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. 2021 Feb 15;11:606893. doi: 10.3389/fimmu.2020.606893

Figure 2.

Figure 2

TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p < 0.05, ** p < 0.01, ***p < 0.005. (C–F) Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR exploiting a Bio-Plex assay and phospho-specific antibodies. Primary human neutrophils were pre-incubated with or without (Ctrl, black) the TRPM7 inhibitor NS8593 (30 µM, red), the TRPM7 kinase blocker TG100-115 (20 µM, blue), or a combination of IPI and NEM (160 and 100 nM, gray), respectively, for 30 min prior to stimulation with 10 ng/ml LPS. Phosphorylation status of human neutrophils upon stimulation with 10 ng/ml LPS for 30 min of (A) NFκB p65 (Ser536), (B) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C) Akt (Ser473), and (D) mTOR (Ser2448), were analyzed. Data were normalized to protein content and presented as mean ± s.e.m.; biological n = 3 and measured in duplicates. Statistics: one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.005.