effect that was significantly reduced in SOCS2 siRNA-transfected cells both at the level of mRNA (Fig. 2A) and protein (Fig. 2B, C). To study the effect of SOCS2 on the MyD88-dependent signaling pathway, proteins from control or SOCS2 siRNA transfected DCs were extracted. LPS activation of this pathway results in a cascade of kinase dependent phosphorylations. The kinetics of the phosphorylation of SAKP/JNK, P38, ERK and IkBa was measured by Western Blotting. In cells with SOCS2 siRNA, LPS treatment led to a reduced phosphorylation of SAKP/JNK, P38, ERK and IkBa (Fig. 3A). When nuclear proteins were extracted from control or SOCS2 siRNA treated DCs the SOCS2 silenced DCs displayed a weaker NF-kB band compared to control DCs (Fig. 3B). This indicates that less NF-kB was translocated into the nucleus after SOCS2 elimination, supporting the notion that SOCS2 silencing inhibits the MyD88-dependent signaling pathway in human DCs by inter- rupting the normal SOCS2 dependent kinase cascade. Protein extracts from control or SOCS2 siRNA transfected DCs were then tested for the effect of SOCS2 on the MyD88- independent signaling pathway. IRF3, a kinase that is phosphor- ylated and translocated into the nucleus following LPS stimulation, was phosphorylated to a lesser extent in the SOCS2 knock-down DCs (Fig. 3C). This supports the notion that TLR4 signaling is SOCS2 dependent in human DCs, not only via the MyD88- dependent but also via the MyD88-independent signaling pathway. In initial studies, we tested two different SOCS2 siRNAs having different sequences. These experiments showed that there were no differences in the extent of SOCS2 reduction and effect between the two SOCS2 siRNAs. For this reason we selected one SOCS2 siRNA in subsequent experiments. LPS induced gene expression is dependening on SOCS2 Activation of the TLR4 signaling pathway induces, for example, TNF-a, IL-6, IL-1b and CCL-4 via the MyD88-dependent pathway [30,31] and IFN-b, CXCL-9, and CXCL-10 via the MyD88-independent pathway [32,33]. To investigate if the inhibition of TLR4 signaling by SOCS2 silencing caused subsequent changes in mRNA expression levels of downstream mediators in this pathway, we measured LPS inducible gene expression by quantitative PCR. SOCS2 knock-down significantly decreased mRNA expression of TNF-a, IL-6, IL-1b and CCL-4 (Fig. 4A), and similar effects were seen on the expression of INF-b, CXCL-9, and CXCL-10 (Fig. 4B). These findings suggests that SOCS2 influences LPS inducible gene expression via both the MyD88-dependent and independent signaling pathway. Role of SOCS2 in DC maturation in response to inflammatory signals As the LPS effect on DC maturation is mediated via TLR4 signal transduction we decided to investigate a possible role of SOCS2 in DC maturation. SOCS2 knock-down DCs were stimulated with LPS 24 h after siRNA transfection and harvested for flow cytometry analysis. LPS stimulation of SOCS2 siRNA transfected iDCs led to an impairment in DC maturation. This was demonstrated by changes in surface molecule expression. CD83, the marker for mature DCs [34], was significantly decreased in SOCS2 siRNA transfected DCs. Moreover, these DCs showed a weaker expression of the co-stimulatory molecules CD40, CD86, whereas no evident effect on CD80 and HLA-DR was demonstrated, based on the percentage of positive cells (% positive cells) compared with control group (Fig. 5). Cells transfected with SOCS2 siRNA showed a decrease in mean fluorescence intensity around 40%, for the CD83 marker and SOCS2 and DC Maturation Figure 2. SOCS2 siRNA inhibits SOCS2 mRNA and protein expression in DCs during maturation. Enriched monocytes were cultured in the presence of GM-CSF and IL-4 for 6 days. iDCs were transfected with SOCS2 siRNA or control siRNA and incubated for 24 hours. (A). SOCS2 mRNA gene expression from DC transfected with SOCS2 silencing RNA during maturation. Transfected cells were exposed to LPS (200 ng/ml) for 2 hours, the cells were then harvested for the measurement of SOCS2 RNA expression by qRT-PCR. Data shown were mean of duplicate determination from 4 donors. (B, C). SOCS2 protein level expression from DCs transfected with SOCS2 silencing RNA during maturation. Transfected cells were exposed to LPS (200 ng/ml) for 8 hours, after which whole-cell lysates were extracted for the measurement of SOCS2 protein levels by Western Blotting. (B) Picture shown is 1 of 4 typical donors. (C) Data shown are mean of SOCS2 bands quantified and normalized to b-actin from four donors. Values for the control were set to 1. Error bars illustrate s.d. * P,0.05; ** P,0.01. doi:10.1371/journal.pone.0007178.g002 PLoS ONE | www.plosone.org 3 September 2009 | Volume 4 | Issue 9 | e7178