SHOCK-D-18-00340-1.pdf

Background: Neutrophil dysfunction plays an important role in inflammation-induced tissue injury. Previously,we identified protein kinase C-d (PKCd) as a critical controller of neutrophil activation and trafficking but how PKCd isregulated in inflammation has not been delineated. PKCd 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 (O2 ) and neutrophil extracellular traps (NETs) were measured in bonemarrow neutrophils (BMN) isolated from wild type (WT) and PKCdY155F knock-in mice (PKCd tyrosine 155 ! phenylalanine).Our novel 3D biomimetic microfluidic assay (bMFA) was used to delineate PKCd-mediated regulation of individualsteps in neutrophil adhesion and migration using WTand PKCdY155F BMN and mouse lung microvascular endothelial cells(MLMVEC). In-vivo studies – WT and PKCdY155F knock-in mice underwent sham or cecal ligation and puncture surgeryand the lungs harvested 24 h post-surgery. Results: In vitro – PKCdY155F BMN had significantly reduced O2 and NETsrelease compared with WT. WT BMN, but not PKCdY155F BMN, demonstrated significant adhesion and migration acrosstumor necrosis factor-activated MLMVEC in bMFA. PKCd inhibition significantly reduced WT BMN adhesion and migrationunder low shear and near bifurcations, but had no effect on PKCdY155F BMN. In vivo – mutation of PKCd tyrosine 155significantly decreased neutrophil migration into the lungs of septic mice. Conclusions: PKCd tyrosine 155 is a keyphosphorylation site controlling proinflammatory signaling and neutrophil–endothelial cell interactions. These studiesprovide mechanistic insights into PKCd regulation during inflammation.