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Clinical Trial
. 2015 Mar 3;112(9):2817-22.
doi: 10.1073/pnas.1414055112. Epub 2015 Feb 17.

SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx

David Nobuhiro Douda  1 , Meraj A Khan  2 , Hartmut Grasemann  3 , Nades Palaniyar  4
Affiliations
Clinical Trial

SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx

David Nobuhiro Douda et al. Proc Natl Acad Sci U S A. .

Abstract

Neutrophils cast neutrophil extracellular traps (NETs) to defend the host against invading pathogens. Although effective against microbial pathogens, a growing body of literature now suggests that NETs have negative impacts on many inflammatory and autoimmune diseases. Identifying mechanisms that regulate the process termed "NETosis" is important for treating these diseases. Although two major types of NETosis have been described to date, mechanisms regulating these forms of cell death are not clearly established. NADPH oxidase 2 (NOX2) generates large amounts of reactive oxygen species (ROS), which is essential for NOX-dependent NETosis. However, major regulators of NOX-independent NETosis are largely unknown. Here we show that calcium activated NOX-independent NETosis is fast and mediated by a calcium-activated small conductance potassium (SK) channel member SK3 and mitochondrial ROS. Although mitochondrial ROS is needed for NOX-independent NETosis, it is not important for NOX-dependent NETosis. We further demonstrate that the activation of the calcium-activated potassium channel is sufficient to induce NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is essential for the NOX-dependent pathway, whereas its activation is not essential for the NOX-independent pathway. Despite the differential activation, both NOX-dependent and -independent NETosis require Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into previously unknown mechanisms for NOX-independent NETosis.

Keywords: NADPH oxidase; NETosis; SK channels; neutrophil extracellular traps; neutrophils.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Calcium ionophores induce rapid NETosis. (A) NET release in response to A23187 or PMA was measured using a plate reader assay (n = 5). A23187- and PMA-mediated NETosis in human neutrophils follow different kinetics of NET release. Ionophore-induced NETosis is faster than that of PMA-induced NETosis. (B and C) Neutrophils were activated with A23187 (B) or ionomycin (C) in the presence or absence of calcium. NETosis was measured using a plate reader assay. NET release is expressed as percentage of total DNA (n = 3). Calcium ionophore-induced NETosis requires extracellular calcium. (D) Neutrophils were incubated in the presence or absence of A23187 (4 µM) or ionomycin (5 µM) for 300 min. Cells were stained for DNA (green) and MPO (red). Immunofluorescence imaging shows that the calcium ionophore A23187 and ionomycin induce NETosis. (Scale bar, 10 µm.) Images are representative of three independent experiments. A23, A23187; Ca2+, calcium chloride; Io, ionomycin. *P < 0.05; **P < 0.01; ***P < 0.001 (A–C, two-way ANOVA).
Fig. 2.
Fig. 2.
Calcium ionophore-mediated NETosis is NOX2 independent. (A–D) Human neutrophils were loaded with cytosolic ROS indicator DHR123 and activated with PMA or A23187. Live cell fluorescence image analysis of DHR123 loaded cells treated with control buffer (A), PMA (B), or A23187 (C). The presence of cytosolic ROS is indicated by the green fluorescence, and the cells were counterstained with Hoechst 33342 live cell DNA stain (blue) (n = 4). (Scale bar, 20 µm.) (D) Representative flow cytometric analysis of cytosolic ROS production in neutrophils loaded with DHR123 and activated with either PMA or A23187, in the presence or absence of DPI (n = 3). (E and F) DHR123-based ROS detection plate reader assays show that PMA induces a significantly greater cytosolic ROS production compared with A23187. (G–I) Neutrophils were activated by PMA, A23187, or ionomycin in the presence or absence of DPI (20 μM) and NET release was measured using a plate reader assay. The results are expressed as percentage of reduction in the presence of DPI compared with the activating agonist alone. DPI significantly reduces PMA-mediated NETosis (G), whereas it fails to inhibit A23187 (H)- and ionomycin (I)-mediated NETosis (n = 3). DPI → PMA, preincubation with DPI and activated with PMA; A23, A23187; DPI → A23, preincubation with DPI and activated with A23187; Io, ionomycin; DPI → Io, preincubation with DPI and activated with ionomycin. *P < 0.05 (E, two-way ANOVA; F–I, Student’s t test).
Fig. 3.
Fig. 3.
Immunoblot analysis. (A–D) Human neutrophils were harvested after an activation with PMA, A23187, or negative control (-ve) for the indicated times. The negative (-ve) control samples were incubated at 120 min in the absence of any activator. (A) Immunoblots show that A23187, but not PMA, induces citrullination of histone H3 (citH3) (n = 4). (B–D) The activation of kinases was assessed by immunoblotting for phospho (p)-ERK (B, n = 3), p-Akt (C, n = 3), and p-p38 (D, n = 2). Total kinases t-Akt, t-ERK, and t-p38 were used as loading controls. ERK is highly activated during PMA-induced NETosis. Akt is activated in both forms of NETosis, albeit a moderate level of activation in A23187-induced NETosis. p38 is equally activated in both forms of NETosis.
Fig. 4.
Fig. 4.
A23187-mediated NETosis requires mitochondrial respiration. (A and B) Human neutrophils were loaded with a mitochondrial ROS indicator, MitoSox, and activated with PMA or A23187. A time-course fluorescence plate reader assay for MitoSOX fluorescence shows a larger mitochondrial ROS production in cells activated by A23187 as opposed to PMA-activated cells. A mitochondrial uncoupler, DNP, abolishes mitochondrial ROS production induced by A23187 (n = 3). (B) MitoSOX assays show that A23187-activated cells produce a significantly higher levels of mitochondrial ROS at the peak production at the 25-min time point, compared with the cells activated by PMA (n = 3). (C) A23187-mediated NETosis is reduced in the presence of DNP (750 µM) as shown by time course analysis (n = 7). (D) PMA-mediated NETosis is not significantly reduced in the presence of DNP (750 µM) as shown by time course analysis (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 compared with the agonist alone (A, C, and D, two-way ANOVA; B, Student’s t test).
Fig. 5.
Fig. 5.
SK channel is required for ionophore-mediated NETosis. (A) Human neutrophils were activated with A23187 in the presence of SK channel inhibitor NS8593 (100 µM). The plate reader assay shows that NETosis is significantly reduced in the presence of NS8593 (n = 3), suggesting that A23187-induced NET DNA release in human neutrophils requires the activation of the SK channel. (B) Neutrophils were activated with PMA in the presence of NS8593. The plate reader assays show that NETosis is not inhibited in the presence of NS8593 (100 µM; n = 3), indicating that PMA-mediated NETosis does not require the activation of the SK channel. (C and D) Human neutrophils were activated with A23187 (C) or ionomycin (D) in the presence of SK channel inhibitor apamin (200 nM; n = 3). The plate reader assays show that NETosis is reduced significantly in the presence of apamin; hence, A23187- and ionomycin-induced NETosis requires the activation of the SK channel. A23, A23187; Io, ionomycin. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the agonist alone (two-way ANOVA).
Fig. 6.
Fig. 6.
SK3 is required for the NOX-independent NETosis. (A) The plate reader assay shows that A23187- or ionomycin-mediated NETosis is significantly reduced in siRNA (si)-transfected dHL-60 cells, compared with scrambled RNA (sc)-transfected control cells at 240 min postactivation (n = 3). Shown above the graph is the result of an immunoblot analysis illustrating a successful knockdown of SK3 protein expression after siRNA transfection (n = 3). Therefore, knockdown of the SK3 channel by siRNA significantly reduces calcium ionophore-induced NETosis. (B and C) SK channel activator 1-EBIO induces NETosis. (B) The plate reader assays were performed to monitor NET release over time in response to the activation with 1-EBIO (n = 5). (C) Immunofluorescence staining confirms that compared with DMSO control, the activation of potassium channels by 1-EBIO induces NET release as shown by the colocalization of DNA (green) and MPO (red) after 300 min. sc, scrambled RNA-transfected cells; si, SK3 siRNA-transfected cells. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the agonist alone (A, one-way ANOVA; B, two-way ANOVA).
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