6b). as and mice compared to control littermates. The relative proportions of CD4 or CD8 single positive, double positive, and double negative thymocytes were comparable between and control littermates (Supplementary Fig. 1a). Splenic CD4 and CD8 T cell numbers were also comparable (Supplementary Fig. 1b). Although na?ve CD4 T cells from both groups differentiated comparably into Th1, Th2, and Treg cells in vitro (Fig. 1a, 1b), the differentiation of and were also down-regulated in CD4 T cells (Fig. 1d). Th17 cells also had lower mRNA amounts for GW 5074 NR4A2, a well-established CREB/CRTC2 target that has been shown to regulate Th17 differentiation and autoimmune disease (Fig. 1d)18, 21. Open in a separate window Physique 1 Decreased Th17 differentiation in CRTC2-mutant mice(a and b) Wild type and CRTC2 mutant na?ve CD4 T cells skewed towards Th1, Th2, Treg, and Th17 cells. Cytokine levels determined by flow cytometry. Plots are gated on live CD4 T cells. Number in each quadrant indicates cell frequency. Data are representative of 3 experiments. Bar graphs represent the mean of percentage (right) +/? SEM, n = 3, from 3 impartial experiments with comparable results. (c) Decrease in IL-17A and IL-17F secreting Th17 cells from CRTC2-mutant or wild type littermates under Th17 differentiation conditions. IL-17A and IL-17F levels determined by flow cytometry. The electronic gate indicates the frequency of IL-17A (top) or IL-17F (bottom) CD4 T cells. Data are representative of 3 experiments. Bar graphs represent the mean percentage (right) +/? SEM, n = 3, from 3 impartial experiments with comparable results. (d) mRNA amounts for IL-17A,IL-17F, and NR4A2 in CRTC2-mutant T cells under Th17 differentiation conditions. Treatment with PGE2 indicated. Data represent the mean +/? SEM2, n = 3, from 3 impartial experiments with comparable results. (e) Immunoblot showing effect of PGE2 and forskolin (FSK) on CRTC2 dephosphorylation and CREB phosphorylation in CD4 T cells under Th17 differentiation conditions. Statistical analysis were performed with unpaired Student’s t-test. Differences were considered statistically significant at P 0.05 (* P 0.05; ** P 0.005 and *** P 0.0005). Despite these effects, mRNA amounts GW 5074 for other Th17 genes, including the retinoic acid receptor-related orphan receptor (ROR)-yt ((Supplementary Fig. 2). We reasoned that loss of Crtc2 could interfere with either the proliferation or survival of Th17 cells. Supporting this notion, disruption of CREB activity has been shown to stimulate apoptosis and to reduce proliferation 16, 17. When placed under Th17 differentiation conditions, however, CD4 T cells showed comparable Annexin-V staining relative to wild type cells (Supplementary Fig. 3). Moreover, mRNA amounts for the anti-apoptotic factors Bcl2 and Bcl-xL appeared Rabbit polyclonal to ACC1.ACC1 a subunit of acetyl-CoA carboxylase (ACC), a multifunctional enzyme system.Catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis.Phosphorylation by AMPK or PKA inhibits the enzymatic activity of ACC.ACC-alpha is the predominant isoform in liver, adipocyte and mammary gland.ACC-beta is the major isoform in skeletal muscle and heart.Phosphorylation regulates its activity. comparable between wild type and cells. Arguing as well against an effect on proliferation, CRTC2-mutant CD4 T GW 5074 cells actually showed a slight increase in mitotic index compared to wild type (Supplementary Fig. 3b). The prostaglandin PGE2, a product of activated innate immune cells, has been found to promote the differentiation of human and murine Th17 cells9. Consistent with these findings, exposure to PGE2 stimulated CRTC2 dephosphorylation (Fig. 1e), and it increased and mRNA amounts in wild type but not CRTC2-mutant Th17 differentiated cells (Fig. 1d). Exposure to PGE2 also increased mRNA amounts for and gene blocks Th17 cell differentiation even in the absence of exogenous PGE2, we considered that T cells may themselves produce PGE2 endogenously. Supporting this notion,.