Akt shifts NETosis to apoptosis in human neutrophils. (A) Immunoblot analysis for the activation of Akt as determined by its phosphorylation. Cells were lysed after 1 hour of PMA (25 nM) activation. DPI (20 μM) was used for inhibiting NOX2-mediated ROS production. Total Akt and glyceraldehyde-3-phosphate dehydrogenase were used as loading control (n = 6 donors; −ve, negative control; DPI → PMA, neutrophils were pretreated with DPI [20 μM] and then activated with PMA). (B) Analysis of cells for their production of ROS by flow cytometry. Prior to the activation with PMA (25 nM), cells were preincubated for 30 or 60 minutes in the presence or absence of Akt inhibitor XI (Akt-i XI; 10 μM; Akt-i XI → PMA) or DPI (20 μM; DPI → PMA) for 30 minutes. In all conditions, cells were also incubated with dihydrorhodamine 123 as a probe for ROS production. Cells were gated using forward and side scatter and confirmed with Hoechst 3342 as a counterstain. Shown is a representative of 4 independent experiments with cells from 4 individuals. (C-D) Fluorescence plate reader assay for NETosis. Cells were cultured in a 96-well culture plate (3 × 10⁵ cells per well) in the presence of Sytox Green (5 μM; Invitrogen), a cell impermeable DNA-binding dye, to monitor release of NET DNA. Varying concentrations (0-10 μM) of 2 different Akt-i, (C) Akt inhibitor XI (Millipore), and (D) MK2206 (Sellekchem), were added to the cells 30 minutes prior to the activation of cells with PMA (25 nM). Numbers beneath the graphs represent the concentration of Akt-i used prior to activation with PMA. Extracellular DNA release was monitored at t = 0, 30, and 60 minutes and every hour for a total of 5 hours. Shown is the fluorescence intensity at 5 hours. NETotic index was calculated as percentage of total fluorescence given off by PMA-only positive control. (n = 4-7 donors). (E) Differential quantification of live, NETotic, and apoptotic (pyknotic) nuclei. Cells were cultured in an imageable special optics 96-well plate in the presence or absence of Akt-i (0-10 μM) for 30 or 60 minutes prior to the activation of PMA (25 nM). The numbers in parentheses represent the concentration of Akt-i used prior to activation with PMA. Live and apoptotic nuclei are not stained by Sytox Green dye unless the cells are fixed. Thus, the cells from the plate reader assay were fixed at the end of the assay in the presence of the dye, and cells were differentially quantified on the basis of their nuclear morphology. Representative images of live, NETotic, and apoptotic nuclei quantified are shown below the graph. At least 100 cells were quantified in each condition (n = 4-5 individual donors). (F) Immunofluorescence staining for myeloperoxidase (MPO) and cCasp3. Neutrophils were incubated with H₂O₂ (8 mM) to induce necrosis. In other conditions, cells were activated with PMA (25 nM) with or without preincubation for 30 minutes with Akt inhibitor XI. Cells were stained with MPO (mouse α-MPO, 1:250; Abcam) as a marker for NETs and cCasp3 (rabbit α-cleaved caspase 3, 1: 150; Cell Signaling) as a marker for apoptosis. Arrows, NETotic DNA and nuclei; arrowheads, cCasp3-positive cells. Bar: 10 µm (n = 4 donors). All data are expressed as mean ± standard error of the mean where appropriate. *P < .05 and ***P < .001 compared with PMA only controls (C-D). Analysis of variance with (C-D) Dunnett's or (E) Bonferroni post-tests was used for determining statistical significance. (G) Proposed role of Akt in regulating the switch between NETosis and apoptosis.