FIGURE 1.

SphK1 is essential for TLR7/9-mediated type I interferon response. A-C, CAL-1 cells pre-treated with SK1-I (10 μM) or vehicle followed by stimulation with R848 (10 μg/mL), CpG-ODN 2006 (1 μM), CpG-ODN 2216 (3 μM) for 3 h (A, B) or 18 h (C) as indicated. IFN-α (A) and IFN-β mRNA (B) measured by qPCR and the mRNA levels normalized to β-actin. IFN-β easured by ELISA (C). Data are mean ± SD of three independent experiments. D-E, Flt3L-derived mouse pDCs pre-treated with SK1-I or vehicle and then stimulated with the indicated ligands. Levels of IFN-α (D) and IFN-β (E) measured by ELISA. Data are representative of four independent experiments. F, Flt3L-derived mouse pDCs treated with PF543 (10 μM), followed by R848 for 18 h. Levels of IFN-α and β in cell supernatant determined by ELISA. G, Expression of SphK1 determined in Flt3L-pDCs derived from SphK1^+/+ and SphK1^−/− mice by immunoblotting (H) Flt3L-derived mouse pDCs isolated from SphK1^+/+ and SphK1^−/− mice and treated with R848 (10 μg/mL) and MCMV (MOI = 1) and levels of IFN-α and IFN-β measured by ELISA. Data are mean ± S.D from four independent experiments. I, SphK1^+/+ and SphK1^−/− mice (n = 4/group) intraperitoneally injected with R848 (2 μg). One group of SphK1^+/+ mice (n = 4) pre-treated with SK1-I (10 mg/kg) for 1 h before challenging with R848. IFN-α and IFN-β in serum measured by ELISA. J, PBMCs isolated from healthy human subjects (n = 10) stimulated with R848 (10 μg/mL) and CpG-ODN (1 μM) in the presence and absence of SK1-I IFN-β mRNA measured by qPCR and normalized to β-actin. K, Human pDCs isolated from peripheral blood of healthy human subjects treated with R848 either with or without SK1-I pretreatment. ELISA analyses of IFN-α and β in cell supernatants after 18 h of ligand treatment. Statistical significance was determined by ANOVA followed by post hoc Tukey test. (A-F, H, I, K), Wilcoxon paired t test (J). *P < .05, **P < .005, ***P < .001