Fig 2: IL-10 from invading immune cells is neuroprotective. MRI was used to quantify infarct volume at day 3 and cortical atrophy volume at day 14 (A, B) after tMCAO in the chimeric mice (representative T2 image). C Composite neurological score was performed on day 14 after tMCAO. D Relative gene expression of Il10 in brain resident CD45intermed/CD11b+ microglia and central nervous system-infiltrating CD45high/CD11b+/CD11c−/MHCII−/Ly6g−/F4/80+ macrophages purified 3, 7, and 14 days after tMCAO by fluorescence activated cell sorting from ischemic hemispheres of C57Bl/6 mice. Expression levels were normalized to corresponding levels of microglia after sham surgery or blood macrophages. E Flow cytometric analysis of IL-10 produced by CD4+ Tregs (Foxp3+) or non-Tregs (Foxp3−). Infarct and atrophy data are presented as mean ± SEM of 16 WT mice which received Il10−/− and 10–11 Il10−/− mice which received WT bone-marrow cells (A, B), neurological score as mean ± SEM of 15 WT mice which received Il10−/− and 10 Il10−/− mice which received WT bone-marrow cells (C), RT-qPCR gene expression data as mean ± SEM of 3–7 (D) and flow cytometric data as mean of ± SEM of 5–10 mice in each group (E). Significances analyzed by Student t test (A, B), Mann–Whitney U test (C), and 1-way ANOVA with Bonferroni post hoc test (D, E). *P < 0.05, **P < 0.01, ***P < 0.01 and ****P < 0.0001. MΦ macrophages, MG microglia

Fig 3: Neutralization of IL-17A abolishes the worse outcome of Il10−/− mice. Triphenyltetrazolium chloride staining was used for evaluation of infarct volume at day 7 of IL-17A antibody (A) or IgG control (C) treated Il10−/− and WT control mice. Bederson score was performed 7 days after tMCAO IL-17A antibody (B) or in IgG control (D) treated group. Infarct data are presented as mean ± SEM of 9–11 WT and 6–9 Il10−/− mice per group. Bederson score as mean ± SEM of 9–11 WT and 7–10 Il10−/− mice per group. Statistical significances were analyzed by Student t test (A, C) and Mann–Whitney U test (B, D). *P < 0.05

Fig 4: Clinical and immunological impact of the adoptive transfer of trained-macrophages on A20 cells B lymphoma tumor growth in mice. (A) Schematic representation of the in vivo induction of B lymphoma in mice and scheduling of trained macrophage infusion. Mice (n = 7 in each group) were subcutaneously injected with 2 × 106 A20 cells and sacrificed at day 34 in one experiment. (B) Tumor volume evolution in the different groups of A20-challenged mice (n = 7). Tumor growth was monitored three times a week with a microcaliper, and tumor volumes were calculated as length × width2 × 0.5236 and expressed in mm3. Statistics are shown between the BCG-macrophages infused (A20-BCG) and the untreated A20 groups. Differences were detected at sacrifice using the Mann–Whitney test. **p ≤ 0.01. (C) Cytokine production in the supernatant of splenic cells harvested at sacrifice, stimulated with 5 μg/ml of Concanavalin A and incubated for 48 h at 37 °C with 5% CO2. Levels of TNF-α, IL-6, and IL-10 were assessed by ELISA and concentrations are expressed in pg/ml. Data represent the mean ± SEM from the duplicate of at least n = 5 biologically independent samples. (D, E) Flow cytometric analysis of splenic and tumoral CD4+ (C) and CD8+ (D) T-cell phenotype at day 34 of the experiment. Tumor infiltrating T cells were gated on CD3 and CD4 or CD8 positive cells among CD45 positive population. Data represent the mean fluorescence intensity of CD69 ± SEM, reflecting the activation status of the assessed cells, obtained from at least n = 5 biologically independent samples. (F, G) RT-qPCR assessment of fizz1, inos, il10, tgfb, foxp3, il4, il6, and infg mRNA levels in the spleen and tumor lesions of the different groups of A20-challenged mice at day 36 of the experiment. Results were expressed as fold increase versus the untreated A20-mice group, derived from at least n=5 biologically independent samples. Unpaired T-test was used to detect the differences between the groups. NS, non-significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Fig 5: Il10 deficiency leads to increased infarct sizes, enhanced brain atrophy, and poorer long-term outcome following tMCAO. MRI was used to quantify infarct volume at day 3 (A) and cortical atrophy volume at day 14 (C) after tMCAO in WT control and Il10−/− mice (representative T2 image). B Triphenyltetrazolium chloride (TTC) staining was used for the evaluation of infarct volume at day 7 (representative TTC brain slices). D Composite neurological score was performed on day 14 after tMCAO. E Il10/GFP-positive cells were visualized in the penumbra area of the ischemic hemisphere by GFP counterstaining in FIR-tiger mice 14 days after tMCAO (green, Il10/GFP-positive cells; red, CD45-positive cells; blue, 40,6-diamidino-2-phenylindole nuclear staining; scale bar 20 µm). Infarct data are presented as mean ± SEM of 8–10 WT and 6–10 Il10−/− mice (A, B), atrophy data as mean ± SEM of 11 WT and 13 Il10−/− mice (C), and neurological score of 12 WT and 11 Il10−/− mice (D). Statistical significances were analyzed by Student t test (A–C) and Mann–Whitney U test (D). *P < 0.05 and ***P < 0.001