SOCS2 Influences LPS Induced Human Monocyte-Derived Dendritic Cell Maturation Jin Hu1,2,4, Ola Winqvist2, Amilcar Flores-Morales1, Ann-Charlotte Wikstro¨ m3, Gunnar Norstedt1* 1 Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden, 2 Unit of Clinical Allergy Research, Department of Medicine Solna, Karolinska Institute, Stockholm, Sweden, 3 Department of Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden, 4 Department of Trauma Orthopedic & Hand Surgery, The First Affiliated Hospital of Guangxi Medical University, Guangxi Medical University, Nanning, Guangxi, P.R. China Abstract Dendritic cells (DCs) are highly specific antigen presenting cells, which link innate and adaptive immune responses and participate in protecting hosts from invading pathogens. DCs can be generated in vitro by culturing human monocytes with GM-CSF and IL-4 followed by LPS induced DC maturation. We set out to study the suppressor of cytokine signaling (SOCS) proteins during maturation and activation of human monocyte-derived DCs from peripheral blood in vitro. We found that the expression of SOCS2 mRNA and protein is dramatically up-regulated during DC maturation. Silencing of SOCS2 using siRNA, inhibited DC maturation as evidenced by a decreased expression of maturation markers such as CD83, co-stimulatory molecules CD40, CD86 and HLA-DR. Furthermore, silencing of SOCS2 decreased LPS induced activation of MAP kinases (SAKP/JNK, p38, ERK), IRF3, decreased the translocation of the NF-kB transcription factor and reduced downstream gene mRNA expression. These results suggest a role for SOCS2 in the MyD88-dependent and -independent TLR4 signaling pathways. In conclusion, our results demonstrate that SOCS2 is required for appropriate TLR4 signaling in maturating human DCs via both the MyD88-dependent and -independent signaling pathway. Citation: Hu J, Winqvist O, Flores-Morales A, Wikstro¨ m A-C, Norstedt G (2009) SOCS2 Influences LPS Induced Human Monocyte-Derived Dendritic Cell Maturation. PLoS ONE 4(9): e7178. doi:10.1371/journal.pone.0007178 Editor: Derya Unutmaz, New York University School of Medicine, United States of America Received April 16, 2009; Accepted September 2, 2009; Published September 25, 2009 Copyright: 2009 Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Swedish cancer society, the Cancer and Allergy society, the Lundberg foundation, the Gustav V foundation, the So¨ derberg foundation and the Selander foundation, and also supported by the Chinese Guangxi Education Department and the First Affiliated Hospital of Guangxi Medical University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Gunnar.Norstedt@ki.se Introduction The innate immune system is the first line of defense protecting the host from invading pathogens. Dendritic cells (DCs) serve as highly specific APCs and play a crucial role connecting the induction of innate immunity and the subsequent development of the adaptive immune response [1,2]. In this process, DC maturation serves as the critical switch from maintenance of self- tolerance to the induction of immunity [3]. Mature DCs increase the expression of co-stimulatory molecules, as well as MHC I and II and diverse immune regulative molecules that stimulate naive Th cells to differentiate into Th1 or Th2 cells [4,5]. DCs also secrete large amounts of pro-inflammatory cytokines that activate innate lymphocytes to kill infected cells that have been invaded by pathogens [4,6]. DC’s recognize pathogen-associated molecular patterns by various pattern recognition receptors. Among these receptors, TLRs expressed on APCs, such as DCs and macrophages, serve as key pattern recognition receptors [7]. There are 11 human and 13 mouse TLRs identified to date, and each TLR member precisely recognizes distinct pathogen-associated molecular patterns derived from various microorganisms and activate inflammatory cytokines, chemokines, IFNs and upregulate the expression of co-stimulatory molecules [1]. LPS is a gram negative bacterial cell wall component and a TLR4 ligand [8,9]. Ligand-induced dimeriza- tion activates the TLR4, and adapters are recruited via their Toll- interleukin 1 receptor (TIR) domains. MyD88 is a universal adaptor and acts to recruit the interleukin 1-associated kinas (IRAK) family, TNF receptor-associated factor (TRAF) 6 and IkBa kinases which leads to the activation of the transcription factor, NF-kB and also MAP kinases (JNK, p38, ERK) [10]. MyD88-adaptor like (MAL) is also recruited by TLR4 and stabilize MyD88 in the complex [10]. The above pathway is termed the MyD88-dependent pathway. In addition the MyD88- independent signaling pathway activates a TIR domain-contain- ing adaptor (TRIF), which needs another bridge adaptor, the TRIF-related adaptor molecule (TRAM), and this leads to activation of TRAF3 that contributes to the activation of interferon regulatory factor (IRF) 3 [10,11] and the late phase activation of NF-kB and MAP kinases [12]. Monocytes have been shown to be important DC precursor cells both in vitro and in vivo [13,14]. Monocyte-derived DCs can be generated by monocyte cultivation with GM-CSF and IL-4 [15,16] or IL-13 [17] in vitro, and this makes it possible to generate large quantities of DCs providing a model to investigate the effect of self or environmental agents on the differentiation and maturation pathways of DCs. Suppressor of cytokine signaling (SOCS) family includes eight members, characterized by the presence of a Src homology 2 domain and a C-terminal conserved domain called the SOCS box [18], Each family member plays a unique role in attenuating cellular signaling [19,20]. SOCS1 and SOCS3 have recently been demonstrated to negatively regulate TLR signaling in macrophage and DC maturation [21,22,23]. Although SOCS2 is a well known PLoS ONE | www.plosone.org 1 September 2009 | Volume 4 | Issue 9 | e7178