Fig. 5

M2-Exos inhibit PMN recruitment and NET formation through LXA4 upregulation. a Ex vivo NET formation assay with PMNs isolated from healthy volunteers or septic patients activated by septic plasma (SP) and then cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h with or without BOC-2 (10 µM). Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. b 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. c Transwell analysis of PMN migration capacity isolated from healthy volunteers or septic patients. One-way analysis of variance with Tukey’s multiple comparisons test was used for the analysis. n = 4. d–l WT C57BL/6 mice were administered M2-Exos (300 μg/mouse) derived from mouse Raw264.7 macrophages via intraperitoneal injection 1 h after CLP. To block the LXA4 receptor, mice were treated with 50 µg/kg BOC-2 i.p. 30 min before CLP. d and e Quantification of dsDNA and circulating NET structures in the plasma of mice using PicoGreen fluorescent dye and MPO-DNA-ELISA, respectively. f Representative images showing the presence of NETs (MPO, red; citrullinated H3, green) in the lung tissues, as indicated by white arrows. Nuclei were counterstained with DAPI (blue). Scale bar, 40 μm. g Flow cytometry detection of the percentage of systemic circulating PMNs by staining with CD11b and Gr-1. h Absolute neutrophil number in peripheral blood. i and j Ly6G⁺ cells in the lung tissues were detected by immunofluorescence and immunohistochemistry. Scale bar, 40 μm. k Evaluation of lung histology by H&E staining (magnification × 400). Red arrows indicate neutrophils in the alveolar and interstitial space, blue arrows indicate alveolar macrophages, and green arrows indicate proteinaceous debris filling. Scale bar, 50 μm. Lung injury scores were assessed. l Detection of inflammatory cytokine mRNA (IL-1β, IL-6, TNF-α) expression in lung tissues by RT‒qPCR. Student’s t test was used for the analysis. Graphs represent means ± standard deviations, n = 6; *P < 0.05, **P < 0.01 compared within two groups