Akt is essential to induce NADPH-dependent NETosis and to switch the neutrophil death to apoptosis

Neutrophil extracellular traps (NETs) are DNA-based structures that trap and kill microbes but can also injure host tissues. NETs have been implicated in thrombosis, vasculitis, systemic lupus erythematosus, autoimmunity, pneumonia, sepsis, and transfusion-related acute lung injury. Regulating NETosis is therefore important to prevent pathology. The molecular mechanisms that switch neutrophil death from proinflammatory NETosis to anti-inflammatory apoptosis are not clearly established. Because NOX2-dependent reactive oxygen species (ROS) can induce either NETosis or apoptosis, and Akt is a known inhibitor of apoptosis, the study examines whether Akt functions as a molecular switch controlling the NETosis–apoptosis axis.

Prior work showed that PMA induces autophagy and that both autophagy and PMA-mediated ROS are required for NETosis; inhibition of protein kinase C with wortmannin reduced autophagy and NETosis. Rapamycin studies demonstrated that mammalian target of rapamycin regulates NETosis via hypoxia-inducible factor 1α. However, identities of other key kinases that regulate NETosis–apoptosis pathways were unclear. Akt/PKB is known to regulate neutrophil apoptosis.

Human neutrophils were activated with phorbol 12-myristate 13-acetate (PMA, 25 nM) to induce NOX2-dependent ROS and NETosis. NOX2 activity was inhibited with diphenyleneiodonium (DPI, 20 µM). Akt activity was inhibited using two pharmacological inhibitors: Akt inhibitor XI (0–10 µM; preincubation 30–60 min) and MK2206 (0–10 µM; preincubation 30 min). ROS production was measured by flow cytometry using dihydrorhodamine 123, with cell gating confirmed by forward/side scatter and Hoechst 33342. Akt activation was assessed by immunoblotting for phosphorylated Akt after 1 h PMA stimulation; total Akt and GAPDH served as loading controls. NETosis was quantified in a Sytox Green (5 µM) fluorescence plate reader assay using extracellular DNA release over 5 h in 96-well plates (3×10^5 cells/well), reporting fluorescence at 5 h as NETotic index relative to PMA control. Cell death modalities were differentially quantified by imaging fixed cells stained with Sytox Green and classifying nuclei as live, NETotic, or apoptotic (pyknotic); at least 100 cells per condition were counted. Immunofluorescence microscopy stained for myeloperoxidase (MPO) and cleaved caspase-3 (cCasp3) to distinguish NETosis and apoptosis. Hydrogen peroxide (H2O2, 8 mM) was used as a control to induce necrosis. Donor numbers per assay varied (n=4–7 donors). Statistical analyses used ANOVA with Dunnett’s or Bonferroni post-tests, with significance thresholds of P<.05 or P<.001.

- PMA activated Akt in neutrophils during induction of NETosis; this activation was abrogated by DPI, indicating Akt activation depends on NOX2-mediated ROS (immunoblot, Figure 1A). - Flow cytometry confirmed PMA-induced ROS; DPI, but not Akt inhibitor XI, suppressed ROS generation (Figure 1B). - Two distinct Akt inhibitors (MK2206 and Akt inhibitor XI) dose-dependently inhibited extracellular DNA release/NETosis in the Sytox Green assay (Figures 1C–D). NETotic index at 5 h was significantly reduced versus PMA-only controls (P<.05 to P<.001; n=4–7 donors). - Akt inhibition redirected cell fate from NETosis to apoptosis: preincubation with Akt inhibitor XI increased apoptotic (pyknotic) nuclei and decreased NETotic cells in a dose-dependent manner (quantitative imaging, Figure 1E; n=4–5 donors). - Immunofluorescence showed that Akt inhibition increased cCasp3-positive cells and reduced MPO-decorated extracellular DNA, confirming a switch from NETosis to apoptosis (Figure 1F). - H2O2-induced necrosis did not activate caspase-3 or produce MPO-coated DNA, distinguishing necrosis from NETosis and apoptosis (Figure 1F).

The findings demonstrate that NOX2-dependent activation of Akt is required for PMA-induced NETosis and that Akt simultaneously suppresses apoptosis. Pharmacologic inhibition of Akt does not impair ROS generation but prevents NET formation and promotes caspase-dependent apoptosis, indicating NETosis and apoptosis are opposing, Akt-regulated pathways in neutrophils. Identifying Akt as a molecular switch provides a mechanistic basis for manipulating neutrophil death modalities. Therapeutically, targeting Akt could reduce pathological NETosis and favor resolution via apoptosis in NET-associated hematologic and inflammatory diseases.

Akt is essential for NOX2-dependent NETosis in human neutrophils, and its inhibition switches neutrophil death from NETosis to apoptosis. These results position Akt as a bona fide molecular switch governing the NETosis–apoptosis axis and suggest that modulating Akt activity could be a strategy to treat NET-driven hematologic and inflammatory conditions. Future work should delineate upstream and downstream components linking NOX2-derived ROS to Akt activation and apoptosis, and evaluate in vivo efficacy and safety of targeting Akt to modulate NETosis.