negative regulator of some signaling pathways such as the JAK/ STAT pathway [24], there is little knowledge about the role of SOCS2 in TLR signaling. One study has demonstrated that SOCS2 mRNA expression increased during differentiation and maturation of mouse DCs [25], which suggested a possible SOCS2 involvement in DC function, but subsequent over-expression of the SOCS2 protein did not influence TLR signaling in mouse macrophages [26]. Another study showed that SOCS2 might be involved in the regulation of the immune response upon infection. SOCS2 mRNA and protein were induced by lipoxin (LXA4), an eicosanoid mediator with potent anti-inflammatory properties in DCs, and SOCS2 knock-out mice showed decreased microbial proliferation, leukocyte infiltration, production of pro-inflamma- tory cytokines, and a high mortality upon infection [27]. These findings suggest that SOCS2 may have an important role in the immune response to diverse infectious agents. In this study, we generated DCs from human monocytes cultured with GM-CSF and IL-4 and used them to investigate the role of SOCS proteins and TLR4 signaling pathways in DC maturation. Results Gene expression of SOCS family members during maturation of human monocyte-derived DCs Previous studies have shown that cytokine-inducible SH2- domain protein (CIS), SOCS1, SOCS2 and SOCS3 gene expressions are regulated by LPS stimulation in mouse DCs or macrophages [25,26]. In addition, IL21 [28] and protein allergens [29] can change expressions of SOCS 1 and SOCS 3 in humans DCs. We first studied the gene expression of SOCS members during DC maturation. Human monocyte-derived immature DCs (iDCs) were obtained by culturing with GM-CSF and IL-4. Subsequently the cells were treated with LPS at day 6 of differentiation and harvested for mRNA measurement after 24 h. As shown in Fig. 1A, SOCS2, SOCS3 and SOCS6 changed significantly after LPS treatment, whereas the other SOCS family members CIS, SOCS1, 4 and 5 did not change in expression levels. Based on the regulatory changes of SOCS2 mRNA expression in our experiment, we decided to further focus on the role of SOCS2 in the process of DC maturation. Real-time PCR analysis of total RNA revealed that SOCS2 mRNA levels start to increase at 2 h after LPS treatment and became significant from 4 h till 24 h (Fig. 1B). Western Blotting analysis of whole cell protein extracts showed that also SOCS2 protein levels increased after LPS treatment in a manner that corresponded to the changes seen in mRNA levels. The effect was weak at 4 h, but became significant at 8 h and increased dramatically up to 16 h where the expression was sustained for at least 24 h (Fig. 1C, D). SOCS2 influences MyD88-dependent and MyD88- independent signaling in the TLR4 signaling pathway LPS affects iDC function through activation of the TLR4 signaling pathway, which downstream consists of the MyD88- dependent and the TRIF-dependent pathway, the latter is also called the MyD88-independent pathway [11]. First, we wanted to test whether SOCS2 induced by LPS can affect the TLR4 signaling pathway. A specific silencing RNA (siRNA) targeting human SOCS2 or negative control siRNA with no homology to any known mammalian gene were transfected into iDC, and SOCS2 mRNA expression was then monitored. Low expression levels of SOCS2 was detected in untreated DCs, and as expected LPS treatment induced SOCS2 expression in control cells, an SOCS2 and DC Maturation Figure 1. Expressions of SOCS family members during human monocyte-derived DC maturation. (A). mRNA expression of SOCS family members during DC maturation. Enriched monocytes were cultured in the presence of GM-CSF and IL-4 for 6 days. Quantitative real-time PCR was used to measure SOCS mRNA expression in iDC at d6 and at d7, after exposure to LPS for 24 h. Data shown are the mean of duplicate determination from four donors, fold changes were obtained by comparing effects before and after of LPS treatment. (B, C, D). SOCS2 mRNA and protein expression levels during iDC maturation. Quantita- tive real-time PCR for mRNA and Western Blotting analysis for protein of SOCS2 expression in iDCs exposed to LPS for different time periods. The data shown in (B) are the mean of triplicate determination from four donors. Values for 0 time point were set to 1. The picture shown in (C) represents one Western Blot from a typical donor sample. The data shown in (D) are the mean of SOCS2 bands quantified from Western blots and normalized to b-actin from four donors. Values for 0 time point were set to 1. Error bars illustrate s.d. * P,0.05; ** P,0.01. doi:10.1371/journal.pone.0007178.g001 PLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e7178