Fig. 4: XKR8-mediated lipid scrambling necessitates the activation of mechanosensitive Ca2+ channels for NET formation.
a,b, Quantification of PMA-induced NET formation in WT and XKR8-deficient BMNs, treated with or without EGTA from the beginning (1 mM) (n = 10) (a), or treated with EGTA (1 mM) at indicated time points after PMA stimulation (n = 9) (b). c, Evaluation of membrane tension using Flipper-TR by FLIM imaging. Representative images of Flipper-TR and annexin V in PMA-induced NETs (left) and quantification of Flipper-TR lifetime (τ) in PS-exposed (annexin V+) versus non-exposed (annexin V−) cells (right) (n = 50 in WT control, n = 57 in KO control, n = 59 in PMA-WT-annexin V+, n = 62 in PMA-WT-annexin V−, n = 53 in PMA-KO). Red indicates annexin V-AF594; green shows Flipper-TR. Scale bars, 5 μm. d, Schematic diagram showing calcium channels highly expressed on mouse neutrophils along with their respective inhibitors or agonists. e–i, Quantification of NET formation in WT and XKR8-deficient neutrophils treated with inhibitors and/or agonists listed in d. e, PMA-induced NET formation with inhibitors targeting TRPM2 (JNJ-28583113, 10 μM), TRPM4 (9-phenanthrol, 30 μM), TRPM7 (carvacrol, 100 μg ml−1), TRPV2 (SKF96365, 20 μM), TRPV4 (HC067047, 1 μM), Piezo1 (GsMTx-4, 10 μM), and TRPC3 (Pyr3, 5 μM) for 6 h (n = 10). f, PMA-induced NETs in WT and XKR8-deficient neutrophils treated with the agonists targeting TRPM2 (bilirubin, 10 μM), TRPM7 (naltriben (NTB), 50 μM), TRPV2 (cannabidiol (CBD), 10 μM), TRPV2 (probenecid, 1 mM), TRPV4 (GSK1016790A, 100 nM), Piezo1 (Yoda1, 10 μM), and TRPC3 (GSK1702934A, 100 μM) for 6 h (n = 10). g, PMA-induced NETs in WT and XKR8-deficient neutrophils with both inhibitors and agonists against TRPV2 (SKF96365 and CBD), TRPV4 (HC067047 and GSK1016790A), Piezo1 (GsMTx-4 and Yoda1), TRPC3 (Pyr3 and GSK1702934A) for 6 h (n = 9). h,i, As in f, quantification of LPS-induced NETs (h) or C. albicans-induced NETs (i) in WT and XKR8-deficient neutrophils treated with the agonists targeting TRPV2 (CBD, 10 μM), TRPV2 (probenecid, 1 mM), TRPV4 (GSK1016790A, 100 nM), Piezo1 (Yoda1, 10 μM) and TRPC3 (GSK1702934A, 100 μM) for 6 h (n = 10). NETs were measured by the area of decondensed chromatin DNA (a,b,e–i). Each dot represents one individual field of view (a,b,e–i) or a single cell (c). Data are presented as mean ± s.e.m. from one representative experiment of three (c,e–i) and four (a,b) independent experiments. Statistical analysis was performed using a two-way ANOVA (a,b,e–i) and one-way ANOVA (c). P values are shown on the graphs.
