Fig 1: 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 2: ZQT induced apoptosis in G-MDSCs in TME and inhibited its immunosuppressive activity by activating the STAT3/S100A9/Bcl-2/caspase-3 signaling pathway. Mice were treated with anti-Ly6G neutralizing antibody or isotype control by intraperitoneal injection for 14 days before surgery, after which model mice were given maintenance treatment every other day. (a, b) The bioluminescence images of model mice on day 7 and day 28 after surgery. (c) Survival curves. (d, e) The percentage of G-MDSCs, CD3+ T cells, CD4+ T cells, and CD8+ T cells in tumor tissue were analyzed by flow cytometry. (f, g) CD3+ T cells isolated from the tumor tissue of tumor-bearing C57BL/6 mice in different groups were cocultured with LLC cells for 4 h before they were incubated with CD3, CD8, and CD107α antibodies. Events shown were finally gated on CD3 and CD8. (h) ELISA analysis for the expression of IFN-γ in cells from tumor tissue. (i) RT-qPCR analysis of Arg-1, iNOS, and S100A9 gene expression and flow cytometry for ROS production. (j) IHC staining for Ly6G and CD8 in tumor tissue (scale bar: 50 μM). n = 5. Data are expressed as the mean ± SD. n/s: nonstatistical significance. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Fig 3: Depletion of neutrophils reduces TNF-⍺‒induced edema and neutrophil infiltration over 4 hours. (a) The number of Ly6G+ neutrophils and (b) Ly6Chi monocytes measured in mice blood after i.p. injection with either IgG control or anti-Ly6G antibody for 24 hours. Results are shown as mean ± SEM (n = 6). t-test was performed. ∗∗∗P < 0.001 compared with control IgG. (c) Representative images. (d) Edema volume and (e) neutrophils infiltration measured at dorsal skin sites injected with either Tyrode and TNF-⍺ over 4 hours or SP + CGRP over 30 minutes in neutrophils-depleted mice and control mice. Results are shown as mean ± SEM (n = 5). Two-way ANOVA followed by Tukey’s posthoc test was performed. ∗∗∗∗P < 0.0001 compared with control mice. CGRP, calcitonin-gene related peptide; i.p., intraperitoneal; SP, substance P.
Fig 4: Mitochondrion-containing EVs from neutrophils exhibit Sod2-dependent antithrombotic function.a–b Confocal images of Sod2 (Red), Mito-Dendra2 (Green) and Ly6G (Grey) among blood leukocytes from PhAMfloxed × Mrp8-Cre mice. Scale bar in a, 3 μm; Scale bar in b, 2 μm. c–d Confocal images of SOD2 (Red), TOM22 (Green) and CD16 (Grey) among blood leukocytes from healthy donors. Scale bar in c, 3 μm; Scale bar in d, 2 μm. e Schematic of sequential centrifugation to isolate EVs from mouse vasculature lavage fluid. f Western blot analysis of Sod2, COX IV and Ly6G protein levels in different pellets (P1-P5) purified from mice 4 h after PBS or LPS. g Representative transmission electron microscope (TEM) images of the isolated EVs from P1 (left) and P2 (right). Scale bar, 2 μm. h Representative TEM images of isolated EVs containing small mitochondria (red arrows). Scale bar, 0.2 μm. i Representative TEM images of a liver section showing a mitochondrion-containing EV (red arrow) within an intravascular neutrophil. Scale bar, 2 μm. The boxed area is enlarged at the bottom panel. Scale bar, 1 μm. j, m Representative imaging of liver functional vessel in the indicated recipient mice 1 h after lethal LPS. FITC-Dextran (green) labeled blood vessels, and DID-labeled red blood cells (red) indicated blood flow. Scale bar, 100 μm. k Mean ratios of the functional vessel area to the total vessel area in mice 1 h after lethal LPS. Mice were pre-transferred with LPS-primed EVs (LPSEVs) (n = 6), PBS-primed EVs (PBSEVs) (n = 7) or PBS only (n = 7). p = 0.0396. l Survival of mice after lethal LPS (20 mg/kg) challenge. Mice were pre-transferred with LPS-primed EVs (n = 21), PBS-primed EVs (n = 21) or PBS only (n = 21). n Mean ratios of the functional vessel area to the total vessel area in mice 1 h after lethal LPS. Mice were pre-transferred with LPS-primed Sod2+/− PB EVs (LPSEVsSod2+/−) (n = 6), LPS-primed EVs (LPSEVsWT) (n = 7) or PBS only (n = 7). 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 i. Two-tailed unpaired t tests were used for statistical analyses in k and m. *p < 0.05.
Fig 5: Neutrophils release mitochondrion-containing EVs during migration and reduce endothelial ROS accumulation.a–b Representative images from intravital imaging of neutrophils (Ly6G-Red, red arrows) within liver vasculature (Dextran-Green) in wild type mice. The white arrow indicates a vesicle released by a migrating neutrophil. Scale bar in a, 30 μm; Scale bar in b, 5 μm. c–d Representative images from intravital imaging of neutrophils (Ly6G-Red) within liver vasculature (CD144-Blue) in PhAMfloxed × Mrp8-Cre mice. Mitochondria of neutrophils were labeled by Mito-Dentra2 (Dendra2-Green). The white arrow indicates a mitochondrion-containing vesicle released by a migrating neutrophil. Scale bar in (c), 15 μm; Scale bar in d, 5 μm. e Flow analysis of ROS levels in hepatic endothelial cells 1 h after PBS or lethal LPS in the indicated recipient mice. f M.F.I. of ROS levels in hepatic endothelial cells 1 h after PBS or lethal LPS in the indicated recipient mice (n = 5 per group). 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. Two-tailed unpaired t tests were used for statistical analyses. *p < 0.05, **p < 0.01, ***p < 0.001.
from Cell Signaling Technology for Ly-6G (E6Z1T) Rabbit mAb