**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** *Leishmania species (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, 21–23, 26, 32–35, 52–54].

#### *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) [57–60].

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

Evs' biological functions. Proteomic analysis indicate that parasite EVs are enriched in proteases [33, 45, 63–65], 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 *The Role of Extracellular Vesicles in Immunomodulation and Pathogenesis of* Leishmania*… DOI: http://dx.doi.org/10.5772/intechopen.101682*

(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

#### **Figure 1.**

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

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].

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

#### **Figure 2.**

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

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

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].

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 shockedstationary 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].
