VARGAS, P. | Search bioRxiv

bioRxiv2026

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 (H2O2) 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 H2O2 production. We show that H2O2 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, H2O2 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.

Cell Biology↗

bioRxiv2025

Fast adaptation of Myosin II activity to confinement sustains neutrophil migration in capillaries

As the first responders of the immune system, neutrophils rapidly and abundantly reach inflamed tissues through blood capillaries. The diameter of capillaries can be as narrow as two microns, imposing considerable deformations on neutrophils. Notably, capillary obstruction due to neutrophil retention causes vascular dysfunction and contributes to the pathogenesis of several diseases. However, the cellular mechanisms that allow neutrophils to migrate into small capillaries and to avoid retention remain unknown. In this study, we demonstrate, both in vivo and in vitro, that capillary size does not influence neutrophil migration velocity. During migration into capillaries of different sizes, neutrophils maintain high speed, a phenomenon associated with a global actomyosin cytoskeleton rearrangement in response to confinement strength. In irregular capillaries, neutrophils rapidly adapt their cell contractility via the ROCK-MyoII pathway, which allows them to sustain their migration speed along the vessels despite changes in confinement. At the single cell level, inhibition of ROCK impairs actomyosin cytoskeleton rearrangement and reduces neutrophil migration speed within confined capillaries. At the collective level, ROCK inhibition hampers efficient neutrophil trafficking in a network of small capillaries, resulting in vessel obstruction. These findings reveal a unique capacity of neutrophils to rapidly and dynamically adapt their migration to the confinement strength of capillaries, an ability that might limit vascular dysfunction during inflammation.

cell biology↗