Suppressor of Cytokine Signaling 2 Is a Feedback Inhibitor of TLR

Suppressor of Cytokine Signaling 2 Is a Feedback Inhibitor of TLR-Induced Activation in Human Monocyte-Derived Dendritic Cells This information is current as of September 27, 2021. Gernot Posselt, Harald Schwarz, Albert Duschl and Jutta Horejs-Hoeck J Immunol 2011; 187:2875-2884; Prepublished online 15 August 2011; doi: 10.4049/jimmunol.1003348 Downloaded from http://www.jimmunol.org/content/187/6/2875

Supplementary http://www.jimmunol.org/content/suppl/2011/08/15/jimmunol.100334 http://www.jimmunol.org/ Material 8.DC1 References This article cites 51 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/187/6/2875.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Suppressor of Cytokine Signaling 2 Is a Feedback Inhibitor of TLR-Induced Activation in Human Monocyte-Derived Dendritic Cells

Gernot Posselt, Harald Schwarz, Albert Duschl, and Jutta Horejs-Hoeck

Dendritic cells (DCs) are key players in initiating and directing the immune response. Therefore, their activation state and functional differentiation need to be tightly controlled. The activating stimuli and their signaling networks have long been an area of focus in DC research. Recent investigations have also shed light on the mechanisms of counterregulation and fine-tuning of DC functions. One class of proteins involved in these processes is the family of suppressors of cytokine signaling (SOCS), whose members were originally described as feedback inhibitors of cytokine-induced JAK/STAT signaling. Essential roles in DC function have been assigned to SOCS1 and SOCS3. In this article, we show that SOCS2 also is involved in DC regulation. In human and in murine DCs, Downloaded from SOCS2 is a highly TLR-responsive gene, which is expressed in a time-delayed fashion beginning 8 h after TLR ligation. Functionally, silencing of SOCS2 in DCs results in hyperphosphorylation of STAT3 at later time points. As a consequence, SOCS2-deficient DCs secrete increased amounts of the cytokines IL-1b and IL-10, both being transcriptional targets of STAT3. We propose a model in which SOCS2 acts as a negative regulator of TLR-induced DC activation. The delayed expression of SOCS2 provides a mechanism of late-phase counterregulation and limitation of inflammation-driving DC activity. The Journal of Immunology, 2011, 187: 2875–2884. http://www.jimmunol.org/

endritic cells (DCs) play important roles in immune nisms of counterregulation and fine-tuning in the innate system recognition of invading pathogens during infections. is still in its early stages. One class of molecules that is thought D They bridge the two branches of innate and adaptive to be involved in negative regulation and/or modification of DC- immunity and are thought to be unique in their capacity to prime activating signals contains the suppressors of cytokine signaling naive T cell responses. DCs act as sentinels, responding to (SOCS) proteins (4). evolutionary-conserved microbial structures as indicators of in- The family of SOCS proteins was originally described as fection, using pattern recognition receptors. TLRs are the best comprising feedback inhibitors of cytokine-induced JAK/STAT characterized group of pattern recognition receptors that recognize signaling and consists of eight members, namely, SOCS1-7 and by guest on September 27, 2021 pathogen-associated molecular patterns such as LPS, peptidogly- cytokine-inducible Src homology 2 (SH2)-containing protein cans, flagellin, lipoteichoic acid, or unmethylated CpG DNA, and (CIS) (5). SOCS proteins share a tripartite structure with a central they can stimulate activation of the innate immune system (1, 2). SH2 domain flanked by a conserved C-terminal domain termed As a result, DCs change their activation state, gaining immunos- the SOCS-box and an N-terminal region with low conservation timulatory capacity hallmarked by reinforced migratory homing except for an extended SH2 subdomain and, in SOCS1 and to secondary lymphoid tissues, increased Ag processing and pre- SOCS3, a kinase inhibitory region (KIR). This segmented archi- sentation, expression of costimulatory molecules, and secretion of tecture reflects the functional repertoire of these proteins. SOCS proinflammatory cytokines. These activation-associated changes proteins execute their function in three different ways. First, enable DCs to prime Ag-specific T cells and lead to the initiation SOCS proteins bind competitively to their target proteins, thus of adaptive immune responses (3). The processes of immune ac- displacing and sequestering the signaling intermediates away from tivation are well understood; however, research on the mecha- their respective receptors, thereby inhibiting downstream signaling. The common SH2 domain mediates binding to phospho- tyrosine residues, whereas the extended SH2 subdomain con- Division of Allergy and Immunology, Department of Molecular Biology, Paris Lo- fines the substrate specificity (6). Second, the shared KIR motif of dron University of Salzburg, 5020 Salzburg, Austria SOCS1 and SOCS3 directly inhibits JAK activity, apparently by Received for publication October 8, 2010. Accepted for publication July 11, 2011. acting as a pseudosubstrate for the kinase (7). Third, SOCS pro- G.P. designed and performed research and wrote the manuscript; H.S. performed teins exert their inhibitory action, at least in part, by enhancing research; A.D. helped interpret data and revise the manuscript; J.H.-H. designed research and wrote the manuscript. ubiquitination and subsequent proteasomal depletion of receptors Address correspondence and reprint requests to Dr. Jutta Horejs-Hoeck, Division of or signal-transducing molecules (8). The degradation of interact- Allergy and Immunology, Department of Molecular Biology, Paris Lodron University ing proteins is attributed to the SOCS-box, which interacts with of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria. E-mail address: jutta. and stabilizes complexes of ubiquitin ligases (9). [email protected] SOCS1 and SOCS3 are induced as a consequence of TLR sig- The online version of this article contains supplemental material. naling and are capable of modifying the functional properties of Abbreviations used in this article: CIS, cytokine-inducible SH2-containing protein; DC, dendritic cell; GH, growth hormone; KIR, kinase inhibitory region; moDC, APCs (10–12). Regulatory functions in pathways other than cy- monocyte-derived DC; poly I:C, polyriboinosinic:polyribocytidylic acid; qRT-PCR, tokine signaling have been described. SOCS1 has been shown to quantitative RT-PCR; RPLP0, large ribosomal protein P0; SH2, Src homology 2; interfere with TLR signaling by proteasomal degradation of the siRNA, small interfering RNA; SOCS, suppressor of cytokine signaling. MyD-like adapter molecule Mal (13). In addition, SOCS1 is able Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 to impede autocrine IFN stimulation and is thought to prevent www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003348 2876 SOCS2 CONTROLS TLR-INDUCED DC ACTIVATION overshooting of immune activation (14). SOCS1 deficiency in coats from healthy donors (kindly provided by Transfusionsmedizin der APCs results in hyperactivation and consecutive hyper-Th1 re- Paracelsus Medical University, Salzburg, Austria) over Ficoll-Paque PLUS sponses (15). (GE Healthcare, Uppsala, Sweden) by density gradient centrifugation, according to the manufacturer’s instructions. PBMCs were washed twice In contrast, SOCS3 deficiency furnishes DCs with a toleroge- in RPMI 1640, and cells were allowed to adhere for 90 min. Adherent nic phenotype. In response to many gp130 cytokines, SOCS3- monocytes were washed extensively with warm RPMI 1640 to remove all dependent signal termination limits the duration of STAT3 acti- nonadherent cells and cultured for 7 d in DC medium (RPMI 1640; PAA, vation. Opposed to that, IL-10 signaling is not sensitive to SOSC3 Pasching, Austria), 10% FCS (PAA), 2 mM L-glutamine, 100 U/ml peni- cillin, 100 mg/ml streptomycin, 50 mM 2-ME (all from Life Technologies inhibition and induces long-lasting phosphorylation of STAT3. In Laboratories, Grand Island, NY), and stimulated with 50 ng/ml GM-CSF the absence of SOCS3, a wide array of gp130 cytokines is able to and 50 ng/ml IL-4 (generous gift from Novartis, Vienna, Austria). At day 3, induce prolonged STAT3 activation and to mimic IL-10 stimula- cells were fed with 1 vol DC medium containing fresh cytokines. After 6 d, tion. As a consequence, these cytokines share the anti-inflam- cells were harvested and replated in DC medium without cytokines. At this matory properties of IL-10 (16–18). stage, cells were phenotyped by flow cytometry and were routinely $90% CD1a+ CD14low. SOCS2 was originally described as a feedback inhibitor of the + + growth hormone (GH)/insulin-like growth factor axis, which is Isolation of primary human blood DCs and CD4 CD45Ra reflected by the high growth phenotype of SOCS2-deficient mice naive T cells (19). Surprisingly, SOCS2 transgenic mice also show an increase CD1c+ DCs were isolated from CD19-depleted PBMCs using a BDCA1+ in body weight, suggesting a dual role for SOCS2 in the GH Kit (Miltenyi Biotech, Bergisch Gladbach, Germany), according to the + + signaling cascade. This is supported by in vitro experiments, in manufacturer’s instructions. CD4 CD45RA T cells were isolated using the untouched naive CD4 T cell isolation kit II (Miltenyi Biotech), according to which only low-to-intermediate levels of SOCS2 show inhibitory Downloaded from the manufacturer’s instructions. Isolated cells were phenotyped by FACS. action on GH signaling, whereas high levels of ectopic SOCS2 expression even increase STAT5 activation in response to GH Generation of thioglycolate-induced mouse peritoneal stimulation (20, 21). SOCS2 expression is induced by a number of macrophages different cytokines and hormones in many cell types (22). In ad- cDNAs from both untreated and LPS-stimulated, thioglycolate-induced dition, dioxin and the lipid mediator lipoxin A4 have been de- peritoneal macrophages were kindly provided by G. Schwamberger. Mac-

scribed to stimulate SOCS2 expression in B cells and DCs, rophages were obtained according to standard protocols. In brief, mice were http://www.jimmunol.org/ respectively, each of them dependent on aryl-hydrocarbon re- i.p. injected with 1 ml 1% thioglycolate solution. After 4 d, mice were killed and peritoneal lavage cells were collected. Cells were plated in IMDM ceptor activation (23, 24). Based on forced expression, a regula- (PAA) with antibiotics for 3 h and washed extensively afterward. The tory potential for SOCS2 has been suggested in several pathways; adherent fraction was used for further stimulation as indicated. however, the physiological relevance of these results is contro- Generation of mouse bone marrow-derived DCs versial. Because SOCS2 lacks a KIR domain, the inhibitory function of SOCS2 is dependent on competitive binding via its Bone marrow-derived DCs were generated according to standard protocols. SH2 domain and, even more important, on the proteasomal deg- In brief, bone marrow cells were isolated from femurs of BALB/c mice radation of the proteins with which it interacts (8). Apart from (Charles River). Cells were differentiated in DC medium supplemented with 5% mouse GM-CSF–conditioned supernatant (mouse DC medium) for 9 d. by guest on September 27, 2021 altered GH signaling, SOCS2-deficient mice have also been shown At days 3 and 6, half of the medium was replaced with fresh mouse DC to exhibit alterations in their immune system. They display medium. After 9 d, cells were sedimented and replated for further stim- overshooting immune reactions in a model of toxoplasmosis ulation in mouse DC medium. Cells were routinely phenotyped for CD11c because of a failure to counterregulate DC activation. SOCS2- expression by means of FACS analysis. dependent TRAF6 degradation in APCs was suggested as the TLR ligands and cell culture reagents underlying mechanism (25). However, that study was recently Cells were stimulated with Escherichia coli LPS 055:B5 (Sigma-Aldrich), retracted and the proposed mechanism remains in doubt (26). Pam3CSK4, FSL-1, Flagellin (all from InvivoGen, San Diego, CA), R848 Nevertheless, the main conclusion that SOCS2 plays a regulatory (Alexis Biochemicals, Lausen, Switzerland), immunostimulatory CpG role in DC activation appears to be well supported. oligodeoxynucleotide C274 (as described in Ref. 28, phosphorothioate In this study, we show that SOCS2 is induced as a consequence modified), and polyriboinosinic:polyribocytidylic acid (poly I:C) (Sigma- of TLR stimulation in both human and murine DCs. Compared Aldrich). For the control of STAT3 phosphorylation in response to IL-10 stimulation, cells were incubated with 25 ng/ml recombinant human IL-10 with SOCS1 and SOCS3, which are upregulated within 1 h after (Immunotools, Friesoythe, Germany). Where indicated, experiments were LPS treatment, SOCS2 shows a time-delayed expression starting conducted in the presence of the IL-10R–blocking Ab anti-CDw210 (BD ∼8 h poststimulation. Despite this delay, SOCS2 is a direct tar- Pharmingen) at 20 mg/ml for inhibition of autocrine/paracrine IL-10 get of TLR signaling, for its expression is robust under con- stimulation. The STAT3 inhibitor Stattic (6-nitrobenzo[b]thiophene-1, 1-dioxide) was purchased from Calbiochem (Darmstadt, Germany). ditions of protein-synthesis inhibition. Silencing of SOCS2 in DCs leads to increased cytokine secretion, predominantly of cytokines ELISA with a late and more sustained expression pattern, like IL-10 For each condition, 5 3 105 cells were plated per six wells in 1.5 ml DC and IL-1b. Cytokines with early and transient expression, such medium. Supernatants of DCs were collected and stored at 280˚C until as TNF-a or IL-6, were not affected. Moreover, elevated, IL-10– analysis. Cytokine secretion was measured by using commercially avail- independent, STAT3 phosphorylation was observed in SOCS2- able ELISA kits for TNF-a, IL-6 (both from PeproTech, Eubio, Vienna, deficient DCs. In conclusion, our data suggest an inhibitory Austria), IL-12p70 (BD Pharmingen, Erembodegen, Belgium), IL-1b, and IL-10 (both from R&D Systems, Biomedica, Vienna, Austria). ELISA function for SOCS2 in TLR ligand-induced DC activation. results are shown as normalized secretion. Because of heterogeneous absolute values in different donors, LPS-stimulated secretion in control cells was set as 100%. Materials and Methods All studies involving human material were conducted in accordance with Western blot analysis the guidelines of the World Medical Association’s Declaration of Helsinki. Cells were sedimented, washed, and lysed either directly in 23 SDS sample Generation of human monocyte-derived DCs buffer (Bio-Rad, Vienna, Austria) or in ice-cold Nonidet P-40 lysis buffer (Invitrogen, Lofer, Austria) supplemented with 1 mM PMSF and Complete Monocyte-derived DCs (moDCs) were generated according to the slightly Mini proteinase inhibitor mixture (Roche) for 30 min. Nonidet P-40 lysates modified standard protocol (27). In brief, PBMCs were isolated from buffy were cleared of debris by centrifugation. Equal amounts of protein were The Journal of Immunology 2877

Table I. qRT-PCR primer sequences

Organism Target Gene Strand Sequence Human RPLP0 Forward 59-GGCACCATTGAAATCCTGAGTGATGTG-39 Reverse 59-TTGCGGACACCCTCCAGGAAG-39 Human SOCS1 Forward 59-TTGGAGGGAGCGGATGGGTGTAG-39 Reverse 59-AGAGGTAGGAGGTGCGAGTTCAGGTC-39 Human SOCS2 Forward 59-CCAAATCAACCAAAAAAAGTGACCATGAAGTCCTG-39 Reverse 59-CGGGGATTGAGTTGCACCTGTATAGCATGATATTC-39 Human SOCS3 Forward 59-ATACTATACCTTCCTGTACCTGGGTGGATGGAGCG-39 Reverse 59-TGAGTATGTGGCTTTCCTATGCTGGGTCCCTCT-39 Human SOCS4 Forward 59-TGGAATCACAACTTTAGCTTTGATGCACATGACCC-39 Reverse 59-TGGTTCAAAGAACATACAGGCGCTTGGGTCC-39 Human SOCS5 Forward 59-AAACAGGCGTTTGGAATAGCTGCTGCAATGTAGTC-39 Reverse 59-CACAAAATCATCCTGGGCATAGGAACAGATCCAAC-39 Human SOCS6 Forward 59GCTGAAAAAACTTGCAAAGCAAGGATGGTACTGGG-39 Reverse 59-CGAACAAGAAAAGAACCATCTGGCACGTTTGC-39 Human SOCS7 Forward 59-TCTAAAGGAAGCGCAGCTCATTTCCAAACAGAAGC-39 Reverse 59-TCTTCAAAGCTGGAGCTTGGCAACCAAATGC-39 Human TNFA Forward 59-CAAGCCTGTAGCCCATGTTG-39 Reverse 59-GAGGTTGACCTTGGTCTGGTA-39 5 -GTACCTGAGCTCGCCAGTGA-3 Human IL-1B Forward 9 9 Downloaded from Reverse 59-TCGGAGATTCGTAGCTGGATG-39 Human IL-6 Forward 59-GTACATCCTTCGACGGCATCTC-39 Reverse 59-GGCAAGTCTCCTCATTGAATC-39 Human IL-10 Forward 59-AGGGCACCCAGTCTGAGAACA-39 Reverse 59-CGGCCTTGCTCTTGTTTTCAC-39 Mouse Rplp0 Forward 59-TGCACTCTCGCTTTCTGGAGGGTG-39 Reverse 59-AATGCAGATGGATCAGCCAGGAAGG-39 Mouse Socs2 Forward 59-GCTCAGTCAAACAGGATGGTACTGGGGAAGTATG-39 http://www.jimmunol.org/ Reverse 59-TCTGAATTTCCCATCTTGGTACTCAATCCGCAGG-39 separated by 4–12% SDS-PAGE (Invitrogen) under reducing conditions, glass fiber filters (MACH III M cell harvester; TOMTEC), sealed with blotted on nitrocellulose membrane (Bio-Rad), and blocked with 5% scintillation sheets (MeltiLex), and analyzed in a Microbeta 1450 scin- nonfat dry milk (Carl-Roth, Karlsruhe, Germany) in TBS with 0.05% tillation counter (PerkinElmer). Analogous to the ELISA results, Tween 20. All Abs were purchased from Cell Signaling Technology (Danvers, MA) and used according to the manufacturer’s instructions. Western blot quantification was done with ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/ij/). by guest on September 27, 2021 Quantitative real-time PCR Quantitative RT-PCR (qRT-PCR) was done as described previously (29). In brief, total RNA was isolated with TRIzol (Invitrogen, Lofer, Austria) or a Nucleospin RNA II kit (Macherey-Nagel, Du¨ren, Germany), according to the manufacturer’s instructions. For qRT-PCR, 2 mg total RNA was reverse-transcribed using RevertAid H Minus M-MuLV reverse tran- scriptase (MBI Fermentas, St. Leon-Roth, Germany), following the manufacturer’s protocol. qRT-PCR was performed in a Rotorgene 3000 (Corbett Research) with ready-to-use 2x iQ SYBR Green Supermix (Bio- Rad). Primers were designed by using Vector NTI software (Invitrogen) to amplify targets ranging in size from 120 to 200 nucleotides with annealing temperatures of 65˚C. Target specificity was assessed by product se- quencing and routine recording of melting curves. Sequences of the primers are listed in Table I. mRNA content (x) was calculated using the formula x =22Dct, where Dct represents the difference between the gene of interest and the reference gene, large ribosomal protein P0 (RPLP0). The fold-change value represents the quotient of the mRNA content of the induced sample and that of the corresponding noninduced sample.

Small interfering RNA and MLR For knockdown experiments, we used the validated SOCS2 stealth RNAi Duopack (Invitrogen) and a scrambled sequence oligo with similar GC content as a control oligo. Cells were transfected with Lipofectamine RNAiMax reagent (Invitrogen), according to the manufacturer’s guidelines. In brief, at day 6 of differentiation, 5 3 105 cells were plated in antibiotic-free DC medium and transfected with 100 pmol/well small interfering RNA (siRNA). After overnight incubation, the medium was FIGURE 1. Expression profile of SOCS family members in human supplemented with antibiotics and the cells were used for further moDCs. moDCs were harvested and replated at day 7 of differentiation in . experiments. Transfection efficiency was routinely 90%, as assessed DC medium at 2.5 3 105 cells/ml. Cells were either left untreated or by flow cytometry with fluorescent control siRNA oligonucleotides stimulated with 100 ng/ml E. coli LPS for 24 h. mRNA expression levels (BlockIT; Invitrogen). Knockdown efficacy was analyzed by means of qRT-PCR and Western blotting. For allogeneic MLRs, 1 3 105 naive of all SOCS family members are shown as relative expression to RPLP0 CD4+CD45RA+ T cells were cultured with DCs at a ratio of 30:1 (T cells/ (A) by means of qRT-PCR. Fold-change mRNA values were calculated for DCs) in 96-well, round-bottom plates. After 4 d of coculture, cells were the upregulated SOCS1-3 and human CIS (CISH) (B). Data show repre- pulsed with 1 mCi [3H]thymidine for 16 h. Cells were transferred onto sentative results from one of five donors. 2878 SOCS2 CONTROLS TLR-INDUCED DC ACTIVATION proliferation results are shown as normalized proliferation. Proliferation SOCS2 levels continued to accumulate up to 48 h (Fig. 2B). To stimulated by LPS-treated control cells was set as 100%. exclude potential contributions of residual impurities in our DC + Flow cytometry cultures, we also analyzed expression in CD1a FACS-sorted DCs (.99%) and obtained the same results (Supplemental Fig. 1). All FACS analyses were done on a FACS Canto II instrument (Becton Because moDCs represent inflammation-induced DCs and do not Dickinson) using FACS Diva software for acquisition and analysis. FACS Abs to human Ags (CD1a, CD14, CD40, CD80, CD86, HLA DR, PD-L1, share all features with conventional DCs (31), we chose to isolate PD-L2, CCR4, CCR7) were all purchased from BD Pharmingen. Staining primary CD1c+ blood DCs to confirm the results obtained with the was done according to standard procedures. Cells were then washed in PBS/ moDCs (Fig. 3A,3B). FCS and fixed in 0.1% paraformaldehyde until analysis. For further validation of our data, we also measured SOCS2 expression in murine APCs. LPS stimulation of mouse bone Results marrow-derived DCs and thioglycolate-induced mouse peritoneal SOCS2 is expressed in APCs in response to TLR ligands macrophages resulted in a significant upregulation of SOCS2 SOCS1, SOCS3, and CIS were described to be TLR-responsive mRNA levels. The time course follows a similar delayed expression genes, and specific functions for these proteins in DCs have pattern (Fig. 3C,3D). The delayed timing of SOCS2 expression been established (10, 11, 13–16, 30). Therefore, we screened suggested the possibility that the MyD88-independent TLR4 human moDCs for expression of all SOCS family members (for pathway, which induces delayed activation of NF-kB and IFN primer sequences, see Table I). In a long-term qRT-PCR kinetics regulatory factor 3 (32), could be responsible for SOCS2 ex- experiment, we observed that, in addition to SOCS1, SOCS3, and pression. To test this hypothesis, we investigated whether SOCS2

CIS, SOCS2 is a highly LPS-responsive gene. For SOCS4-7, no expression is triggered solely by the TLR4 pathway or is equally Downloaded from signiﬁcant expression was detectable in LPS-treated DCs (Fig. 1). activated by other TLRs that exclusively use the MyD88- In contrast with SOCS1 and SOCS3, SOCS2 was upregulated in dependent pathway. In addition to the TLR4 ligand LPS, we a delayed fashion, beginning ∼8 h after LPS stimulation and tested a whole panel of commercially available TLR ligands. peaking at 24 h after LPS treatment (Fig. 2A). To verify these moDCs were stimulated with Pam3CSK4 (for TLR1/2), FSL-1 results, we also analyzed the expression of SOCS2 at the protein (TLR2/6), poly I:C (TLR3), ﬂagellin (TLR 5), R848 (TLR7,

level, and the results fully agree with the data from the initial qRT- TLR8), and type C CpG-containing oligonucleotides (28) (TLR9). http://www.jimmunol.org/ PCR screen: SOCS2 protein remained undetectable up to 8 h; SOCS2 expression was observed in response to stimulation of thereafter, increasing protein expression was detectable, and TLR1/2, TLR2/6, TLR4, and TLR7/TLR8, albeit to varying by guest on September 27, 2021

FIGURE 2. Time-course experiments show delayed expression of SOCS2. moDCs were harvested and replated at day 7 of differentiation in DC medium at 2.5 3 105 cells/ml. Cells were stimulated with 100 ng/ml E. coli LPS for the indicated times. mRNA expression levels of SOCS1-3 are shown as relative expression compared with RPLP0 by means of qRT-PCR and are representative of all donors analyzed throughout the study (A). Error bars indicate the SD of two independent experiments for the same donor. B, DCs were stimulated with E. coli LPS as indicated, and total cell lysates were subjected to Western blot analyses for SOCS2 and for STAT1 as a control for equal loading. All results are representative of ﬁve independent experiments. The Journal of Immunology 2879 Downloaded from

FIGURE 4. SOCS2 expression is induced by MyD88-dependent TLRs. moDCs were either left untreated or stimulated with LPS (100 ng/ml), Pam3CSK (10 mg/ml), FSL-1 (1 mg/ml), poly I:C (10 mg/ml), ﬂagellin (1 mg/ml), R848 (10 mg/ml), or CPG ODN (1 mg/ml). Cells were then ana- FIGURE 3. Human primary DCs and murine APCs show SOCS2 ex- lyzed for SOCS2 mRNA expression (upper panel) by qRT-PCR. In addi- http://www.jimmunol.org/ + pression in response to LPS. Human CD1c DCs (A, B) were isolated with tion, total cells lysates of the same experiments were analyzed for SOCS2 5 a BDCA-1 kit (Miltenyi), and 1 3 10 cells were stimulated for the in- protein expression by means of Western blot. Results show mean and SD of dicated times with 100 ng/ml E. coli LPS. Cells were then analyzed for three independent PCR experiments and one of three similar Western blots. SOCS2 expression by qRT-PCR in relation to RPLP0. A, One donor yielding enough cells provided the opportunity to perform a time-series (data not shown). To further address this question, we stimulated experiment. B, In total, three independent donors were analyzed, all the cells with LPS in the presence of the protein synthesis in- yielding similar results. C, cDNAs of thioglycolate-induced mouse peritoneal macrophages were analyzed for Socs2 expression in relation to hibitor cycloheximide, and even though the time kinetics was slightly changed, SOCS2 expression was robust in these settings

Rplp0. Results show mean and SD of three mice in three independent by guest on September 27, 2021 experiments. D, cDNAs of murine bone marrow-derived DCs were ana- (Supplemental Fig. 2). Because DCs are rather sensitive to cy- lyzed for Socs2 expression in relation to Rplp0. Results show mean and SD cloheximide treatment, we had to use a relatively low concen- of two independent DC preparations. tration of the drug (20 mM); nevertheless, protein synthesis inhibition was still efficient, as verified by inhibition of TNF-a extents (Fig. 4). These observations are in line with previous production. Nevertheless, a potential contribution of preformed reports that human moDCs do not express TLR5, TLR7, and intracellular or membrane attached cytokines (38) cannot abso- TLR9, but do express TLR1-4, TLR6, and TLR8 (33, 34). In- lutely be excluded. terestingly, TLR3 stimulation resulted in increased mRNA levels Silencing of SOCS2 in DCs leads to increased secretion of but failed to produce significant levels of SOCS2 protein. Taken IL-10 and IL-1b together, these results suggest that SOCS2 is a feedback molecule that is induced by all TLRs that signal through the MyD88- To investigate the functional role of SOCS2 in DCs, we conducted dependent pathway, and that its expression is not reliant on the RNA interference experiments. To avoid unintended adverse ef- alternative signaling pathway of the LPS receptor TLR4. Notably, fects, for example, cellular stress responses like the PKR/IFN re- although SOCS2 is manifestly induced in all primary human and sponse, we used chemically modified oligonucleotides applying the mouse APCs in response to TLR stimulation, we were not able to Stealth RNAi technology. Silencing was performed with two dif- observe induction of SOCS2 mRNA in response to LPS in any of ferent oligos targeting SOCS2 mRNA to minimize the risk for the tested monocytic-like, macrophage-like, or DC-like cell lines both sequence-dependent and off-target effects. Neither the SOCS2 (THP-1, Monomac-1; RAW, Jaws II). Therefore, primary human oligos nor the control oligo caused any DC maturation themselves, moDCs were used for all subsequent experiments. as determined by FACS analyses (CD83; CD86) and cytokine ELISA (TNF-a), and showed no transcription of the type I IFN SOCS2 is a direct downstream target of TLR ligation IFN-a2 compared with untransfected cells (data not shown). Si- Although delayed expression of SOCS2 has been repeatedly re- lencing efficacy of the siRNA oligos on mRNA levels was ∼70%, ported in other studies (35–37), the deferred induction raised the as determined by qRT-PCR (data not shown); however, SOCS2 question whether SOCS2 is a direct downstream target of TLR protein was hardly detectable, as shown in Western blot analysis of signaling or is stimulated via secreted factors in an autocrine/ the corresponding cell lysates (Fig. 5B). Analysis of the surface paracrine fashion. To exclude potential contributions of auto- expression of the DC activation-associated markers CD40, CD80, crine cytokine stimulation, we treated immature DCs with dif- CD86, CD83, CCR4, and CCR7 failed to identify significant different candidate cytokines that are produced by activated DCs ferences between SOCS2-silenced DCs and control cells 24 h after themselves (IL-1, IL-6, IL-12, IL-10, TNF-a); however, none of LPS stimulation (data not shown). At the same time point, we the tested cytokines nor the combination of them was able to in- analyzed supernatants by ELISA and observed increased levels duce significant levels of SOCS2 mRNA in the absence of LPS of IL-1b (100% = 115 pg/ml) and IL-10 (100% = 26 pg/ml) in 2880 SOCS2 CONTROLS TLR-INDUCED DC ACTIVATION

FIGURE 5. Silencing of SOCS2 results in increased secretion of IL-1b and IL-10. moDCs were transfected with control siRNA or with SOCS2 siRNA duplex S2 #1 and S2 #2, and either left untreated (black bars) or stimulated with E. coli LPS for 24 h (gray bars). Cytokine secretion was determined by

means of ELISA (A). Normalized secretion Downloaded from was calculated with secretion of LPS-treated control cells (CTRL-LPS) equals 100%. Results represent mean and SD of four independent donors (*p , 0.05, **p , 0.005). At the same time points, total cell lysates were prepared and analyzed for SOCS2 and

STAT1 as controls for equal loading by http://www.jimmunol.org/ means of Western blot (B). by guest on September 27, 2021

SOCS2-deficient DCs, whereas TNF-a (100% = 1.3 ng/ml) and IL- involved in regulating the expression of both cytokines. A prom- 6 (100% = 1.4 ng/ml) levels remained unaffected (Fig. 5A). For ising candidate was STAT3, because it was previously described to a better understanding of the observed changes in cytokine pro- play vital roles in the regulation of IL-6, IL-10, and IL-1b in reduction, we conducted time-course experiments and measured the sponse to LPS stimulation (41, 42). To test whether STAT3 acti- actual mRNA levels by qRT-PCR, as well as the cumulative cy- vation is necessary for the expression of these cytokines in human tokine secretion quantified by ELISA for each time point. As moDCs, we treated the cells with the STAT3-specific inhibitor reported previously, TNF-a andIL-6showedanearly-to- compound Stattic and found concentration-dependent repression intermediate transcription profile (39) (data not shown), which of IL-1b, IL-6, and IL-10 transcription, whereas TNF-a was not was completely extinguished before substantial amounts of sensitive toward STAT3 inhibition (Supplemental Fig. 3). SOCS2 were produced in control cells. In contrast, IL-1b and IL- To analyze potential alterations of transcription factor activation, 10 showed delayed and sustained transcription. IL-1b and IL-10 we repeated the time-series experiments and subjected cell lysates to levels were increased after SOCS2 silencing, both at the tran- Western blotting for analyses of phospho-STAT3, phospho-STAT1, scriptional level and in terms of cumulative protein secretion (Fig. phospho-ERK1/2, phospho-p38, and phospho-IkB. As hypothe- 6). The observations corroborate our hypothesis that SOCS2 sized, significantly enhanced phosphorylation of STAT3 was ob- specifically regulates time-delayed processes during DC matura- served, predominantly at late time points (Fig. 7A,7B). Although tion, whereas the early response, including IL-6 and TNF-a se- different donors showed slightly variant time kinetics of STAT3 cretion, is not affected. In addition, a slight reduction of IL-12p70 hyperphosphorylation, a robust effect was observed for all donors protein level was observed under SOCS2 knockdown, most likely at 24 h of LPS treatment (Fig. 7C). This also correlates with our because of heightened IL-10 secretion into the culture medium cytokine-secretion data, where significant enhanced production of (40). IL-1b and IL-10 is exclusively observed at later time points (Fig. 6B). The absent effect of overloud STAT3 activation in SOCS2- SOCS2-deficient DCs show hyperactivation of late-phase silenced DCs on IL-6 production is explained by the different STAT3 timing of SOCS2 and IL-6 transcription. As a matter of fact, IL-6 The observed increased cytokine secretion with specificity for IL-10 transcription peaks and returns to basal levels before significant and IL-1b could indicate that SOCS2 targets a transcription factor expression of SOCS2 is detected (data not shown) (39). We were The Journal of Immunology 2881

FIGURE 6. SOCS2 diminishes the late phase of cytokine production. moDCs were transfected with control siRNA (bold lines; black bars) or with SOCS2 siRNA duplex #2 (dotted lines; gray bars) and either left untreated or stimulated with E. coli LPS for the

indicated times. Cells were harvested, and the actual Downloaded from mRNA levels of SOCS2, TNF-a, IL-10, and IL-1b for each time point were measured by qRT-PCR (A). At the same time points, supernatants were harvested and cumulative cytokine secretion of TNF-a, IL-10, and IL- 1b was measured by ELISA (B). Results show one donor representative of four independent experiments. http://www.jimmunol.org/ by guest on September 27, 2021

not able to observe any differences in early activation of the NF- is no influence of the SOCS2 knockdown in the proliferation kB or MAPK pathway (data not shown). These results are con- of T cells cocultured with immature DCs, for there is virtually no sistent with the time course of SOCS2 expression and the absence expression of SOCS2 in DCs in absence of LPS treatment (Fig. 2). of effects on early targets of TLR signaling such as TNF-a. By contrast, LPS-activated SOCS2 knockdown DCs induced a In interpreting these results, it has to be considered that the 17% increased T cell proliferation compared with LPS-activated interrelationship between IL-10 and STAT3 is ambiguous; IL-10 control DCs (100% = 2165 cpm), as determined by [3H]thymidine is not only regulated by STAT3, it also uses STAT3 as a major incorporation (Fig. 8). In these allogeneic in vitro settings, effector molecule for signal transduction. To test whether increased we were not able to observe any differences in the production of pSTAT3 levels are the origin of increased IL-10 and IL-1b pro- T cell-derived cytokines. Cultures with both control DCs and duction and not a consequence of heightened IL-10 secretion, the SOCS2-deficient DCs resulted in an IFN-g–dominated Th1 re- experiments were repeated in the presence of an IL-10R–blocking sponse (data not shown). Ab. Consistent with our hypothesis, STAT3 hyperphosphorylation in SOCS2-deficient cells persisted despite abrogated IL-10 sig- Discussion naling; although the absolute values show reduced levels in case of The mechanisms underlying the activation of DCs have been anti–IL-10R treatment, the effect of STAT3 hyperphosphorylation a subject of intense research for decades and are now reasonably cannot be attributed to autocrine IL-10 stimulation (Fig. 7D,7E). well understood. More recently, greater attention has been paid to the counterregulation of these activation processes. Balancing of SOCS2-deficient DCs induce enhanced proliferation in naive + the level of immune activation, which is necessary for the clearance CD4 T cells of pathogens and the control of inflammation, is crucial to prevent SOCS2-deficient DCs are characterized by both increased secre- excessive damage to the host. Therefore, understanding the mech- tion of IL-1b, a proinflammatory cytokine, and an increased se- anisms that limit inflammation is of utmost importance for ther- cretion of IL-10, an anti-inflammatory cytokine. To analyze the apeutic approaches that target immune pathologies caused by dys- consequences of this dichotomy in the cytokine profile on sub- regulated immune responses. sequent T cell activation, we performed allogeneic proliferation TLR signaling is one of the major pathways for inducing innate assays with naive CD4+CD45RA+ T cells. As expected, there immune activation. Mechanisms of counterregulation target each 2882 SOCS2 CONTROLS TLR-INDUCED DC ACTIVATION

FIGURE 8. LPS-induced, SOCS2-silenced DCs stimulate increased proliferation in naive allogeneic T cells. For coculture experiments, moDCs were transfected with control siRNA or with SOCS2 siRNA duplex #2 and transferred to 96-well ﬂat-bottom plates. DCs were either left untreated or stimulated with E. coli LPS for 8 h before the addition of 1 3 105 MACS puriﬁed allogeneic T cells. [3H]thymidine incorporation was measured in triplicates for each condition. Data represent mean and SD of four independent experiments. Downloaded from

level of the signaling cascade and include soluble decoy receptor variants (43), alternatively spliced adaptor molecules (MyD88s) (44, 45), and a kinase-inactive form of the IL-1R–associated kinase family (IRAK-M) (46). More recently, SOCS1 was shown to

be crucially involved in the regulation of TLR4 signaling, because http://www.jimmunol.org/ it uses proteasomal depletion of the adaptor molecule MAL as a regulatory mechanism (13). Furthermore, SOCS3 is able to tag the TRAF6/TAK1 complex for proteasomal degradation, thereby limiting signal duration (47). Whereas SOCS1 and SOCS3 have been extensively studied in immune cells, most studies of SOCS2 addressed its function in GH/insulin-like growth factor-mediated STAT5 activation (22). It has been shown that SOCS2 binds to phospho-tyrosine residues of the GH receptor, and that STAT5 inhibition is dependent on the SOCS-box (48). Crystal structures by guest on September 27, 2021 show interaction of the SOCS-box with elongin B and C, thus forming a ternary ubiquitin ligase complex able to mark inter- acting proteins for degradation (49). In this study, we showed that SOCS2 expression in DCs is induced in direct response to TLR activation. Although time-delayed expression appears to be a characteristic of SOCS2 (35–37), we determined that SOCS2 expression is independent of indirect autocrine stimulation, for it requires no de novo protein synthesis. Moreover, SOCS2 expression could be observed in response to multiple TLR ligands, indicating that only MyD88-dependent signaling is necessary for its transcription. Despite limited data pertaining to the function of SOCS2 in the FIGURE 7. SOCS2-silenced DCs show augmented STAT3 activation. A immune system, SOCS2 was recently described as the main me- and B, moDCs were transfected with control siRNA or with SOCS2 siRNA diator of the anti-inflammatory capacities of both natural and duplex #2 and either left untreated or stimulated with E. coli LPS for the aspirin-triggered lipoxins. Furthermore, SOCS2-deficient mice suf- indicated times. Total cell lysates were subjected to Western blot analyses fer from exuberant immune activation in a toxoplasmosis model of pSTAT3 (p-Tyr705), SOCS2, and ERK1/2 as control for equal loading (24, 25). Our data support the immune-dampening function of (A). Results were quantified in reference to ERK1/2 signals using ImageJ software (B). Results are representative of three independent donors. C, SOCS2 in human DCs rather than a coactivating function in TLR Mean and SD for 24-h LPS treatment (*p , 0.05, **p , 0.005) of three signaling, as suggested in a recent publication (50). In our ex- donors are shown. D, moDCs were transfected with control siRNA or with perimental setup, the early transcriptional targets of TLR acti- SOCS2 siRNA duplex #2 and either left untreated or stimulated with E. vation such as TNF-a or IL-6 remained unaffected; however, coli LPS for 24 h in the presence of either an IL-10R–blocking Ab (aIL- SOCS2-silenced DCs displayed increased secretion of IL-10 and 10R) or an isotype control. Cells were harvested, and total cell lysates were IL-1b, two cytokines with a late and sustained expression pattern analyzed for pSTAT3 and SOCS2. Blots were then reprobed with total and both transcriptional targets of STAT3 (41, 42). In allogeneic STAT3 Ab. E, Results from D were quantified and normalized for STAT3 proliferation assays, we could show that the proinflammatory levels. F, moDCs were preincubated with either an isotype control or aIL- activity of IL-1b dominates over the IL-10 effect, for SOCS2- 10R–blocking Ab (10 mg/ml) and stimulated with recombinant human IL- deficient DCs induce hyperproliferation in allogeneic naive 10 (20 ng/ml) for 1 h. Total cell lysates were then analyzed for total and phospho-STAT3 (pTyr705) by Western blot. T cells (Fig. 8). Moreover, we were able to demonstrate that, in SOCS2-deficient DCs, the late activation of STAT3 is enhanced and is independent of autocrine IL-10 signaling, which suggests The Journal of Immunology 2883 that SOCS2 targets an upstream kinase or signaling intermediate 11. Bode, J. G., A. Nimmesgern, J. Schmitz, F. Schaper, M. Schmitt, W. Frisch, responsible for STAT3 activation. It is unlikely that SOCS2- D. Haüssinger, P. C. Heinrich, and L. Graeve. 1999. LPS and TNFalpha induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages. mediated degradation of SOCS3 protein (37) contributes to this FEBS Lett. 463: 365–370. effect, for it would lead to decreased levels of STAT3 activation. 12. Stoiber, D., P. Kovarik, S. Cohney, J. A. Johnston, P. Steinlein, and T. Decker. 1999. Lipopolysaccharide induces in macrophages the synthesis of the sup- The proposed regulatory function of SOCS2 in STAT3 activation pressor of cytokine signaling 3 and suppresses signal transduction in response to is in line with a recent study demonstrating that augmented STAT3 the activating factor IFN-gamma. J. Immunol. 163: 2640–2647. activation correlates with hypermethylation-induced gene silenc- 13. Mansell, A., R. Smith, S. L. Doyle, P. Gray, J. E. Fenner, P. J. Crack, S. E. Nicholson, D. J. Hilton, L. A. O’Neill, and P. J. Hertzog. 2006. Suppressor ing of SOCS1 and SOCS2 in ovarian and breast carcinomas (51). of cytokine signaling 1 negatively regulates Toll-like receptor signaling by However, in opposition to a recent report showing hyperactivation mediating Mal degradation. Nat. Immunol. 7: 148–155. of early MyD88-dependent and -independent TLR signaling (50), 14. Baetz, A., M. Frey, K. Heeg, and A. H. Dalpke. 2004. Suppressor of cytokine signaling (SOCS) proteins indirectly regulate toll-like receptor signaling in in- we were not able to detect any significant changes in activation nate immune cells. J. Biol. Chem. 279: 54708–54715. of the NF-kB and ERK1/2 pathways (data not shown). Our own 15. Hanada, T., K. Tanaka, Y. Matsumura, M. Yamauchi, H. Nishinakamura, observations are corroborated by the delayed expression profile of H. Aburatani, R. Mashima, M. Kubo, T. Kobayashi, and A. Yoshimura. 2005. Induction of hyper Th1 cell-type immune responses by dendritic cells lacking the SOCS2, in which neither elevated SOCS2 mRNA nor protein was suppressor of cytokine signaling-1 gene. J. Immunol. 174: 4325–4332. detectable earlier than 8 h after LPS treatment. The discrepancies 16. Lang, R., A. L. Pauleau, E. Parganas, Y. Takahashi, J. Mages, J. N. Ihle, might, at least in part, be explained by the use of different methods R. Rutschman, and P. J. Murray. 2003. SOCS3 regulates the plasticity of gp130 signaling. Nat. Immunol. 4: 546–550. for siRNA transfection and the types of siRNA used. Furthermore, 17. Yasukawa, H., M. Ohishi, H. Mori, M. Murakami, T. Chinen, D. Aki, T. Hanada, we suspect that the reduction in all parameters as observed by the K. Takeda, S. Akira, M. Hoshijima, et al. 2003. IL-6 induces an anti- previous authors might be indicative of incomplete target speci- inflammatory response in the absence of SOCS3 in macrophages. Nat. Immu- Downloaded from nol. 4: 551–556. ficity in their silencing experiments. These results could also be 18. Matsumura, Y., T. Kobayashi, K. Ichiyama, R. Yoshida, M. Hashimoto, attributed to siRNA- or electroporation-induced preactivation of T. Takimoto, K. Tanaka, T. Chinen, T. Shichita, T. Wyss-Coray, et al. 2007. the DCs and, therefore, an altered maturation response toward Selective expansion of foxp3-positive regulatory T cells and immunosuppression by suppressors of cytokine signaling 3-deficient dendritic cells. J. Immunol. 179: LPS treatment. 2170–2179. In conclusion, our data provide evidence that SOCS2 is a 19. Metcalf, D., C. J. Greenhalgh, E. Viney, T. A. Willson, R. Starr, N. A. Nicola, D. J. Hilton, and W. S. Alexander. 2000. Gigantism in mice lacking suppressor of feedback inhibitor of TLR-induced DC maturation. The delayed http://www.jimmunol.org/ cytokine signalling-2. Nature 405: 1069–1073. expression allows for a period of unrestricted immune activation 20. Favre, H., A. Benhamou, J. Finidori, P. A. Kelly, and M. Edery. 1999. Dual followed by a phase of counterregulation and limitation of inflam- effects of suppressor of cytokine signaling (SOCS-2) on growth hormone signal mation-driving activity. However, the molecular target that inter- transduction. FEBS Lett. 453: 63–66. 21. Greenhalgh, C. J., D. Metcalf, A. L. Thaus, J. E. Corbin, R. Uren, P. O. Morgan, connects SOCS2 expression with limited STAT3 activation, and L. J. Fabri, J. G. Zhang, H. M. Martin, T. A. Willson, et al. 2002. Biological consequently limited cytokine secretion, remains elusive and needs evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling. J. Biol. Chem. 277: 40181–40184. to be addressed in further studies. 22. Rico-Bautista, E., A. Flores-Morales, and L. Fernańdez-Pe´rez. 2006. Suppressor of cytokine signaling (SOCS) 2, a protein with multiple functions. Cytokine Acknowledgments Growth Factor Rev. 17: 431–439.

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