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. 2021 Sep 9;9:718586. doi: 10.3389/fcell.2021.718586

FIGURE 1.

FIGURE 1

Lipid peroxidation is involved in NET formation in vitro and in vivo. (A) NETosis profiling of human neutrophils treated with cell death inhibitors. Peripheral blood neutrophils obtained from two healthy volunteers were stimulated with 50 nM PMA, 5 μM ionomycin, or 3 μg/mL ICs for 4 h in the presence or absence of various cell death inhibitors. Cells were stained with SYTOX Green and Hoechst 33342. The proportion of NETosis was determined by counting the number of SYTOX Green+ cells using a high-content analysis system. The degree of inhibition of each inhibitor is shown by a heatmap. Representative data of two independent experiments are shown. (B–D) Inhibitory effects of trolox on NETosis induction and NET formation induced by PMA or ICs. (B,C) Human neutrophils were stimulated with either 50 nM PMA or 3 μg/mL ICs for 4 h in the presence or absence of 1.6 mM trolox. The proportion of NETosis was determined using the same method as that in panel (A). Average values and s.d. of triplicated samples in a single experiment are shown (B). ***P < 0.005, one-way ANOVA. Representative images are shown (C). Data are representative of two independent experiments. (D) Human neutrophils were stimulated with PMA or ICs in the presence or absence of 1.6 mM trolox. Cells were stained using DAPI and visualized by fluorescence microscopy. (E,F) Lipid peroxidation levels in neutrophils undergoing NETosis. Human neutrophils were stimulated with PMA or ICs for 1 h in the presence or absence of 1.6 mM trolox, and then incubated with BODIPY 581/591 C11. The accumulation of oxidized phospholipids was analyzed by flow cytometry. The higher peak is indicated by arrow. A representative flow cytometry plot is shown (E). Average values of median fluorescent intensity with s.d. of twelve samples in four experiment are shown (F, left). Average values of mean fluorescent intensity with s.d. of triplicate samples in a single experiment are shown (F, right). *P < 0.05, **P < 0.01, one-way ANOVA. (G,H) C57BL/6 mice were administrated PBS (control) or 10 μg of LPS intranasally. Lungs were resected 24 h after injection. (G) Lung sections were analyzed by immunofluorescence analysis with anti-MPO antibody (MPO, green), anti-citH3 antibody (CitH3, red), and DAPI (blue). Data are representative of two independent experiments. (H) Western blot analysis of citH3 protein levels in lungs of mice treated as indicated. (I,J) Inhibitory effects of trolox on citH3 expression in lungs. Mice were injected intraperitoneally with 40 mg/kg trolox at 0 and 24 h prior to intranasal instillation of LPS. Twenty-four hours after instillation, the lungs were analyzed by Western blot with anti-citH3 antibody. Representative images of three mice in each group are shown (I). CitH3 expression levels were normalized to GAPDH expression levels using Evolution Capt Software. Average values and s.d. of citH3 protein levels in each group (without trolox: n = 10, with trolox: n = 10) are shown (J). **P < 0.01, unpaired t-test.