SHOCK-D-18-00340-1.pdf (588.87 kB)
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posted on 2019-03-04, 18:47 authored by Fariborz Soroush, Yuan Tang, Kimberly Guglielmo, Alex Engelmann, Elisabetta Liverani, Akruti Patel, Jordan Langston, Shuang Sun, Satya P. Kunapuli, Mohammad F. Kiani, Laurie E. KilpatrickBackground: 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 is
regulated 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 bone
marrow 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 individual
steps 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 surgery
and the lungs harvested 24 h post-surgery. Results: In vitro – PKCdY155F BMN had significantly reduced O2
and NETs
release compared with WT. WT BMN, but not PKCdY155F BMN, demonstrated significant adhesion and migration across
tumor necrosis factor-activated MLMVEC in bMFA. PKCd inhibition significantly reduced WT BMN adhesion and migration
under low shear and near bifurcations, but had no effect on PKCdY155F BMN. In vivo – mutation of PKCd tyrosine 155
significantly decreased neutrophil migration into the lungs of septic mice. Conclusions: PKCd tyrosine 155 is a key
phosphorylation site controlling proinflammatory signaling and neutrophil–endothelial cell interactions. These studies
provide mechanistic insights into PKCd regulation during inflammation.
we identified protein kinase C-d (PKCd) as a critical controller of neutrophil activation and trafficking but how PKCd is
regulated 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 bone
marrow 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 individual
steps 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 surgery
and the lungs harvested 24 h post-surgery. Results: In vitro – PKCdY155F BMN had significantly reduced O2
and NETs
release compared with WT. WT BMN, but not PKCdY155F BMN, demonstrated significant adhesion and migration across
tumor necrosis factor-activated MLMVEC in bMFA. PKCd inhibition significantly reduced WT BMN adhesion and migration
under low shear and near bifurcations, but had no effect on PKCdY155F BMN. In vivo – mutation of PKCd tyrosine 155
significantly decreased neutrophil migration into the lungs of septic mice. Conclusions: PKCd tyrosine 155 is a key
phosphorylation site controlling proinflammatory signaling and neutrophil–endothelial cell interactions. These studies
provide mechanistic insights into PKCd regulation during inflammation.
