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. 2023 Apr 24;8(8):e166044. doi: 10.1172/jci.insight.166044

Figure 6. Effects of Plg and Pla on NETs release in vivo and in vitro.

Figure 6

(A) Plg+/+ and Plg–/– mice (n = 6) were subjected to nonsevere (30G needle) CLP and the plasma levels of H3cit were determined by ELISA. WT C57BL/6J mice (n = 4–7) were subjected to an i.p. injection of LPS (10 mg/kg) and then treated with Pla (10 μg/mouse i.p.) 3 hours later. (B and C) NETs release (MPO/DNA) in plasma (B) and peritoneal lavages (C) was determined 12 hours after LPS injection. Bone marrow neutrophils obtained from C57BL/6J mice were pretreated with Plg (4 μg/mL), Pla (4 μg/mL), or with Pla preincubated with the inhibitors (D-Val-Phe-Lys chloromethyl ketone [VPLCK] 22.5 nM or TXA 0.1 M) by 1 hour before stimulation with ultrapure LPS (1 μg/mL) for an additional 4 hours. (D) Quantification of NETs release (MPO/DNA) in supernatant. (E) WT C57BL/6J mice were subjected to an i.p. injection of LPS (10 mg/kg) and then treated with Pla (10 μg/mouse, i.p.) 3 hours later. TXA (100 mg/kg, i.p.) was injected 30 minutes before Pla. Plasma was collected 12 hours after LPS injection for NETs release (MPO/DNA) measurement by ELISA. **P < 0.01, ***P < 0.001 when comparing untreated (Unt) or PBS-injected mice with LPS-stimulated or injected and treated groups. P values are indicated in the graphs when comparing CLP-WT versus CLP-KO mice (unpaired 2-tailed Student’s t test) or vehicle with Pla-treated mice/cells by 1-way ANOVA with post hoc Newman-Keuls (multiple groups). Outliers were removed from the graphs when detected. (F) Representative fluorescence images of NETs stained for DNA (DAPI, blue), H3cit (green), and MPO (red) are shown. Scale bar: 50 μm at ×630 magnification.