Fig. 3.

Myeloid-specific loss of Casp8 augments Th1 responses in the CNS of EAE mice. (A) Flow cytometry analysis of myeloid cells in the CNS of EAE mice. Mononuclear cells were isolated from spinal cords of WT and C8KO^LysM mice 2 wk after MOG_35–55 immunization. The population of infiltrated macrophages (CD11b⁺ CD45^high) was significantly greater in C8KO^LysM than WT mice. Microglia (CD11b⁺ CD45^int), neutrophil (CD11b⁺Ly6G⁺), and dendritic cell (CD11c⁺CD11b⁺) populations were not significantly different between genotypes. Data are presented as the percentage of CD11b⁺ cells as well as total cell numbers of specified cell populations. n = 3 to 4 mice per genotype. (B) Flow cytometry analysis of MHCII surface expression levels and MHCII⁺ myeloid cell populations in WT and C8KO^LysM mice. n = 7 mice per genotype. (C) Immunohistochemistry analysis of macrophages/microglia in the lumbar spinal cord of WT and C8KO^LysM EAE mice. (Upper) Quantitative CD68⁺ and MHCII⁺ cell analysis. (Lower) Representative images of lumbar spinal cord sections stained with anti-CD68 and MHCII antibodies. n = 4 mice per genotype. (Scale bar, 200 μm.) (D and E) Flow cytometry and immunohistochemistry analysis of CD4⁺ T helper cells showing increased CD4⁺ T cell infiltration into the CNS of C8KO^LysM mice as compared to WT. Boxes denote the lesion area shown at a higher magnification and quantified. n = 4 mice per genotype. (Scale bar, 50 μm.) (F) Antigen-specific responses of mononuclear cells isolated from spinal cords of WT and C8KO^LysM mice at preclinical stage of EAE (10 d postinfection). The CNS mononuclear cells were restimulated with or without MOG_35–55 (30 μg/mL) for 72 h, and the levels of cytokines/chemokines in the supernatant were analyzed by multiplex immunoassay. n = 4 mice per genotype. Data are a representative of three independent experiments. For IL-1β and IFN-γ, the difference between genotypes was analyzed using nonparametric Mann–Whitney U test. Data present mean ± SEM, *P < 0.05; **P < 0.0.1; ns, not significant.