*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, 82–84]. 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 complementmediated 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

### *The Role of Extracellular Vesicles in Immunomodulation and Pathogenesis of* Leishmania*… DOI: http://dx.doi.org/10.5772/intechopen.101682*

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 *The Role of Extracellular Vesicles in Immunomodulation and Pathogenesis of* Leishmania*… DOI: http://dx.doi.org/10.5772/intechopen.101682*

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].
