Neutrophil extracellular vesicles modulate neutrophils response to Leishmania parasites

In Leishmania infection, neutrophils are the first cells to migrate to the infected site, where they can either kill the parasite or exacerbate the infection. Extracellular vesicles (EVs), released by nearly all cells, including neutrophils, regulate various physiological processes, potentially affecting tissue homeostasis or spreading infectious agents. Neutrophils release EVs in response to inflammatory stimuli, playing immunomodulatory roles. While neutrophil’s role in Leishmania infection has been well-studied, the impact of EVs released by these cells in response to the parasite remains underexplored. This study aimed to characterize EVs released by human neutrophils stimulated by Leishmania amazonensis promastigotes and to examine their effects on neutrophil modulation in vitro and infection in vivo. Neutrophils isolated from healthy individuals were stimulated with L. amazonensis promastigotes, and small extracellular vesicles (SEVs) were obtained by ultracentrifugation. Nanoparticle tracking analysis (NTA) and electron microscopy showed that SEVs form a homogeneous population of approximately 150 nm vesicles, with parasite stimulation increasing vesicle production. The vesicles were found to be non-toxic to neutrophils and did not alter parasite phagocytosis. However, the stimulated SEV fraction led to increased NET production, independent of ROS and dependent on PAD4. Both SEVctrl and SEVLa reduced IL-8 production by neutrophils, but SEVLa-treated neutrophils showed enhanced migration towards this chemokine. In vivo, SEVs worsened ear lesions caused by Leishmania infection and increased parasite load as well. Ou results show that EVs released by neutrophils modulate neutrophil’s function, impacting in vivo infection by Leishmania parasites. Future studies will focus on characterizing the vesicle content and exploring the complex mechanisms by which they influence infection outcomes.