The Role of Extracellular Vesicles in Immunomodulation and Pathogenesis of Leishmania and Other Protozoan Infections

Abstract

Extracellular vesicles (EVs) have lately emerged as crucial mediators in parasite infections. Recent research suggests that protozoan parasites, including Leishmania, employ EVs as transport vehicles to deliver biologically active effector molecules such as parasitic virulence factors to modulate the host immune system and their microenvironment. The immunomodulatory effects of EVs play an essential role in the formation and progression of parasitic diseases. The immunomodulatory strategies applied by EVs of protozoan origin have similarities to the development and progression of other infections or diseases such as cancer. In this chapter, we will provide recent insights into the role of EVs in host-pathogen interactions, intercellular-communication, immunomodulation and pathogenesis of Leishmania and other protozoan parasites, including Plasmodium spp., Toxoplasma spp. and Trypanosoma spp. In addition, biologically inspired by the immunomodulation strategies of protozoan parasites, new immunotherapeutic models are being currently investigated to implement EVs more intensively in both therapy and diagnostics. Therefore, besides highlighting the role of EVs in protozoan infections, this chapter sheds light briefly on new immunotherapeutic approaches utilizing the strategies of protozoan EVs in medicine.

1. Introduction

Cellular communication is essential for all life forms to observe, comprehend and affect their surroundings [1, 2, 3, 4, 5, 6]. One pathway that cells employ for the transfer of information is the use of extracellular vesicles (EVs) – lipid-bilayered secreted vesicles that carry lipids, nucleic acids and proteins that can cause physiological changes in other cells. The use of EVs for cellular communications is a highly conserved process of life. The EV secretion was observed in all types of cells and organisms studied up to date, including plants [7, 8, 9], prokaryotes [3, 10, 11] and protozoans [12, 13, 14, 15, 16, 17, 18, 19, 20, 21]. Moreover, evidence suggests that EVs can affect cells of different species, even across different kingdoms [10, 11, 13, 16, 20]. Cross-kingdom EV interactions were shown to take part in the pathogenesis of some parasitic diseases such as those caused by protozoan parasites [22, 23].

Protozoan parasites, also known as first animals, are single-celled organisms that display diversity among unicellular eukaryotic organisms with a complex life cycle on the host system [20]. They have developed many strategies not only to provide their survival and reproduction, but also to enable the invasion into the hosts by means of immune strategies including change in host antigens, development of self-tolerance, immune inactivation, immunosuppression and intervention of molecule-mimetic mechanisms between parasites and host antigens [16, 24, 25]. Recent studies propose that the parasites actually utilize the extracellular vesicles as one infection strategy [18, 20, 21, 26, 27, 28, 29, 30, 31], where the questions are arisen on how EVs modulate the host immune system and ultimately cause the infection. Based on the cell of origin, the release mechanisms of EVs from different protozoan parasites, including Apicomplexa and Kinetoplastids such as Leishmania species (spp.) [22, 23, 26, 32, 33, 34, 35], Plasmodium spp. [31, 36, 37, 38, 39, 40, 41], Toxoplasma spp. [36, 42, 43] and Trypanosoma spp. [44, 45, 46, 47, 48, 49] were described, where the parasitic infections were studied in detail for leishmaniasis, malaria, toxoplasmosis and Chagas disease independently.

Among the many species and subspecies of protozoa, Leishmania are digenetic intracellular protozoan parasite that cause leishmaniasis through the localization either in mononuclear phagocytes of vertebrates as amastigote form or in the sandfly vector as promastigote form. There are three main forms of leishmaniasis, including a localized form- cutaneous leishmaniasis (CL) or mucocutaneous leishmaniasis (MCL), and a life-threatening form – visceral leishmaniasis (VL) (also known as “Kala-azar”) [50].

The EVs released from parasites or infected cells play a significant role in host-pathogen communications and thus contribute to pathogenesis [12, 13, 15, 16, 18, 19, 20, 21, 51]. Studies indicated that Leishmania exosomes can modulate the host immune system through monocyte cytokine production occurring in response to Leishmania infection, which in return further exacerbates Leishmania infection [14, 21, 22, 23, 26, 32, 33, 34, 35, 52, 53, 54]. Likewise, Evs’ role in the occurrence of infection was also confirmed later for more protozoan family members such as Plasmodium spp. [31, 36, 37, 38, 39, 40, 41], Toxoplasma spp. [36, 42, 43] and Trypanosoma spp. [44, 45, 46, 47, 48, 49], which further directed the attention of researchers on protozoan EVs and their mechanism of action.

This chapter largely focuses on the role of EVs in Leishmania-host interaction, immunomodulation of the host immune system by Leishmania EVs, manipulation of the cellular microenvironment in favor of Leishmania species. In addition, the role of EVs in the pathogenesis of other protozoan parasites including Plasmodium spp., Toxoplasma spp. and Trypanosoma spp. are discussed and compared at the biological level to get a better insight on strategies in immunomodulation mechanisms. At the end of the chapter, novel and potential immunotherapeutic approaches utilizing the strategies of protozoan EVs are briefly discussed.

2. Extracellular vesicles (EVs)

Extracellular vesicles are nano-sized messengers secreted by all cell types. They consist of a lipid bilayer membrane, proteins, nucleic acids and other biomolecules, which together make up the “message” to be conveyed to other cells. The composition of molecules that control the message differs in different cell types, and under different physiological conditions.

EVs’ size ranges between 20 and 1000 nm in diameter, and they can be produced through a variety of different biogenesis pathways, with different physical and structural properties. Budding from the cellular membrane generally forms larger vesicles called microvesicles – however, this biogenesis pathway may also form vesicles that are smaller than 200 nm. Small extracellular vesicles can also be formed through the invagination of the cellular membrane into endosomes, collected and secreted together in multivesicular bodies (MVBs), or so-named exosomes [55]. However, it should be noted that most of the EV isolation methods used today cannot separate exosomes from small EVs formed through membrane budding, resulting in mixed populations of EVs in the working medium. The full extent of the biogenesis pathways remains to be unknown to researchers, and this is even more apparent in non-mammalian EVs [12]. However, evidence indicates that parasites secrete EVs through both the membrane budding and the multivesicular body pathways, mimicking the previously studied EV secretion pathways of mammalian cells [45].

3. Immunomodulation and pathogenesis by EVs from Leishmania species and other protozoan parasites

While the study of EVs in eukaryotes other than mammalians has been gaining momentum, the methods used in these studies were developed with mammalian EVs in mind. The International Society for EVs has listed the minimal requirements for categorizing a particle as an extracellular vesicle as reporting the size distribution of the population at a single-vesicle resolution, and detecting the presence of transmembrane and cytosolic proteins in the sample while testing for a non-vesicle related protein as negative control [6, 12]. While the physical characteristics of non-mammalian EVs do not differ greatly from their mammalian counterparts, the literature lacks the necessary amount of data to decide on protein biomarkers for most non-mammalian samples. These experimental results are also required for the characterization of Leishmania EVs and other protozoan parasites, including Plasmodium spp., Toxoplasma spp. and Trypanosoma spp.

3.1 Leishmaniaspecies (spp.)

Leishmania spp. are protozoan parasites belonging to the Trypanosomatidae family in the Kinetoplastidae order, belonging to the characteristics of a kinetoplast. They are obligated intracellular parasites that primarily infect macrophages in the mammalian through the transmission of the bite of an infected sand fly and cause leishmaniasis. Moreover, they are digenetic organisms that survive and replicate either as the promastigote, i.e., the extracellular form existing in the insect midgut or as the amastigote, i.e. intracellular form lodged within phagolysosome-like vacuoles inside the macrophages [50, 56].

The promastigote form of parasites inoculate in the dermis by the bite of a sandfly (Lutzmoyia spp., Phlebotomus spp.) are thought to infect macrophages and/or dendritic cells (DCs) of the skin where they transform into amastigotes and might protect their host cell from apoptosis [25]. Studies have shown that exosomes released from Leishmania spp. promastigote and amastigotes play a crucial role in host-pathogen interactions and intercellular communication, leading to the development of infection (pathogenesis) and immunomodulation [14, 21, 22, 23, 26, 32, 33, 34, 35, 52, 53, 54].

3.1.1 Leishmaniasis

Leishmaniasis is a neglected tropical disease caused by vector-borne parasites of the genus Leishmania. There are over 20 species of Leishmania that cause life-threatening disorders widely distributed in 98 tropical and subtropical regions including Asia, South America, Northern Africa, Southern Europe and the Middle East. According to the recent WHO report, more than 350,000 people are estimated at risk and 1.3 million new cases of leishmaniasis occur every year [50].

Leishmaniasis can be grouped into three main clinical forms: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), also known as “Kala-azar”, and mucocutaneous leishmaniasis (MCL), depending on which species is involved in the infection [50]. CL is a benign but often disfiguring condition that is caused by the multiplication of Leishmania in the phagocytes of the skin and has a tendency toward spontaneous resolution. The coexistence of these clinical forms in the same patient is rare. MCL is a metastatic form of localized CL infections occurring during the first episode of CL within 5 years. Lymphatic or hematogenous dissemination of the amastigotes from the skin to the naso-oropharyngeal mucosa results in the destruction of the nose and mouth to the pharynx and larynx. Untreated infections can result from severe disfiguration or even death. VL is a severe condition that results from the dissemination of Leishmania in the phagocytes, mainly macrophages, and is fatal in almost all cases if left untreated. VL is characterized by irregular bouts of fever, substantial weight loss, swelling of the spleen and liver and serious anemia [50].

The outcomes of the infection are highly dependent on both host and pathogen factors involved in a molecular battle where the fittest survive and continue. In this context, it is well established that macrophages play an important role in defense against various parasites by regulating their invasion and progression within the potential host. However, like other pathogens, most Leishmania species have developed effective strategies to circumvent the innate immune response in the early moments of infection, provided by rapidly blocking the induction and regulation of major host cell functions including nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12), radical oxygen species (ROS) [57, 58, 59, 60].

Recent studies have investigated that EVs released from Leishmania can involve in the pathogenesis by delivering the virulence factors – GP63, Elongation Factor 1-alpha (EF-1α) and others – to mammalian host cells, modulating their microenvironment and inferring on host signaling pathways [26, 34, 61, 62].

3.1.2 Secretion of EVs containing Leishmania proteins

EVs carry biological messages in the form of the lipids, proteins and nucleic acids they are composed of. Both the cargo enclosed within the EV and the structural molecules of the EV itself can initiate cellular responses. The lipids and membrane proteins of EVs are capable of interacting with the surface receptors of a recipient cell, allowing the EV to initiate cell-to-cell contact-dependent responses by acting as a surrogate to their cell-of-origin. Cells tailor the cargo of their EVs for them to initiate the desired response on recipient cells [55].

Protein interactions are one of the primary ways for EVs to affect target cells. Hence, the proteomic analysis of protozoan EVs becomes crucial in determining Evs’ biological functions. Proteomic analysis indicate that parasite EVs are enriched in proteases [33, 45, 63, 64, 65], stress response proteins [45, 64, 66] and transcription factors [45, 67].

One of the most common types of proteins found in parasite EVs are proteases. Proteases are a large family of hydrolytic enzymes that take part in a large majority of biological processes. Through the breakdown of specific peptides, proteases allow the activation and removal of various proteins, regulating biological reactions associated with them [68]. Proteases are considered as one of the virulence factors of parasites increasing the infectivity by inactivating the complement system and cleaving transcription factors that aid macrophage activation. Leishmania parasites and other trypanosomatids employ Leishmania virulence factors, such as metalloprotease GP63 and other immunosuppressive proteins, as well as the ER/Golgi-mediated secretion pathway to exit the host cell post-transfection [21]. An example of this process was shown with L. mexicana, where cysteine proteases were sorted into lysosomes and subsequently released via the flagellar pocket when they reached the Golgi apparatus [21, 29].

Initial clues for the existence of EV-mediated non-conventional protein secretion in parasites came from a study of the Leishmania parasites, where hydrophilic acylated surface protein B (HSAPB) was found to be present on the parasites’ membrane despite not having a signal peptide, transmembrane domain or GPI-anchor site [21]. A study by Denny et al. discovered a novel sequence of 18 amino acids that act as a “special” signal peptide, which allows the transfer of the protein to the cellular membrane [21]. The study also showed that the transfer of HSAPB continued even after the transfection of mammalian cells, with the protein being observed on the cell surface. This non-conventional secretion pathway of proteins is a characteristic feature of EVs and is crucial for the ability of parasite EVs in manipulating the hosts’ microenvironment.

The evidence of Leishmania exosome secretion was demonstrated in the study of L. mexicana exoproteome associated with proteases [69]; however, the first report on the certain secretion of Leishmania exosomes was issued by Silverman et al. [54]. Also, proteomic analysis of parasite EVs reveals that different types of proteases are among the most abundant type of proteins in their proteome [62, 64, 65]. The enrichment of proteases in EVs occurs during the entire lifecycle of the parasites during the avirulent procyclic and virulent metacyclic phases [62]. However, metacyclic parasite EVs were shown to contain a higher concentration of proteases than EVs of avirulent procyclic parasites, suggesting a link between proteases and infectivity (34). Another study showed that Leishmania species can also hijack host proteases through plasminogen binding proteins that bind plasmin-precursor plasminogen to the parasite cell membrane. One such plasminogen binding protein, discovered in Leishmania mexicana EVs, is enolase, a highly conserved EV protein that may allow immune avoidance and parasite dissemination [63].

On the other hand, the EVs of different parasites have similar physical and biochemical properties with each other as well as with EVs of mammalian origin [54]. TEM micrographs captured the secretion of Leishmania exosomes through the fusion of MVBs with the parasite membrane [53] and orthologues to key proteins commonly associated with EV formation, such as Rab GTPases, Alix, and ESCRT proteins were found in the proteome of Leishmania EVs.

Another category of proteins commonly found in parasite EVs are stress-response proteins. Parasites face various stress conditions in both their insect and vertebrate hosts, and the proteomic profile of the parasite reflects that suitably. Oxidoreductase proteins may protect the parasite from the free radicals of the immune system [45], while chaperone proteins such as the ER chaperone glucose-regulated protein (GRP), heat shock protein 70 (HSP70) are commonly reported as upregulated in parasite EVs [45, 66]. Their presence in the EVs may be due to the elevated expression of these proteins in the parasite itself, instead of an EV-specific sorting mechanism.

Transcription and translation factors detected in parasite EVs may also have roles in parasite infectivity and resilience against stress factors [45, 67]. While it is not clear whether or not if these factors are specifically packaged into EVs for a function, or present due to their abundance in the cytoplasm, studies note that proteins such as EF 1 or 2 were shown to be pro-infective in the parasite itself [70].

A recent study indicated that Leishmania donovani infection led to a quantitative and qualitative change in the protein profile of EVs released by the infected macrophages, confirmed by mass spectrometry and western blot analysis. Through the protein analysis, 59 parasite-derived proteins in EVs were found, which promote angiogenesis by inducing endothelial cells to release angiogenesis-promoting mediators [32].

EVs’ role in exposed drug resilience of particular strains was also investigated. L. infantum strains resistant to various Leishmania drugs were found to secrete EVs with different physical and proteomic profiles and secreted more EVs than wild-type parasites [67]. Different histone and ribosomal proteins were found to be enriched in the EVs of drug-resistant strains, which might be a non-specific adaptation of the parasite to increase its fitness in general. This knowledge may be used to diagnose whether or not a patient is infected with a drug resilient strain of the parasite, and could potentially allow identification and prediction of the drug-resistance mechanism of the strain before starting the therapy [45, 67].

3.1.3 The evidence of the EVs released from Leishmania spp.

Leishmania parasites secrete EVs both in vitro and in vivo in the sandfly midgut [53] and these EVs display immunomodulatory and signal-triggering events on the host system, associating with the parasite virulence factors. Studies with mice and immune cells showed that EVs released from Leishmania spp. and infected cells may affect and contribute to the clinical form and severity of the disease regarding the multitude of factors [21].

Originally, the presence of exosomes-like vesicles secreted from Leishmania parasites was suggested in the supernatant of infected macrophage cultures by proteomic analysis of the secretome of Leishmania donovani [64]. Silverman and colleagues proposed that L. donovani utilizes the alternative non-classical secretion pathways and targeting mechanism rather than the classical secretion signal to direct the secreted protein export [64]. Based on this study, exosomes from Leishmania parasites are involved in the delivery of proteins into host target cells [54, 64].

On the other hand, the first report on the release of the exosomes from the protozoan pathogens and their use as a vehicle for protein secretion and uptake by macrophages was established by Silverman et al. [30]. This study demonstrated that L. donovani and L. major can release exosomes that were detected in cytosol of the infected macrophages and selectively induced secretion of IL-8 from macrophages [30]. Furthermore, exosome release was significantly detected in the culture supernatant of L. donovani, L. mexicana and L. major spp., under high temperature (37°C) and low pH in which condition required for promastigote differentiation into amastigotes. In another study, using Leishmania expressing green fluorescent protein (GFP), they found a release of Leishmania GFP+ vesicles into infected cells and an uptake fluorescence vesicles by non-infected cells, with the collection of GFP and parasite proteins in structures consistent with MVBs within the cytosol of infected macrophages [30].

In addition to studies on EVs from Leishmania within mammalian hosts, the secretion of EVs from Leishmania residing within the sandfly midgut was also demonstrated by Atayde et al. [53]. Moreover, the detailed characterization of EVs isolated from infected sandfly midguts was investigated. Leishmania EVs isolated from infected sandfly midguts were also compared with previously described in vitro-isolated Leishmania EVs.

3.1.4 Host manipulation and immunomodulation by EVs from Leishmania spp.

Leishmania inhibits normal macrophage functions and also interferes with the innate and acquired (both cell-mediated and humoral) immunity [60]. The uptake of promastigotes by the host-immune cells involves several different strategies that allow the parasite’s protective mechanism to evade their immune systems [71]. To survive and evade the host defense mechanism, transmission begins with the differentiation of the intracellular amastigote form of Leishmania that replicates within macrophages in the vertebrate hosts to the extracellular promastigote form in the sandfly vector [60, 72].

Briefly, the life cycle of Leishmania begins with an infection of the female sandflies after ingesting blood meal in Leishmania-infected vertebrate hosts, as illustrated in Figure 1. In the sandfly vector, within the midgut, ingested amastigotes proliferate and then migrate to the foregut to differentiate into metacyclic promastigotes presented on the salivary glands of the sandfly vector. Once delivered to a vertebrate host by the bite of an infected sandfly, promastigotes attach to phagocytic cells, macrophages, and are readily engulfed. Parasite-containing parasitophore vacuoles fuse with lysosomes forming a “phagolysosomes” in which promastigotes differentiate into the vertebrate stage, a flagellate form of amastigote [60, 73] (Figure 1). When a sandfly ingests a blood meal from an infected host, amastigotes differentiate back into promastigotes and become metacyclic. The metacyclic promastigotes that inoculate in the dermis by the bite of a sandfly (Lutzmoyia spp., Phlebotomus spp.) are thought to infect macrophages and/or DCs of the skin, where they transform into amastigotes into macrophages and might protect their host cell from apoptosis [74].

Figure 1.

The lifecycle of Leishmania parasites. Biorender software was used to create this figure under an academic license.

Once Leishmania metacyclic promastigotes (infective form) with sandfly saliva components are delivered into the mammalian hosts by an infected sandfly, promastigotes have to evade the complement-mediated cell-lysis before being eliminated by phagocytosis and must survive the impact of the innate immune system (Figure 2). For phagocytosis, macrophages are the main immune population involved in the elimination and clearance of the parasites. Although macrophages are the main host cell for Leishmania parasites, monocytes, DCs and neutrophils can be infected and contribute differentially to the immune response and the outcome of the infection [75] (Figure 2). As the first cell to be recruited to the infection site, neutrophils have delivered promastigotes to the macrophages through facilitating a silence entry, proposed as “Trojan Horse” [76] (Figure 2). Neutrophils infiltration and recruitment are contributed by various factors such as the leishmania chemotactic factor inducing IL-8 secretion by human neutrophils or interleukin-17 (IL-17), a hallmark of T helper 17 (Th17) inflammation in later phases of mucocutenous infection [77, 78]. Although parasites can readily be found in neutrophils, it is within mononuclear phagocytes that there is the best evidence for their replication and long-term survival. In a previous study, two-photon intravital imaging of mouse skin following needle injection of L. major has revealed that promastigotes were taken up by resident DCs like Langerhans within the first 4 h of infection and stimulating the activation of cytotoxic CD8-T cells [79]. DCs play a critical role in development of the immune response and coordinating an effector T helper 1 (Th1) adaptive immunity over the secretion of cytokines. Pro-inflammatory cytokines such as interleukin-2 (IL-2), interferon-gamma (IFN-γ) and TNF-α can activate the anti-parasitic mechanisms of the macrophages, leading to parasite inactivation and secretion of the cytokines such as IL-4, IL-5 and IL-13 to control the infection [71] (Figure 2). On the other hand, as the numbers of DCs and resident macrophages in the skin are too limited to sustain parasite multiplication, the progression of infection requires the recruitment of monocytes (Figure 2). DCs can become monocyte-derived DCs (moDCs) that express the major histocompatibility complex class II (MHC class II) molecules, which are critical for the secretion of IL-12 leading to the activation of a host-protective Th1- type response [80].

Figure 2.

The interaction of innate immune cells during Leishmania infection. Biorender software was used to create this figure under an academic license.

Several studies indicated that Leishmania exosomes can modulate monocyte cytokine production in response to Leishmania infection by influencing the innate and adaptive immune systems [22, 26, 30, 52, 54, 61] (Figure 2). Silverman and colleagues found that L. donovani exosomes could be predominantly immunosuppressive regarding cytokine responses on IFN-γ inhibition and IL-10 production by human moDCs [54]. In addition, exosomes released from heat shock protein 100 (HSP100) null Leishmania donovani in contrast to wild type L. donovani exosomes, are highly proinflammatory on immune cells, enabling the differentiation of naive CD4 lymphocytes into Th1 cells [54]. Similarly, pretreatment of mice with L. donovani- and L. major-released exosomes led to exacerbated infection and pathogenesis in vivo, related with IL-10 production and impaired generation of inflammatory Th2 cell response for parasite elimination and clearance [54].

In addition, studies on Leishmania EVs showed that EVs can involve in the pathogenesis by modulating the microenvironment of the mammalian hosts which is at a high temperature and a low pH than the midgut of the sandfly, and thus causing the disease [30, 61, 69]. Regarding the effect of the host microenvironment on Leishmania EVs, three independent studies have reported on temperature-dependent vesicle release from Leishmania spp. with different perspectives [30, 69, 81]. Accordingly, the release of L. donovani EVs was increased 3-fold by heat shocked-stationary phase promastigotes at a temperature mimicking the human body (37°C) [30]. In another study, increased temperature triggered the secretion of vesicles with the exposure of 4 h heat shocks [69]. However, contrary to temperature-induced vesicle release, Barbosa and colleagues indicated that the temperature shift (ambient temperatures of 25–26°C and 37°C) reduced the secretion of EVs from promastigotes and increasing temperature decreased parasite viability and morphology, hence affecting the release of EVs [81].

Up-regulation of EV secretion induced by infection-like temperatures suggested that these vesicles are released into the extracellular environment, before the invasion of a host such as macrophage, neutrophil, or DC occurs. These EVs may be secreted from either inoculated metacyclic promastigotes within the sand-fly salivary gland, free amastigotes in the mammalian hosts, or both [26, 32, 53, 64]. A study of Atayde et al. [53] demonstrated that in vivo secreted Leishmania EVs in the sand fly midgut were egested by the sand fly during the bite, and these vesicles may have a role in the establishment and pathology of the CL [53]. Co-injection of mice footpads with metacyclic L. major promastigotes plus midgut-isolated or in vitro-isolated L. major EVs led to a significant increase in footpad swelling, and produce exacerbated lesions up to 6 weeks post-infection through over induction of inflammatory cytokines, in particular IL-17a (which is related to neutrophil infiltration) [53, 78]. On the other hand, a recent study indicates that L. donovani infection may promote angiogenesis by inducing endothelial cells to release angiogenesis promoting mediators including IL-8, G-CSF/CSF-3 and VEGF-A. This study shows the changes in the composition of EVs from infected cells resulted from Leishmania infection and suggests that EVs from infected cells could promote the vascularization in Leishmania infections [32].

3.1.5 Host manipulation and immunomodulatory properties of Leishmania EVs associated with parasite virulence factors

Protozoan parasites have developed numerous effective strategies to improve their protective mechanisms to escape from the immune system by modulation of the hosts’ immune response and signaling pathways, as well as virulence factor secretion [20, 25, 71, 75, 82, 83, 84]. Moreover, they secrete EVs containing various parasitic factors and signaling molecules to modify the hostile microenvironment of their hosts to their benefit [26, 29, 33, 52]. By secreting EVs with proteases, parasites suppress the initial immune response raised at the point of infections for long enough to establish a foothold in their hosts [26, 29, 33, 52].

Leishmania utilizes multiple virulence factors including lipophosphoglycan (LPG) and surface acid proteinase (GP63), which trigger the modulation with the activation of protein tyrosine phosphatases (PTP), inhibition on pro-inflammatory transcription factors NF-κB, AP-1 and STAT-1 as well as other signaling molecules such as JAK-2, IRAK-1 and MAP kinases to successfully deactivate and infect on their host macrophages [52].

Together with the parasite surface molecules, multiple host cell receptors (complement receptor type 1 and type 3 (CRl, CR3), mannose-fucose-receptor, fibronectin receptor, macrophage receptor for advanced glycosylation end products) play a crucial role in the attachment and uptake of promastigotes by the immune cells [25].

Leishmania metacyclic promastigotes (infective) have to evade the complement-mediated cell-lysis via parasitic virulence factors such as GP63 and LPG, before being eliminated by phagocytosis. Moreover, they are resistant to complement activation in contrast to procyclic promastigotes (non-infective) that are extremely sensitive to the complement system, explained by the role of surface LPG. The surface LPG plays a central role in the parasite’s entry and survival in host cells. In the metacyclic promastigotes, LPG is longer than non-infective procyclic forms and is almost completely absent in amastigotes, resulted in inhibiting the attachment of the C5b-C9 complement system subunits to the parasite surface [85]. In addition, surface protein kinases were indicated to phosphorylate the complement system, therefore, hampering the cascade. The surface protein, gp63, a zinc-dependent metalloprotease, is 10-fold less abundant than LPG, as an important Leishmania virulence factor that is expressed at the surface of the parasite via a glycophosphatidylinositol (GPI) anchor, or is directly secreted to the extracellular environment. GP63 promotes parasite survival by the stimulation of immunomodulation on the macrophages, and thus, plays a crucial role in pathogenesis. Previous studies on the action of GP63 in parasitic infections reported that GP63 can protect L. amazonensis and L. major against cell-lysis by converting the C3b complement subunit into C3bi which accumulates on the surface of the parasites [85]. Fixation of C3 by the parasite increases the recognition of parasites by the macrophages’ complement receptors 1 (CR1) and complement receptors 3 (CR3) allowing intracellular survival [86]. Thus, it appears that Leishmania not only inhibits activation of the lytic membrane attack complex (CSb-C9), but instead exploits C3 for “silent” invasion of host macrophages [25].

Experiments on mice and macrophages showed that these exosomes exhibit immunomodulatory activity, confirming the presence of parasite virulence factors in their content such as the surface metalloprotease GP63 [15, 26, 30, 33, 52, 54, 69, 87]. Hassani et al. previously showed that the contents of the macrophage exosomes undergo changes following LPS stimulation or Leishmania infection. Furthermore, they indicated that exosomes released from Leishmania-infected cells display unique signatures regarding composition and abundance of several functional groups of proteins such as plasma-membrane associated proteins, chaperons and metabolic enzymes [26]. In this study, surface metalloprotease GP63 was shown in the contents of the exosomes from Leishmania-infected macrophages, which could induce signaling molecules such as MAP kinases (except JNK) and immune-related gene expression like NF-kB associated with the immune system in naive macrophages [26]. The induction of phosphorylation of signaling proteins and translocation of activatory transcription factors into the nucleus was determined within 15 min and up to 1 h after treatment of exosomes isolated from LPS and Leishmania-induced macrophages and in particular in pro-inflammatory nuclear translocation of NF-kB and AP-1 and early tyrosine phosphorylation of MAP kinases ERK and P38. So, the overall effect of macrophage-infected exosomes in naive macrophages can be claimed as the down-regulation of pro-inflammatory genes and suppression of macrophage activation.

Another study comparing the EVs of wild-type and GP63-knockout Leishmania parasites showed the importance of GP63 in the modulation of macrophage responses [52]. While the wild-type EVs were capable of downregulating several genes associated with the immune response, GP63-knockout parasite EVs alteration of immune response genes occurred in a different pattern and had significantly reduced immunosuppressive capabilities. Furthermore, the lack of GP63 altered the proteome of EVs, suggesting that GP63 may have roles in the cargo-determinacy of parasite EVs [26, 52]. In addition, evidence suggests that exosomes secreted from Leishmania-infected cells containing GP63, may down-regulate the generation of specific host miRNAs and facilitate infection of the liver [87]. In one study, EVs secreted by L. donovani were shown to reduce miR-122 activity in hepatic cells, which reduced serum cholesterol levels and increased the infectivity of the parasite. The GP63 proteins of parasites EVs were suggested as the agent behind this alteration, as they could target the miRNA processor Dicer1 [87]. All these studies indicate that EVs from Leishmania spp. display a wide range of targets in mammalian hosts and, have an immune-hampering role.

3.2 Other protozoan parasites

3.2.1 Toxoplasma spp.

Toxoplasma gondii is a globally protozoan pathogen that uses felids (cats) as their primary host. When infecting other mammalians, the parasite infects the hosts’ brain tissues, forming cysts. Infected rodents exhibit behavioral changes, such as reduced aversion of felines [88]. The effects of the parasite in humans are less understood, however, studies link T. gondii infection with neural diseases such as Alzheimer’s [89].

T. gondii EVs carry several virulence factors that aid their infectivity. In one study, complete mRNAs of neurologically active proteins, as well as various miRNAs were found in T. gondii EVs, which may have the capacity to affect the neural cells that they enter. The most enriched mRNA’s belonged to various neurologically active proteins, Rab-13, eukaryotic translation EF 1-α1, thymosin beta 4 and LLP homolog [90]. One mRNA observed in the study, e.g. eukaryotic translation elongation factor 1, was also reported to be present in Leishmania EVs and associated with autism [90, 91]. Furthermore, immunoregulatory miRNA miR23-b was observed in the EVs, which regulates the secretion of IL-17. In addition to mRNA and miRNA components, T. gondii EVs were also shown to carry several proteins under the excreted/secreted antigens family, such as surface antigens, microneme proteins, dense granule antigens and rhoptry proteins, which are known to regulate the immune response of their hosts [42, 92].

3.2.2 Plasmodium spp.

Malaria is one of the deathliest protozoan parasitic diseases in the world and the leading cause of mortality in sub-Saharan Africa. It is caused by the family of Plasmodium parasites, which are spread through infected Anopheles mosquitoes, leading to fatal conditions such as cerebral malaria or severe malarial anemia. When passed to a human, the parasite infects red blood cells, allowing it to evade the immune response and penetrate deep tissues. The infected red blood cells increase vascular permeability and cause the apoptosis of endothelial cells, which both increase the severity of the disease and facilitate the spread of the parasite throughout the body.

As with other parasites, EVs secreted by malaria parasites modulate the hosts’ immune system to increase the survivability of the Plasmodium parasite. When parasites were blocked from secreting EVs, they had reduced virulence and lessened symptoms in models of cerebral malaria [93]. Secretion of EVs continues after the infection of red blood cells. Studies show that the parasite hijacks the EV secretion in infected red blood cells, modifying their cargo. Infected red blood cells secrete EVs enriched in parasite surface antigens, and contain proteins associated with immunosuppression [94]. One study observed 120 plasmodial RNAs in infected red blood cells, which coded for proteins involved in drug resistance, as well regulatory small RNAs. The presence of these modified EVs can be used as a marker for the diagnosis of malaria [31]. In another study, infected red blood cells were shown to secrete EVs with parasite-specific proteins and RNA. Furthermore, proteins and miRNA that can alter gene expressions in endothelial cells, such as Ago2, were observed in these EVs. These infected EVs may explain malaria-associated vascular dysfunction [95].

3.2.3 Trypanosoma spp.

Trypanosomatids are insect-borne parasites that cause fatal diseases such as Chagas’ disease [96] or African trypanosomiasis, “the sleeping sickness” [97]. EVs secreted by trypanosomes were shown to increase virulence in various studies. Proteins associated with metabolism, parasite survival and virulence were observed in parasite EVs [45]. In one study, EVs of Trypanosoma brucei rhodesiense were shown to carry serum resistance-associated protein – a key protein for human infectivity- as well as flagellar proteins that increase virulence. Furthermore, the parasite EVs were shown to have the capacity to induce rapid erythrocyte clearance and anemia, suggesting a parasite-free pathogenesis pathway [44]. Another study observed that the parasite uses EVs to increase infectivity and survivability. Secreted vesicles enhanced parasite cyclogenesis, and lead to up to five times increased infection rates on susceptible cells [46].

4. EVs as diagnostic and therapeutic tools for protozoan parasitic infections

EVs offer exciting clinical opportunities in many diseases as diagnostic tools, drug delivery vehicles, or therapeutic agents – and parasitic infections are no exception. Both protozoan and host cell EVs are used in clinical applications against parasitic diseases. Moreover, immune cells infected with parasites also produce EVs that can induce inflammatory responses through the secretion of cytokines and chemokines in vitro and in vivo [21, 22, 54, 98, 99]. Considering their immunomodulatory effects, EVs could be potential vaccine candidates as components for infectious diseases [100, 101, 102, 103, 104, 105, 106].

EVs take part in the complex web of interactions that happen between immune cells. In particular, EV secreted by regulator immune cells like dendritic or T cells mimic the actions of their parental cell and prime the immune system against pathogens. When antigens of L. major are given to DCs, when administered, EVs secreted by those DCs were observed to protect mice from the parasite to great effect [100]. The EVs reduced footpad swelling and were capable of inducing antigen-specific T-cell responses [100]. A similar approach was also successful in inducing antigen-specific T-cell response against T. gondii [101, 102]. Using EVs instead of whole cells has several advantages, such as increased stability in freeze-thaw situations, and cannot alter their antigen-presentation, which may sometimes be the case with freeze-thawed DCs [103].

In addition to pulsing immune cells with protozoan antigens, protozoan EVs can also be used to induce the immune system, similar to vaccines. EVs from Plasmodium yoelii- infected reticulocytes were found to be capable of immunizing mice against the protozoan. Immunized mice were capable of producing IgG antibodies that could target the infected reticulocytes [39]. Similarly, EVs isolated from L. amazonensis-infected macrophages induce the production of the proinflammatory cytokines IL-12, IL-1b and TNF-α by neighboring macrophages, which contributes to modulate the immune system in favor of a Th1 immune response as well as the elimination of the Leishmania, and therefore, control of the infection [23].

As an image of the secreting cell, EVs have considerable potential as a diagnostic tool against parasitic diseases. The protein and miRNA cargo of EVs can allow a non-invasive biopsy of the parasite and may allow the determination of any drug resistance [104]. Regrettably, there are few examples of the use of EVs for the diagnosis of parasitic infections. One study of Trigonoscuta cruzi EV proteome revealed enrichment of antigen proteins used for the diagnosis of the parasite. Moreover, one category of proteins, retrotransposon hot spot proteins, do not cause any cross-reactivity with parasites of other diseases such as malaria, leishmaniasis or others, and may allow a definitive diagnosis of Chagas disease [105].

The natural ability of EVs to deliver cargo between cells gives makes them an attractive candidate for drug delivery applications. It has been shown that encapsulating drugs within EVs may grant them cell-specific targeting, reduced toxicity, increased circulation times and increased biodistribution with the ability to pass through tissue barriers such as the blood-brain barrier. However, the field of EV-mediated drug delivery is still at its infancy [106], with few studies done on delivering anti-protozoan drugs. The one study available to the field showed that antimalarial drugs atovaquone and tafenoquine were more effective in inhibiting the growth of P. falciparum when loaded into vesicles isolated from malaria-infected red blood cells [38].

5. Conclusion

With the expansion of knowledge in parasitic diseases, the critical function of EVs became more evident in the development of the diseases. EVs applies many strategies not only to provide the survival and reproduction of Leishmania parasites inside the host, but also to enable the invasion by means of immune strategies including change in host antigens, development of self-tolerance, immune inactivation, immunosuppression and intervention of molecule-mimetic mechanisms between parasites and host antigens [16, 24, 25]. Recent studies propose that the parasites actually utilize the EVs as one infection strategy [18, 20, 21, 26, 27, 28, 29, 30, 31], where the questions are arisen on how EVs modulate the host immune system and ultimately cause the infection. Based on the cell of origin, the release mechanisms of EVs from different protozoan parasites, including Apicomplexa and Kinetoplastids such as Leishmania species (spp.) [22, 23, 26, 32, 33, 34, 35], Plasmodium spp. [31, 36, 37, 38, 39, 40, 41], Toxoplasma spp. [36, 42, 43] and Trypanosoma spp. [44, 45, 46, 47, 48, 49] were described, where the parasitic infections were studied in detail for leishmaniasis, malaria, toxoplasmosis and Chagas disease independently.

Several studies indicated that Leishmania exosomes can modulate monocyte cytokine production in response to Leishmania infection by influencing the innate and adaptive immune systems using parasitic virulence factors [22, 26, 30, 52, 54, 61]. Silverman and colleagues found that L. donovani exosomes could be predominantly immunosuppressive regarding cytokine responses on IFN-γ inhibition and IL-10 production by human moDCs [54]. In another study, macrophage-infected exosomes in naive macrophages were shown to downregulate the pro-inflammatory genes and suppression of macrophage activation [26]. Similarly, EVs secreted by the malaria parasite modulate the hosts’ immune system to increase the survivability of the Plasmodium parasite. When parasites were blocked from secreting EVs, they had reduced virulence and lessened symptoms in models of cerebral malaria [93].

In addition to cytokine response, studies indicated that EVs can involve in the pathogenesis by modulating the microenvironment of the mammalian hosts which is at a high temperature and a low pH than the midgut of the sandfly and thus causing the disease [30, 61, 69]. Up-regulation of EV secretion induced by infection-like temperatures suggested that these vesicles were released into the extracellular environment, before the invasion of a host such as macrophage, neutrophil or DC occurs.

While EVs play such a multifaceted role in immunomodulation and disease development at protozoan diseases, the application potential of EVs as therapeutic agents or drug delivery vehicles in therapy or as a biomarker at diagnostics attracts the researchers’ attention working on these fields. Considering their immunomodulatory effects, EVs could be potential vaccine candidates as components for infectious diseases [100, 101, 102, 103, 104, 105, 106] and the application of protozoan EVs in the clinic may be expected in the near future.

Acknowledgments

The authors thank Department of Genetics and Bioengineering Yeditepe University for financial support.

Conflict of interest

No conflict of interest was declared by the authors.

Abbreviations

CRl	Complement receptor type 1
CR3	Complement receptor type 3
CL	Cutaneous leishmaniasis
DC	Dendritic cell
EF 1-α	Elongation factor 1-alpha
EVs	Extracellular vesicles
HSAPB	Hydrophilic acylated surface protein B
GFP	Green fluorescent protein
IL	Interleukin
IFN-γ	Interferon-gamma
L.	Leishmania
Leishmania spp.	Leishmania species
LPG	Lipophosphoglycan
MHC class II	Major histocompatibility complex class II
moDCs	monocyte-derived dendritic cells
MCL	Mucocutaneous leishmaniasis
NO	Nitric oxide
PTP	Protein tyrosine phosphatases
ROS	Radical oxygen species
Th1	T helper 1
Th17	T helper 17
TNF-α	Tumor necrosis factor-alpha
T. gondii	Toxoplasma gondii
VL	Visceral Leishmaniasis