Fig. 3.

Systemic atRA therapy skewed neutrophil towards N2 phenotype and facilitated neutrophil clearance by microglia / macrophage. a Schematic diagram showing that neutrophils in ipsilateral brain were magnetically sorted with anti-Ly6G antibodies and subjected to qPCR. b Heat map showing the phenotypic shift of neutrophil in stroke lesion of atRA pre-treated mice. Multiple N1 (orange characters) and N2 (blue characters) markers were analyzed with qPCR. In the heat map, data are displayed as fold change to neutrophils from PBS-treated group. Black, expression un-altered, fold change = 1; Red, expression up-regulated, fold change > 1; Green, expression downregulated, fold change < 1. c Quantification of phenotypic markers that showed significant mRNA expression alteration in neutrophil from atRA-treated mice compared with those from PBS-treated mice. Quantification of markers that without significant difference between the two groups were displayed in Additional file 1: Figure S2. N = 8 mice per group. *P ≤ 0.05, ***P ≤ 0.001, versus PBS group in t test. d–f Phenotypic shift of neutrophil from brain lesion after atRA treatment was evaluated with flow cytometry at 1–3 days after tMCAO. Representative flow panels and quantification of the percentage of CD206⁺ (d) (percentage among CD11b⁺CD45^hiLy6G⁺ neutrophil), Ym1/2⁺ (e) (mean fluorescence intensity (MFI) among CD11b⁺CD45^hiLy6G⁺ neutrophil), and Arg1⁺ (f) (percentage among CD11b⁺CD45^hiLy6G⁺ neutrophil) of neutrophil were shown. N = 4 mice per group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, versus PBS-treated group in two-way ANOVA. g–h Clearance of neutrophil by microglia / macrophage was assessed with immunofluorescent staining and flow cytometry at 3d after tMCAO. g Left: Representative images demonstrating neutrophil (Ly6G⁺, green) engulfed by microglia/macrophage (Iba1⁺, red) in infarct penumbra at 3d after tMCAO. The engulfed neutrophil by microglia/macrophage (Ly6G⁺Iba1⁺, yellow) was emphasized with white arrows. Right: Quantification of Ly6G⁺Iba1⁺ engulfed neutrophil by microglia/macrophage. N = 4 mice per group. *P ≤ 0.05, versus PBS group in t test. h Neutrophil (CD11b⁺CD45^hiLy6G⁺) engulfed by macrophage (CD11b⁺CD45^hiF4/80⁺) in stroke lesion at 3 days after tMCAO was gated as CD11b⁺CD45^hiLy6G⁺F4/80⁺ cells. Representative flow panels and quantification of CD11b⁺CD45^hiLy6G⁺ F4/80⁺ engulfed neutrophil among CD11b⁺CD45^hi cells were shown. N = 6 mice per group. **P ≤ 0.01, versus PBS group in t-test. MACS, magnetic cell sorting