Spatiotemporal dynamics of hydrogen peroxide during neutrophil swarming in 3D

Neutrophil swarming enables the coordinated recruitment of large numbers of cells to sites of infection. Although reactive oxygen species (ROS) are key mediators of neutrophil function, their dynamics during collective behavior remain poorly defined. Here, we developed a 3D ex vivo swarming system combined with HyPer7-expressing dHL-60 cells to visualize intracellular hydrogen peroxide (H ₂ O ₂ ) dynamics in real time at single-cell resolution. This approach enabled us to reliably measure NADPH oxidase 2 (NOX2) activity during neutrophil swarming, revealing dynamic H ₂ O ₂ production. We show that H ₂ O ₂ production is spatially confined to cells at the stimulation site and temporally coupled to swarming initiation and distal cell recruitment. ROS production depends on NOX2 activity and calcium signaling but is not required for swarm formation or amplification. Notably, H ₂ O ₂ accumulation was detected throughout activated cells rather than being restricted to phagosomes, suggesting a broader intracellular oxidative response. Together, these findings reveal a coordinated oxidative program associated with neutrophil swarm initiation and raise the possibility that ROS contribute to signaling functions beyond their established role in phagocytosis.