Neutrophil extracellular traps (NETs) are web-like structures released by neutrophils that play a crucial role in the innate immune response. While NETs are effective in trapping and killing pathogens, their excessive or dysregulated release can contribute to various inflammatory diseases, including thrombosis, vasculitis, lupus, autoimmunity, pneumonia, sepsis, and transfusion-related acute lung injury. Understanding the mechanisms regulating NET formation, known as NETosis, is therefore critical for developing therapeutic strategies. NETosis is a distinct form of cell death, different from apoptosis. Apoptosis is a programmed cell death process that is anti-inflammatory, whereas NETosis is a pro-inflammatory process. This study focuses on identifying key molecules responsible for switching neutrophil cell death from the pro-inflammatory NETosis to the anti-inflammatory apoptosis pathway. Previous studies have implicated reactive oxygen species (ROS) and autophagy in NETosis, but the precise role of kinases remains largely unclear. Akt, a serine/threonine-specific protein kinase, is a well-known inhibitor of apoptosis and a promising candidate for regulating the NETosis-apoptosis switch. This study aims to investigate the role of Akt in this crucial regulatory process.

Several studies have highlighted the role of NETs in various inflammatory diseases. Previous research has established a link between NOX2-dependent ROS production and NETosis, suggesting a critical role for ROS in triggering this process. The involvement of autophagy, a cellular self-cleaning process, in NETosis has also been demonstrated, implying that it is a necessary component of NET formation. Studies using pharmacological inhibitors such as wortmannin (a protein kinase C inhibitor) and rapamycin (an mTOR inhibitor) have provided insights into the signaling pathways involved. Wortmannin's ability to inhibit both autophagy and NETosis underscores the importance of autophagy in this process. Rapamycin's role in suppressing mTOR and subsequently affecting NETosis via HIF-1α further supports the complexity of the regulatory network. Despite these advances, the precise mechanisms underlying the transition between NETosis and apoptosis remain poorly understood. Akt, a well-established regulator of apoptosis, emerges as an attractive candidate given its role in suppressing programmed cell death. The hypothesis is that Akt serves as a molecular switch, influencing the balance between NETosis and apoptosis in neutrophils.

The study employed a multifaceted approach combining immunoblotting, flow cytometry, fluorescence plate reader assays, and immunofluorescence microscopy to investigate the role of Akt in regulating NETosis and apoptosis in human neutrophils. First, immunoblotting was used to assess Akt activation following stimulation with PMA, a potent inducer of NETosis, and in the presence or absence of DPI, a NOX2 inhibitor. This allowed the researchers to determine the relationship between NOX2-mediated ROS production and Akt activation. Flow cytometry was employed to measure ROS production in neutrophils under various conditions, including PMA stimulation and Akt inhibition. This confirmed the role of NOX2 in ROS generation and the effects of Akt inhibition. Two distinct Akt inhibitors, Akt inhibitor XI and MK2206, were used in a Sytox Green plate reader assay to quantify NET DNA release, providing a direct measure of NETosis. Dose-response experiments helped determine the impact of Akt inhibition on NET formation. Finally, immunofluorescence microscopy was utilized to visualize and quantify the number of apoptotic versus NETosis cells, employing markers like MPO and cleaved caspase-3. This enabled direct visualization of the switch between NETosis and apoptosis upon Akt inhibition. The use of H2O2 as a positive control for necrosis provided a further point of comparison.

The study's key findings demonstrate that PMA, a known inducer of NETosis, activates Akt in a NOX2-dependent manner. This indicates that ROS production triggers Akt activation. Importantly, the use of two different Akt inhibitors, Akt inhibitor XI and MK2206, revealed that Akt is essential for PMA-induced NETosis; inhibition of Akt significantly reduced NET DNA release in a dose-dependent manner. This finding strongly suggests that Akt activation is a crucial step in the NETosis pathway. Moreover, the researchers observed that Akt inhibition shifted the balance towards apoptosis, leading to a decrease in NETosis and an increase in apoptotic cells. This was confirmed by both quantitative analysis and immunofluorescence microscopy visualizing the co-localization of MPO (NET marker) and cleaved caspase-3 (apoptosis marker). These results demonstrate that Akt acts as a molecular switch, promoting NETosis when activated and allowing apoptosis when inhibited. The study found no activation of apoptotic caspase 3 or MPO coating of DNA prior to release in neutrophils undergoing necrosis.

The findings of this study provide compelling evidence that Akt plays a crucial role in regulating the balance between NETosis and apoptosis in neutrophils. The observed NOX2-dependent activation of Akt suggests that ROS production acts as an upstream signal that activates Akt, thereby triggering NETosis. The successful manipulation of this switch through Akt inhibition opens up exciting possibilities for therapeutic intervention. Targeting Akt could potentially offer a novel strategy to manage inflammatory diseases associated with excessive NET formation. By shifting the balance from NETosis to apoptosis, it may be possible to reduce inflammation and tissue damage. The study's findings have implications for various inflammatory conditions where excessive NET formation contributes to pathology. Further investigation is needed to explore the translational potential of Akt inhibition in clinical settings.

This research conclusively demonstrates that Akt serves as a pivotal molecular switch regulating the decision between NETosis and apoptosis in neutrophils. Activation of Akt, downstream of NOX2 and ROS generation, promotes NETosis, while Akt inhibition redirects neutrophil death towards apoptosis. This discovery highlights Akt as a promising therapeutic target to control NET-mediated inflammation, opening up avenues for new treatment strategies in various inflammatory disorders.

The study primarily used in vitro experiments with human neutrophils. The findings may not perfectly translate to the complex in vivo environment, where other factors could influence the NETosis-apoptosis balance. Further in vivo studies are needed to validate these findings and assess their clinical relevance. Additionally, the study focused on a limited set of Akt inhibitors. Future research could explore the effects of a wider range of Akt inhibitors to confirm the specificity of the observed effects. Finally, the mechanism through which Akt regulates NETosis requires further investigation, possibly involving the identification of downstream Akt targets in the NET formation pathway.