DENV infection sequentially induces CREB activation and CREB-mediated IL-10 production through PKA- and PI3K-regulated PKB.

<p>A: A Western blot showing the time-kinetic phosphorylation of CREB (pCREB Ser133) in THP-1 cells infected with DENV serotype 2 PL046 (DENV 2, MOI = 1). ** P<0.01, compared with untreated cells. B: Immunostaining followed by confocal microscopy (with z-stacks) revealing the expression and intracellular localization of the DENV E protein (green) and CREB (red) 24 h after infection with DENV 2 (MOI = 1) or treatment with UV-iDENV 2; DAPI (blue) was used for nuclear staining. Scale bar indicates 5 µm. Images are representative of three independent experiments and a semiquantitative analysis of CREB nuclear cytoplasmic redistribution was performed using the line profile of Leica Confocal Software. Fluorescence intensities along the white lines sectioning the cells through the cytoplasm and nucleus are plotted for DENV E, CREB, and DAPI. C: CREB expression was silenced in THP-1 cells using small interfering RNA (siRNA-CREB clone #1 and clone #2); a scrambled siRNA was used as a control. siRNA-transfected THP-1 cells were infected with DENV 2 (MOI = 1) for 48 h. ELISA was used to quantify IL-10 production. ***P<0.001, compared with the scrambled siRNA. Western blots showing the expression of CREB and SOCS3. D: THP-1 cells were pre-treated with or without the PKA inhibitor H-89, the PI3K inhibitor LY294002, or the PKC inhibitor bisindolylmaleimide-1 (Bis) for 0.5 h, and then infected with DENV 2 (MOI = 1) for 48 h. ELISA and Western blotting analyses were used to detect the expression of IL-10 and the indicated proteins. ***P<0.001, compared with untreated cells; ###P<0.001, compared with DENV-infected cells. PKA fluorescent assays (E) and PIP3 precipitated assays (F) were used to detect the time-kinetic activities of PKA and PI3K, respectively, in THP-1 cells infected with DENV 2 (MOI = 1). The data are shown as fold changes with respect to the normalized values of the controls. **P<0.01, compared with untreated cells. A Western blot showing the time-kinetic phosphorylation of PKB (pPKB Ser473) in DENV 2 (MOI = 1)-infected THP-1 cells (G) and in THP-1 cells pre-treated with or without H-89, LY294002, or Bis for 0.5 h, and then infected with DENV 2 (MOI = 1) for 48 h (H). For the Western blotting analyses, the arrow indicates a non-specific binding signal, and β-actin was the internal control. One set of representative data obtained from three independent experiments is shown. The ratios of phosphorylated CREB and PKB to total proteins and the relative densities of CREB and SOCS3 are shown. For all experiments, the quantitative data shown represent mean ± SD values of three independent experiments.