A microphysiological system of sterile injury demonstrates neutrophil reverse migration via macrophage-derived extracellular vesicle crosstalk.

Persistent neutrophilic inflammation can lead to tissue damage and chronic inflammation, contributing to non-healing wounds. The resolution phase of neutrophilic inflammation is critical to preventing tissue damage, as observed in diseases characterized by influx of neutrophils such as atherosclerosis and non-healing wounds. Animal models have provided insight into resolution of neutrophilic inflammation via efferocytosis and reverse migration (rM); however, species-specific differences and complexity of innate immune responses make translation to humans challenging. Thus, there is a need for in vitro systems that can elucidate mechanisms of resolution of human neutrophilic inflammation. Here, we developed a human microphysiological system (MPS) to mimic an inflammatory sterile injury (SI) microenvironment to study the role of macrophage derived extracellular vesicles (M-EVs) in determining the resolution of inflammation via neutrophil rM. The MPS integrates a human umbilical vein endothelial cell (HUVEC) lined lumen, injury site spheroid, human neutrophils, macrophages and macrophage derived EVs to investigate the role of M-EVs in neutrophil rM in vitro. The key features of the MPS enabled us to demonstrate that EVs derived from macrophage subsets modulate migratory behavior in primary neutrophils differently in specific inflammatory microenvironments. Importantly, we identified a new mechanism underlying neutrophil rM via M-EV, where neutrophils exposed to M2-EV-derived IL-8 migrate away from the SI site upon reaching the site, using the SI MPS. Overall, our SI MPS system demonstrates a reverse migratory pattern in human primary neutrophils, advancing the study of the resolution of inflammation via M-EVs.