Extracellular vesicles as mediators of in vitro neutrophil swarming on a large-scale microparticle array

Neutrophils combat infections and promote healing of damaged tissues while protecting the surrounding healthy tissue through a process called swarming. Swarming neutrophils release soluble factors that recruit additional neutrophils and shape the inflammation response. Additionally, neutrophils release extracellular vesicles (EVs), which are gaining attention as important intercellular mediators. We developed a large-scale array of bioparticles on a glass substrate that triggers neutrophil swarming in vitro in a spatially and temporally controlled manner that facilitates the analysis of neutrophil migration. Our platform can generate 30 000 neutrophil swarms on a glass slide in a highly reproducible manner (98% patterning efficiency), which produces an EV-rich supernatant that enables quantitative characterization of inflammation-specific EVs. Healthy neutrophils were able to form uniform swarms across the bioparticle array, which demonstrates a high degree of intercellular coordination. However, neutrophils swarming on the bioparticle array tended to have a lower radial velocity than neutrophils swarming toward a single target. After collecting and isolating EVs released by swarming and non-swarming neutrophils, we found that neutrophils constitutively release exosomes and microvesicles. Furthermore, EVs released by swarming neutrophils cause neutrophil activation and contain the proinflammatory mediator galectin-3, suggesting that EVs have an active role during neutrophil swarming. Ultimately, understanding EVs' role in intercellular communication during swarming will improve understanding of the complex signaling pathways involved in the regulation of inflammation.