Capsule summary
19Hydrolases perinuclearly accumulate in activated neutrophils of CGD patients or after inhibition 20 of PI3-kinase dependent ROS production, preventing their nuclear access and the resulting NETosis was induced by PMA (Fig 1, A) or Candida albicans (Fig 1, B) stimulation 50 together with PI3-kinase inhibitors in human HD and CGD neutrophils, and DNA release was 51 followed over time by SYTOX assays. Both stimuli resulted in NETosis in HD neutrophils.
52However, only inhibition of PI3-kinases through 3-methyladenine (3-MA) caused a significant 53 decrease in NETosis induced by both stimuli. Previously, nuclear NE translocation was suggested 54 to initiate chromatin decondensation and subsequent NETosis 1 . NE's nuclear translocation, as 55 assessed by confocal microscopy, was also reduced with 3-MA after opsonized yeast and PMA 56 incubation (Fig E1, A). Therefore, the PI3-kinase inhibitor 3-MA, but to a lesser extent other PI3-57 kinase inhibitors, like wortmannin and spautin-1, can inhibit NETosis. reduce autophagosome formation when compared to untreated cells (Fig 1, C). In addition, 63 membrane association of LC3B, detected as LC3-II levels by Western blotting, was not 64 significantly different ± 3-MA (Fig E1, B). Along these lines, neutrophils of NETosis-65 incompetent CGD patients induced macroautophagy to similar extent as HDs after stimulation 66 with opsonized yeast or PMA (Fig E2). In contrast, PI3-kinase inhibition with 3-MA consistently 67 reduced both yeast-and PMA-induced ROS production (Fig 1, D), suggesting that the negative 68 effect of 3-MA on NETosis might be related to the ineffectiveness of cells to generate ROS rather 69 than blocking macroautophagy. Together, these results point to a macroautophagy-independent,
70but ROS dependent mechanism of PI3-kinase to control NETosis in human neutrophils.
71To further investigate the steps leading to NETosis, we looked into the kinetics of NE 72 localization. Confocal microscopy of NE in PMA-treated HD and CGD neutrophils confirmed 73 the absence of extracellular or intra-nuclear NE staining in CGD neutrophils (Fig 2, A (Fig 1, E; Fig E3). As a second measure of neutrophil hydrolases' access to the 80 nucleus, we assessed proteolysis of histone H4 (Fig 2, C; Fig E4). After PMA-activation, HD 81 neutrophils initiated NETosis and H4 was degraded to allow DNA decondensation (Fig E4, A), -5 -whereas in CGD cells, H4 levels remained stable 3 h and 4 h after PMA-induction (Fig E4, B).
83This indicates that neutrophils deficient in phagosome-associated NOX2-mediated ROS 84 production are unable to induce NE nuclear translocation, but can nevertheless accumulate NE 85 around their nuclei.
86To confirm that the classical macroautophagy machinery was not involved in the transport 87 of vesicular NE to the nucleus, we assessed perinuclear co-localization of LC3B and NE in 88 neutrophils isolated from HDs by confocal microscopy. As previously observed (Fig 2), NE 89 translocated to the area around the nucleus 1h post-stimulation with PMA ( Fi...