Fig 2: Serpine1 reversed the chemotaxis inhibition of dauricine on neutrophils. (A) Diagram of primary neutrophils from the bone marrow of mice. (B) The purity of primary neutrophils isolated from the bone marrow of mice was detected by flow cytometry with APC-CD11b and PE-Ly6G antibodies. (C) mRNA expression of serpine1 was detected via quantitative real-time PCR after 48 h of plasmid transfection in primary microglia. n = 3 mice per group. Vector group vs serpine1 overexpression group, ***p < 0.001; unpaired Student’s t-test. (D) Primary neutrophils were prestrained with DiO (cell membrane green fluorescent probe) and then migrated for 3 h with an inducer from microglia. Representative pictures of DiO-stained primary neutrophil migration for 3h from the control+vector, LPS+vector+DMSO, LPS+vector+dauricine, and LPS+serpine1+sauricine groups and were counted (E), n = 5 per group. Control+vector vs LPS+vector+DMSO group, ***p < 0.001; LPS+vector+DMSO vs LPS+vector+dauricine group, ###p < 0.001; LPS+vector+dauricine vs LPS+serpine1+dauricine group, &&p < 0.01; unpaired Student’s t-test. (F) Ligand receptor docking was performed with AutoDock and analyzed with PyMOL. The top panel shows the joint diagram of dauricine and serpin-1 protein. The lower panel shows the energy data under natural conditions and molecular docking.

Fig 3: Dauricine reduces neutrophil infiltration into the MCAO-R brain, and inhibits the serpine1 activity and expression. Immunofluorescence staining of ly6G protein in mouse slices of sham (A), MCAO (B) and MCAO + dauricine (C), and the ly6G positive cells were counted (D). Microglia cell induced by LPS or dauricine for 3h, and stained with serpine1 and DAPI (E), mean fluorescence intensity of serpine1 was counted (F). n = 5 per group. Control group vs LPS group, *p < 0.05; LPS group vs (LPS + dauricine) group, ##p < 0.01; unpaired Student’s t-test.

Fig 4: Impaired neutrophil recruitment in Fpr2-deficient mice during GBS-infection. (A) Cell counts in peripheral blood samples after i.v. challenge with GBS at 0h, 3h, and 6h; kinetics of recruitment of cells positive for CD11b, Ly6G(neutrophils), Ly6C(monocyte/macrophages), CD19 (B lymphocytes) and CD3 (T lymphocytes) in WT and Fpr2-defective mice. (B) Representative images of liver tissue sections labelled with mIHC(Left), neutrophil number analyzed by inform software (Right Up) and Flow cytometry (Right down). Three-color, multiplex immunofluorescent images of mice liver tissue sections displaying the spatial distribution of different immune lineages and markers. The technique labels three channels as follows: Ly6G (green), CXCL1(red)and DAPI (blue). (C) Survival and numbers of bacterial in blood of WT and Fpr2–/– mice pretreated with anti-Ly-6G antibody or the isotype control after i.v. challenge with 2×108 CFU/ml of GBS. Data are representative of three independent experiments (n = 5 mice in the per group) and are shown as mean ± SD. *P < 0.05, ns means non sense.

Fig 5: Transfer of Sod2-rich EVs alleviates the coagulopathy in neutropenic mice upon lethal LPS challenge.a Survival of α-Ly6G (1A8) (n = 15), or isotype antibody (Rat IgG2a) (n = 15) treated mice after lethal LPS. p = 0.0078. b Survival of DT-treated PMNDTR (ROSA26-iDTRKI × Mrp8-Cre) (n = 15) mice and PMNWT (Mrp8-Cre) (n = 15) littermates after lethal LPS. p = 0.0185. c Representative imaging of liver functional vessel in the neutropenic mice 1 h after PBS or lethal LPS. FITC-Dextran (green) labeled blood vessels, and DID-labeled red blood cells (red) indicated blood flow. Scale bar, 100 μm. d Mean ratios of the functional vessel area to the total vessel area in α-Ly6G (1A8) (n = 4), or isotype antibody (n = 4) treated mice after PBS. Mean ratios of the functional vessel area to the total vessel area in α-Ly6G (1A8) (n = 8), or isotype antibody (n = 6) treated mice after lethal LPS. Mean ratios of the functional vessel area to the total vessel area in α-Ly6G (1A8) treated mice after lethal LPS. Mice were pre-transferred with LPS-primed EVs (LPSEVs) (n = 5). e Western blot analysis of Ly6G protein levels in different pellets (P1-P5) purified from WT or TSPAN9−/− mice 4 h after LPS. f Survival of mice after lethal LPS challenge. Mice were pretransferred with LPS-primed TSPAN9−/− PB neutrophils (LPSneutrophilsTSPAN9−/−) (n = 6), LPS-primed WT PB neutrophils (LPSneutrophilsWT) (n = 10) or PBS only (n = 10). Source data are provided as a Source Data file. Data are representative of, or pooled from at least three independent experiments. Data are mean ± SD. log-rank (Mantel–Cox) test was used for statistical analyses in a, b, f. Two-tailed unpaired t tests were used for statistical analyses in d. *p < 0.05, **p < 0.01, N.S. not significant.