Fig. 8

Exosomal PGE2 from M2 macrophages is necessary for 15-LO upregulation and PMN inhibition. a PGE2 levels in M0-Exos/M2-Exos/exosomes from celecoxib (20 µM)-treated PBMC-differentiated M2 macrophages (Cel-M2-Exos) were examined by ELISA. PMNs isolated from healthy volunteers were activated by septic plasma (SP) and then cocultured with M2-Exos/Cel-M2-Exos/Cel-M2-Exos + PGE2 (100 nM) for 5 h. b Immunoblot analysis of 15-LO in PMNs. c and d LXA4 and LTB4 concentrations in the supernatant of PMNs were detected by ELISA. e 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. f Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. g Transwell analysis of neutrophil chemotaxis towards IL-8. Flow cytometry detection of CXCR2 (h) and ROS (i) expressions in cocultured PMNs. One-way analysis of variance with Tukey’s multiple comparisons test was used for the analysis. n = 4–6. j–u WT C57BL/6 mice were administered M2-Exos/Cel-M2-Exos (300 μg/mouse) derived from mouse Raw264.7 macrophages via intraperitoneal injection 1 h after CLP. j 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. k and l Quantification of dsDNA and circulating NET structures in the plasma of mice using PicoGreen fluorescent dye and MPO-DNA-ELISA, respectively. m Flow cytometry detection of the percentage of systemic circulating neutrophils by staining with CD11b and Gr-1. n Absolute neutrophil number in peripheral blood. o and p Ly6G⁺ cells in the lung tissues were detected by immunohistochemistry and immunofluorescence. Scale bar, 40 μm. CXCR2 (q) and ROS (r) expressions in peripheral blood neutrophils were detected by flow cytometry. s Evaluation of lung histology by H&E staining (magnification × 400). Red arrows indicate neutrophils in the alveolar and interstitial space, and black arrows indicate thickening of the alveolar walls. Scale bar, 50 μm. Lung injury scores were assessed. t 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. u Survival rate of CLP mice treated with M2-Exos or Cel-M2-Exos (n = 8); the log-rank test was used for the analysis. *P < 0.05, **P < 0.01 compared within two groups