Role of Neutrophil-Derived Extracellular Vesicles in Modulating Human Macrophage Responses During Leishmania amazonensis Infection

Extracellular vesicles (EVs) are lipid bilayer structures that carry proteins, genetic material, and other cellular components, facilitating intercellular communication. They play a crucial role in various biological processes, including parasite-host interactions. During Leishmania amazonensis infection, neutrophils are the first responders and significantly influence subsequent immune responses. Albeit Leishmania primarily infects macrophages, it is known to also interact with neutrophils and dendritic cells. Given neutrophils role in L. amazonensis infection and their ability to produce EVs, this study aimed to investigate how EVs from neutrophils, with or without L. amazonensis stimulation, affect macrophage infection and immune modulation. We characterized EVs using protein quantification, nanoparticle tracking analysis (NTA), dot blot, and transmission electron microscopy (TEM) for four types of vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs), stimulated (LEVLa and SEVLa) or not (LEVct and SEVct) with L. amazonensis. Nanoparticle tracking analysis (NTA) and electron microscopy analyses demonstrated that LEV fractions have structures ranging in size from 250 to 360 nm, while the SEV fraction is more homogeneous, with vesicles of 150 nm. All procedures were approved by the ethics committee CAAE:49889621.7.0000.5257. Results showed increased protein content and EV numbers (NTA) in stimulated neutrophils. SEVs were positive for the CD63 marker, and TEM analysis confirmed their similarity to described neutrophil EVs in the literature. We then examined the impact of these EVs on infected macrophages. In a killing assay, our preliminary results showed that LEVs promoted parasite replication. ELISA analysis of TNF-α and IL-6 levels in macrophage supernatants revealed that EVs from stimulated neutrophils increased cytokine release. These findings suggest that neutrophil-derived EVs may modulate the immune response in macrophages.