Figure 3: Rac1 genetic deficiency reduces IL-17A expression and improves EAE symptoms.

(a) Naive CD4⁺ T cells were isolated from mice with conditional deletion of Rac1 in CD4⁺ T cells (CD4-Cre x Rac1^fl/fl) or control floxed mice (Rac1^fl/fl) and T cells were stimulated under Th1, Th2 and Th17 conditions and cytokine profile was analyzed by intracellular staining. (b) Heat map of relative mRNA expression of Th17-associated molecules by Taqman PCR from CD4-Cre x Rac1^fl/fl and control Rac1^fl/fl mice. (c) Cytokine expression by Luminex. Naive CD4⁺ T cells were isolated from CD4-Cre x Rac1^fl/fl or control Rac1^fl/fl mice and T cells were stimulated in vitro under pathogenic Th17 cell condition with IL-6 and TGF-β1 for 4 days, rested for 2 days, and followed by restimulation with the addition of IL-23 for another 2 days. (d) Mean EAE clinical score (±s.e.m.) measured in CD4-Cre x Rac1^fl/fl and littermate control Rac1^fl/fl mice (n=10 mice per group) that were immunized with MOG_35–55/CFA. The mean maximal score of CD4-Cre x Rac1^fl/fl versus control Rac1^fl/fl mice was compared by two-tailed Mann–Whitney U test, P=0.008. (e,f) ELISPOT analysis of the frequency of IL-17A and IFNγ-producing cells out of 400,000 (e) spleen and (f) LN cells isolated from CD4-Cre x Rac1^fl/fl and Rac1^fl/fl immunized mice that were re-challenged with MOG_35–55 peptide (10 μg ml⁻¹) for 36 h and positive cells were quantified (n=3–5 mice per group). (g) MOG_35–55-specific proliferation of splenocytes that were isolated from CD4-Cre x Rac1^fl/fl and Rac1^fl/fl mice and re-challenged with MOG_35–55 peptide (0, 5 and 25 μg ml⁻¹) 10 days after mouse immunization. (h) Heat map showing relative cytokine expression of CNS-infiltrated CD4⁺ T cells. Infiltrated cells were prepared from the spinal cords of recipient mice (3 mice per group) followed by Taqman PCR. (i) Histopathology of H&E-stained spinal cord sections of mice described in d on DPI 14. Boxed area in top row is enlarged below. Original magnification × 10 (top row) and × 40 (bottom row) with indicated calibration bars corresponding to 200 μm (top row) and 50 μm (bottom row). Results are representative of two independent experiments with two mice per group. Data are shown as mean±s.e.m.*P<0.05; **P<0.01 by Student t-test. NS, non-significant.