ROLE OF EXTRACELLULAR VESICLES RELEASED BY HUMAN NEUTROPHILS STIMULATED WITH Leishmania amazonensis IN ENDOTHELIAL CELL ACTIVATION.

Neutrophils rapidly infiltrate Leishmania-infected tissue in vivo and interact with parasites, impacting disease outcome. Studies have shown that neutrophils can release extracellular vesicles (EVs) containing membrane receptors, adhesion molecules, granule proteins, and cytokines. These EVs have multiple functions in neutrophil responses. During inflammation, neutrophil migration from the bloodstream to tissues is orchestrated by interactions with endothelial cells. Our work aims to understand how EVs from Leishmania-stimulated neutrophil influence these interactions. We purified EVs from neutrophils, activated or not with Leishmania amazonensis, via ultracentrifugation (CEP: 49889621.7.0000.5257). EVs were characterized by nanoparticle tracking analysis (NTA), transmission microscopy, and western blot. Leishmania stimulation increased the release of small EVs from Leishmania-stimulated neutrophil (SEVLa) compared to controls (SEVct). Dot blot assays showed positive labeling for CD63 and argonaute protein, with stronger staining in SEVLa, and negative labeling for cytochrome C in both groups. To assess the effect of EVs on vascular permeability, we carried out a transwell assay using TEER (transepithelial/ transendothelial electrical resistance) and observed that treatment of HBMEC with SEVLa resulted in increased endothelial cells permeability compared to both positive control (bradykinin) and negative control. Moreover, neutrophils treated with SEVLa adhere more to HBMEC monolayer in comparison to SEVct treated neutrophils. Next steps include evaluating adhesion molecule expression on HBMEC and neutrophils, and cytokine and angiogenic factor production (e.g., TNF-alpha, IL-8, VEGF) by these cells after treatment with EVs. Our results suggest that SEVLa could significantly influence cell migration to inflamed tissues.  Additionally, the presence of argonaute protein suggests a potential role for microRNAs in modulating endothelial cell activation.