Fig. 7

M2-Exos promote LXA4 production in PMNs by increasing 15-LO expression. a–i PMNs isolated from healthy volunteers were activated by septic plasma (SP) and then cocultured with M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h with or without PD146176 (1 µM) and LXA4 (100 nM), as indicated in the figures. DMSO was used as a negative control. a Representative images of 15-LO in PMNs detected by immunofluorescence. (b left panel) 15-LO and 5-LO expressions in PMNs were detected by Western blot. (b right panel) PMNs isolated from septic patients were cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h. 15-LO and 5-LO expressions in PMNs were detected by Western blot. c and d LXA4 and LTB4 concentrations in the supernatant of PMNs were detected by ELISA. e Transwell analysis of neutrophil chemotaxis towards IL-8. f Typical images of NET formation using SYTOX Green (green). Scale bar, 50 μm. NET formation was quantified as the percentage of neutrophils forming NETs and the NET area per microscopic field. g Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. Flow cytometry detection of CXCR2 (h) and ROS (i) expressions in cocultured PMNs. Student’s t test (c and d) or one-way analysis of variance with Tukey’s multiple comparisons test (e–i) was used for the analysis. Graphs represent means ± standard deviations, n = 4–6; *P < 0.05, **P < 0.01 compared within two groups