Introduction
Neutrophil granulocytes are key cells of the innate immune system with a primary function of killing invading microorganisms such as bacteria, fungi and parasites to prevent pathogenic spread and invasion[
1,
2]. Once identified, neutrophils phagocytose and destroy microbes inside the phagolysosome by localised disgorgement of granule contents and the generation of reactive oxygen species (ROS)[
3]. Engulfment of the microorganism allows killing to take place in a confined area within the cell and not in the extracellular space. Neutrophils may also liberate granule contents and ROS into the surrounding extracellular space to destroy nearby foreign pathogens. Dysregulation of these processes may cause histotoxic damage surrounding host cells. More recently a further extracellular killing mechanism available to neutrophils has been described known as neutrophil extracellular trap (NET) formation[
3,
4]. NETs are formed by the mixing of cytoplasmic contents with nuclear histones and DNA to form a network which is propelled to the exterior of the cell. Microbes are caught in this mesh and killed by the neutrophil proteins and histones contained in the NETs. This process of NET formation leads to a form of cell death, NETosis, that has been characterised as being different from either apoptosis or necrosis[
5].
NET formation is known to be stimulated by specific cytokines (e.g., interleukin 8 (IL-8)), bacterial products (e.g., lipopolysaccharide (LPS)) and importantly by clinically relevant pathogens such as
Shigella flexneri,
Staphylococcus aureus,
Salmonella thyphimurium,
Streptococcus pneumoniae and the fungus
Candida albicans[
6]. Stimulation and activation of neutrophils with the diacylglycerol (DAG) mimetic phorbol 12-myristate 13-acetate (PMA) also results in the production of NETs and has given important clues as to the possible mechanism involved in the formation of such structures. It is clear that NET formation following PMA stimulation is dependent on ROS production (via the NADPH oxidase system) and this is likely to follow the activation of protein kinase C (PKC) as well as other pathways such as raf-MEK-ERK[
7].
The PKC isozyme family is comprised of conventional, novel and atypical isoforms[
8]. There are at least four conventional isozymes: PKCα, PKCβI, PKCβII and PKCγ. The novel isozyme group has four subtypes: PKCδ, PKCε, PKCη and PKCθ. The third group, atypical isozymes, consists of PKCζ and PKCι[
9]. PMA stimulates conventional (α, βI, βII, γ) and novel (δ, ε, η, θ) PKC by mimicking the activating ligand DAG[
8]. PKC isoforms of all classifications have been reported in neutrophils from healthy donors[
10]. Given that PMA activation triggers NET formation, we hypothesised that specific isoform(s) of PKC are a key modulator of the NET formation pathway. To address this hypothesis we evaluated a panel of PKC inhibitors on NET formation.
Material and methods
Reagents
Dihydrorhodamine (DHR), dimethyl sulfoxide (DMSO), diphenyliodonium (DPI), Phorbol 12-myristate 13-acetate (PMA), Ro-31-8220, PKCζ pseudosubstrate myristoyl trifluoroacetate (PKCζ inhibitor) and SYTOX green were purchased from Sigma-Aldrich (Dorset, UK); Rottlerin, Gö 6976 and LY333531 were from Calbiochem (Merck) (Darmstadt, Germany).
Isolation of human neutrophils
Peripheral blood neutrophils were isolated from healthy human volunteers according to Lothian Research Ethics Committee approvals #08/S1103/38 via dextran sedimentation and Percoll™ discontinuous gradients as described[
11,
12]. Informed written consent was obtained from all subjects. Purity of the neutrophils was assessed by examination of cytocentrifuge preparations and was greater than 95%.
Neutrophils (5×104 cells/well) in HBSS containing Ca2+, Mg2+ and Hepes (20 mM) were aliquoted (180 μl) into 96 well plates and left to settle for 30 min at 37°C. The inhibitors Ro-31-8220, DPI, rottlerin, PKCζ inhibitor, Gö 6976 and PKCβ inhibitor were added at appropriate concentrations to wells in duplicates and incubated for 30 min before adding PMA. The final volume in each well was 200 μl. Plates were incubated for 4 h and then SYTOX green (6 μM final concentration), a cell-impermeable nucleic acid stain, with an excitation/emission maxima of 504/523 nm to give a green fluorescent light, was added and NET formation was observed by measuring mean fluorescence in 96 well plates. In some experiments 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to stimulate cells in place of PMA. Results were evaluated by measuring the mean fluorescence in 96 well plates after the subtraction of background fluorescence. Cells were also visualised by fluorescent microscopy carried out on a Zeiss Axiovert S100 fluorescent microscope (Carl Zeiss, Germany) and an Evos fl inverted microscope (AMG, Bothwell, WA).
Statistical analysis
Data were assessed by one way ANOVA followed by a post-hoc Dunnett’s test. The data were expressed as mean ± standard error of the mean (SEM), and values of p < 0.05 were considered statistically significant. All statistics were performed using GraphPad Prism 5 software (GraphPad, CA, USA).
Discussion
The results clearly show that NET formation induced by PMA is PKC and NADPH oxidase dependent. NET formation was blocked by both Pan-PKC inhibition and conventional-PKC inhibition. Furthermore, a specific PKCβ inhibitor (LY333531) also blocked NET formation. LY333531 has high selectivity for PKCβ over other conventional isoforms (IC50 of around 5 nM) with a 60 fold selectivity for PKCβ over PKCα[
15]. At higher concentrations specific inhibitors may have non-selective effects on other PKC isoforms. The IC50 of LY333531 for PKCα is around 300 nM, suggesting that the majority of the effect of this compound at the concentrations utilised in our study is via the inhibition of PKCβ and not PKCα; this is evidenced by the significant reduction in NET formation with 100 nM LY333531. The intracellular concentration of LY333531 within the neutrophil following incubation is unknown but it is unlikely to be fully absorbed and as such again we would suggest the effects are due to inhibition of PKCβ. Previous work has demonstrated that PKCβ accounts for 50% of the neutrophil response to PMA further underlining the likely predominant role of PKCβ in NET production[
16].
Oxidative burst and the generation of reactive oxygen species including superoxide anions (0
2-) and nitric oxide (NO) are fundamental responses of the neutrophil to inflammatory stimuli and pathogens. NET formation is dependent on NADPH oxidase activation and consequently on the generation of 0
2- which can be blocked by DPI. DPI inhibits NADPH oxidase by binding to specific subunits in the enzyme complex and preventing electron flow and 0
2- production[
17]. The main component of NADPH oxidase is the flavocytochrome b558, a dimer of p22phox and gp91phox, which is an active transporter of electrons across the membrane. Coupled to these are proteins p40phox, p47phox, p67phox and p21rac which are crucial to electron translocation[
18]. These proteins assemble when activated to produce 0
2- which are then spontaneously converted to H
2O
2. Interestingly, p47phox has to be phosphorylated to acquire a conformational rearrangement to expose the domains that are important for the NADPH oxidase function, and this phosphorylation is mediated by PKC[
19]. This is consistent with our findings that PKC is involved in PMA induced NET formation and furthermore that PKCβ is the isoform crucially involved. This is further underlined by the finding that oxidative burst is reduced by concentrations of LY333531 that reduce NET formation.
The beneficial anti-microbial effects of NET formation have been described in several studies[
4,
20‐
25]. Indeed, this is perhaps most pertinently displayed in restoration of NADPH oxidase function in chronic granulomatous disease by gene therapy leading to an increased resistance to fungal infection and clinical improvement secondary to the restoration of the ability to form NETs[
22]. Several studies however have demonstrated a pro-inflammatory potential of NETs in a diverse range of diseases including systemic lupus erythematosus[
26‐
28], cystic fibrosis[
1,
29,
30] and psoriasis[
31]. Therefore the modulation of NET production may be a viable anti-inflammatory target. Inhibition of PKC activity represents one such target as PKC inhibitors have been in development for many years as potential anti-cancer therapies, many of which are orally bioavailable[
9]. Furthermore the relative redundancy in PKC function due to multiple isoforms may allow the targeting of specific PKCs in specific cell types at specific organ sites. PKCβ knock out in a murine model has been demonstrated to modulate ischemia reperfusion injury
in vivo[
32], however these mice may also be immunodeficient[
33] and thus caution must be exercised in any strategy to specifically target PKC. Extracellular traps from both neutrophils and mast cells have been demonstrated in psoriatic skin lesions and from purified neutrophils from psoriasis patients in association with IL-17 and MPO, directly implicating extracellular traps in the pathogenesis of disease[
31]. A previous study of a PKC inhibitor AEB071 with specificity for PKC α, β, and θ in psoriasis demonstrated not only
in-vitro effects on T cell proliferation and cytokine production but also a clinical improvement in psoriatic lesions in treated patients[
34]. We may infer that some of this effect may be due to a direct effect of PKC inhibition on NET formation and thus inflammation in the skin lesions of these patients. Further studies will of course be required to support this hypothesis.
In summary, NET formation in response to PMA and DAG analogues is dependent on PKC activation. Furthermore, we demonstrate that conventional PKC and in particular PKCβ is the predominant isoform responsible for NET formation under these conditions. Although NETs have been demonstrated to entrap and kill various microorganisms there is burgeoning evidence implicating a role for these structures in inflammatory disease and potential modulation of NET production (by PKC inhibition) may offer a novel anti-inflammatory strategy.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RDG, CDL, AM, FL and KM carried out the experiments. RDG, CH, DJD and AGR designed the experiments and provided a critical review of methods. RDG drafted the manuscript. All authors read and approved the final manuscript.