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Front Microbiol
2018 Jan 01;9:3056. doi: 10.3389/fmicb.2018.03056.
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Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells.
Barbosa FMC
,
Dupin TV
,
Toledo MDS
,
Reis NFDC
,
Ribeiro K
,
Cronemberger-Andrade A
,
Rugani JN
,
De Lorenzo BHP
,
Novaes E Brito RR
,
Soares RP
,
Torrecilhas AC
,
Xander P
.
Abstract
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.
FIGURE 1. Leishmania amazonensis promastigotes spontaneously release EVs from their surface after incubation at 26, 34, and 37°C for (A–C) 1, (D–F) 2, (G–I) 4, and (J–L) 24 h. Parasites are seen by SEM at different magnifications. The arrows indicate EVs on the surface of the parasites. Scale bars = 1 μm.
FIGURE 4. Purification of EVs released by L. amazonensis by SEC. Titration of the L. amazonensis EV-specific antibody response in BALB/c mice. Blood samples were collected before immunization (non-immunized mice), 15 days after the first immunization and 15 days after the second immunization. The level of IgG was measured in (A) animals immunized with EVs prepared after 4 h of parasite incubation at 26°C and in (B) animals immunized with EVs prepared after 4 h of parasite incubation at 37°C. Analyses were performed using ELISA with twofold serially diluted sera. The result is depicted as the mean optical density (OD) at 492 nm of five mice per group. (C) SEC. The promastigotes were cultured for 4 h at 26, 34, or 37°C. The presence of EVs was measured in each fraction by ELISA with anti-EV polyclonal antibodies. Seventy samples were collected, and each fraction was analyzed by ELISA. The graph shows the overlap of the measurements performed for the L. amazonensis EVs released at 26, 34, and 37°C. (D) Size profile of L. amazonensis EVs obtained by SEC. The particles were analyzed by NTA.
FIGURE 5. Enzyme-linked immunoassay (ELISA) and dot blotting analysis of EVs released by L. amazonensis promastigotes. The parasites were cultured at 26, 34, or 37°C for 4 h. The presence of gp63 and LPG were evaluated by specific mAbs. (A) Dot blotting of EVs (4 μg per sample) probed with the mAb anti-gp63. (B) The detection of LPG was performed by ELISA with mAb CA7AE.
FIGURE 8. Development of infection in BALB/c mice experimentally infected with L. amazonensis promastigotes in the presence or absence of EVs from the parasite (L. ama+EVs and L. ama, respectively). BALB/c mice were infected in the footpad with 1 × 106
L. amazonensis promastigotes in the presence or absence of 1 × 107 EVs. (A) The lesion size was evaluated weekly for 7 weeks. The graph shows the measurement of the paw size and each point is representative of the mean of the measurements. (B) The parasite burden was evaluated by limiting dilution in the footpads (n = 5). Bars denote the average of 5 measurements, and error bars denote the SD. Student’s t-test ∗P < 0.05. (C) Histopathological findings in lesions after 7 weeks post-infection of BALB/c mice infected in the absence of EVs and (D) mice infected in the presence of EVs. The paws were removed, fixed, and 2 tissue samples from each animal were cut into 5 μm sections for staining with hematoxylin and eosin. The data are representative of 3 independent experiments.
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