Fig. 7: Siglec-F^high late brain infiltrating neutrophils promote endothelial migration and can be induced by GM-CSF.

A Siglec-F^highCD101^high neutrophils were quantified in the brain, n = 6 (sham 24 h), n = 7 (sham 7 d), n = 8 (HI 24 h/ 7 d). B Projection of Siglec-F^highCD101^high neutrophils onto the UMAP of all cells from Fig. 6E or of brain neutrophils from Fig. 6H. C Surface marker expression on Siglec-F^high compared to Siglec-F^low brain-derived neutrophils. Only mice with >2% Siglec-F^high neutrophils at day 7 are displayed, n = 8 (24 h), n = 6 (7 d). D Multiplex immunohistochemistry of the ipsilateral hippocampus at day 7. Distance of Siglec-F^low and Siglec-F^high neutrophils to the next adjacent vessel was quantified for neutrophils from n = 3 mice, two-tailed Mann-Whitney test. Scale bar: 50 µm (E) qPCR of angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), VEGFA and IL-4 in isolated neutrophils at day 7, n = 6 biological replicates, details are provided in the source data file. F Wound healing response in scratch assays of bEND.3 in co-culture with brain neutrophils in presence or absence of 5 µg/ml anti-VEGF. Relative wound-healing compared to untreated controls was quantified, n = 10 (SiglecF^low/SiglecF^high), n = 6 (anti-VEGF w/o neutrophils), n = 4 (anti-VEGF + SiglecF^high neutrophils) biological replicates, i.e. wells, each with 10,000 neutrophils from 1-4 mice / well. Details are provided in the source data file. G GM-CSF protein abundance in brain tissue lysates, determined in proteome profiler antibody arrays (Fig. 2A), n = 3 (sham 24 h/ 7 d), n = 4 (HI 24 h), n = 7 (7 d). H Expression of Siglec-F, CD11b and CXCR4 after incubation of isolated Siglec-F^low blood neutrophils with 0.5 µg/ml GM-CSF for 18 h, two-tailed unpaired t-test, n = 6 mice. I Relative survival of isolated Siglec-F^low blood neutrophils after incubation with 0.5 µg/ml GM-CSF for 18 h, two-tailed unpaired t-test, n = 6 mice. J Wound healing response in scratch assays with GM-CSF pre-treated neutrophils or their supernatant, n = 9 vehicle (3x incubation with neutrophils (white circles), 6x incubation with supernatant (grey circles)), n = 3 incubation with GM-CSF-pretreated neutrophils, n = 6 incubation with supernatant of pretreated neutrophils. Bar graphs in A/F/G-J and centers in D/E show median values. A/E/F/G/J: One-way ANOVA followed by Šídák’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Individual values and exact p-values are given in the source data file.