Sphingosine-1-phosphate reduces CD4+ T-cell activation in type 1 diabetes through regulation of hypoxia-inducible factor short isoform I.1 and CD69

OBJECTIVES-Non-obese diabetic (NOD) mice develop spontaneous type 1 iabetes. We have shown that sphingosine-1-phosphate (S1P) reduces activation of NOD diabetic endothelium via the S1P1 receptor. In the current study, we tested the hypothesis that S1P could inhibit CD4(+) T-cell activation, further reducing inflammatory events associated with diabetes. RESEARCH DESIGN AND METHODS-CD4(+) T-cells were isolated from diabetic and nondiabetic NOD mouse splenocytes and treated in the absence or presence of S1P or the S1P1 receptor-specific agonist, SEW2871. Lymphocyte activation was examined using flow cytometry, cytokine bead assays, and a lymphocyte:endothelial adhesion assay. RESULTS-Diabetic T-cells secreted twofold more gamma-interferon (IFN-gamma) and interleukin-17 than nondiabetic lymphocytes. Pretreatment with either S1P or SEW2871 significantly reduced cytokine secretion by similar to 50%. Flow cytometry analysis showed increased expression of CD69, a marker of lymphocyte activation, on diabetic T-cells. Both S1P and SEW2871 prevented upregulation of CD69 on CD4(+) cells. Quantitative RT-PCR showed that lymphocytes from diabetic NOD mice had 2.5-fold lower hypoxia-inducible factor (HIF)-1 alpha short isoform I.1 (HIF1 alpha I.1) mRNA levels than control. HIF1 alpha I.1 is a negative regulator of lymphocyte activation. S1P significantly increased HIF1 alpha I.1 mRNA levels in both control and diabetic groups. IFN-gamma production and surface CD69 expression was significantly increased in lymphocytes of HIF1 alpha I.1-deficient mice. S1P did not reduce either CD69 or IFN-gamma expression in lymphocytes from HIF1 alpha I.1-deficient mice. CONCLUSIONS-S1P acts through the S1P1 receptor and HIF1 alpha I.1 to negatively regulate T-cell activation, providing a potential therapeutic target for prevention of diabetes and its vascular complications.