PROTEIN KINASE C-DELTA (PKCδ) TYROSINE PHOSPHORYLATION IS A CRITICAL REGULATOR OF NEUTROPHIL-ENDOTHELIAL CELL INTERACTION IN INFLAMMATION

Background: Neutrophil dysfunction plays an important role in inflammation-induced tissue injury. Previously, we identified protein kinase C-delta (PKC delta) as a critical controller of neutrophil activation and trafficking but how PKC delta is regulated in inflammation has not been delineated. PKC delta activity is regulated by tyrosine phosphorylation on multiple sites. Tyrosine155 is a key regulator of apoptosis and gene expression, but its role in proinflammatory signaling is not known. Methods: In-vitro studies-superoxide anion (O-2(-)) and neutrophil extracellular traps (NETs) were measured in bone marrow neutrophils (BMN) isolated from wild type (WT) and PKC delta Y155F knock-in mice (PKC delta tyrosine 155 -> phenylalanine). Our novel 3D biomimetic microfluidic assay (bMFA) was used to delineate PKC delta-mediated regulation of individual steps in neutrophil adhesion and migration using WTand PKC delta Y155F BMN and mouse lung microvascular endothelial cells (MLMVEC). In-vivo studies - WT and PKC delta Y155F knock-in mice underwent sham or cecal ligation and puncture surgery and the lungs harvested 24 h post-surgery. Results: In vitro-PKC delta Y155F BMN had significantly reduced O-2(-) and NETs release compared with WT. WT BMN, but not PKC delta Y155F BMN, demonstrated significant adhesion and migration across tumor necrosis factor-activated MLMVEC in bMFA. PKC delta inhibition significantly reduced WT BMN adhesion and migration under low shear and near bifurcations, but had no effect on PKC delta Y155F BMN. In vivo - mutation of PKC delta tyrosine 155 significantly decreased neutrophil migration into the lungs of septic mice. Conclusions: PKC delta tyrosine 155 is a key phosphorylation site controlling proinflammatory signaling and neutrophil-endothelial cell interactions. These studies provide mechanistic insights into PKC delta regulation during inflammation.