Fig. 5

Myeloid-specific ikkβ gene deletion inhibits the mRNA expression of co-stimulatory molecules in macrophages and regulates the proliferation of T cells by co-culture with microglia. a-b Spleen mononuclear cells obtained from WT and LysM-Cre/Ikkβ ^F/F mice at 15–18 days after immunization were incubated with MOG_35–55 peptide and PLP peptide for 48 and 72 h. The cells were pulsed for 18 h with solution containing ³H-methylthymidine and thymidine incorporation was measured. The results were expressed as counts per minute (mean ± SEM). c-e Macrophages obtained from WT and LysM-Cre/Ikkβ ^F/F mice were stimulated with lipopolysaccharide for 6 h. The mRNA expression levels of CD40 (c), CD80 (d), and CD86 (e) were determined using real-time RT-PCR. Data are represented as mean ± SEM. (ANOVA test; *p < 0.05 and **p < 0.01 versus supernatant from WT mice; ##p < 0.01 versus supernatant from normal control mice). f Microglial cells were isolated from brains of postnatal 1–3 day WT or LysM-Cre/Ikkβ ^F/F mice. At 15 days after EAE induction, CD4⁺ T cells (95 % pure) were harvested from lymph nodes of each group using anti-mouse CD4 magnetic beads. MOG_35–55 peptide-specific T cells were co-cultured with microglia for 24 h and incubated with APC anti-mouse CD4, PE anti-mouse IFN-γ, FITC anti-mouse IL-17A, PE anti-mouse CD25, and APC anti-mouse Foxp3 antibodies. Far left, CD4⁺ T cell gate (1 × 10⁴ cells in R2) used to identify CD4⁺/IFN-γ⁺, CD4⁺/IL-17⁺, and CD4⁺/CD25⁺/Foxp3⁺ T cells. Representative data and mean ± SEM values from 3 independent experiments are shown in spot plot graphs. (ANOVA test; *p < 0.05 and **p < 0.01 versus WT microglia and WT T cell group)