Fig 1: CS2164 decreases the frequencies of MDSCs and TAMs in both spleen and tumor tissue. The frequencies of spleen (a), CD11b+ Gr-1+ MDSC cells (b), and F4/80+ MHC-II+ TAMs (c) in tumor tissues from vehicle-treated or CS2164-treated mice described in Fig. 1 were analyzed by flow cytometry. The representative plots are shown and the accumulative data are expressed as mean ± SD from one of two independent experiments. *P < 0.05; **P < 0.01 compared with the vehicle control group. MDSC, myeloid-derived suppressor cell; TAM, tumor-associated macrophage.
Fig 2: Mast cells influence phagocytosing activity of macrophages during resolution phase of zymosan-induced inflammation. (A) FACS analysis for the number of neutrophils, macrophages, eosinophils and dendritic cells in Mcpt5-DTA-Cre- and Cre+ mice 48 h after injection of zymosan (10 µl, 12 mg/ml in PBS). Data are shown in % of all cells as mean ± S.E.M. (n=5–9), unpaired two-tailed t-test. (B) Number of M1-like (CD86+) macrophages and M2-like (CD206+) macrophages 48 h after injection of zymosan (10 µl, 12 mg/ml in PBS). Data are shown in % of all F4 80+ cells as mean ± S.E.M. (n=7–9), unpaired two-tailed t-test. (C) Gating strategy for flow cytometry analysis of phagocytosis of pHrodo Red zymosan by macrophages (F4 80+) and neutrophils (F4 80-/Ly6G+). (D, E) Phagocytosis of pHrodo Red zymosan (10 µl, 12 mg/ml in PBS) by neutrophils (panel D) or macrophages (panel E) in Mcpt5-DTA-Cre- and Cre+ mice 48 h after zymosan injection. Data are mean ± S.E.M. (n=7–9), unpaired two-tailed t-test, *p<0.05. (F) Gating strategy for flow cytometry analysis of phagocytosis of neutrophils by macrophages. Intracellular staining of Ly6G+ neutrophils was performed. (G) Decreased phagocytosis of neutrophils (intracellular Ly6G) by macrophages (F4 80+). Data are mean ± S.E.M. (n=7–9), unpaired two-tailed t-test, *p<0.05.
Fig 3: The effect of sunitinib treatment on intratumoral MDSC, Tregs, CD8+ T cells and activation status of the CD8 T cell population. C57Bl6 mice were subcutaneously inoculated with TC-1 tumor cells (n = 3–6 mice/group). On day 15 after tumor inoculation, sunitinib treatment was started, daily, i.p., for a period of 9 consecutive days. Three increasing dosages of sunitinib were used: 20, 40 and 60 mg/kg body weight. Mice were sacrificed and tumors and spleens were harvested. The levels of (A) MDSCs (CD11b+Gr1+), (B) Tregs (CD4+FoxP3+), (C) CD8+ T cells (CD8+; black bars) and the activation status of CD8+ T cells (CD69+; white bars) were analyzed by immunostaining and multicolor fluorescence cytometry. Experiments were repeated twice. Shown here are averages and SD for each experimental group (*p < 0.05; **p < 0.01; ***p < 0.001).
Fig 4: M1 macrophage phagocytosis of neutrophils is reduced in mast cell-deficient Mcpt5-DTA Cre+ mice compared to Cre- control mice. Images show representative BH t-SNE analysis from Mcpt5-DTA Cre+ or Cre- mice 48 h after injection of zymosan. Plots on the left are colored by cell clusters defined by PhenoGraph analysis (Figure S4). Plots on the right are heatmaps for the expression of the neutrophil marker Ly6G. The position of cell phenotype clusters containing CD86+ M1 macrophages (M1 MF) or neutrophils (Ly6G+/F4 80-) is indicated.
Fig 5: Loss of Cemip Enhances Local Skin Inflammation in Response to S. aureus(A–F) Flow cytometry analysis of single-cell suspensions from the skin showing expression of CD11c/MHCII, Ly6G/CD11b, and LyG-C cells isolated from control, Cemip-/- control infection, and Cemip-/- infection. Cells were gated on CD3-negative. Numbers represent the percentages of the cells in the indicated gate.(G) Representative sections of skin from control and Cemip-/- mice at 3 days after S. aureus infection. Tissues are stained with red with Gr-1 antibody and blue with DAPI. Scale bar, 20 µm.(H) qRT-PCR of the relative abundance of transcripts for IL-6 as normalized to ß-actin (n = 4 for normal condition, n = 8 for infection mice/group).All error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (t test).
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