NK1R-DCs downregulate CREB1/TORC2 nuclear translocation resulting in decreased IL-10 and increased IL-12p70 secretion by LN-DCs. (A) Expression of luciferase (relative luminic units) by BMDCs transfected with plasmid DNA encoding the reporter protein under the transcriptional control of CREB1-, NFκB-binding enhancer elements, or the CMV promoter. BMDCs were left untreated (control) or cultured with the NK1R agonist SarSP, LPS, or agonist CD40 Ab. (B) Expression of CREB transcripts analyzed by quantitative RT-PCR in untreated-DCs, NK1R-DCs, Ag-DCs, and Ag-NK1R-DCs. Means ± 1 SD of triplicates of 1 representative experiment of 2 are illustrated. (C-D) Negative regulation of TORC2 translocation in BMDCs nuclei by NK1R signaling. (C) Western blot analysis of TORC2 content in cytoplasmic and nuclear protein lysates prepared from untreated-DCs, NK1R-DCs, Ag-DCs, and Ag-NK1R-DCs, resolved by SDS-PAGE, and probed for TORC2. (D) Percentages TORC2 translocation based on normalized density values of 3 independent experiments. (E) Secretion of IL-12p70 and IL-10 (by ELISA) in 24-hour supernatants of cocultures of (1) IL-12p35^KO BMDCs, left untreated (DC), NK1R-DCs, the latter transduced or not with RAd–IL-10, and (2) DCs freshly-isolated from LNs (LN-DC) stimulated or not (control) with IFN-γ and agonist CD40 Ab. (A-E) Means ± 1 SD of triplicates from 1 representative experiment of 3 are shown.