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