Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A

Abstract

Excessive or aberrant generation of neutrophil extracellular traps (NETs) has recently become implicated in the underlying aetiology of a number of human pathologies including preeclampsia, systemic lupus erythromatosus, rheumatoid arthritis, auto-antibody induced small vessel vasculitis, coagulopathies such as deep vein thrombosis or pulmonary complications. These results imply that effective pharmacological therapeutic strategies will need to be developed to counter overt NETosis in these and other inflammatory disorders. As calcium flux is implicated in the generation of reactive oxygen species and histone citrullination, two key events in NETosis, we analysed the roles of both extra- and intracellular calcium pools and their modulation by pharmacological agents in the NETotic process in detail. Interleukin-8 (IL-8) was used as a physiological stimulus of NETosis. Our data demonstrate that efficient induction of NETosis requires mobilisation of both extracellular and intracellular calcium pools. Since modulation of the calcineurin pathway by cyclosporine A has been described in neutrophils, we investigated its influence on NETosis. Our data indicate that IL-8 induced NETosis is reduced by ascomycin and cyclosporine A, antagonists of the calcineurin pathway, but not following treatment with rapamycin, which utilizes the mTOR pathway. The action of the G protein coupled receptor phospholipase C pathway appears to be essential for the induction of NETs by IL-8, as NETosis was diminished by treatment with either pertussis toxin, a G-protein inhibitor, the phospholipase C inhibitor, U73122, or staurosporine, an inhibitor of protein kinase C. The data regarding the calcineurin antagonists, ascomycin and cyclosporine A, open the possibility to therapeutically suppress or modulate NETosis. They also provide new insight into the mechanism whereby such immune suppressive drugs render transplant patients susceptible to opportunistic fungal infections.

Figure 1. Detection of IL-8 induced NETosis and calcium mobilization.

A. Upper panel: Detection of NETs induced by IL-8, PMA or ionomycin by fluorescent microscopy using SYTOX Green. Magnification: 20x. Middle panel: Detection of NETs induced by IL-8, PMA or ionomycin by fluorescent immunohistochemistry for the presence of MPO (myeloperoxidase) or citrullinated histone H3 (citH3) on NETs structures. Magnification: 20x; scale bar: 50 nm Lower panel: Detection of NETs induced by IL-8, PMA or ionomycin scanning electron microscopy (SEM). NETs were induced by treatment with rIL-8 (100 ng/ml), PMA (50 nM) or ionomycin (5 µM). Scale bar: 10 µm. B. Quantification of NETs release from activated neutrophils by fluorimetry. NETs were induced by treatment with rIL-8 (100 ng/ml), PMA (50 nM) or ionomycin (5 µM) and monitored over a period of 180 minutes. NET formation (100%) is normalized to that mediated by ionomycin (5 µM) or PMA (50 nM) treatment after 180 minutes, indicating a reduced NETotic response triggered by IL-8 (ng/ml). C. Examination of intracellular calcium release in activated neutrophils by fluorescence microscopy. Detection of calcium mobilization by fluorescent microscopy using Fluo-3 AM loaded neutrophils. Mobilization of intracellular calcium was noted in cells treated with ionomycin or IL-8, while only minimal flux was detected in PMA treated cells. Magnification: 40x; scale bar: 20 nm. D. Quantitation of intracellular calcium levels. Fluorimetric quantitation of intracellular calcium flux using Fluo-3 AM loaded neutrophils. Both IL-8 and ionomycin treatment lead to substantial increases in free intracellular calcium, unlike PMA, where only a minimal increase was noted.

Figure 2. Role of intra- and extracellular calcium pools in the induction of NETosis by IL-8. A. Examination of intracellular calcium mobilization by fluorescence microscopy in IL-8 stimulated neutrophils treated with TMB-8 and EGTA.

Calcium mobilization was triggered by treatment with IL-8. Intracellular calcium utilization was antagonized by TMB-8, while extracellular calcium was chelated with EGTA. Mobilization of intracellular calcium in cells treated with IL-8 was greatly reduced by chelation of external calcium by EGTA or binding of internal stores by TMB-8, and largely abolished by both agents in combination. Magnification: 40x; scale bar: 20 nm. B. Detection of NETosis by fluorescent microscopy using SYTOX Green under calcium sequestering conditions. NETosis induced by IL-8 is greatly reduced by EGTA, to a lesser extent by TMB-8, and completely abolished by the action of both agents. NETosis triggered by PMA was largely unaffected by the action of TMB-8, being more sensitive to the chelation of extracellular calcium by EGTA. Magnification: 10x. NETs generation was normalized as 100% for 10 ng/ml IL-8 or 50 nM PMA treatment. C. Detection of NETs by fluorescent immunohistochemistry following calcium sequestration or treatment with cyclosporine A. The combined action of TMB-8 and EGTA effectively blocked IL-8 induced NETs as detected by the presence of citH3 or MPO. In a similar manner, treatment with the calcineurin antagonist CSA, greatly diminishes IL-8 induced NETs generation. Magnification: 20x; scale bar: 50 nm. Inserts indicate higher magnification view in panel immediately below. D. Quantitative fluorimetric detection of IL-8 or PMA induced NETosis following calcium sequestration. Upper panel illustrates NETosis induced by IL-8, while lower panel illustrates NETosis induced by PMA. For this analysis, neutrophils (1×10⁵/well) were plated in 96-well plates in the appropriately modified medium and activated with increasing doses IL-8 (0–100 ng/ml). Following 1 hr culture, NETs were quantified fluorimetrically and the data normalized as described in Materials and Methods. An analysis from 4 different experiments is shown, and the data are presented as mean ± SEM. Two-way ANOVA with Bonferroni correction were applied to assess differences between multiple groups. Statistically significant P values are indicated in the graphs as follows: ****, P<0.0001 and ***, P<0.001. E. Influence of calcium sequestration on ROS production. ROS was measure via Luminol detection after induction with rIL-8 (100 ng/ml) or PMA (50 nM) or ionomycin (5 µM). H₂O₂ (50 µM) served as a relative control and considered 100%. DPI (25 µM), a specific inhibitor of NADPH mediated ROS generation, was used to block ROS production and was used as negative control. Extracellular calcium was chelated with EGTA and release of intracellular calcium was antagonized with TMB-8. An analysis from 6 different experiments is shown, and the data presented as mean ± SD (standard deviation). Two-way ANOVA with Bonferroni correction were applied to assess differences between multiple groups. Statistically significant P values are indicated in the graphs as follows: ****, P<0.0001; ***, P<0.001; **, P<0.01 and *, P<0.05. F: Chelation of intracellular calcium reduces NETosis. Neutrophils (1×105/well) were plated in the 96-well plates in triplicates. Cells were pre-treated with 10 µM BAPTA-AM, a cell permeable calcium chelator, for 15 minutes, washed extensively and then activated with either 100 ng/ml IL-8, or with 50 nM PMA, or with 5 µM ionomycin. NETs were quantified fluorimetrically following 1 hour culture as described. An analysis from 8 different experiments is shown, and the data are presented as mean ± SD (standard deviation). Unpaired t-test was used to calculate two-tailed P value to estimate statistical significance of differences between two groups. Statistically significant P values are indicated in the graphs as follows: Statistically significant P values are indicated in the graphs as follows: ****, P<0.0001. The data were normalized as 100% NET formation as induced by treatment with rIL-8 (100 ng/ml), PMA (50 nM) or ionomycin (5 µM) in the absence of any inhibitors.

Figure 3. Inhibition of IL-8 induced NETosis by ascomycin and cyclosporine A. A. Quantitative fluorimetric detection of NETosis modulated by ascomycin, cyclosporine A or rapamycin.

1×10⁵ cells/well were plated in triplicates in 96-well plates. Cells were pre-incubated with the indicated increasing doses (0–200 ng/ml) of calcineurin antagonists for 15 min at 37°C prior to the addition of 100 ng/ml IL-8 (upper panel), or 50 nM PMA (middle panel), or 5 µM ionomycin (lower panel). NETs were quantified fluorimetrically after 1 hour culture using SYTOX Green dye. An analysis from 8 different experiments is shown, and the data presented as mean ± SD (standard deviation). NET formation was normalized to 100% as induced by treatment with rIL-8 (100 ng/ml), PMA (50 nM) or ionomycin (5 µM) in the absence of any inhibitors. Two-way ANOVA with Bonferroni correction were applied to assess differences between multiple groups. Statistically significant P values are indicated in the graphs as follows Statistically significant P values are indicated in the graphs as follows: ****, P<0.0001 and *, P<0.05. B. Monitoring of cell viability by WST-1 assay. In this representative experiment cells (1×10⁵ cells/well) were pre-treated with Ascomycin for 15 minutes, following which they were either left without further stimulus, or stimulated with 100 ng/ml IL-8, 50 nM PMA or 5 µM ionomycin. Following addition of the WST-1 reagent, cell viability was monitored over a period of 4 hours according to the manufacturer's instructions. This assay indicates that the vast majority of cells were metabolically active over the entire culture period. No significant changes were noticed between any of the groups.

Figure 4. Involvement of the GPCR-PLC pathway in IL-8 induced NETosis. A. Treatment with pertussis toxin inhibits IL-8 induced NETosis.

Isolated neutrophils were pretreated with pertussis toxin (PTX) (100 ng/ml) for 15 minutes and then cultured with activators, rIL-8 (100 ng/ml), PMA (50 nM), ionomycin (5 µM) for 1 hour. NETosis was detected by fluorescent microscopy for SYTOX Green stained extracellular DNA structures. Magnification: 10x. B. Fluorimetric detection of NETosis reduction by pertussis toxin treatment in IL-8 stimulated neutrophils. This figure illustrates a quantitative reduction in IL-8 induced NETosis by treatment with PTX (15 minute pre-treatment) (upper panel), while that triggered by PMA (lower panel), was largely unaffected. An analysis from 6 different experiments is shown, and the data presented as mean ± SD (standard deviation). NET formation was normalized to 100% as induced by treatment with rIL-8 (100 ng/ml) or PMA (50 nM) in the absence of any inhibitors. Unpaired t-test was used to calculate two-tailed P value to estimate statistical significance of differences between two groups. Statistically significant P values are indicated in the graphs as follows: **, P<0.01. C. Influence of staurosporine treatment on IL-8 induced NETosis. This figure illustrates a quantitative reduction in IL-8 induced NETosis by treatment with the protein kinase C inhibitor, staurosporine (STS) (15 minute pre-treatment) (upper panel), and almost complete abolition of that triggered by PMA. An analysis from 6 different experiments is shown, and the data presented as mean ± SD (standard deviation). NET formation was normalized to 100% as induced by treatment with rIL-8 (100 ng/ml) or PMA (50 nM) in the absence of any inhibitors. Unpaired t test was used to calculate two-tailed P value to estimate statistical significance of differences between two groups. Statistically significant P values are indicated in the graphs as follows: **, P<0.01. D. Inhibition of Phospholipase C reduced IL-8 induced NETosis. Following pre-treatment with U73122 for 15 minutes, an inhibitor of phospholipase C, neutrophils were cultured for 1 hour in the presence of 100 ng/ml IL-8, or 50 nM PMA, or 5 µM ionomycin. These data indicate that only IL-8 induced NETosis was significantly affected. NETs were quantified fluorimetrically after 1 hr culture using SYTOX Green dye and the data normalized as described. An analysis from 4 different experiments is shown, and the data presented as mean ± SD (standard deviation). Unpaired t-test was used to calculate two-tailed P value to estimate statistical significance of differences between two groups. Statistically significant P values are indicated in the graphs as follows: Statistically significant P values are indicated in the graphs as follows: ****, P<0.0001.

Figure 5. Schematic representation of the calcium-dependent signal transduction pathway triggered by IL-8.

The action of calcineurin is antagonized by Cyclosporine A and Ascomycin, that of the G protein coupled IL-8 receptor by PTX and that of PLC by U73122. External calcium is chelated by the action of EGTA, while intracellular calcium is sequestered by the cell permeable chelator BAPTA-AM. The release of intracellular calcium from the endoplasmic reticulum is antagonized by TMB-8. DPI inhibits the action of the NADPH oxidase required for ROS production, a key step in NETosis.