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

NETosis, the process by which neutrophils release neutrophil extracellular traps (NETs), contributes to host defense but is increasingly implicated in inflammatory and autoimmune diseases. Two mechanistic classes of NETosis have been described: NOX2-dependent (requiring NADPH oxidase-derived ROS) and NOX-independent (e.g., induced by calcium ionophores), but the regulatory mechanisms for the latter remain unclear. Given prior evidence that small-conductance Ca2+-activated K+ (SK) channels in neutrophils can trigger mitochondrial ROS and apoptosis, the study asks whether calcium influx-induced, NOX-independent NETosis is mediated by mitochondrial ROS and SK channels, and how its signaling differs from NOX-dependent NETosis.

Prior studies established that NOX2-derived ROS is essential for NETosis induced by stimuli such as PMA, and patients with CGD show impaired NET formation to certain stimuli, substantiating the NOX-dependent pathway. Reports also document NOX-independent NETosis induced by calcium ionophores and some microbes. SK channels are the predominant Ca2+-activated K+ channels in neutrophils and were shown to mediate NOX-independent ROS and apoptosis (Fay et al., 2006). Potassium flux is important for neutrophil antimicrobial functions, yet its role in NETosis had not been examined. Additionally, signaling via ERK, Akt, and p38 has been implicated in PMA-induced (NOX-dependent) NETosis, and histone H3 hypercitrullination has been associated with calcium ionophore-induced NETosis via PAD4.

- Cells: Human peripheral blood neutrophils isolated from healthy donors via PolymorphPrep; some assays used differentiated HL-60 neutrophil-like cells (dHL-60). - NETosis assays: Plate reader assay with Sytox Green (cell-impermeable DNA dye) to quantify extracellular DNA kinetics; immunofluorescence microscopy to confirm NETs by colocalization of extracellular DNA and myeloperoxidase (MPO). - Stimuli: PMA (NOX-dependent comparator), calcium ionophores A23187 and ionomycin (to induce NOX-independent NETosis). Extracellular calcium depletion experiments assessed Ca2+ dependency. - ROS measurements: Cytosolic ROS (NOX-derived) measured using DHR123 by confocal imaging, flow cytometry, and plate reader; mitochondrial ROS measured using MitoSOX in plate reader assays. - Inhibitors and modulators: DPI (NOX inhibitor); DNP and FCCP (mitochondrial uncouplers) to inhibit mitochondrial ROS; kinase inhibitors FR180204 (ERK), Akt inhibitor XI, SB202190 (p38); SK channel inhibitors NS8593 and apamin; scyllatoxin (preferential SK2 blocker); MK886 (TRPM7 inhibitor); SK channel activator 1-EBIO. - Signaling: Western blotting for phospho-ERK, phospho-Akt, phospho-p38, total kinases, and citrullinated histone H3 (citH3) over time courses up to 120 min post-stimulation. - Genetics: siRNA-mediated knockdown of SK3 (KCNN3) in dHL-60 cells, validated by immunoblot; NETosis quantified post knockdown upon A23187 or ionomycin stimulation. - Statistics: Data as mean ± SEM; comparisons by Student’s t test or ANOVA with appropriate post tests; significance threshold P ≤ 0.05. - Kinetics and requirements compared side-by-side for PMA vs ionophores, with and without extracellular Ca2+, DPI, mitochondrial uncouplers, kinase and channel modulators.

- Calcium ionophores (A23187, ionomycin) induce rapid NETosis with kinetics distinct from PMA-induced NETosis. Removal of extracellular Ca2+ significantly reduces ionophore-induced NETosis (P < 0.001), but not PMA-induced NETosis. - NOX dependency: PMA robustly induces cytosolic ROS (DHR123) abolished by DPI; ionophores induce minimal cytosolic ROS. DPI significantly suppresses PMA-induced NETosis (P < 0.001) but does not significantly reduce A23187- or ionomycin-induced NETosis, establishing NOX independence for the ionophores. - Signaling: PMA strongly activates ERK and Akt; A23187 shows drastically reduced ERK activation and moderate Akt activation; p38 activation is similar in both. Functionally, ERK inhibition reduces PMA-induced NETosis but not A23187-induced; Akt inhibition reduces both PMA- and A23187-induced NETosis; p38 inhibition does not significantly affect NETosis. - Mitochondrial ROS: A23187 induces significantly higher mitochondrial ROS (MitoSOX) than PMA. Mitochondrial uncouplers (DNP, FCCP) abolish mitochondrial ROS and significantly, dose-dependently reduce A23187-induced NETosis (P ≤ 0.001), but do not significantly reduce PMA-induced NETosis. Thus, mitochondrial ROS is required for NOX-independent, but not NOX-dependent, NETosis. - SK channels: SK channel inhibitors NS8593 and apamin significantly reduce A23187- and ionomycin-induced NETosis in time- and concentration-dependent manners, but do not inhibit PMA-induced NETosis. SK2 targeting (scyllatoxin) and TRPM7 inhibition (MK886) do not reduce ionophore-induced NETosis. - SK3 specificity: siRNA knockdown of SK3 (KCNN3) significantly reduces NET release triggered by A23187 and ionomycin, confirming SK3 requirement. - Sufficiency: The SK channel activator 1-EBIO rapidly induces NETosis in human neutrophils, confirmed by Sytox Green kinetics and MPO–DNA colocalization, demonstrating that SK channel activation is sufficient to drive NOX-independent NETosis. - Additional marker: Histone H3 hypercitrullination (citH3) is observed in ionophore-induced but not PMA-induced NETosis, consistent with PAD4-dependent signatures of the NOX-independent pathway.

The study delineates mechanistic distinctions between NOX-dependent and NOX-independent NETosis. Calcium ionophore-driven NETosis proceeds rapidly, requires extracellular calcium, depends on mitochondrial (but not NOX2-derived) ROS, and is mediated by the SK3 small-conductance Ca2+-activated K+ channel. Signaling profiles differ: ERK activation is robust and necessary in NOX-dependent NETosis, while ERK activation is minimal and dispensable in NOX-independent NETosis; Akt activity is required in both, and p38 activation does not translate into a requirement in either pathway under these conditions. The data indicate limited cross-talk between mitochondrial ROS and NOX activation during NETosis, with mitochondrial superoxide not triggering NOX-derived cytosolic ROS in the NOX-independent context. Functionally, direct pharmacological activation of SK channels is sufficient to trigger NETosis, positioning SK3 upstream of mitochondrial ROS generation in this pathway. These mechanistic insights help explain stimulus-specific NETotic responses and suggest new intervention points for diseases where excessive NETosis contributes to pathology.

This work identifies a calcium influx–driven, NOX-independent NETosis pathway that is rapid and requires mitochondrial ROS and the SK3 small-conductance K+ channel. It contrasts with the classical PMA/NOX2-dependent pathway in both signaling requirements and ROS sources: ERK is essential only for NOX-dependent NETosis, while Akt is required in both. Pharmacological activation of SK channels is sufficient to induce NETosis, and SK channel blockade or mitochondrial uncoupling suppresses NOX-independent NETosis. These findings broaden the mechanistic framework of NETosis and suggest therapeutically targetable nodes—mitochondrial ROS generation and SK3 channel activity—for conditions with dysregulated NET formation. Future studies should define the precise coupling between SK3 activity and mitochondrial ROS production in neutrophils, assess potential mitochondrial SK channels, map upstream calcium dynamics and PAD4 regulation, and evaluate in vivo relevance and therapeutic modulation in disease models.

- The study primarily uses in vitro human neutrophils and dHL-60 cells; in vivo validation of the pathway and therapeutic modulation was not addressed. - Depletion of extracellular calcium only partially reduced ionophore-induced NETosis, likely due to mobilization of intracellular calcium stores; precise contributions of store-operated pathways were not dissected. - While mitochondrial ROS is implicated via uncouplers, specific mitochondrial ROS sources and mitochondrial potassium channel identities in neutrophils remain undefined; mitochondrial SK channels have not been identified in neutrophils. - Reliance on pharmacological inhibitors/activators may introduce off-target effects; although multiple tools (e.g., two uncouplers; two SK inhibitors; genetic SK3 knockdown) were used to mitigate this. - The study indicates signaling requirements (ERK, Akt, p38) but the downstream effectors linking these kinases to chromatin decondensation and NET release were not fully elucidated.