Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and 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

Inhibition of claudin-10 expression in B16F10 inhibits the influence of B-1 lymphocytes in melanoma aggressiveness.

<p>B16F10 cells were transfected for 24h with stealth claudin-10 siRNA1. The inhibition of claudin-10 expression in B16F10 blocks <b>A)</b> activation of ERK pathway and claudin-10 expression and, <b>B)</b> impairs the B16F10 aggressiveness induced by the contact with B-1 lymphocytes. (Bars represent the mean number of lung tumor per experimental condition ±SD (<i>n</i> = 3). **p < 0.01; ***p < 0.001, using one-way ANOVA with Tukey’s post hoc test.</p

IL-10 deficiency is associated with impaired expression of claudin-10 in B-1 lymphocytes.

<p>Total RNA of B-1 lymphocytes enriched culture from wild-type (B-1WT) or IL-10^-/- knockout (B-1IL-10^-/-) mice was submitted to microarray analysis. <b>A)</b> Heatmap showing differential expression of eleven transcripts between B-1WT and B-1IL-10^-/- lymphocytes, and the down-regulation of claudin-10 (cldn-10) in B-1IL-10^-/- samples. <b>B)</b> Western blot analysis of cldn-10 protein in whole-cell extracts from B-1WT and B-1IL-10^-/-. β-actin protein was used as internal control.</p

Claudin-10 expression on B-1 lymphocytes mediates the alterations in B16F10 melanoma cell behavior.

<p><b>A)</b> Expression of claudin-10 on B-1 lymphocytes from peritoneal cell culture transfected for 24 h with stealth claudin-10 siRNA1 (B-1 cldn-10 siRNA1), siRNA2 (B-1 cldn-10 siRNA2), siRNA3 (B-1 cldn-10 siRNA3). <b>B)</b> The number of lung colonies in mice injected with B16F10 melanoma cells from single culture (B16F10) or after co-cultivation with B-1 lymphocytes (B16F10+B1), transfected or not with claudin-10 siRNA1 (B16F10 + B-1 cldn-10siRNA1), siRNA2 (B16F10 + B-1 cldn-10 siRNA2), siRNA3 (B16F10 + B-1 cldn-10 siRNA3). <b>C)</b> Inhibition of claudin-10 expression impairs the activation of ERK pathway and the claudin-10 expression in B16F10 cells after B-1 contact. GAPDH was used as internal control. Bars represent the mean number of lung colonies per experimental condition ±SD. *p < 0.05; **p < 0.01;***p < 0.001, using one-way ANOVA with Tukey’s post hoc test.</p

B-1 lymphocytes, but not other peritoneal cells, induce the pro-metastatic effect on B16F10 melanoma cells.

<p><b>A)</b> Adherent peritoneal cells (apc) from wild-type mice, but not from B cell-deficient mice (BKO), obtained from enriched peritoneal cell cultures increases B16F10 metastatic potential upon cell-to-cell contact. <b>B)</b> The same results were obtained with cell-sorting technique, confirming that B-1 lymphocytes are responsible for changes in the melanoma behavior. (Bars represent the mean number of lung tumor per experimental condition ±SD (<i>n</i> = 4); **p < 0.01; ***p < 0.001, using one-way ANOVA with Tukey’s post hoc test.</p

Endogenous IL-10 drives the influence of B-1 lymphocytes in B16F10 behavior.

<p><b>A)</b> The deficiency in IL-10 production did not affect the phenotype and the number of B-1 lymphocytes recovered from the culture of peritoneal cavity cells; <b>B)</b> but affected their ability to increase the metastatic potential of B16F10 melanoma cells. Bars represent the mean number of lung colonies per experimental condition ±SD. **p <0.001, using one-way ANOVA with Tukey’s post hoc test.</p