Figure 4: Pharmacological inhibition of Tiam1/Rac1 reduces murine IL-17A expression and ameliorates EAE symptoms.

(a) Flow cytometry staining of CD4⁺ T cells treated with NSC23766. Naive CD4⁺ T cells were isolated from WT mice and were polarized under Th1, Th2 and Th17 cell conditions in the presence of NSC23766 (94 μM) or control solvent. Cytokine expression was measured on day 4 after differentiation. Numbers in outlined areas indicate percentage of positive cells. (b) Heat map of mRNA expression of Th17-associated cytokines in Th17 cells treated with NSC23766. Naive CD4⁺ T cells were differentiated in vitro with IL-6 and TGF-β1 in the presence or absence of NSC23766 for 4 days and cytokine profile was assessed by Taqman PCR. (c,f) Mean EAE clinical score (±s.e.m.) measured in WT mice that received NSC23766 (c), EHT1864 (f) or solvent (n=15–20 mice per group). Mice were immunized with MOG_35–55/CFA and received 8 daily injections of (c) NSC23766 (5 mg kg⁻¹) or (f) EHT1864 (40 mg kg⁻¹) i.p. starting on the day of immunization. Control mice received PBS treatment according to the same regimen. (d,g) ELISPOT assay of the frequency of MOG_35–55-reactive IL-17A and IFNγ-producing cells in immunized mice. Spleens were isolated from NSC23766 (d) or EHT1864 (g) and control mice on day 10 after immunization and were stimulated with MOG_35–55 peptide (10 μg ml⁻¹) for 36 h and positive cells were quantified (n=3–5 mice per group). (e,h) MOG_35–55-specific proliferation of splenocytes isolated from NSC23766 (e) or EHT1864 (h) and control mice on day 10 after immunization. Cultures were re-challenged with MOG_35–55 peptide (0, 5 and 25 μg ml⁻¹) and proliferation was measured by [³H]-thymidine incorporation reported as CPM (+s.d.) from triplicate wells (n=3–5 mice per group). Data are representative of three experiments with similar results and are shown as mean±s.e.m. *P<0.05; **P<0.01 by Student t-test. NS, non-significant.