Fig 1.
Activated microglia/macrophages accumulate in the perilesional areas after MCAO.
(A) TTC-stained brain section indicating the stroke area (pale color marked by asterisk). (B) Upper panels show glial scar at day 4 in the perilesional area stained for microglia (Iba1) and astroglia (GFAP) that do not strictly overlap. Lower panels show quantifications of Iba1 and GFAP reactive cells in the glial scar region for different time points. Asterisks mark the infarct area. (See S2 Fig for different coronal slices of stroke brain). (C) Progressive accumulation of activated microglia/macrophages in the glial scar region over 4 days. (D) Representative images of Iba1/CD68 co-staining and quantification at day 4 after ischemic injury. (E) Representative images of Ki67/ CD68 immunofluorescent co-staining and quantification at day 4 after ischemic injury. Scale bars in B and C represent 32 μm and data are presented as average counts ± SEM per field of 0.1 mm2. Scale bars in D and E represent 18 μm and field sizes of 0.125 mm2. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 2.
M2 microglia/macrophages are recruited early after MCAO and progress towards M1 phenotype.
(A–B) Upper panels are representative double-labeled immunohistochemistry for iNOS+CD68+ (M1, A) and Arg1+CD68+ (M2, B) cells in the infarct border after stroke. Arrows indicate double positive cells and arrowheads indicate CD68+ cells not stained with Arg1 or iNOS, respectively. Scale bars represent 16 μm. The quantification of these cells at different time points are presented in the lower panels. Data are presented as average (Av) counts ± SEM per field of 0.125 mm2 (C) iNOS+CD68+ and Arg1+CD68+ cells shown as a proportion of total CD68+ cell population. (D) The dramatic increase in M1 cells by 4 days after stroke was confirmed by brain transcript analysis of M1 marker Cd86/B7-2. A–D: Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 3.
CD8 signaling in post-stroke brain.
(A) Expression of innate immune receptors and of (B) signal transduction molecules of respective pathways (indicated in brackets) involved in M1 polarization analyzed 4 days after stroke in the perilesional areas of the ischemic hemisphere. (C) Quantification of CD8+CD68+ cells as average (Av) counts ± SEM per field of 0.125 mm2 (D) Proportion of CD68+ cells expressing CD8 for MCAO animals. Sham animals are not plotted that lacked either or both CD8+ or CD68+ cells showed ratio was either 0 or a/0. A-D: Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 4.
CD8-expressing cells in post-stroke brain.
(A–B) Upper panel shows representative triple-labeled immunostaining of CD8+CD68+ cells expressing iNOS or Arg1 in the perilesional areas of the ischemic hemisphere at 6 h, and at days 1, 2, 3 and 4 after stroke. Lower panel shows quantification data. (C) Proportion of CD8+CD68+ cells expressing either Arg1 or iNOS at 4 d after stroke. Data in A–C is presented as average ± SEM. Scale bars represent 32 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 5.
CD8 stimulation induces M1 phenotype.
(A) Left panel shows increased iNOS+ and decreased Arg1+ staining in cultured peripheral macrophages after 24h with CD8-stimulating ligand, compared to the IgG1 isotype control. Right panel shows quantification of iNOS+ and Arg1+ cells presented as percentage increase or decrease respectively over the IgG1 control condition. (B) Increased phagocytosis of E. coli particles after CD8 or CD8-LPS stimulation by macrophages in in vitro assays. (C) IL-4-stimulated cells, when further stimulated with a CD8-stimulating ligand, showed increased iNOS and decreased Arg1 expression (left panel). Right panel shows quantification of iNOS+ and Arg1+ cells presented as percentage increase or decrease respectively over the IgG1 control condition. (D) Increased phagocytosis of E. coli particles with IL-4-CD8-stimulated macrophages compared to the IL-4-IgG1 isotype control. (E) Increased expression of signal molecules Syk and Raptor after CD8 stimulation. Data presented in A–E are normalized to control and presented as average ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. A, C, D: Scale bars represent 18 μm.