Background: Neutrophil dysfunction plays an important role in inflammation-induced tissue injury. Previously,<br>we identified protein kinase C-d (PKCd) as a critical controller of neutrophil activation and trafficking but how PKCd is<br>regulated in inflammation has not been delineated. PKCd activity is regulated by tyrosine phosphorylation on multiple sites.<br>Tyrosine155 is a key regulator of apoptosis and gene expression, but its role in proinflammatory signaling is not known.<br>Methods: In-vitro studies – superoxide anion (O2<br>) and neutrophil extracellular traps (NETs) were measured in bone<br>marrow neutrophils (BMN) isolated from wild type (WT) and PKCdY155F knock-in mice (PKCd tyrosine 155 ! phenylalanine).<br>Our novel 3D biomimetic microfluidic assay (bMFA) was used to delineate PKCd-mediated regulation of individual<br>steps in neutrophil adhesion and migration using WTand PKCdY155F BMN and mouse lung microvascular endothelial cells<br>(MLMVEC). In-vivo studies – WT and PKCdY155F knock-in mice underwent sham or cecal ligation and puncture surgery<br>and the lungs harvested 24 h post-surgery. Results: In vitro – PKCdY155F BMN had significantly reduced O2<br> and NETs<br>release compared with WT. WT BMN, but not PKCdY155F BMN, demonstrated significant adhesion and migration across<br>tumor necrosis factor-activated MLMVEC in bMFA. PKCd inhibition significantly reduced WT BMN adhesion and migration<br>under low shear and near bifurcations, but had no effect on PKCdY155F BMN. In vivo – mutation of PKCd tyrosine 155<br>significantly decreased neutrophil migration into the lungs of septic mice. Conclusions: PKCd tyrosine 155 is a key<br>phosphorylation site controlling proinflammatory signaling and neutrophil–endothelial cell interactions. These studies<br>provide mechanistic insights into PKCd regulation during inflammation.