Fig 2: ATP release after excitotoxic neuronal cell death induces NETosis via a P2X7R-Ca2+-PKC signaling pathways. (A) Primary cortical neuron cultures were treated with 100 µM NMDA for various duration. NMDA-conditioned media (NCM) was prepared after 90 min of NMDA treatment and used to treat blood PMNs. (B) Primary cortical neuron cultures were treated with 100 µM NMDA for 30, 60, 90, or 120 min, and the ATP levels in primary cortical neuron culture media were subsequently measured using an ATP assay kit. (C) HMGB1 levels in culture media were determined after treating primary cortical neurons with NMDA (100 µM) for 30, 60, 90, or 120 min by immunoblotting. (D,E) Levels of PAD4 and CitH3 in NCM-treated blood PMNs were determined at the indicated times by immunoblotting. (F,G) Blood PMNs were pretreated with A438079 (20 µM), BAPTA (2.5 µM), Go6983 (2 µM), apocynin (20 µM), or Trolox (20 µM) for 20 min prior to treatment with NCM for 6 h, with the levels of PAD4 and CitH3 subsequently determined using immunoblotting. Immunoblots are representative of 2~4 independent experiments. (H) dsDNA release was measured after pretreating blood PMNs with A438079 (20 µM), BAPTA (2.5 µM), Go6983 (2 µM), apocynin (20 µM), or Trolox (20 µM) for 20 min prior to treatment with NCM. (I) Blood PMNs were treated with NCM or NMDA-untreated control media (con-M) for 3 or 9 h, and ATP levels in culture media of blood PMNs were determined using an ATP assay kit. Results are presented as mean ± SEM (n = 3). ** p < 0.01, * p < 0.05 versus the PBS-treated controls, ## p < 0.01 versus NCM only-treated cells, $ p < 0.05 between indicated group. Numbers under the blot indicate band intensities.

Fig 3: ATP-P2X7R mediates NETosis induction in ischemic brains. (A) Apyrase (0.2 U/g, i.v.) and BzATP (5 mg/kg, i.v.) was administered at 6 h post-MCAO and Trolox (2.5 mg/kg, i.v.) and apocynin (2.5 mg/kg, i.v.) was administered at 9 h post-MCAO. Immunoblotting and immunofluorescence staining was carried out at 12 or 24 h post-MCAO. (B–F) PAD4 and CitH3 levels in blood PMNs or cortical penumbras of ischemic hemispheres were determined at 12 or 24 h post-MCAO, respectively, by immunoblotting with or without apyrase (0.2 U/g, i.v.) treatment at 6 h post-MCAO (B–D), and immunofluorescence staining of brain tissues sections with CitH3 antibody was carried out at 24 h post-MCAO (E,F). (G,I,J) Apocynin (2.5 mg/kg) or Trolox (2.5 mg/kg) was administered 9 h post-MCAO and levels of PAD4, CitH3, and NET formation in blood PMNs purified at 12 h post-MCAO were assessed by immunoblotting (G) or immunofluorescence staining with CitH3 antibody (I,J), respectively. (H) PAD4 and CitH3 levels in cortical penumbra of the ischemic hemisphere were determined at 12 post-MCAO after treating BzATP at 6 h post-MCAO and with or without Trolox (2.5 mg/kg, i.v.) and apocyanin (2.5 mg/kg, i.v.) administration at 9 h post-MCAO. Immunoblots are representative of 2 or 3 independent experiments. Scale bars in (E,I) represent 50 µm. Results are presented as mean ± SEM (n = 3). ** p < 0.01 versus sham control, ## p < 0.01 versus MCAO. Numbers under the blot indicate band intensities.

Fig 4: Intracellular Ca2+ influx and PKCa activation in ATP-P2X7R-induced NETosis. (A) Blood PMNs were pretreated with A438079 (10 µM), BAPTA (2.5 µM), or Go6983 (1 µM) for 20 min prior to treatment with BzATP (50 µM) for 30 min. Intracellular Ca2+ levels were visualized by Fura 2-AM and quantified using ImageJ (http://rsbweb.nih.gov/ij/, accessed on 16 October 2020). Quantification of intracellular Ca2+ was normalized relative to PBS-treated controls. (B,C) Blood PMNs were pretreated with BAPTA (2.5 or 5.0 µM) for 20 min prior to treatment with ATP (100 µM) or BzATP (50 µM) for 4 h. Levels of PAD4 (B) or CitH3 (C) were subsequently determined by immunoblotting. (D) Blood PMNs were treated with ATP (100 µM) for the indicated duration or with 50, 100, or 200 µM of ATP for 30 and levels of PKCa and phospho-PKCa were examined by immunoblotting. (E,F) Blood PMNs were pretreated with A438079 (10 or 20 µM) or with BAPTA (2.5 or 5 µM) for 20 min prior to treatment with ATP (100 µM) for 30 min, then the levels of PKCa and phospho-PKCa were subsequently determined by immunoblotting. (G,H) Blood PMNs were pretreated with Go6983 (1 or 2 µM) for 20 min prior to treatment with ATP (100 µM) or BzATP (100 µM) for 4 h, then PAD4 or CitH3 levels were subsequently determined by immunoblotting. Immunoblots are representative of 2~4 independent experiments. (I,J) Blood PMNs were pretreated with BAPTA (2.5 µM) or Go6983 (2 µM) for 20 min prior to treatment with 100 µM of ATP for 12 h, with amounts of extracellular DNA complexed with neutrophil elastase (NE-DNA) subsequently assessed using Quant-iT PicoGreen dsDNA assay kit (I) and visualized using Sytox Green (J). Arrows in j indicate the released DNA and scale bars represent 50 µm. Results are presented as mean ± SEM (n = 3). ** p < 0.01, * p < 0.05 versus the PBS-treated control, ## p < 0.01 between indicated group.

Fig 5: Adenosine triphosphate (ATP) accumulation and ATP-mediated induction of PAD4 and CitH3 in blood neutrophils and cerebral cortical tissue after MCAO in male Sprague-Dawley (SD) rats. (A) ATP levels were measured using ATP luminescence kit in plasma collected at 6 and 12 h post-MCAO. (B,C) Levels of PAD4 and CitH3 in blood neutrophils (blood PMNs) or in cortical penumbra were examined at 3, 6, 12, and 24 h post-MCAO by immunoblotting. (D) BzATP (0.5, 1, 2.5, or 5 mg/kg, i.v.) was administered 6 h post-MCAO and PAD4 and CitH3 levels were examined in cortical penumbra at 12 h post-MCAO by immunoblotting. (E) ATP (5, 10, 20, or 50 mg/kg, i.v.) was administered to normal rats and PAD4 and CitH3 levels were examined in blood PMNs isolated 4 h after ATP administration by immunoblotting. (F) BzATP (5 mg/kg, i.v.) was administered to normal rats and PAD4 and CitH3 levels were examined in blood parenchyma and in circulating neutrophils (PMNs) isolated 1, 3, or 6 h after BzATP administration by immunoblotting. Immunoblots are representative of 2 or 3 independent experiments. Numbers under the blot indicate band intensities. ATP levels were presented as mean ± SEM (n = 4). ** p < 0.01 versus sham controls.