Cytosolic S100A8/A9 promotes Ca²⁺ supply at LFA-1 adhesion clusters during neutrophil recruitment

S100A8/A9 is an endogenous alarmin secreted by myeloid cells during many acute and chronic inflammatory disorders. Despite increasing evidence of the proinflammatory effects of extracellular S100A8/A9, little is known about its intracellular function. Here, we show that cytosolic S100A8/A9 is indispensable for neutrophil post-arrest modifications during outside-in signaling under flow conditions in vitro and neutrophil recruitment in vivo, independent of its extracellular functions. Mechanistically, genetic deletion of S100A9 in mice caused dysregulated Ca²⁺ signatures in activated neutrophils resulting in reduced Ca²⁺ availability at the formed LFA-1/F-actin clusters with defective β₂ integrin outside-in signaling during post-arrest modifications. Consequently, we observed impaired cytoskeletal rearrangement, cell polarization, and spreading, as well as cell protrusion formation in S100a9 compared to wildtype (WT) neutrophils, making S100a9 cells more susceptible to detach under flow, thereby preventing efficient neutrophil recruitment and extravasation into inflamed tissue.