Neutrophils are critical innate immune cells, employing various mechanisms to eliminate pathogens, including oxidative burst, phagocytosis, and the release of neutrophil extracellular traps (NETs) via NETosis. NETs, composed of DNA and antimicrobial proteins, effectively trap and kill microbes. However, excessive NET formation is implicated in various inflammatory and autoimmune conditions, including rheumatoid arthritis, vasculitis, thrombosis, cystic fibrosis, and acute respiratory distress syndrome. Therefore, understanding the mechanisms regulating NETosis is crucial for developing effective therapies. Two main NETosis pathways are recognized: NOX-dependent, requiring NADPH oxidase 2 (NOX2) and its ROS production; and NOX-independent, triggered by stimuli like calcium ionophores. While the NOX-dependent pathway is relatively well-characterized, the mechanisms governing NOX-independent NETosis remain unclear. This study aimed to elucidate the molecular mechanisms underlying calcium ionophore-induced, NOX-independent NETosis, focusing on the roles of mitochondrial ROS and calcium-activated potassium channels.

Previous research has established the importance of NOX2 and ROS in NOX-dependent NETosis. Inhibition of NOX2 or deficiencies in NOX-mediated ROS production, as seen in chronic granulomatous disease (CGD), impair NET formation. Conversely, NOX-independent NETosis has been observed with stimuli such as calcium ionophores. Studies have shown that the calcium-activated potassium channel of small conductance (SK channel), mainly SK3 in neutrophils, plays a role in NOX-independent neutrophil apoptosis and mitochondrial ROS production. The involvement of potassium channels in NET formation, however, remained unexplored. This gap in knowledge provided the impetus for this current investigation into the role of mitochondrial ROS and SK channels in calcium ionophore-mediated NETosis.

Human neutrophils were isolated from healthy donor blood. NETosis was induced using phorbol 12-myristate 13-acetate (PMA) for NOX-dependent NETosis and the calcium ionophores A23187 and ionomycin for NOX-independent NETosis. NET release was quantified using a plate reader assay with Sytox Green, a cell-impermeable DNA dye. Cytosolic and mitochondrial ROS production were measured using dihydrorhodamine (DHR) 123 and MitoSOX, respectively. Kinase activation (ERK, Akt, p38) was assessed via immunoblotting. The roles of NOX2, mitochondrial ROS, and SK channels were investigated using pharmacological inhibitors (DPI, DNP, FCCP, NS8593, apamin) and siRNA-mediated knockdown of SK3. A SK channel-specific activator, 1-Ethyl-2-benzimidazolinone (EBIO), was used to determine the sufficiency of SK channel activation for NETosis induction. Differentiated HL-60 cells (dHL-60 cells) were used in some assays. Statistical analysis employed Student's t-tests or ANOVA with post-hoc tests, with p<0.05 considered statistically significant.

The study revealed several key findings: 1. Calcium ionophore-induced NETosis is significantly faster than PMA-induced NETosis. 2. Calcium ionophore-induced NETosis is NOX-independent, as demonstrated by the lack of significant inhibition by the NOX inhibitor DPI. 3. Calcium ionophore-induced NETosis exhibits substantially lower ERK and moderate Akt activation compared to PMA-induced NETosis, while p38 activation is similar in both. 4. Inhibition studies revealed that Akt activity is essential for both NOX-dependent and -independent NETosis, but ERK is crucial only for NOX-dependent NETosis. 5. Mitochondrial ROS production is necessary for NOX-independent NETosis, but not for NOX-dependent NETosis, as shown by experiments with mitochondrial uncouplers DNP and FCCP. 6. Inhibition studies using NS8593 and apamin, along with siRNA knockdown of SK3, confirm the critical role of the SK3 potassium channel in NOX-independent NETosis. 7. Activation of the SK channel by 1-EBIO is sufficient to induce NETosis, demonstrating its causal role in this pathway.

This study clearly distinguishes NOX-dependent and NOX-independent NETosis pathways. The NOX-independent pathway, triggered by calcium influx, is rapid and relies on mitochondrial ROS generation and SK3 channel activation. The differential kinase activation profiles—particularly the non-essential role of ERK in the NOX-independent pathway—highlight distinct signaling mechanisms. The finding that Akt is essential for both pathways underscores its importance in NETosis regulation. The identification of mitochondrial ROS and the SK3 channel as key mediators of NOX-independent NETosis opens new avenues for therapeutic intervention. Targeting these components might provide novel strategies for managing diseases characterized by excessive NET formation.

This study identifies a novel mechanism for NOX-independent NETosis involving mitochondrial ROS and the SK3 potassium channel. The distinct signaling characteristics and the identification of these key components offer valuable insights into NETosis regulation and potential therapeutic targets for diseases associated with dysregulated NET formation. Future research could explore the precise interactions between SK3, mitochondrial ROS, and other signaling molecules in greater detail, furthering our understanding of NETosis and facilitating the development of specific therapies.

The study primarily used in vitro models, which may not fully reflect the complexity of in vivo conditions. While the study convincingly implicates SK3, further investigation into the specific role of other SK channel subunits and potential crosstalk with other ion channels or signaling pathways may be warranted. The use of pharmacological inhibitors may have off-target effects, and although the results are highly suggestive of a direct role for SK3, further studies employing alternative gene editing techniques could definitively confirm its role.