Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication.
Keywords: alveolar macrophages; extracellular vesicles; high sensitivity flow cytometry; nanosafety.