability to bind to endothelium and placental tissue and form rosettes is related to severity of the disease, it has been a dogma that P. vivax is unable to undergo cytoadherent phenomena. However, some studies have demonstrated that red blood cells (RBCs) infected by P. vivax can cytoadhere to host cells, while the molecules participating in this host–parasite interaction are still a matter of speculation. In the present overview, we address the evidences currently supporting the adhesive profile of P. vivax and, additionally, discuss the putative role of phosphatidylserine—a cell membrane phospholipid with cytoadhesive properties that has been detected on the surface of Plasmodium-parasitized RBCs.

It is known that the main pathological phenomenon related to high virulence of P. falciparum is the sequestration of parasitized red blood cells (pRBC) to vascular endothelium and placenta, which allows late-stage forms of parasite to evade splenic phagocytosis, while provoking host damage by obstructing blood flow and inducing local pro-inflammatory response (9). Additional factors contributing to the pathogenesis of falciparum malaria comprise rosetting of pRBC with non-parasitized red blood cells (nRBC) as well as clumping of pRBC mediated by platelets (10). All these cytoadhesive events of P. falciparum-pRBC are recognized to be mediated by a large and diverse family of parasite antigens, named P. falciparum erythrocyte membrane protein 1 (PfEMP1), that is expressed on the surface of pRBC and shows affinity to several host receptors, including intercellular adhesion molecule 1 (ICAM-1), platelet-endothelial cell adhesion molecule 1, chondroitin sulfate A (CSA), hyaluronic acid (HA), thrombospondin (TSP), and CD36 (10). Since homologous of var genes, which encode PfEMP1, have not been identified in other Plasmodium species and the cytoadherence of pRBC was not consistently documented in non-falciparum malaria, it was believed that the cytoadherence phenomenon of pRBC was restricted to P. falciparum infection (11)(12)(13)(14). However, some evidences currently support that red blood cells (RBCs) infected by P. vivax (Pv-pRBC) also undergo cytoadherence events, as addressed below in the present paper.

eviDenCeS OF P. vivax CYTOADHeSiOn
Since Pv-pRBC lack adhesive knob structure and, especially, because all parasite stages can be observed in the peripheral blood of patients, it has been a dogma that P. vivax lacks the ability to cytoadhere and, therefore, to sequester. However, in 2010 it was demonstrated, for the first time, that Pv-pRBC are able to cytoadhere in vitro to host cells (15). In this study, Pv-pRBC obtained from Brazilian non-severe patient samples were tested by static and flow cytoadhesion assays using human lung endothelial cells (HLEC), monkey brain endothelial cells, and human placental cryosections. Although the number of Pv-pRBC adhered under static conditions was lower than that observed for pRBC infected by P. falciparum, the strength of interaction with endothelium was similar. Moreover, it was shown using transfected Chinese Hamster Ovarian (CHO) cells that the binding of Pv-pRBC to ICAM-1-expressing cells was twice as high as to non-transfected cells or CD36-expressing cells and that the adherence to placental cryosections and HLEC was inhibited by soluble CSA, suggesting involvement of both ICAM-1 and CSA in adhesive processes of P. vivax. In fact, the adhesive capacity of Pv-pRBC to HLEC and the involvement of ICAM-1 were later recorded in studies using P. vivax isolates from Colombia (16). Moreover, a treatment with chondroitinase reversed the adherence of P. vivax isolates from the Asia-Pacific region to immobilized CSA, while it was additionally observed that hyaluronidase disturbed Pv-pRBC adhesion mediated by HA (17). Nevertheless, the degree of commitment of each host adhesive receptor studied until now is still a matter of speculation. For instance, in the study with Thai patients described above, all P. vivax isolates were adherent to immobilized CSA and HA, but none adhered to ICAM-1 (17), and when P. vivax isolates from Brazilian Amazon region was evaluated, a low frequency of pRBC adhesion to ICAM-1 and CSA was observed (18).
Corroborating the adhesive profile of P. vivax, it was recently reported that the schizont stage was absent in the peripheral circulation in more than half of Brazilian patients diagnosed with P. vivax malaria by blood smears and, even when P. vivax schizonts were detected, they were mostly present at low frequency (19). Moreover, in vitro maturation of P. vivax isolates provided a greater ability of Pv-pRBC to cytoadhere to HLEC than the same isolates before maturation, revealing a higher adhesive capacity of mature forms. These data indicate that P. vivax might be sequestered in the deep vasculature and that maturation of late stages of P. vivax occur outside peripheral circulation. Actually, more than 50 years ago sequestration of Pv-pRBC was proposed by Field et al. (20), who showed a disappearance of schizonts from the peripheral blood of a P. vivax patient. In addition, recent discoveries showing accumulation of P. vivax schizonts and gametocytes in the bone marrow (21); detection of a large number of intact Pv-pRBC in the spleen (22); and presence of Pv-pRBC within pulmonary microvasculature from a patient with negative blood smear at the time of death (23) support the hypothesis that P. vivax has the ability to sequester.
Although P. vivax does not present any protein homologous to PfEMP1, a group of variable proteins (VIR proteins) is expressed by this parasite species (24). In contrast to PfEMP1, VIR proteins are not clonally expressed and can additionally be found within pRBC, indicating initially that these P. vivax antigens have different functions from PfEMP1 ones (25). However, based on their variant nature and presence on pRBC surface, the role of VIR antigens in Pv-pRBC adhesion has been evaluated. Thus, computational analysis using a P. falciparum-based algorithm revealed putative adhesive protein motifs in VIR proteins (26), which could explain the capacity of vir gene (VIR-14) to mediate adhesion of pRBC to ICAM-1 when transfected into a non-adhesive P. falciparum line (27). Also, consistent with the participation of VIR proteins in the sequester phenomenon of Pv-pRBC, it was previously demonstrated that antibodies against variants of VIR proteins (VIR-A4 and VIR-E5) partially inhibit adhesion of Pv-pRBC to HLEC (15).
Therefore, there is now a growing body of evidence supporting that P. vivax parasites possess adhesive phenotypes. Indeed, besides adhesion to endothelium and placental tissue, it is known that P. vivax has the ability to form rosettes, which are defined by the binding of a pRBC with two or more nRBC. Rosetting formation in P. vivax infection was described more than 20 years ago (28) and has been shown to be more frequent than in P. falciparum infection (29,30), but few studies have been conducted to investigate this P. vivax phenomenon; largely due to the absence of a P. vivax continuous culture method. Notwithstanding, it was already demonstrated that rosettes in P. vivax infection are formed by interaction of pRBC containing trophozoites, schizonts, or gametocytes with mature RBCs (normocytes), a process that involves glycophorin C receptor present on nRBC surface (30). Furthermore, P. vivax rosettes were shown to be stable even under high physiological shear stress and rosette formation was closely associated with induction of an increased rigidity of Pv-pRBC, P. vivax Cytoadherence and PS Frontiers in Immunology | www.frontiersin.org September 2017 | Volume 8 | Article 1148 possibly contributing to sequestration of P. vivax in the microvasculature (31). However, differently from P. falciparum, both host and parasite antigens intricate on P. vivax rosetting as well as the relation of this adhesive phenomenon to the pathogenesis of vivax malaria remain unknown.

THe ROLe OF PHOSPHATiDYLSeRine (PS) in CYTOADHeSive PHenOMenA
While some research efforts have been dedicated to identifying P. vivax antigens participating in cytoadhesion of pRBC, little attention has been given to host RBC factors that have adhesive potentiality, such as PS. PS is a cell membrane phospholipid usually restricted to the inner leaflet of the lipid bilayer (32), but during apoptotic cell death processes PS is exposed on cell surface, promoting recognition and clearance of dying cells by phagocytes (33). Externalization of PS also occurs in activated platelet and transiently in activated lymphocytes and mast cells, where it is associated with procoagulant activity, homing to sites of inflammation and cell degranulation, respectively (34)(35)(36). Furthermore, it has been shown that the presence of PS on external leaflet of cell membrane is a hallmark of suicidal erythrocyte death, named eryptosis (37). Eryptosis occurs in senescent RBC and can precociously be triggered by a variety of endogenous and xenobiotics stimuli (38,39). Similarly to apoptosis of nucleated cells, eryptotic processes are characterized by many morphological and biochemical changes, i.e., Ca 2+ influx, cysteine protease activity, PS exposure, cell shrinkage, and plasma membrane microvesiculation, with externalized PS rendering RBC susceptible to clearance by splenic phagocytes (40). Accordingly, overinduction of PS-exposing eryptotic RBC is believed to contribute to the development of anemia related to several clinical disorders, as reported in both experimental and human malaria (41,42). But, additionally, PS on RBC surface is also considered one of the factors responsible for thrombo-occlusive events in pathologies such as sickle cell disease, chronic renal failure, retinal vein occlusion, and diabetes; in part, by mediating RBC adherence to endothelium as well as cell aggregation (43)(44)(45)(46).
Indeed, adherence of PS-exposing RBC to endothelium is observed in vitro under dynamic flow conditions mimicking venular wall shear stress (47,48) and takes place through interaction of PS with the scavenger receptors CD36 or CXC chemokine ligand 16 (CXCL16) expressed on endothelial cell membrane, as well as with TSP, which is found in the basement membrane and extracellular matrix of endothelium and that can be exposed by vascular injury (45,48,49). Moreover, soluble plasma TSP can interact with CD36 and, in this manner, could operate as a bridge to adherence of PS-exposing RBC (50,51). Such interactions involving PS, CD36, TSP, and CXCL16 have also been involved in RBC-platelet aggregation, agreeing with the presence of CD36 and CXCL16 in platelet membrane (52)(53)(54). Thus, if parasite antigens able to provide pRBC adhesiveness are absent, P. vivax could explore host adhesive molecules to mediate cytoadhesive events of pRBC.
Interestingly, it has previously been demonstrated that intraerythrocytic plasmodia development progressively induces PS exteriorization on pRBC, with larger exposure at the late stages of parasite maturation (55,56), which possibly result from eryptosis stimulation. Schizogonic process is described to activate non-selective cation channels in host pRBC membrane, allowing the entry of Ca 2+ necessary for parasite intracellular growth, which, in turn, leads to the activation of phospholipid scramblases responsible for PS exposure (57). Although PS externalization has not been evaluated in P. vivax infection, it was already detected by flow cytometry in RBC infected by P. falciparum, P. berghei, and P. yoelii (41,58,59) and in P. falciparum, the binding of late-stage pRBC exposing PS to CD36-expressing cells as well as immobilized CD36 and TSP was inhibited by annexin V, PS-containing liposomes or glycerophosphorylserine-a soluble form of PS (60), indicating that PS could, at least in part, support cytoadhesive phenomena of pRBC in malaria. Consistent with this possibility, a relationship between cytoadhesive activity and PS exposure was also reported when knobby and knobless P. falciparum strains, which differentially induce PS externalization on late-stage pRBC, were studied (60) and, more recently, it was also shown that PS-expressing RBC can operate as nuclei for RBC aggregation induced by P. falciparum-conditioned medium (61).
Importantly, studies conducted on P. berghei ANKA experimental infection with CD36-deficient rodents have demonstrated that CD36 is an essential receptor for sequestration of schizont-stage pRBC, which occurred mostly in the capillaries of lungs and adipose tissue, but not in the brain, where endothelial expression of CD36 is low or absent (62). Indeed, besides being incriminated in acute tissue injury induced by P. berghei ANKA-pRBC accumulation in lung (63), CD36 is known as an important receptor mediating pRBC sequestration, non-related to brain and placental tissue in P. falciparum malaria (64), and its expression on surface of platelets and RBC has been implicated in clumping and rosetting processes of P. falciparum-pRBC, respectively (65,66). However, it is noteworthy that, in contrast to P. falciparum, which expresses the adhesin PfEMP1, but similarly to Plasmodium chabaudi, whose late-stage forms undergo CD36-dependent cytoadhesion in vitro (67), no putative parasite ligand for CD36 has been identified in genome of P. berghei, or even other species of Plasmodium displaying cytoadherence phenotypes, such as P. vivax (14,68), reinforcing the premise that alternative pathways, not based on the expression of parasite adhesins, could mediate CD36-dependent cytoadhesion of latestage pRBC.
Additional evidences for this proposition are also documented in P. vivax malaria. First, a reduction in Pv-pRBC adhesion to HLEC was achieved in the presence of anti-CD36 antibodies, although the small number of samples limited the statistical analysis concerning extension of CD36 participation in P. vivax cytoadhesion (15). Second, while studying cellular trafficking and the adhesive propriety of P. vivax VIR proteins in P. falciparum transgenic lines, it was shown that only one variant of VIR proteins (VIR-14) was exposed at the surface of pRBC, mediating cytoadherence to CHO cells through ICAM-1, but not CD36 (27). Thus, it is tempting to speculate that if antigens encoded by vir genes participate in Pv-pRBC adhesive events, it seems that it does not take place through a CD36-dependent mechanism, in which PS could play a role (Figure 1). In view of this possibility, In turn, PS mediates sequestration of pRBC to microvasculature through interaction with receptors expressed on endothelial cells, such as CD36, or thrombospondin (TSP) exposed in injured endothelium. In a CD36-dependent manner, PS-exposing pRBC can promote rosetting of non-parasitized red blood cells (RBCs) as well as autoagglutination (clumping) by binding to platelets. Additionally, von Willebrand factor (vWF) can indirectly contribute to the sequestration of PS-exposing pRBC by mediating platelet adhesion at sites of vascular damage (69).

AUTHOR COnTRiBUTiOnS
PT and SL wrote the paper. Both authors read and approved the final version of the manuscript.