Overall, the levels of all secreted cytokines were significantly decreased in a dose-dependent manner in stressed as well as in nonstressed mice, demonstrating the known immunosuppressive Belnacasan cell line effects of the drug (Fig. 5). Notably however, splenocytes harvested from stressed mice were less responsive to the immunosuppressive effects of MP as compared with splenocytes harvested from nonstressed mice. Specifically, reduced immunosuppressive effect of MP

on splenocytes harvested from stressed mice was found for IL-2, IFN-γ, IL-17A, and IL-10, but not for IL-4 (Fig. 5). Moreover, a comparison of the IFN-γ/IL-4 ratio in the presence of increasing MP concentrations revealed that MP at 100 ng/mL tended to shift the activated lymphocytes toward a Th2 response in nonstressed

but not in stressed mice (Fig. 5E). A similar comparison of the IL-17/IL-4 ratio revealed that MP did not affect this ratio in nonstressed mice but significantly shifted the activated lymphocytes toward a Th17 response in stressed mice (Fig. 5F). Such steroid learn more resistance was also evident for the innate proinflammatory factors TNF-α and MCP-1 (Fig. 5H and I). To further investigate the effect of CVS on immune effector functions, cytokine production was measured following stimulation of splenocytes from stressed and nonstressed mice with anti-CD3 or MOG35-55, 9 days following MOG35-55 injection. Anti-CD3 stimulation induced higher levels of secreted IFN-γ but not of IL-17A (Fig. 6A and E) and MP was isothipendyl significantly less

suppressive of their production in splenocytes from stressed compared to splenocytes from nonstressed mice (Fig. 6A and B, E and F). Although only a trend of increased levels of IFN-γ was detected following MOG35-55-induced T-cell activation (Fig. 6C), IL-17A was significantly increased in splenocytes from stressed mice compared with splenocytes from nonstressed mice (Fig. 6G). MP completely abolished T-cell activation of splenocytes from both stressed and nonstressed mice (Fig. 6C, D, G and H), possibly due the markedly lower amounts of cytokines secreted compared to anti-CD3 stimulation. Our data demonstrate an increase in proinflammatory cytokine levels induced by MOG35-55 immunization following CVS. However, it is yet not clear whether the CD4+CD25+ Treg population, which can strongly impact the progression of EAE, is affected by CVS. We initially found that the frequency of CD4+ T cells was decreased by 8% in the spleen and by 33.7% in circulating PBL in stressed compared with nonstressed female mice (Supporting Information Fig. 3A and B). The effect of CVS on the frequency of CD4+ Treg cells was examined by either intracellular staining of Foxp3 or surface staining of CD127 as a potential bio-marker of Treg cells ([34] and Supporting Information Fig. 3 and 4).