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Leishmania infection causes considerable human morbidity and may develop into a deadly visceral form in endemic regions. The parasite infects macrophages where they can replicate intracellularly. Furthermore, they modulate host immune responses by using virulence factors (lipophosphoglycan, glycoprotein-63, and others) that promote survival inside the cells. Extracellular vesicles (EVs) released by parasites are important for cell-cell communication in the proinflammatory milieu modulating the establishment of infection. However, information on the ability of EVs from different Leishmania species to modulate inflammatory responses is scarce, especially from those species causing different clinical manifestations (visceral vs. cutaneous). The purpose of this study was to compare macrophage activation using EVs from three Leishmania species from New World including L. infantum, L. braziliensis, and L. amazonensis. EVs were released from promastigote forms, purified by ultracentrifugation and quantitated by Nanoparticle Tracking Analysis (NTA) prior to murine macrophage exposure. NTA analysis did not show any differences in the EV sizes among the strains. EVs from L. braziliensis and L. infantum failed to induce a pro-inflammatory response. EVs from both L. infantum WT and LPG-deficient mutant (LPG-KO) did not show any differences in their interaction with macrophages, suggesting that LPG solely was not determinant for activation. On the other hand, EVs from L. amazonensis were immunomodulatory inducing NO, TNF-α, IL-6, and IL-10 via TLR4 and TLR2. To determine whether such activation was related to NF-κB p65 translocation, THP-1 macrophage cells were exposed to EVs. In the same way, only EVs from L. amazonensis exhibited a highly percentage of cells positive for NF-κB. Our results suggest an important role of EVs in determining the pattern of immune response depending on the parasite species. For L. infantum, LPG was not determinant for the activation.

Extracellular vesicles (EVs) shed by trypomastigote forms of Trypanosoma cruzi have the ability to interact with host tissues, increase invasion, and modulate the host innate response. In this study, EVs shed from T. cruzi or T.cruzi-infected macrophages were investigated as immunomodulatory agents during the initial steps of infection. Initially, by scanning electron microscopy and nanoparticle tracking analysis, we determined that T. cruzi-infected macrophages release higher numbers of EVs (50–300 nm) as compared to non-infected cells. Using Toll-like-receptor 2 (TLR2)-transfected CHO cells, we observed that pre-incubation of these host cells with parasite-derived EVs led to an increase in the percentage of infected cells. In addition, EVs from parasite or T.cruzi-infected macrophages or not were able to elicit translocation of NF-κB by interacting with TLR2, and as a consequence, to alter the EVs the gene expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and STAT-1 and STAT-3 signaling pathways. By proteomic analysis, we observed highly significant changes in the protein composition between non-infected and infected host cell-derived EVs. Thus, we observed the potential of EVs derived from T. cruzi during infection to maintain the inflammatory response in the host.

B-1 cells are considered an innate-like B cell population that participates in effective innate and adaptive responses to pathogens. B-1 cells produce immunoglobulins, cytokines, chemokines, migrate to inflammatory sites, and differentiate into mononuclear phagocyte-like cells. Murine B-1 cells phagocytosed Leishmaniain vitro and in vivo and participate in immunity against Leishmania. Our group showed that B-1 cells or their extracellular vesicles (EVs) led to a resistance to experimental infection by L. amazonensis. However, the B-1 cells’ responses to Leishmania or EVs isolated from parasites are still poorly characterized. Studying the activation and differentiation of B-1 cells in vivo can contribute to a better understanding of how these cells participate in immunity to L. amazonensis. Thus, we evaluated the expression of myeloid (M-csfr, G-csfr, Spi-1) and lymphoid (EBF, E2A, IL-7R) lineage commitment factors, Toll-like receptors (TLRs), activation cell surface markers, nitric oxide (NO) and reactive oxygen species (ROS) production in murine peritoneal B-1 cells collected after 24 or 48 h post-infection with Leishmania (Leishmania) amazonensis promastigotes or EVs released by the parasites. Our results demonstrated that L. amazonensis infection did not stimulate the expression of CD40, CD80, CD86, F4/80, and MHC II in B-1 cells, but a significant decrease in the production of NO and ROS was observed. The infection induced a significantly higher arginase expression in B-1 cells, but the stimulation with EVs led to a decrease in this gene expression. TLR-2 and TLR-6 had significantly higher expression in B-1 cells from mice intraperitoneally stimulated with the parasite. The TLR-9 expression was higher in animals infected or stimulated for 48 h with EVs. Interestingly, in B-1 cells the stimulus with L. amazonensis led to a substantial increase in the expression of myeloid restricted transcription factors. Thus, our study suggests that the parasites or EVs differently modulated the activation and differentiation of B-1 cells.

The extracellular vesicles (EVs) released by Leishmania can contribute to the establishment of infection and host immunomodulation. In this study, we characterized the shedding of EVs from Leishmania (Leishmania) amazonensis promastigotes. This species is the causative agent of cutaneous leishmaniasis, and its role during interactions with bone marrow-derived macrophages (BMDMs) and peritoneal B-1 cells was evaluated. Leishmania amazonensis promastigotes cultivated in vitro at different times and temperatures spontaneously released EVs. EVs were purified using size-exclusion chromatography (SEC) and quantitated by nanoparticle tracking analysis (NTA). NTA revealed that the average size of the EVs was approximately 180 nm, with concentrations ranging from 1.8 × 108 to 2.4 × 109 vesicles/mL. In addition, the presence of LPG and GP63 were detected in EVs obtained at different temperatures. Naïve BMDMs stimulated with EVs exhibited increased IL-10 and IL-6 expression. However, incubating B-1 cells with parasite EVs did not stimulate IL-10 expression but led to an increase in the expression of IL-6 and TNFα. After 7 weeks post-infection, animals infected with L. amazonensis promastigotes in the presence of parasite EVs had significant higher parasite load and a polarization to Th2 response, as compared to the group infected with the parasite alone. This work demonstrated that EVs isolated from L. amazonensis promastigotes were able to stimulate macrophages and B-1 cells to express different types of cytokines. Moreover, the immunomodulatory properties of EVs probably contributed to an increase in parasite burden in mice. These findings suggest that the functionality of L. amazonensis EVs on immune system favor of parasite survival and disease progression.