Protein Kinase C-Delta (PKCδ) Tyrosine Phosphorylation is a Critical Regulator of Neutrophil-Endothelial Cell Interaction in Inflammation

Abstract

Background: Neutrophil dysfunction plays an important role in inflammation-induced tissue injury. Previously, we identified protein kinase C-δ (PKCδ) as a critical controller of neutrophil activation and trafficking but how PKCδ is regulated in inflammation has not been delineated. PKCδ 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 PKCδY155F knock-in mice (PKCδ tyrosine 155 → phenylalanine). Our novel 3D biomimetic microfluidic assay (bMFA) was used to delineate PKCδ-mediated regulation of individual steps in neutrophil adhesion and migration using WT and PKCδY155F BMN and mouse lung microvascular endothelial cells (MLMVEC). In-vivo studies - WT and PKCδY155F knock-in mice underwent sham or cecal ligation and puncture surgery and the lungs harvested 24 h post-surgery.

Results: In vitro - PKCδY155F BMN had significantly reduced O2 and NETs release compared with WT. WT BMN, but not PKCδY155F BMN, demonstrated significant adhesion and migration across tumor necrosis factor-activated MLMVEC in bMFA. PKCδ inhibition significantly reduced WT BMN adhesion and migration under low shear and near bifurcations, but had no effect on PKCδY155F BMN. In vivo - mutation of PKCδ tyrosine 155 significantly decreased neutrophil migration into the lungs of septic mice.

Conclusions: PKCδ tyrosine 155 is a key phosphorylation site controlling proinflammatory signaling and neutrophil-endothelial cell interactions. These studies provide mechanistic insights into PKCδ regulation during inflammation.

Figure 1. Generation of PKCδY155F knock-in model

(A.) Schematic representation of targeted generation of PKCδY155F knock-in mice. Exons 2–9 are represented as grey vertical lines. PKCδ tyrosine 155 is located on Exon 5 and is indicated with an asterisk. Identification of wildtype and PKCδY155F knock-in mice using (B.) gtF/gtR primer pair (indicated in panel A) and (C.) primers that specifically recognize PKCδY155F site by PCR.

Figure 2. The bMFA mimics a physiologically relevant microvascular environment

Microvascular network maps obtained in vivo are reproduced on PDMS to assemble the biomimetic microfluidic assay (scale bar 1 cm) (A). Microvascular endothelial cells, WT (scale bar 50 μm) (B) and KI155 (scale bar 50 μm) (C), formed a complete lumen in the vascular channel of bMFA (green indicates F-actin; red indicates cell nuclei).

Figure 3. PMA- and fMLP-stimulated Superoxide anion generation (O₂⁻)

Superoxide onion generation by WT BMN and KI 155 BMN in response to PMA or fMLP was measured as SOD-inhibitable cytochrome c reduction. The measurements indicate significant quantities of superoxide anion generation in response to PMA and fMLP. While WT BMN O₂⁻ generation in response to either PMA or fMLP was similar, lack of PKCδ Tyr 155 phosphorylation in KI155 BMN decreased O₂⁻ generation significantly in response to fMLP, but not PMA. (N=11; Mean±SEM; * p<0.05 student t-test)

Figure 4. TNF-stimulated O₂⁻ Generation

Superoxide anion generation in WT BMN with or without PKCδ-TAT peptide inhibitor (PKCδ-i) treatment and KI 155 BMN in response to TNF-α measured as SOD-inhibitable cytochrome c reduction. TNF-α stimulated O₂⁻ generation was significantly decreased in KI155 BMN and in WT BMN treated with the PKCδ-i (A). V_max of the reaction was also significantly reduced with KI155 BMN and WT BMN treated with the PKCδ-i as compared to WT BMN (B). There was a significant increase in the lag time to O₂⁻ production in both the KI155 BMN and WT BMN treated with the PKCδ inhibitor as compared to WT BMN (C). (N=24 for WT group, N=9 for KI155 group, N=5 for WT+PKCδ-i group; Mean±SEM; * p<0.05, ** p<0.01 one-way ANOVA)

Figure 5. Role of PKCδ in Neutrophil Extracellular Trap (NET) Formation

NET formation was measured fluorometrically in BMN by monitoring DNA release. NET formation in WT BMN in response to IL-1 (A) (N=5) and fMLP (B) (N=5) increases over 4 hrs. Inhibition of PKCδ with the inhibitor decreased NET formation in WT BMN significantly over the course of 4hrs ((A) and (B)). NET formation was also significantly attenuated in response to IL-1 and TNF-α in KI155 BMN as compared to WT BMN (C) (N=5 for WT group, N=4 for WT+PKCδ-i and KI155 groups). (Mean±SEM; * p<0.05 two-way ANOVA)

Figure 6. Role of PKCδ Tyr155 phosphorylation on permeability of MLMVEC

WT MLMVEC permeability increased significantly after TNF-α activation. Pharmacological inhibition of PKCδ in WT MLMVEC using PKCδ-i reduced the permeability to No Treatment levels. In the absence of PKCδ Tyr 155 phosphorylation in KI155 MLMVEC, permeability was not altered in response to TNF-α activation or after treatment with PKCδ-i. (N=3, Mean±SEM, ** p<0.01 compared to No Treatment; ## p<0.01 compared to TNF-α, two-way ANOVA)

Figure 7. Migration of WT and KI155 BMN across MLMVEC in bMFA

There was a significant increase in migration of WT BMN across TNF-activated WT MLMVEC in response to fMLP. Pharmacological inhibition with the PKCδ-TAT inhibitor (PKCδ-i) significantly decreased the number of migrated WT neutrophils across MLMVEC (A). Migration of TNF-activated KI155 BMN across WT MLMVEC in response to fMLP was not significantly different as compared to No Treatment levels. PKCδ inhibition did not change migration of KI155 BMN significantly (B). Migration of KI155 BMN across TNF-activated KI155 MLMVEC in response to fMLP did not increase compared to No treatment levels. Treatment with PKCδ-i did not significantly impact KI155 BMN migration across KI155 MLMVEC (C). (N=3; Mean±SEM; *** p<0.001 compared to No Treatment; ## p<0.01, ### p<0.001 compared TNF-α, one-way ANOVA)

Figure 8. Adhesion of WT and KI155 BMN to MLMVEC

There was a significant increase in adhesion of WT BMN to TNF-α activated WT MLMVEC in the presence of fMLP, especially at low shear rates and near bifurcations. Pharmacological inhibition with the PKCδ-TAT inhibitor (PKCδ-i) significantly reduced the adhesion level of WT BMN to WT MLMVEC to No Treatment levels (A). TNF-α activation of KI155 BMN did not increase adhesion to WT MLMVEC above the No Treatment levels. PKCδ-i treatment did not significantly change the adhesion level of KI155 BMN to WT MLMVEC (B). Adhesion of KI155 BMN to TNF-activated KI155 MLMVEC was not significantly different from No Treatment. Treatment with PKCδ-i did not significantly alter the adhesion level of KI155 BMN to KI155 MLMVEC (C) (N=3; Mean±SEM; * p<0.05, ** p<0.01, *** p<0.001, compared to No Treatment; # p<0.05, ## p<0.01, compared to TNF-α, one-way ANOVA)

Figure 9. Role of PKCδ Tyr155 phosphorylation on Pulmonary MPO Activity in Sepsis

MPO analysis was performed in mouse lung samples harvested 24h post-surgery. There was a significant increase in MPO activity in the lungs from WT septic mice (WT-CLP, n=4) as compared to sham surgery WT mice (WT-sham, n=4). MPO activity was significantly lower in lungs from KI155 septic mice (KI155, n=3) as compared to WT septic mice (WT-CLP). Values are expressed as MEAN±SEM in relative fluorescence units (RFU)/min/mg. (** p<0.01, WT-CLP vs. WT-sham and WT-CLP vs. KI155-CLP, one-way ANOVA)