Salp15 Binding to DC-SIGN Inhibits Cytokine Expression by Impairing both Nucleosome Remodeling and mRNA Stabilization (original) (raw)
Figure 3
DC Cytokine Inhibition by Salp15 Is Raf-1-Dependent
(A) Salp15 induces phosphorylation of Raf-1. DCs were pretreated with medium or with blocking anti-DC-SIGN antibodies (AZN-D2) before addition of 25 μg/ml Salp15. Intracellular phosphorylation of Raf-1 was measured by flow cytometry using rabbit anti-phospho-c-Raf (Ser338) mAb and rabbit anti-c-Raf (Tyr341) pAb.
(B) Raf-1 was specifically silenced by siRNA. DCs were transfected with 100 nM siRNA (Raf-1 or non-targeting siRNA as a control). Raf-1 and B-Raf mRNA was quantified by real-time PCR using gene-specific primers. Relative mRNA expression was corrected for GAPDH expression.
(C) RNAi-mediated silencing of Raf-1 abrogates Salp15-mediated cytokine inhibition. siRNA-treated cells were stimulated for 6 h with LPS in the presence or absence of 25 μg/ml Salp15. Cytokine gene expression was measured by real time PCR and corrected for GAPDH expression. Relative mRNA expression of LPS-stimulated non-targeting siRNA-treated cells was set at 1.
(D) Raf-1 inhibition by GW5704 abrogates Salp15-mediated cytokine inhibition. DCs were incubated with 1 μM GW5704, or DMSO as a negative control, for 2 h before stimulation with LPS for 6 h (mRNA) or 18 h (protein) in the presence or absence of 25 μg/ml Salp15. Cytokine gene expression was measured by real time PCR and corrected for GAPDH expression. Relative mRNA expression of LPS-stimulated cells was set at 1. For protein levels, LPS was set at 100%, which is representative of 133 ± 17 pg/ml IL-12p70; 9,188 ± 850 pg/ml TNF-α, and 1,563 ± 800 pg/ml IL-6. GW5704 alone did not induce cytokine production (unpublished data).
(E) MEK1/2-inhibitor U0126 blocks Salp15-mediated cytokine inhibition. DCs were pre-incubated for 2 h with anacardic acid (AA), an inhibitor of the histone acetyltransferases p300 and CBP, U0126, an inhibitor of MEK1 and MEK2, or DMSO as a negative control. Cells were stimulated as described in (C) and cytokine gene expression was measured by real time PCR and corrected for GAPDH expression. Relative mRNA expression of LPS-stimulated cells was set at 1.
(F and G) Salp15 does not induce phosphorylation of ERK. DCs were stimulated for 0 to 45 min with Salp15 (25 μg /ml), LPS, or PMA/Ionomycin as a positive control. Phosphorylation of ERK was determined by flow cytometry using a rabbit anti-phospho-p44/42 MAPK (Thr202/Tyr402) mAb.
Bars represent the mean of at least three independent experiments ± SE. For (C) mRNA expression levels in non-target siRNA-treated DCs activated with LPS + Salp15 were compared to Raf-1 siRNA-treated DCs activated with LPS + Salp15. In (D) and (E) levels in DCs treated with LPS + Salp15 were compared to levels in DCs treated with LPS + Salp15 in the presence of the indicated inhibitor. A two-sided unpaired Student _t_-test was applied and a _p_-value < 0.05 was considered statistically significant. (*) 0.01 < p < 0.05; two asterisks (**) 0.001 < p < 0.01.