Extracellular Vesicles Released by Leishmania : Impact on Disease Development and Immune System Cells

Leishmania spp. release extracellular vesicles (EVs) containing parasite molecules, including several antigens and virulence factors. These EVs can interact with the host cells, such as immune cells, contributing to the parasite–host relationship. Studies have demonstrated that Leishmania -EVs can promote infection in experimental models and modulate the immune response. Although the immunomodulatory effect has been demonstrated, Leishmania -EVs can deliver parasite antigens and therefore have the potential for use as a new diagnostic tool and development of new therapeutic and vaccine approaches. This review aims to bring significant advances in the field of extracellular vesicles and Leishmania , focusing on their role in the cells of the immune system.


Introduction
The host-parasite communication and the parasite's intercellular interactions are crucial in the life cycle of the Leishmania parasites [1,2]. In addition, several bioactive molecules released by the parasites have shown an important role in the parasite's adaptation in the host [3]. In mammalian hosts, molecules released by Leishmania contribute to the parasite's infectivity and the physiopathology of the leishmaniasis, acting by several mechanisms, such as subverting the immune response and favoring the intracellular multiplication of the parasite [3].
Several works have demonstrated that Leishmania species can release proteins and other molecules in extracellular vesicles (EVs) [4][5][6]. EVs is a generic term used to describe particles spontaneously released by prokaryotic and eukaryotic cells [7]. Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, lipids, and cellular metabolites are present in EVs that can deliver information from one cell to another [8]. Thus, EVs are now considered a new mechanism of intercellular communication [7]. pathophysiology of several diseases. In addition, there is a great expectation of the applications of EVs in diagnostics, treatments, and vaccine development.
Currently, there is a consensus that EVs play an important role in cell-cell communication being a vehicle for transporting molecules between cells, even cross-kingdom [8,18,20]. The effects on the recipient cells depend on the cell type, the origin of EVs, their content, and EVs can act locally and/or systemically. The changes in the recipient cells include modulation of the intracellular signaling pathways, gene regulation, post-transcriptional regulation, activation, or inhibition of different cell types [21][22][23]. After target cell recognition, EVs can interact with surface receptors, followed by fusion with the plasma membrane for releasing their content, and signaling different intracellular events. However, EVs can also be endocytosed by target cells or collapse after their secretion, delivering their contents into the intracellular space [8,15].
In parasitic diseases, EVs have brought an exciting field to investigate since they can act as mediators in parasite-host interaction, allowing the transfer of virulence factors and effector molecules from the parasites to the host [24][25][26]. Parasites EVs are related to the pathogen adhesion, the spread of the parasites, and play a role in regulating the host's immune system. In addition, immune cells infected and/ or stimulated with parasite components can release EVs [23] containing messenger RNA (mRNA), small noncoding RNAs (microRNA), chromosomal and mitochondrial DNA, retrotransposons, parasites antigens, and major histocompatibility complex (MHC) I and II [23,27]. The effects in immunity are diverse, including modulation of innate immune response and antigen presentation.
The production and releasing of EVs by parasites or parasitized cells have been described and characterized in several parasitic infections [25]. For example, in Leishmania, several biological markers and virulence factors have been described in EVs released by the parasites [10,28]. Thus, EVs released by these pathogens can have a role in the disease progression and the host's immune response to the parasite, contributing to the strategy to bypass the immune system.

EVs released by Leishmania spp
Leishmania species can release proteins and other molecules in EVs. Although the mechanisms for exosome/MVs secretion in Leishmania are still unclear, proteomics analysis of EVs has shed light on the functions and properties of these particles. Initial work showed that Leishmania donovani could use EVs as a protein transport vehicle [29]. Additional studies confirmed that L. donovani releases EVs. Leishmania major, Leishmania mexicana, and Leishmania amazonensis also used EVs as an important mechanism for protein secretion [4,5,30]. The presence of EVs in the intestinal lumen of sandflies and their release together with the parasites during the blood meal reinforce the hypothesis that these EVs contribute to the process of infection and development of leishmaniasis [6].
The release of EVs by Leishmania is related to the temperature. Promastigotes of L. mexicana and L. donovani increased the release of EVs after parasite cultivation at 37°C (mammalian host temperature), compared to the EVs obtained from parasites incubated at 26°C (vector temperature) [30]. Furthermore, to L. donovani, differences in the content of the EVs obtained at 37°C and 26°C [4] were also observed, suggesting a possible parasite strategy for establishment in the host. However, L. amazonensis showed a different pattern in EVs releasing since a higher number of particles were detected after cultivation at 26°C, compared to the parasite incubated at 34°C or 37°C [5]. Altogether these observations suggest that Leishmania species can adapt differently to the release of EVs.

Leishmania -One of the Most Diverse Anthroponotic Parasitic Diseases
Proteomic studies showed the presence of the metalloprotease GP63 in EVs released by Leishmania cultivated in vitro and by the parasite infecting sandflies. GP63 is the main surface glycoprotein of Leishmania and is considered a virulence factor since it contributes to the parasite escape of immune response [31][32][33][34]. Evaluating the proteomic profile of EVs released by Leishmania infantum in three different phases (logarithmic, stationary, and metacyclic stages) showed that the metacyclic phase had a higher abundance of GP63. In contrast, EVs of parasites in the logarithmic phase had the lowest abundance [35]. In a similar approach, higher concentrations of GP63 were detected in EVs released by L. infantum in the stationary phase while parasites in the logarithmic phase showed enrichment of ribosomal proteins [36]. However, proteomic analysis of EVs from Leishmania infantum chagasi showed no significant biological differences in EVs released by parasites in logarithmic or stationary phases [37].
Besides GP63, other proteins have already been identified in L. donovani-EVs, such as elongation factor-1α (EF-1α), fructose-1,6-bisphosphate aldolase FBA, HSP70, and HSP90 [4]. A comparative study of L. infantum-EVs from drug-resistance parasites identified differences in their morphology, size, distribution, and protein content. Identifying proteins related to drug resistance in EVs from resistant parasites can bring new possibilities to predict prognostics and treatments in leishmaniasis [38].
The presence of small noncoding RNAs was identified in EVs released by L. donovani and Leishmania braziliensis, suggesting the regulatory role of these EVs in the host cells [39]. Additional studies to address the EVs content from different Leishmania species may clarify the role of these particles in visceral and cutaneous leishmaniasis. Furthermore, these studies may provide the use of Leishmania-EVs in diagnostics, the development of a vaccine, and promising therapeutic alternatives.

Leishmania-EVs and immune response
Some evidence have pointed that Leishmania-EVs present immunomodulatory effects, altering the immune response and contributing to the disease progression. The treatment of human monocytes with L. donovani-EVs induced the production of interleukin 10 (IL-10) and inhibited the tumor necrosis factor-alpha (TNF-α) production, even after challenging with interferon-gamma (IFN-γ) [11]. Similar effects were observed in dendritic cells (DC) treated with these EVs since the production of cytokines IL-12p70, TNF-α and IL-10 were inhibited and there was impaired in the ability of these cells to stimulate the differentiation naive CD4 T lymphocytes into T helper 1 (Th1) profile [11]. On the other hand, EVs released by L. amazonensis increased the expression of IL-10 and IL-6 in bone marrowderived macrophages (BMDM) [5]. In fact, EVs released by different Leishmania species seem to induce different responses in human macrophages [40]. EVs from L. infantum and L. braziliensis failed to induce an inflammatory response in human macrophages. However, L. amazonensis-EVs stimulated human macrophages to produce nitric oxide (NO), TNF-α, IL-6, and IL-10 via Toll-like receptor 4 (TLR4) and TLR2 (Figure 1A) [40].
Few studies have proposed mechanisms of intracellular signaling pathways activated by Leishmania-EVs into phagocytes cells. EVs released by L. amazonensis amastigotes containing DNA fragments were capable of inducing the CD200 expression in macrophages [41]. The high expression of this molecule leads to the inhibition of NO production, contributing to the parasite survival [41]. In addition, evidence suggests that the composition of EVs can influence the outcome of cell signaling. Leishmania EVs-containing Leishmania RNA virus (LRV1) released by Leishmania guyanensis trigger TLR3/TRIF (TIR domain-containing adaptor inducing interferon-β signaling), inducing inflammatory cytokines (pro-IL-1b, TNF-a, and IL-12), and the autophagy by impairing NLRP3 (NOD-, LRR-and pyrin domain-containing protein 3) inflammasome network [42,43] (Figure 1B). Thus, these initial studies demonstrated a refined and complex intracellular signaling pathway induced by EVs, which depends on the species and evolutionary form that is releasing the EVs and the presence or absence of Leishmania virus. [44]. Experiments using CD1d specific ligands (glycolipid α-GalactosylCeramide (α-GalCer) suggest that lipids present in L. infantum-EVs and other exocomponents released by the parasites may compete for the CD1 binding site, inhibiting iNKT activation [44]. In addition, our group showed that murine B-1 cells (a subtype of B lymphocytes) stimulated with EVs released by L. amazonensis produced higher levels of NO, compared to non-stimulated B-1 cells [45]. The increase in the expression of TLR-9, TNF-α, and transcriptional factors related to the differentiation of B-1 cells to phagocytes are important changes observed in B-1 cells treated with L. amazonensis-EVs [45]. These data suggest that Leishmania-EVs participate in the modulation of different cells and different levels of the immune response. Interestingly, some mechanisms seem conservated between species, but some specifies are related to Leishmania species making comparative studies necessary.

Besides macrophages and DCs, Leishmania-EVs can modulate other immune cells. EVs released by L. infantum inhibited the expansion of peripheral iNKT (Invariant Natural Killer T) cells and the production of IL-4 and IFN-γ by this cell type
In a mammalian host, Leishmania is an intracellular parasite. Thus, studying changes in infected cells can provide important information about the parasite's biology. Silverman et al. [4] showed Leishmania exosomes and exosomal proteins in the cytosolic compartment of infected macrophages. In addition, EVs released by macrophages infected with L. mexicana containing GP63, and this finding instigated the investigation to uncover the role of these EVs in immunity [46]. Naïve macrophages exposed to EVs from L. mexicana-macrophages infected cells induced the activation of mitogen-activated protein (MAP) kinases (except c-Jun N-terminal kinase -JNK) and the nuclear translocation of nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) [46]. BMDM infected with L. amazonensis released EVs which were able to activate naive macrophages to produce proinflammatory cytokines IL-12, IL-1β, and TNF-α, contributing both to modulate the immune system in favor of a Th1 immune response and to the elimination of the Leishmania, leading, therefore, to the control the infection [47] (Figure 1B). Thus, infected macrophages are able to release EVs that deliver information to activate naïve macrophages, contributing to activate an innate immune response.
Evidence suggests that EVs released by Leishmania-infected cells can stimulate different cells, promoting a response against the parasite. EVs released by macrophages infected with L. donovani stimulated endothelial cells to produce granulocyte colony-stimulating factor (G-CSF)/CSF-3, and vascular endothelial growth factor A (VEGF-A), promoted an increase in epithelial cell migration and induced endothelial cell tube formation [48] (Figure 1B). A study with EVs released by B-1 cells infected with L. amazonensis showed the impact of these EVs on naive macrophages activation and the protective effect on the experimental infection with the parasite [49]. Macrophages treated with EVs from infected peritoneal B-1 cells alter the expression of inducible nitric oxide synthase (iNOS), IL-6, IL-10, and TNF-α [49]. Overall, these studies demonstrated that Leishmania infection changes the content of EVs from infected cells and suggest that these EVs participate in the activation of immune and nonimmune cells, actively participating in the pathophysiology of the Leishmania infection.

EVs and leishmaniasis progression
Experimental models have contributed to better understanding the role of EVs in the leishmaniasis progression. The treatment of mice with L. donovani-EVs before the parasite infection exacerbated the infection and induced IL-10 production in the spleen [11]. Furthermore, mice treated with L. major-EVs before challenge with the parasite showed an increased frequency of IL-4-producing CD4+ T cells in both the spleen and lymph nodes, leading to disease exacerbation [11]. These findings suggest that Leishmania EVs are predominantly immunosuppressive and favor the parasite. In fact, our group demonstrated that L. amazonensis EVs co-injected with the parasite led to disease exacerbation with a predominance of Th2 response in BALB/c mice [5]. Similar results were observed for L. major, but the co-injection of the parasite and related EVs induced an increase in the expression of IL-17 and IL-4 [6].
Changes in the content of EVs may impact the immune response and disease progression [9,11]. Studies performed with genetically modified parasites showed that in a mouse model of air pouch formation (murine air pouch injection) EVs derived from L. major GP63 knockout (KO) (L. major GP63 −/− ) induced greater recruitment of inflammatory cells, compared to EVs derived from wild parasites [9]. Furthermore, EVs derived from L. donovani exhibited an immunosuppressive effect and exacerbated the disease in animals challenged with the parasite, but EVs derived from L. donovani HSP100 KO (L. donovani HSP100 −/− ) were able to induce a pro-inflammatory response and did not exacerbate the disease [11]. Thus, the hypothesis that EVs derived from parasites with different virulence profiles (virulent and attenuated) present relevant alterations in their protein content and can induce distinct immune responses in an experimental immunization model cannot be discarded.
The relevance of EVs in Leishmania infection's biology was shown by the demonstration that Leishmania promastigotes release EVs in the sandflies [6]. The experimental infection with L. major in the presence of EVs released by the parasite in the vector led to higher lesion size and parasite load, associated with impaired effector immune response [6]. Taken together, the in vivo studies suggest that EVs released by Leishmania participate in the infection, favoring the establishment of the parasite and the progression of the disease.

Conclusions
The knowledge acquired studying EVs has allowed understanding that these particles are related to intercellular communication and cross-kingdom relationship. The release of these EVs by Leishmania is related to initial infection, modulation of the immune system, and disease progression in the host (Table 1). However, several aspects of the biology and physiology of these molecules still need to be better investigated. Would releasing these EVs into the vector be related to the parasite's adaptation to that environment? Can EVs contribute to parasite multiplication in the vector? Is there population regulation and/or transfer of resistance factors and immune response escape by EVs between different Leishmania species? Do these transfers occur in the vector and/or in the mammalian host? Can vesicles released by Leishmania be used for the development of vaccines and new diagnostic approaches? Thus, the field of EVs released by Leishmania and other pathogens is fascinating and, there will be significant advances and contributions to the area in the future with the discovery of new therapeutic targets and new players in the host-parasite relationship.