Figure 5: Fibrin induces activation of innate immunity via CD11b/CD18 to induce Th1-cell differentiation.

(a) BMDMs treated with fibrin or in the presence of anti-CD11b or isotype IgG antibody control were co-cultured with naive WT CD4⁺ T cells and analysed for gene expression or by FACS. Gene expression analysis of transcriptional factors indicative of Th1, Th2, Th17 and T_reg cells in CD4⁺ T cells co-cultured with fibrin-stimulated BMDMs. Data are presented as mean±s.e.m. (n=4 independent experiments, *P<0.05 by non-parametric Mann–Whitney U-test). (b) Gated percentage of IFN-γ- or IL-4-expressing CD4⁺ T cells after co-culture with fibrin-stimulated BMDMs in the presence of rat IgG isotype control antibody or anti-CD11b antibody. Data are presented as mean±s.e.m. (n=3 independent experiments, *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA and Bonferroni’s multiple comparisons test). Real-time PCR analysis of Th1-inducing cytokine IL-12p40 in fibrin-stimulated BMDMs in the presence or absence of anti-CD11b antibody. Data are presented as mean±s.e.m. (n=3–4 independent experiments, *P<0.05, **P<0.01 by one-way ANOVA and Bonferroni’s multiple comparisons test). (c) Real-time PCR analysis of transcription factors and cytokines indicative of Th1, Th2, Th17 and T_reg cells in the corpus callosum at 3 days after ACSF or fibrinogen injection. Data are presented as mean±s.e.m. (n=3–5 mice per group). *P<0.05 (non-parametric Mann–Whitney U-test). (d) Comparison of IFN-γ-, IL-4- and IL-17-producing cells in infiltrated CD4⁺ and CD8⁺ T cells isolated from the brains and spleens after ACSF or fibrinogen injection at day 7 post injection. Data are presented as mean±s.e.m. (n=3 independent experiments, *P<0.05, ***P<0.001 by one-way ANOVA and Bonferroni’s multiple comparisons test). (e) IFN-γ and IL-4 expression in CD4⁺ lymph node T cells isolated from saline- or fibrin-depleted (ancrod) mice after PLP_139–151-induced EAE and restimulated in vitro with PLP_139–151. Data are presented as mean±s.e.m. (n=6 mice per group, *P<0.05, **P<0.01 by non-parametric Mann–Whitney U-test). ANOVA, analysis of variance; FACS, fluorescence-activated cell sorting; T_reg, regulatory T cell.