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. 2018 Sep 18:9:2036.
doi: 10.3389/fimmu.2018.02036. eCollection 2018.

"V体育ios版" Physiological Stimuli Induce PAD4-Dependent, ROS-Independent NETosis, With Early and Late Events Controlled by Discrete Signaling Pathways

Olga Tatsiy  1 , Patrick P McDonald  1
Affiliations

Physiological Stimuli Induce PAD4-Dependent, ROS-Independent NETosis, With Early and Late Events Controlled by Discrete Signaling Pathways

Olga Tatsiy et al. Front Immunol. .

Abstract

Neutrophils are known to extrude decondensed chromatin, thus forming NETs (neutrophil extracellular traps). These structures immobilize pathogens, thereby preventing their spreading, and are also adorned with antimicrobial molecules. NETs can also influence pathogenesis in chronic inflammation, autoimmunity, and cancer. Despite the importance of NETs, the molecular mechanisms underlying their formation, as well as the upstream signaling pathways involved, are only partially understood. Likewise, current methodological approaches to quantify NETs suffer from significant drawbacks, not the least being the inclusion of a significant non-specific signal VSports手机版. In this study, we used novel, fluorescent polymers that only bind extruded chromatin, allowing a specific and standardized quantification of NETosis. This allowed us to reliably rank the relative potency of various physiologic NET inducers. In neutrophils activated with such stimuli, inhibition of the Syk or PI3K pathways blocked NETosis by acting upon late events in NET formation. Inhibition of the TAK1, p38 MAPK, or MEK pathways also hindered NETosis, but by acting on early events. By contrast, inhibiting PKC, Src family kinases, or JNK failed to prevent NETosis; cycloheximide or actinomycin D were also ineffective. Expectedly, NET formation was deeply compromised following inhibition of the NADPH oxidase in PMA-activated neutrophils, but was found to be ROS-independent in response to physiological agonists. Conversely, we show for the first time in human neutrophils that selective inhibition of PAD4 potently prevents NETosis by all stimuli tested. Our data substantially extends current knowledge of the signaling pathways controlling NETosis, and reveals how they affect early or late stages of the phenomenon. In view of the involvement of NETs in several pathologies, our findings also identify molecular targets that could be exploited for therapeutic intervention. .

Keywords: NADPH oxidase; extracellular traps; neutrophils; protein arginine deiminase; signaling. V体育安卓版.

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Figures

Figure 1
Figure 1
Detection of NETs using Sytox Green in human neutrophils. (A) Unstimulated neutrophils were cultured in suspension for the indicated times in the presence of increasing concentrations of Sytox Green, prior to the addition of Hoechst 33342 and subsequent fluorescence microscopy analysis of unfixed cells. Total Sytox Green fluorescence values were divided by total Hoechst 33342 fluorescence, to normalize for cell number. Mean ± s.e.m. from duplicate measurements for each experimental condition from a representative experiment, shown on the right at 10X magnification. (B) Neutrophils were cultured for 4 h on poly-L-lysine coated coverslips with the indicated Sytox Green concentrations, in the absence (“ctrl”) or presence of 30 nM fMLP. The cells were then digested or not with DNase I (100 U/ml, 30 min, 37°C), then stained with Hoechst 33342, prior to fluorescence microscopy analysis. A representative experiment is shown (at 40X magnification).
Figure 2
Figure 2
Detection of NETs using MPO or PlaNET reagents in human neutrophils. (A) Neutrophils cultured on poly-L-lysine-coated coverslips were incubated for 4 h in the absence (“ctrl”) or presence of 30 nM fMLP, and digested or not with DNase I, prior to fluorescent microscopy detection of MPO as described in Methods. A representative experiment is shown (40X magnification). (B) Neutrophils cultured on poly-L-lysine-coated coverslips were incubated for 4 h in the absence (“ctrl”) or presence of 30 nM fMLP, and further incubated in the presence or absence of DNase I (100 U/ml, 30 min, 37°C), prior to fluorescent microscopy detection of NETs using PlaNET Green and Hoechst 33342 counterstaining, as described in Methods. A representative experiment is shown (40X magnification). (C) Neutrophils were treated as described in (B); fluorescent microscopy detection of NETs was conducted using PlaNET Blue and propidium iodide counterstaining (5 μM, 20 min), as described in Methods. A representative experiment is shown (40X magnification).
Figure 3
Figure 3
Relative potency of physiological neutrophil agonists or PMA to induce NETosis. (A) Neutrophils cultured on poly-L-lysine-coated coverslips were incubated for 4 h in the absence (“ctrl”) or presence of 30 nM fMLP, 1 nM GM-CSF, 100 U/ml TNFα, or 50 nM PMA. NETosis was then assessed using PlaNET Green as described in Methods. Representative fields are shown at 40X magnification. (B) Quantitative representation of the above experiments, in which PlaNET Green fluorescence values were standardized according to total cell number (i.e., the number of individual events detected using a cell-permeable nuclear stain), thus yielding a NETosis index. Mean ± s.e.m. from at least 5 independent experiments. ***p < 0.001 vs. unstimulated cells. (C) Neutrophils were cultured as described above for 4 h in the absence (“ctrl”) or presence of 30 nM fMLP, 1 nM GM-CSF, 100 U/ml TNFα, 50 nM PMA, 30 nM C5a, 50 nM PAF, 100 U/ml IFNγ, 100 nM LTB4, or 10 nM IL-8. Quantitative representation of these experiments, in which PlaNET Blue fluorescence values were standardized as described in (B). Mean ± s.e.m. from 3 independent experiments. **p < 0.02 vs. unstimulated cells.
Figure 4
Figure 4
Signaling pathways controlling NETosis induced by physiological neutrophil agonists or PMA. Neutrophils cultured on poly-L-lysine-coated coverslips were pre-treated (15 min, 37°C) with the following inhibitors or their diluent (DMSO): 10 μM piceatannol (Syk inhibitor); 10 μM SrcI1 (Src family kinase inhibitor); 10 μM LY294002 (PI3K inhibitor); 1 μM (5Z)-7-oxozeaenol (TAK1 inhibitor); 1 μM SB202190 (p38 MAPK inhibitor); 10 μM U0126 (MEK inhibitor); 10 μM SP600125 (JNK inhibitor); 10 μM JNK inhibitor VIII (a different JNK inhibitor); 5 μM AS601285 (a third JNK inhibitor); 10 μM Gö6976 (a PKC inhibitor). The cells were then further incubated for 4 h in the absence (“ctrl”) or presence of 30 nM fMLP, 1 nM GM-CSF, 100 U/ml TNFα, or 50 nM PMA. NETosis was assessed using PlaNET Green as described in Methods. (A) Quantitative representation of these experiments, expressed as NETosis index. Mean ± s.e.m. from at least 3 independent experiments. *p < 0.05 and **p < 0.01 vs. stimulus alone. (B) Representative fields for each experimental condition are shown at 10X magnification.
Figure 5
Figure 5
Identification of early and late processes underlying NET generation. (A) Neutrophils cultured on poly-L-lysine-coated coverslips were treated either before or after stimulation for the indicated times with the following inhibitors or their diluent (DMSO): 10 μM piceatannol (“pic,” Syk inhibitor); 10 μM LY294002 (PI3K inhibitor); 1 μM (5Z)-7-oxozeaenol (TAK1 inhibitor); 1 μM SB202190 (p38 MAPK inhibitor); 10 μM U0126 (MEK inhibitor). The cells were also stimulated for 4 h in the absence (“ctrl”) or presence of 30 nM fMLP, 1 nM GM-CSF, 100 U/ml TNFα, or 50 nM PMA. NETosis was then assessed using PlaNET Green as described in Methods. Quantitative representation of these experiments, expressed as NETosis index. Mean ± s.e.m. from 3 independent experiments. *p < 0.05 and **p < 0.01 vs. stimulus alone. (B) Neutrophils cultured on poly-L-lysine-coated coverslips were pre-treated (15 min, 37°C) with 20 μg/ml cycloheximide, 5 μg/ml actinomycin D, or their diluent (DMSO), prior to a further incubation of 4 h in the absence (“ctrl”) or presence of 30 nM fMLP, 1 nM GM-CSF, 100 U/ml TNFα, or 50 nM PMA. NETosis was then assessed using PlaNET Green as described in Methods. Quantitative representation of these experiments, expressed as NETosis index. Mean ± s.e.m. from at least 3 independent experiments.
Figure 6
Figure 6
Involvement of endogenous ROS and PAD4 in NET generation. (A) Neutrophils cultured on poly-L-lysine-coated coverslips were pre-treated (15 min, 37°C) with 10 μM DPI or its diluent (DMSO), prior to a further incubation of 4 h in the absence (“ctrl”) or presence of 30 nM fMLP, 1 nM GM-CSF, 100 U/ml TNFα, or 50 nM PMA. NETosis was then assessed using PlaNET Green as described in Methods. Quantitative representation of these experiments, expressed as NETosis index. Mean ± s.e.m. from at least 5 independent experiments. **p < 0.01 and ***p < 0.0001 vs. stimulus alone. (B) Neutrophils cultured on poly-L-lysine-coated coverslips were pre-treated (15 min, 37°C) with 10 μM chloraminidine (“Cl-A,” a general PAD inhibitor), 10 μM GSK484 (a PAD4 inhibitor), or their diluent (DMSO), prior to a further incubation of 4 h in the absence (“ctrl”) or presence of the above stimuli, followed by determination of the NETosis index. Mean ± s.e.m. from at least 4 independent experiments. *p < 0.04, **p < 0.01, and ***p < 0.001 vs. stimulus alone.
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