Fig. 3

M2-Exos inhibit PMN recruitment and NET formation during sepsis in vitro. a and b PMNs from healthy volunteers were preactivated by septic plasma and then cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages. After 5 h, PMNs were collected for migration capacity analysis with IL-8 as a chemokine. After a 2-h incubation, cells in the lower chamber were collected and counted under a microscope. c and d PMNs isolated from septic patients were directly cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h after isolation. Then, PMNs were transferred for the transwell assay. e–h Ex vivo NET formation assay with neutrophils isolated from healthy volunteers or septic patients activated by septic plasma (SP). Plasma from healthy volunteers (HP) was used as a negative control. Typical images of NET formation are presented in e and g using SYTOX Green (green), where white arrows indicate NETs. Scale bar, 50 μm. NET formation was quantified as the percentage of neutrophils forming NETs and the NET area per microscopic field. f and h Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. One-way analysis of variance with Tukey’s multiple comparisons test was used for the analysis. Graphs represent means ± standard deviations; *P < 0.05, **P < 0.01 compared within two groups