The role of extracellular vesicles in malaria biology and pathogenesis

Accumulating evidence suggests that EVs contribute to malaria-associated clinical symptoms, in particular in severe disease (Table 1). Initial studies focused on MVs of host cell origin, largely referred to as microparticles. These host-origin MVs were associated with malaria through their role in cerebral pathogenesis, as increased levels of endothelial-derived MVs were present in patients with severe cerebral malaria [33]. The role of MVs in malaria was further dissected using mouse models of severe disease. ABCA1 knock-out mice, which have reduced ability to produce MVs, were protected from cerebral malaria [34]. In control mice, MVs of host platelet, monocyte and endothelial cell origins were released during infection, but this was significantly reduced in ABCA1 knock-outs. In particular, there were reduced inflammatory hallmarks such as high serum TNF and platelet and leukocyte sequestration in the brain. This in vivo work was in agreement with the association of MVs with parasite cytoadherence and their likely role in severe disease caused by parasite sequestration, particularly in cerebral malaria [35]. The importance of ABCA1 in MV-related malaria pathogenesis was further supported by findings from a field study of human patients infected with Plasmodium falciparum, where MV levels in patients with severe and uncomplicated malaria were tested for association with their ABCA1 promoter haplotypes [36]. The authors reported that MV release increased during malaria infection, MV levels positively correlated to disease severity, and ABCA1 promotor genotypes were associated with susceptibility to severe malaria. These studies demonstrate that host-derived MVs were an important factor contributing to severe malaria.

Recently, a better understanding of the content of host-derived MVs during severe malaria was achieved based on proteomic analysis of MVs from mice with cerebral malaria [4]. There were significant changes in the protein content of MVs from Plasmodium berghei (strain ANKA)-infected mice compared to naïve mice, and network analyses indicated that these proteins were actively involved in cerebral malaria pathogenesis. Specifically, TNF and TGFβ1 were predicted to regulate the cerebral malaria-associated proteins in MVs. These findings are in agreement with the association of MVs with cerebral malaria and the involvement of TNF in this disease [4]. However, the field is still lacking in studies on the nucleic acid content of host-derived EVs in malaria. Transcriptomic investigations are needed to further determine if and how host-derived EVs might modulate pathogenesis in malaria, and contribute to disease severity.

Several field studies have reported increased levels of MVs during active Plasmodium infection, analysed by flow cytometric analysis of surface stained vesicles, which returned to normal after resolution of infection. However, there have been some differences in reported MV origin, and occurrence during severe disease (Table 1). In P. falciparum-infected patients in Cameroon, there was a significant increase in circulating MVs in patients with cerebral malaria, but not in non-cerebral severe or uncomplicated malaria, compared to controls [37]. Platelet-derived EVs in particular were positively associated with severity of cerebral symptoms [37]. Similar results were reported from a field study in India, where MVs originating from platelets, erythrocytes and endothelial cells were increased during infection [38]. In this case, however, the authors reported increased MV levels in cerebral and non-cerebral severe malaria, but not in uncomplicated malaria. Furthermore, high MV levels also correlated with high serum TNF, and levels of MVs and TNF both returned to normal ranges after resolution of infection.

In addition to endothelial cells playing a central role in P. falciparum cytoadherence and disease severity, platelet involvement in cerebral malaria is also well established [39]. In vitro, platelet MVs can bind to P. falciparum infected red blood cells (iRBC) in a PfEMP1-dependent manner, transfer platelet antigens to iRBC, and induce iRBC cytoadherence to endothelial cells [40]. This has provided mechanistic insights into the involvement of MVs in cerebral malaria, suggesting that EVs can promote cerebral pathology by stimulating iRBC cytoadhesion in the brain. A recent study has investigated the fate and effect of MVs in P. berghei-infected mice [41]. They found that when MVs isolated from infected mice were transferred to recipient mice, these MVs localized to cerebral microvessels in infected recipient mice, but not in uninfected recipient mice. Furthermore, transfer of MVs from TNF-stimulated endothelial cells induced brain histopathology similar to cerebral malaria, indicating MVs might be active contributors to the pathologies associated with severe malaria. Notably MV localization to the brain only occurred when the recipient mice were also infected with P. berghei, suggesting that the adhesion of these MVs to host organs requires iRBC presence/interaction.

Although most reports have focused on EVs in P. falciparum, a study of Plasmodium vivax infected individuals in Brazil showed that active infection was also associated with increased MV release, but these MVs originated from platelets, leukocytes and erythrocytes, and not from endothelial cells [42]. Platelet-derived EVs in particular were correlated with high fever, suggesting these host-derived EVs might play a central role in the inflammatory symptoms of P. vivax infection [42]. It is interesting to note that endothelial-derived MVs were consistently found to be increased with P. falciparum infection (Table 1), which is a cytoadhering parasite, but not with P. vivax, which does not cytoadhere. The presence of endothelial cell MVs in P. falciparum infection likely reflects the central involvement of this cell type in development of cytoadherent-dependent severe disease symptoms.

Placental malaria is a complication of infection caused by accumulation of iRBC in the placental intervillous space in pregnant women [43], mediated by parasite cytoadherence. A recent study investigated potential links between host-derived MVs and placental malaria [44]. In contrast to severe malarial anaemia or cerebral malaria, there were no changes in total MV or placental trophoblast-specific MV release in women with placental malaria compared to uninfected, indicating that although this severe disease involves parasite sequestration, host-derived MVs are not involved in this specific pathological process during pregnancy-associated malaria. However, there was an overexpression of microRNA miR-517c in MVs from malaria-infected women. miR-517 has been implicated in regulating trophoblast and placental function [45], suggesting that MVs could have applications as biomarkers in placental malaria, and that further research is warranted.

Despite several published reports, there has not been extensive characterization of host-derived MVs from humans infected with malaria. These experiments would be challenging given the high heterogeneity of human samples, but the field would benefit from a more thorough descriptive analysis of vesicles size and composition in these sample types. Furthermore, as shown in Table 1, there are discrepancies in the methodology used to isolate MVs, e.g. some studies investigate the supernatant from 13,000×g centrifugation [37, 38], whereas others investigate the pellets from higher centrifugation speeds [36, 40, 41]. These differing methods of sample processing will likely result in varying compositions of the materials being investigated, and cause differing results. Furthermore, many reports did not analyse the morphology of the vesicles being studied, in particular their size, making it difficult to determine exactly what types of vesicles were investigated. Therefore, a consensus on sample preparation is required to ensure that the same vesicle types are being investigated between groups.

Despite these discrepancies, the findings of these studies collectively demonstrate that MVs are specifically released by host cells during malaria infection, and that these MVs mediate a variety of pathological effects. Their correlation with increased inflammation and iRBC cytoadherence indicates that MVs might be important causative agents of severe cerebral pathogenesis in particular. Currently, it remains difficult to determine whether malaria-associated inflammation causes increased MV release, and the MVs themselves are key mediators of pathogenesis; or if the malaria-associated pathology occurs first and the MV release is a secondary outcome of activated cells. Further work, in particular more detailed proteomic and genomic analyses of human-derived MV content, is still required to tease out effects that might be causative versus correlated between host-derived MV release and severe malaria.

Mouse models of malaria have allowed further understanding of the origins, roles, and effects of EVs in Plasmodium infection. Although host-derived EVs have been extensively studied in malarial disease, there have also been investigations into EVs of parasitic origin. A report by Couper et al. showed that MVs from the serum of P. berghei ANKA-infected mice had a strong pro-inflammatory effect, stimulating macrophage CD40 surface expression and TNF release in a dose-specific manner [46]. Furthermore, these MVs were mostly of iRBC origin because they contained P. berghei antigens. Using a variety of knock-out mice, the authors demonstrated that the stimulatory effects of MVs were not due to a generalized inflamed state, but specific to high density parasitic infection. Furthermore, the stimulatory effect of MVs was found to be dependent on TLR signaling, as there was no response from MyD88-/- and TLR4-/- macrophages, indicating that ligands that stimulate the TLR pathway are present in MVs [46]. However, this study only looked at TNF release and CD40 upregulation as a measure of macrophage activation, and it is likely that other pathways (including other TLRs) are also activated by MVs and induce release of cytokines that were not tested for.

Much of the work into EVs in malaria has focused on MVs, rather than exosomes. However, a study on mice infected with Plasmodium yoelii 17X, which preferentially invades reticulocytes, similarly to P. vivax, showed that infected reticulocytes released EVs that had exosome-like markers and contained parasite-derived proteins [47]. They also reported that immunization of mice with the iRBC-derived EVs induced iRBC-specific antibodies and protection from lethal infection. Furthermore, this was the first demonstration that exosome-like vesicles, rather than the larger MVs, are released from Plasmodium-parasitized cells.

To date, there has been only one study of Plasmodium-derived EVs from human field samples. Nantakomol et al. investigated RBC-derived MVs in patients infected with P. falciparum, P. vivax and Plasmodium malariae [48]. The authors distinguished MVs of iRBC origin from MVs of uninfected RBCs based on the presence of parasite protein RESA, which is inserted on the membrane of parasitized RBC, and showed that iRBC released >tenfold more MVs than uninfected RBC, suggesting an active shedding of vesicles from iRBC. In agreement with previous work, MV numbers increased upon malaria infection, and high MV concentrations correlated with higher parasitaemia and severe disease. This study also investigated P. falciparum MV release in vitro, and showed that MV release increased as the parasite matured within the RBC. Additionally, the authors linked induction of MV release to the presence of hemin or parasite products, but this was only shown for uninfected RBC and not with iRBC. This work demonstrated that MVs were released from various human-infective Plasmodium species in vivo, and that these contained parasite-derived proteins. However, there are still no reported investigations into what biological functions these Plasmodium-derived EVs might have during active infection.

The majority of reports on EVs in human malaria investigated host-derived MVs, but two recent studies have looked at the function of parasite-derived rather than host-derived vesicles, and independently reported that P. falciparum EVs can mediate cell–cell communication between parasites [49, 50]. Regev-Rudzki et al. utilized genetically modified parasites to demonstrate intra-parasitic exchange of genetic material via vesicles. They co-cultured two different parasite strains, each containing plasmids encoding different fluorescent markers and separate drug resistance genes (for resistance to WR99210 or blasticidin), in the presence of both WR and blasticidin. They found that, despite each strain having resistance exclusively to one drug, parasites were able to survive treatment with both drugs, and the next generation of parasites harbored both drug resistance genes and fluorescent markers. This plasmid transfer was found to occur via extracellular exosome-like vesicles released from the iRBC, and plasmid transfer was observed to occur most efficiently during early ring stage of the asexual life cycle. The authors showed that drug resistance could be transferred to drug-sensitive iRBC by incubation with vesicles purified from the co-culture supernatants. Furthermore, they demonstrated that induction of this vesicle-mediated cell–cell communication also led to increased gametocytogenesis in vitro, suggesting that parasite-derived EVs might provide a means of quorum sensing to trigger gametocytogenesis.

Mantel et al. extensively described P. falciparum MV production and composition. In contrast to Regev-Rudzki et al. they investigated vesicles produced by late-stage trophozoites/schizonts that were larger in size. The MVs were profiled by proteomics and Western blotting, and were found to contain parasite-specific proteins, such as SBP1 and RESA, indicating that these MVs are of parasite origin, though the contribution from host cells in this process remains unclear. Similar to Regev-Rudzki et al. the authors also reported that vesicles derived from P. falciparum cultures induced gametocytogenesis in recipient cultures. However, it is important to note that these two studies used different methodology to isolate vesicles, and therefore the EVs investigated for each study could represent different subpopulations. Furthermore, the method by which vesicles induced gametocytogenesis was not determined in either study. Moreover, Mantel et al. demonstrated that when monocyte-derived macrophages were stimulated with MVs, they upregulated the expression of IL-1β, IL-6, IL-10 and IL-12, and specifically released IL-10 and TNF, showing that vesicles had an effect on host cells.

Further in vitro studies by Mantel et al. [51] revealed that P. falciparum iRBC-derived EVs could affect host endothelial cells through transfer of functional microRNA. Specifically, the authors showed that EVs from iRBC contain host-derived microRNA and Argonaute2 protein, a member of the RNA-induced silencing complex, and that these EVs can be taken up by human endothelial cells. It was further demonstrated that EV-delivered microRNAs reduced expression levels of target proteins and affected the barrier function of endothelial cells. This work suggests a potential targeted effect of EVs on host cell function, which could have important implications for the understanding of clinical disease. Still, the role of parasite-derived vesicles in P. falciparum biology and host-related functions in vivo remains largely undetermined.