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Immunity

Article C-Type Lectin DC-SIGN Modulates Toll-like Receptor Signaling via Raf-1 Kinase-Dependent Acetylation of Transcription Factor NF-kB Sonja I. Gringhuis,1,2 Jeroen den Dunnen,1,2 Manja Litjens,1 Bert van het Hof,1 Yvette van Kooyk,1 and Teunis B.H. Geijtenbeek1,* 1

Department of Molecular Cell Biology and Immunology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands These authors contributed equally to this work. *Correspondence: [email protected] DOI 10.1016/j.immuni.2007.03.012

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SUMMARY

Adaptive immune responses by dendritic cells (DCs) are critically controlled by Toll-like receptor (TLR) function. Little is known about modulation of TLR-specific signaling by other pathogen receptors. Here, we have identified a molecular signaling pathway induced by the C-type lectin DC-SIGN that modulates TLR signaling at the level of the transcription factor NF-kB. We demonstrated that pathogens trigger DC-SIGN on human DCs to activate the serine and threonine kinase Raf-1, which subsequently leads to acetylation of the NF-kB subunit p65, but only after TLR-induced activation of NF-kB. Acetylation of p65 both prolonged and increased IL10 transcription to enhance anti-inflammatory cytokine responses. We demonstrated that different pathogens such as Mycobacterium tuberculosis, M. leprae, Candida albicans, measles virus, and human immunodeficiency virus-1 interacted with DC-SIGN to activate the Raf-1-acetylationdependent signaling pathway to modulate signaling by different TLRs. Thus, this pathway is involved in regulation of adaptive immunity by DCs to bacterial, fungal, and viral pathogens. INTRODUCTION Dendritic cells (DCs) are very efficient antigen-presenting cells that play a central role in orchestrating adaptive immune responses to pathogens (Banchereau and Steinman, 1998). DCs recognize pathogens through pattern recognition receptors (PRRs) that are triggered by highly conserved structures expressed by micro-organisms, leading to DC maturation. PRR-induced DC maturation enhances their ability to activate T cells and also leads to the production of immunomodulatory cytokines, which are essential for T cell priming and differentiation (Pasare and Medzhitov, 2004; Sporri and Reis e Sousa, 2005). Im-

portant PRRs are the Toll-like receptors (TLRs) that signal via conserved pathways to induce the activation of several transcription factors, of which nuclear factor-kB (NF-kB) appears pivotal for DC maturation as well as cytokine production (Ouaaz et al., 2002). Recently, it has become evident that other PRRs also play key roles in DC-induced immune responses. A striking example is DC-SIGN, a C-type lectin expressed by DCs that interacts with a large array of pathogens (Geijtenbeek et al., 2000; van Kooyk and Geijtenbeek, 2003). Several pathogens target DC-SIGN to modulate DC-mediated immune responses to promote their survival (van Kooyk and Geijtenbeek, 2003). Strikingly, mycobacteria interact with DC-SIGN to affect TLR4-mediated immune responses by DCs, suggesting that this C-type lectin modulates TLR4 signaling (Geijtenbeek et al., 2003). The ligand responsible for this DC-SIGN-mediated immune modulation is ManLAM, a cell-wall component abundantly expressed by Mycobacterium tuberculosis. Binding of ManLAM to DC-SIGN impairs lipopolysaccharide (LPS)induced maturation of DCs and increases the production of the immunosuppressive cytokine interleukin-10 (IL-10) (Geijtenbeek et al., 2003). In addition, DC-SIGN also modulates immune responses to several other pathogens, and the immunological outcome is specific for the pathogen (Bergman et al., 2004; Smits et al., 2005; Steeghs et al., 2006). These findings support an important role for DC-SIGN as an immunomodulatory receptor. The molecular mechanisms underlying DC-SIGN-mediated modulation of immune responses are poorly defined. DC-SIGN might induce intracellular signaling that converges with the TLR4-signaling pathway. Few clues have emerged in the search for DC-SIGN-initiated signal transduction routes. A role for the tyrosine kinase Syk has been suggested based on its role in signaling induced by other C-type lectins: Dectin-1 and C-type lectin-like receptor-2 (CLEC-2) (Brown, 2006; Rogers et al., 2005; Suzuki-Inoue et al., 2006). The YXXL motif in the cytoplasmic tails of Dectin-1 and CLEC-2 has been shown to recruit Syk; a similar cytoplasmic motif is present in DC-SIGN. Also, triggering of DC-SIGN with a stimulatory antibody has been reported to induce phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB) (Caparros et al., 2006); however, it remains unclear Immunity 26, 605–616, May 2007 ª2007 Elsevier Inc. 605

Immunity DC-SIGN Induces p65 Acetylation via Raf-1

Figure 1. DC-SIGN Signaling Modulates TLR3-, TLR4-, and TLR5-Dependent IL-10 Induction (A) ManLAM induces IL-10 after TLR3, TLR4, and TLR5 triggering. Human iDCs were treated with TLR4 ligand LPS, TLR3 ligand poly(I:C), TLR5 ligand flagellin, ManLAM, or combinations, and IL-10 protein amounts were measured by ELISA after 24 hr. A representative experiment of two donors is shown. Results are presented as the means ± SD from duplicate samples. (B) IL-10 mRNA production is also increased after TLR activation. Cells were treated similar as in (A). Specificity for DC-SIGN was determined by preincubating the cells for 2 hr with the blocking anti-DC-SIGN antibody AZN-D2. After 6 hr of stimulation, relative IL-10 mRNA expression was determined by quantitative real-time PCR analysis. IL-10 mRNA expression in LPS-stimulated cells was set at 1. Results are presented as the means ± SD from at least three independent experiments. (C) ManLAM does not affect IL-10 mRNA stability after LPS stimulation. Cells were stimulated as indicated for 3 hr, before the addition of 10 mg/ml actinomycin D (ActD) to block transcription. At 20 min time periods, mRNA expression was quantified by real-time PCR analysis.

whether these kinases play a role in the immunomodulation induced by pathogenic ligands such as ManLAM. Here, we have identified the mechanism by which DC-SIGN modulates TLR-dependent responses in human DCs. Triggering of DC-SIGN by ManLAM activated the serine and threonine kinase Raf-1, independently of TLR signaling. Activation of Raf-1 led to acetylation of p65, one of the key activating subunits of NF-kB, but only after TLR signaling had activated NF-kB. Strikingly, acetylation of p65 prolonged transcriptional activity of NF-kB and enhanced the transcription rate from the IL10 gene. We further demonstrated that this Raf-1-acetylation-dependent pathway is central to modulation of TLR-specific immune responses elicited by DCs in response to mycobacteria, fungi, and viruses. The identification of this signaling pathway triggered by DC-SIGN will help further our understanding of how pathogens elicit specific immune responses. RESULTS DC-SIGN Signaling Modulates TLR3, TLR4, and TLR5 Signaling Mycobacteria interact with DC-SIGN to affect TLR4mediated immune responses by DCs, suggesting that this C-type lectin modulates TLR4 signaling (Geijtenbeek et al., 2003). We investigated whether binding of the cellwall component ManLAM from Mycobacterium tuberculosis to DC-SIGN also modulated the cytokine production induced through other Toll-like receptors besides TLR4 by stimulating primary human immature DCs (iDCs) with 606 Immunity 26, 605–616, May 2007 ª2007 Elsevier Inc.

either TLR3 ligand poly(I:C), TLR4 ligand LPS, or TLR5 ligand flagellin, alone and in combination with ManLAM. After 24 hr stimulation, ManLAM increased the expression of IL-10 protein by all three TLR ligands (Figure 1A). Similarly, after 6 hr stimulation, ManLAM upregulated IL-10 mRNA by all three TLR ligands (Figure 1B). The upregulation of IL-10 observed at the mRNA level correlated with IL-10 protein expression. Indeed, the stability of existing IL-10 transcripts after LPS stimulation was not affected by ManLAM (Figure 1C), further indicating that the induction of IL-10 is regulated at the transcriptional level. The modulation of TLR signaling by ManLAM was mediated by DC-SIGN, because addition of a blocking antibody against DC-SIGN abrogated the ManLAM-induced upregulation of IL-10 production for each TLR (Figure 1B). Thus, ManLAM binding to DC-SIGN modulates different TLR-mediated cytokine responses. Raf-1 Is Essential for DC-SIGN-Mediated Modulation of TLR Signaling We investigated the molecular mechanism(s) underlying the DC-SIGN-TLR crosstalk by employing a series of specific inhibitors directed against a broad range of potentially important signaling proteins. The NF-kB inhibitor BAY11-7082 completely abrogated the production of IL-10 by LPS-stimulated iDCs (Figure 2A), indicative of the important role of NF-kB in initiating transcription from the IL10 promoter. The p38 inhibitor SB203580 partially blocked the LPS-induced IL-10 mRNA expression, although the ManLAM-induced enhancement of IL-10 mRNA expression was unaffected (Figure 2A). Noticeably,


Figure 2. DC-SIGN-Mediated Modulation of TLR-Dependent IL-10 Induction Requires Raf Kinase Activity (A) Relative IL-10 mRNA expression by human iDCs treated with LPS and/or ManLAM for 6 hr in the presence of either Raf inhibitor GW5074, NF-kB inhibitor BAY11-7082, Syk inhibitor piceatannol, p38 inhibitor SB203580, MEK1/2 inhibitor U0126, PI 3-kinase inhibitor LY294002, PKB inhibitor API-2, PKA/MSK1 inhibitor H89, or 0.1% DMSO as a control; cells were preincubated for 2 hr with inhibitors before stimuli were added. After 6 hr of stimulation, relative IL-10 mRNA expression was determined by quantitative real-time PCR analysis. IL-10 mRNA expression in LPS-stimulated cells was set at 1. Results are presented as the means ± SD from at least three independent experiments. (B) Inhibition of Raf-1 blocks IL-10 upregulation induced by ManLAM after stimulation of DCs with different TLR ligands. Cells were stimulated with ligands and Raf inhibitor GW5074 as in (A). Results are presented as the means ± SD from at least three independent experiments.

the ManLAM-mediated upregulation of LPS-induced IL-10 mRNA expression was completely inhibited by the Raf kinase inhibitor GW5074, whereas it had no effect on LPS-induced IL-10 mRNA expression (Figure 2A). None of the other inhibitors used had any effect on the ManLAM-mediated augmentation of the LPS-induced IL-10 production, including the Syk kinase inhibitor piceatannol (Figure 2A). The Raf inhibitor blocked ManLAMmediated upregulation of IL-10 mRNA expression not only after LPS stimulation, but also after stimulation with TLR3 ligand poly(I:C) and TLR5 ligand flagellin (Figure 2B). These data indicate that Raf kinases are essential in DCSIGN-induced signaling pathways leading to modulation of TLR signaling. We therefore determined whether Raf kinases are activated by binding of ManLAM to DC-SIGN. GW5074 blocks the activities of both Raf-1 and B-Raf (Horiuchi et al., 2006; Lackey et al., 2000). Stimulation of iDCs with LPS for 10 min did not result in Raf-1 kinase activity (Figure 3B), but it did result in substantial B-Raf activity (Figure 3A). Strikingly, stimulation of iDCs with ManLAM alone induced activation of Raf-1 but not B-Raf kinase activity (Figures 3A and 3B). ManLAM-induced activation of Raf-1 was completely abolished in the presence of the DC-SIGN blocking antibody AZN-D2 (Figure 3B). By using RNA interference, we silenced Raf-1 expression in primary iDCs (Figure 3D). Knockdown of Raf-1 abrogated ManLAM-induced upregulation of IL-10 (Figure 3E). Thus, ManLAM binding to DC-SIGN leads to activation of Raf-1 independent of TLR signaling. DC-SIGN Activates Raf-1 through Ras Raf-1 activation requires recruitment of Raf-1 to the membrane through an interaction with the active form of the small GTPase Ras (Baccarini, 2005). We observed that

active Ras in iDCs is induced by ManLAM (Figure 4A). Ras activation alone is, however, insufficient to activate Raf-1, and additional phosphorylation on tyrosine and serine residues is required for activation. In particular, phosphorylation of serine 338 (Ser338) and tyrosines 340 and 341 (Tyr340 and Tyr341) is essential for activation of the kinase activity of Raf-1 (Wellbrock et al., 2004). Indeed, ManLAM induced the phosphorylation of Ser338 and Tyr340 and Tyr341 on Raf-1 (Figure 4B). This is mediated by DC-SIGN, because antibodies against DC-SIGN completely blocked phosphorylation on both Ser338 and Tyr340 and Tyr341 (data not shown). Moreover, cellpermeable Tat-peptides fused to the dominant-negative form of Ras, Tat-RasN17, completely blocked phosphorylation of Raf-1 (Figure 4B) as well as ManLAM-induced upregulation of IL-10 (Figure 4C), demonstrating that Raf-1 activation by ManLAM is dependent on Ras and essential to IL-10 upregulation. Phosphorylation of Tyr340 and Tyr341 and Ser338 has been reported to be carried out by Src family tyrosine kinases and p21-activated kinases (Paks), respectively (King et al., 2001; Wellbrock et al., 2004). We pretreated iDCs with PP2, an inhibitor of Src kinases, and toxin B, an inhibitor of the Rho family GTPases, upstream effectors of Paks. ManLAM-induced phosphorylation of Ser338 and of Tyr340 and Tyr341 was inhibited by PP2 and toxin B, respectively (Figure 4B). Moreover, we detected phosphorylation of both Pak and Src kinases after ManLAM binding (data not shown). These data were confirmed by the block in ManLAM-induced IL-10 production by the combination of PP2 and toxin B (Figure 4C). Thus, DC-SIGN binding by ManLAM activates Ras and the Src and Pak kinases, which results in phosphorylation and activation of Raf-1. Immunity 26, 605–616, May 2007 ª2007 Elsevier Inc. 607


Figure 3. DC-SIGN Signaling Induces Raf-1 Activation, Independent of TLR4 Signaling (A–C) Kinase activity assay of B-Raf (A)-, Raf-1 (B)-, or ERK1/2 (C)-specific immunocomplexes from cell lysates of human iDCs treated with LPS or DC-SIGN ligand ManLAM for 10 min; cells were preincubated for 2 hr with the blocking DC-SIGN antibody AZN-D2 as indicated. Kinase activities in either LPS (A, C)- or ManLAM (B)-stimulated cells were set at 1. Results are presented as the means ± SD from three independent experiments. (D) Raf-1 was specifically silenced by RNA interference. DCs were transfected with 100 nM Raf-1 SMARTpool or control nontargeting siRNAs. At 72 hr after transfection, B-Raf and Raf-1 mRNA was measured by quantitative real-time PCR. Results are presented as the means ± SD from two independent experiments. (E) Raf-1 is essential to ManLAM-induced IL-10 upregulation. Raf-1 or control siRNA knockdown DCs were incubated with LPS alone or in combination with ManLAM, and IL-10 mRNA was determined as described in Figure 1A. Results are presented as the means ± SD from two independent experiments.

The YXXL Motif of DC-SIGN Is Not Essential to Raf-1 Activation DC-SIGN contains an YXXL motif in the cytoplasmic domain that might be involved in signaling. The YXXL motif is also present in Dectin-1 and CLEC-2 and has been shown to recruit Syk, which is essential in signaling of both Dectin-1 and CLEC-2 (Brown, 2006; Rogers et al., 2005; Suzuki-Inoue et al., 2006). To investigate the role of this motif, we transduced Jurkat cells with DC-SIGN in which Tyr31 was replaced by an alanine (DC-SIGN Y31A). ManLAM induced phosphorylation of Raf-1 in Jurkat cells expressing DCSIGN WT, but not in mock transduced Jurkat cells (Figure 4D), demonstrating that DC-SIGN WT induces Raf-1 activation in Jurkat cells similar to iDCs. The YXXL motif in DC-SIGN is not essential for Raf-1 activation, as shown by the fact that DC-SIGN Y31A-expressing cells phosphorylated Raf-1 upon ManLAM stimulation (Figure 4D). In line with this observation, we could not detect a role for Syk in DC-SIGN signaling, because the Syk inhibitor piceatannol did not prevent ManLAM-mediated IL-10 induction (Figure 2A). Moreover, we did not detect active Syk after ManLAM binding to DC-SIGN on iDCs as determined by flow cytometry and a kinase activity assay specific for Syk (Figure S1 in the Supplemental Data available online). These data demonstrate that the YXXL motif in DC-SIGN and Syk activation is not essential to Raf-1 activation. Raf-1 Activation by DC-SIGN Does Not Lead to Extracellular Signal-Regulated Kinase Activation Next, we set out to identify the downstream effectors of Raf-1 that modulate TLR signaling. Several downstream 608 Immunity 26, 605–616, May 2007 ª2007 Elsevier Inc.

effectors of Raf-1 have been described, of which the best characterized are the dual-specificity kinases MEK1 and MEK2, which lead to activation of ERK1 and ERK2 (Baccarini, 2005; Kolch, 2005; Wellbrock et al., 2004). Recently, it has been described that the DC-SIGN-activating antibody MR-1 induces ERK phosphorylation (Caparros et al., 2006). We determined whether ERKs function as downstream effectors of Raf-1 in the DC-SIGN-mediated modulation of TLR signaling by performing a kinase assay specific for ERK1 and ERK2. LPS strongly induced ERK activation in iDCs after 10 min of stimulation, as previously reported (Figure 3C; Arrighi et al., 2001; Kawai and Akira, 2006; Re and Strominger, 2001). Remarkably, we could not detect any activation of ERK kinase activity after ligation of DC-SIGN with ManLAM (Figure 3C). In accordance with these results, we observed that MEK1/2 inhibitor U0126 did not block ManLAM-induced enhancement of the LPS-induced IL-10 mRNA expression (Figure 2A). Our findings demonstrate that DC-SIGN signals through a different Raf-1-dependent effector than the MEK-ERK module. DC-SIGN Signaling Results in Raf-1-Dependent Phosphorylation of Serine Residue Ser276 of p65 Several studies have implied that Raf-1 plays a role in the modulation of the transactivation activity of NF-kB, although the mechanism remains unclear (Baumann et al., 2000; Wellbrock et al., 2004). Signaling through all TLRs leads to activation of NF-kB (Kawai and Akira, 2006), so we investigated whether Raf-1-dependent DC-SIGN signaling modulates TLR-induced NF-kB activity. We first


Figure 4. DC-SIGN Signaling Activates Ras and Src and Pak Kinases to Induce Raf-1 Phosphorylation and Activation (A) DC-SIGN binding to ManLAM induces active Ras. Active GTP-bound Ras was precipitated with Raf-RBD-GST from cell lysates of human iDCs treated with LPS and/or ManLAM for 5 min, resolved by SDS-PAGE, and detected by immunoblotting with anti-Ras. Total Ras was detected in lysates to confirm equal amounts. (B) ManLAM induces the phosphorylation of Ser338 and Tyr340 and Tyr341 on Raf-1 through Ras and the Src and Pak kinases. DCs were stimulated as indicated for 10 min. Cells were pretreated with Tat-RasN17, the dominant-negative form of Ras, the Src kinase inhibitor PP2, or toxin B, an inhibitor of the Rho family GTPases, upstream effectors of Paks. Phosphorylated Raf-1 was measured by intracellular staining of DCs with anti-phospho-c-Raf (Ser338) or anti-c-Raf (p Tyr340 or pTyr341), followed by incubation with PE-conjugated donkey anti-rabbit Ig and flow cytometry. A representative experiment from at least two donors is shown. (C) The Raf-1-mediated IL-10 upregulation by ManLAM is dependent on Ras activation as well as Src and Pak kinases. DCs were treated with different inhibitors as described in (B), and IL-10 mRNA was determined as described in Figure 1A. Results are presented as the means ± SD from at least two independent experiments. (D) The YXXL motif of DC-SIGN is not essential to Raf-1 activation. Jurkat cells transduced with wild-type DC-SIGN and the Y31A mutant were incubated with ManLAM, and phosphorylation of Raf-1 was determined as described in (B).

examined the composition of the active NF-kB dimer, because the components that make up the transcription factor (p50, p52, p65, RelB, c-Rel) have different binding affinities and transactivation features (Hayden and Ghosh, 2004; Saccani et al., 2003). LPS strongly induced the nuclear translocation and DNA binding of p65 and p50, whereas we could detect only a modest increase in binding of c-Rel and a negligible change in p52 and RelB binding in nuclear extracts from LPS-stimulated iDCs (Figure 5A). Stimulation of iDCs with ManLAM alone or in combination with LPS did not induce the nuclear translocation and binding of any of the NF-kB components (Figure 5A). These data demonstrate that DC-SIGN signaling does not induce translocation of NF-kB into the nucleus. NF-kB activity can be regulated by covalent modifications such as phosphorylation and acetylation (Hayden and Ghosh, 2004). We determined the phosphorylation status of the key serine residues Ser276 and Ser536. Phosphorylation of Ser536 within the transactivation domain was detected upon stimulation of iDCs with LPS (Figure 5B). This observation is in accordance with previous reports that phosphorylation of p65 on Ser536 is required

for LPS-dependent NF-kB activation (Yang et al., 2003). Stimulation of iDCs with LPS and ManLAM together did not further enhance phosphorylation of p65 (Figure 5B). We could not detect any phosphorylation of Ser276 as a result of either LPS or ManLAM stimulation (Figure 5C). However, strikingly, combined triggering of TLR4 and DC-SIGN signaling did result in phosphorylation of Ser276 (Figure 5C), indicating that LPS-mediated activation of NF-kB is required in order to allow for ManLAMinduced phosphorylation of p65 at Ser276. Phosphorylation of Ser276 of p65 is a downstream target of Raf-1 in the DC-SIGN signaling pathway, as shown by the abrogation of the observed Ser276 phosphorylation after LPS + ManLAM stimulation in the presence of the Raf inhibitor (Figure 5C). Thus, only when TLR signaling has activated NF-kB will DC-SIGN-mediated signaling result in phosphorylation of p65 at Ser276, supporting our data that ManLAM binding to DC-SIGN induces IL-10 only after TLR activation. DC-SIGN Signaling Triggers Acetylation of p65 Phosphorylation of Ser276 of p65 has been linked to the acetylation of p65 on several lysines, induced by binding Immunity 26, 605–616, May 2007 ª2007 Elsevier Inc. 609


Figure 5. DC-SIGN-Mediated Raf-1-Dependent Signaling Induces p65 Phosphorylation on Ser276 and Acetylation, after Concurrent TLR Signaling (A) DC-SIGN signaling does not influence nuclear translocation of different NF-kB subunits. DNA-binding ELISA of the NF-kB subunits p65, RelB, c-Rel, p50, and p52 in nuclear extracts of human iDCs treated as indicated for 1 hr. NF-kB was allowed to bind to oligonucleotides containing the NF-kB-binding consensus sequence; specific antibodies were used to detect the different subunits within the bound complexes. Results are presented as the means ± SD from two independent experiments. (B and C) DC-SIGN signaling induces p65 phosphorylation on Ser276 through Raf-1. ELISA of phospho-Ser536-p65 (B) or phospho-Ser276-p65 (C) in cell lysates of human iDCs treated as indicated for 30 min; cells were preincubated for 2 hr with Raf inhibitor GW5074 as indicated. Results are presented as the means ± SD from three independent experiments. (D) DC-SIGN induces acetylation of p65. ELISA with acetyl-lysine antibodies on captured p65 from cell lysates of human iDCs treated as indicated for 30 min; cells were preincubated with Raf inhibitor GW5074 as in (C). Results are presented as the means ± SD from three independent experiments. (E) Acetylation of p65 is essential to ManLAM-induced IL-10 upregulation. Relative IL-10 mRNA production by human iDCs treated with different ligands for 6 hr as indicated; cells were preincubated for 2 hr with anacardic acid (AA), an inhibitor of the histone acetyltransferases p300 and CBP. IL-10 mRNA was determined as described in Figure 1A. Results are presented as the means ± SD from three independent experiments.

of the histone acetyltransferases (HATs) CREB binding protein (CBP) and p300 to the phosphorylated Ser276 residue (Zhong et al., 1998, 2002). Indeed, DC-SIGN and TLR4 signaling cooperated in inducing acetylation of p65 (Figure 5D). We could not detect any p65 acetylation after stimulation with either LPS or ManLAM alone (Figure 5D). Similarly to phosphorylation of Ser276, acetylation of p65 could be blocked by the Raf inhibitor (Figure 5D). These findings imply that the Raf-1-dependent signaling pathway induced by DC-SIGN leads first to phosphorylation of p65 on Ser276 and subsequently to its acetylation. Acetylation of p65 has been described to regulate NFkB action on multiple levels (Chen and Greene, 2003). We used anacardic acid, an inhibitor of CBP/p300specific HAT activity (Sun et al., 2006), to establish whether the acetylation of p65 accounts for the modulation of the TLR-induced IL-10 production by DC-SIGN. Strikingly, the upregulation of TLR3-, TLR4-, and TLR5-induced IL-10 mRNA expression after costimulation with ManLAM was completely abrogated in the presence of anacardic acid (Figure 5E), demonstrating that p65 acetylation plays 610 Immunity 26, 605–616, May 2007 ª2007 Elsevier Inc.

a central role in the upregulation of TLR-induced IL-10 production by DC-SIGN. Not only IL10 but also other NF-kB-dependent cytokine promoters are targeted; we observed a similar Raf-1-acetylation-dependent induction of IL-8. Notably, ManLAM binding to DC-SIGN also upregulated the early response genes interferon b (IFNb) and IFN-stimulated gene 15 (ISG15), which are dependent on both NF-kB and IRF3 (Figure S2).

DC-SIGN-Induced Acetylation Prolongs Transcriptional Activity of NF-kB and Enhances Transcription Rate from the IL-10 Gene Acetylation of p65 results in enhanced transcriptional activity of NF-kB (Chen and Greene, 2003). First, we examined the duration of NF-kB activation in DCs after stimulation, by measuring DNA binding p65 in nuclear extracts. After LPS stimulation, NF-kB remained active for as long as 4 hr (Figure 6A). Strikingly, LPS + ManLAM costimulation prolonged the nuclear activity of NF-kB to


Figure 6. DC-SIGN-Induced Acetylation Prolongs Nuclear p65 DNA Binding and Enhances the IL-10 Transcription Rate (A) DC-SIGN signaling prolongs nuclear p65 DNA binding compared to LPS signaling alone. DNA binding was determined as described in Figure 5A. Results are presented as the means ± SD from two independent experiments. (B) Acetylation of p65 prolongs nuclear p65 DNA binding. ELISA of p65 in nuclear extracts of human iDCs treated similar as in (A); cells were preincubated with Raf inhibitor GW5074 and HAT inhibitor anacardic acid (AA) as in Figures 5C and 5E. DNA binding was determined as in (A). Results are presented as the means ± SD from two independent experiments. (C) DC-SIGN signaling enhances the IL10 transcription rate. Relative IL-10 transcription rate in nuclei isolated from human iDCs treated similar as in (A); cells were preincubated for 2 hr with the blocking DC-SIGN antibody AZN-D2 as indicated. Transcription by isolated nuclei was initiated in the presence of 16-biotin-UTP; after 20 min of transcription, nuclei were lysed, RNA with incorporated biotin was isolated, and quantitative real-time PCR analysis was performed to determine the relative IL-10 RNA production. The amount of RNA produced in 20 min is representative of the transcription rate. The IL-10 RNA production by LPS-stimulated cells was set at 1. Results are presented as the means ± SD from two independent experiments.

at least 8 hr (Figure 6A) and was dependent on both Raf-1 activation and acetylation (Figure 6B). The induction of IL-10 mRNA within 6 hr of LPS + ManLAM stimulation (Figure 1B) suggests that the prolonged NF-kB activation from 4 to 8 hr is not the only mechanism involved. Therefore, we determined the effect of DC-SIGN signaling on the transcription rate from the IL10 gene. Stimulation of iDCs with ManLAM enhanced the LPS-induced IL10 transcription rate approximately 3-fold (Figure 6C). We could completely block this ManLAM-induced increase by the addition of the Raf and HAT inhibitors (Figure 6C). Thus, the increase in IL-10 by DCs after ManLAM and LPS stimulation is regulated at the level of NF-kB by prolonging NF-kB activity and enhancing the transcription rate from the IL10 gene, both processes triggered by DC-SIGN through the Raf-1acetylation-dependent pathway. Raf-1-Dependent Acetylation of p65 Is Central to DC-SIGN-Mediated Cytokine Responses to Pathogens Next, we investigated whether intact mycobacteria also induce the Raf-1-acetylation-dependent pathway in DCs through triggering of DC-SIGN. Remarkably, M. tuberculosis, M. leprae, and M. bovis BCG all induced IL10 through DC-SIGN- and TLR-dependent signaling pathways. The DC-SIGN-dependent signaling could be inhibited by the Raf and HAT inhibitors (Figure 7). M. leprae alone induced a low amount of IL-10 that could be further

increased by LPS (Figure 7). Besides mycobacteria, other pathogens also target DC-SIGN on DCs to modulate immune responses (van Kooyk and Geijtenbeek, 2003). Here, we demonstrate that also fungi and viruses modulate cytokine responses through the DC-SIGN-mediated Raf-1-acetylation-dependent pathway (Figure 7). Similarly to the mycobacteria, Candida albicans alone induced IL-10 production through coactivation of DC-SIGN and TLR signaling pathways. S. cerevisiae mannan induced IL-10 only in the presence of LPS (Figure 7). Human immunodeficiency virus-1 (HIV-1) also required prior TLR activation to modulate cytokine responses through DC-SIGN, whereas measles virus alone induced IL-10, because measles virus triggers both DC-SIGN (de Witte et al., 2006) and TLR2 (Bieback et al., 2002) signaling (Figure 7). The pathogens induced the Raf-1-acetylation-dependent pathway to enhance IL-10 production, because the cytokine response was decreased substantially through the Raf and HAT inhibitors. These data demonstrate that the DC-SIGN-mediated Raf-1-acetylation-dependent signaling pathway plays an important role in the regulation of DC-specific cytokine responses to pathogens. DISCUSSION TLRs control multiple dendritic cell functions and activate signals that are critically involved in adaptive immune responses. Little is known about how individual TLR Immunity 26, 605–616, May 2007 ª2007 Elsevier Inc. 611


Figure 7. Mycobacteria, Fungi, and Viruses Target DC-SIGN to Modulate TLR Signaling through a Raf-1- and AcetylationDependent Signaling Pathway iDCs were stimulated as described in Figure 5E with pathogens in combination with LPS as indicated. Raf and HAT inhibitors were used to determine whether these pathogens signal through DCSIGN-mediated Raf-1-acetylation-dependent pathway. IL-10 mRNA was determined as described in Figure 1A. A representative experiment from two donors is shown.

activation translates into the induction of adaptive immunity to a given pathogen and how other PRRs contribute to the TLR-induced signaling pathways. Here we describe a molecular mechanism by which mycobacteria, fungi, and viruses target DC-SIGN to modulate TLR signaling in human DCs. We demonstrate that ManLAM binding to DC-SIGN on human DCs activates the serine and threonine kinase Raf-1, which leads to phosphorylation and subsequent acetylation of p65, the activating subunit of the transcription factor NF-kB (for model, see Figure S3). Acetylation of p65 has a dual effect on transcription activity that strongly enhances IL-10 expression by DCs: p65 acetylation prolongs NF-kB activity and increases the transcription rate from the IL10 gene. Notably, although Raf-1 is activated by DC-SIGN triggering alone, the acetylation of p65 through Raf-1 occurs only when NF-kB is activated by TLRs. Thus, the DC-SIGN-dependent signaling pathway is controlled and can influence DC function only after the DC has sensed pathogens through TLR activation. 612 Immunity 26, 605–616, May 2007 ª2007 Elsevier Inc.

We have identified a role for Raf-1 in controlling adaptive immune responses through activation by DC-SIGN. The role of Raf-1 in proliferation, differentiation, survival, and motility has been studied intensely, often focusing on its role as an effector of Ras in the signaling cascade leading to ERK activation through the activation of antigen, cytokine, or growth factor receptors (Baccarini, 2005; Kolch, 2005; Wellbrock et al., 2004). DC-SIGN triggering results in Ras activation and subsequent activation of Raf-1 through phosphorylation by Src and Pak family kinases. It remains to be determined how ligand binding to DC-SIGN couples to Ras activation. Studies on Dectin-1 and CLEC-2 signaling suggested a possible role for the cytoplasmic YXXL motif through Syk kinase activation (Brown, 2006; Rogers et al., 2005; Suzuki-Inoue et al., 2006). However, our results exclude a role for the YXXL motif and Syk in DC-SIGN-dependent Raf-1 activation. ERK has been implicated as a downstream effector of Raf-1, but our data demonstrate that Raf-1 activation through DC-SIGN affects cytokine responses independently of ERK. This is in line with a previous finding that ERK is not required for Raf-1-dependent activation of NF-kB (Baumann et al., 2000). However, our results contradict data showing that ERK phosphorylation can be induced with a DC-SIGN-activating antibody (Caparros et al., 2006); an explanation might be that the antibodyinduced signaling differs from that of pathogens. A link between NF-kB as the downstream effector of Raf-1 has repeatedly surfaced in studies over the past dozen years (Baumann et al., 2000; Wellbrock et al., 2004), but the underlying mechanism has remained elusive so far. Here, we have identified the molecular mechanism by which Raf-1 affects NF-kB activity: Raf-1 activation through DC-SIGN results in acetylation of the NF-kB subunit p65. DC-SIGN-mediated acetylation of NF-kB leads to both increased and prolonged transcriptional activation from the IL10 promoter. This dual effect of DC-SIGN signaling can be attributed to acetylation of different lysine residues of p65. Acetylation of Lys310 is required for full transcriptional activity of p65, possibly creating a binding site for an as yet unidentified transcriptional coactivator increasing the transcription rate (Chen and Greene, 2003; Chen et al., 2005). Acetylation of Lys218 and Lys221 of p65 impairs the assembly with IkB, preventing the inactivation and nuclear export of NF-kB to the cytoplasm, explaining the observed prolonged NF-kB response. It is tempting to speculate that the recruitment of the HATs CBP and p300 by p65 to the IL10 promoter might also facilitate transcription by changing the local status of histone acetylation. Recently, it has been shown that LPS stimulation is accompanied by chromatin remodeling of secondary response genes, such as IL10, but the histone acetylation levels remain low (Ramirez-Carrozzi et al., 2006). An increase in acetylation could positively influence transcription (Li et al., 2007). Thus, acetylation is crucial to the DC-SIGN-enhanced transcriptional activity in DCs. In contrast to the activation of Raf-1 by DC-SIGN, the acetylation of p65 induced by DC-SIGN occurs only after


simultaneous activation by TLRs. Raf-1 by itself cannot induce NF-kB activation, but enhances its transcriptional activity only after other signaling routes have induced the release of NF-kB from its inhibitor IkB, indicating that the DC-SIGN signaling route functions as a modulatory pathway (Figure S3). Acetylation of p65 is known to be dependent on phosphorylation of a specific serine residue, Ser276 (Chen et al., 2005; Zhong et al., 1998). Indeed, our data demonstrate that activation of Raf-1 via triggering of DC-SIGN leads to the phosphorylation of serine residue Ser276 of p65. However, phosphorylation at this residue occurs only when NF-kB is released from its inhibitor IkB by TLR activation and translocates to the nucleus (Kawai and Akira, 2006). TLR triggering also leads to phosphorylation of Ser536, which is both necessary and sufficient for the LPS-mediated transactivation of NF-kB (Schmitz et al., 2004; Yang et al., 2003). Acetylation of p65 after DC-SIGN triggering is presumably a direct result of the DC-SIGN-induced phosphorylation of Ser276, because recruitment of the HATs p300 and CBP to p65 depends on prior phosphorylation of Ser276 (Chen et al., 2005; Zhong et al., 1998, 2002). Unstimulated DCs already show high p300/CBP acetyltransferase activities (data not shown), suggesting that the acetyltransferases are poised to respond immediately upon TLR-dependent nuclear translocation of NF-kB and DC-SIGN-Raf-1dependent phosphorylation of p65. The identity of the kinase responsible for the phosphorylation of Ser276 of p65 as a result of DC-SIGN-induced Raf-1 activation is still under investigation. Our results point toward a nuclear kinase, because nuclear translocation of active NF-kB that is due to TLR signaling is required before Raf-1-mediated phosphorylation of Ser276 is detected. Protein kinase A (PKA) and mitogen- and stress-activated kinase-1 (MSK1) have previously been reported to phosphorylate Ser276 of p65 (Vermeulen et al., 2003; Zhong et al., 1998); however, an inhibitor of these kinases, H89, did not inhibit the ManLAM-induced IL-10 upregulation. DC-SIGN signaling modulates not only TLR4- but also TLR3- and TLR5-induced cytokine responses. The endosome-resident TLR3 signals through TRIF, while TLR5 on the outer membrane depends on the binding of MyD88, whereas TLR4 induces both MyD88- and TRIF-dependent pathways, all culminating in the activation of the transcription factor NF-kB (Kawai and Akira, 2006). M. tuberculosis, M. leprae, M. bovis BCG, and Candida albicans trigger TLR2- and/or TLR4-mediated signaling through their cell-wall components (Abel et al., 2002; Krutzik et al., 2003; Netea et al., 2006). Our data demonstrate that these pathogens simultaneously activate the different pathogen receptors DC-SIGN and TLR on DCs, thereby inducing pathogen-specific anti-inflammatory cytokine responses. The weak induction of IL-10 by M. leprae is probably due to the activation of only TLR2 (Krutzik et al., 2003), because simultaneous TLR4 triggering by LPS strongly increased the IL-10 response through the Raf-1-acetylation-dependent pathway. This suggests that the cytokine response is dependent on which TLR is triggered. Nota-

bly, TLR activation of monocytes induces the expression of DC-SIGN (Krutzik et al., 2005), providing a further example of how the TLR and DC-SIGN pathways reinforce each other. HIV-1 induces IL-10 upregulation through DC-SIGN only after TLR4 triggering by LPS, whereas measles virus induces IL-10 in the absence of LPS, because it binds to both TLR2 (Bieback et al., 2002) and DC-SIGN (de Witte et al., 2006). Inhibition of Raf-1 activation or p65 acetylation resulted with all the DC-SIGN-pathogen interactions in a decrease of IL-10, suggesting that this Raf-1-dependent acetylation pathway might be a potential target for therapies to prevent immunosuppression during infections. Elevated amounts of IL-10 that can cause some of the immunosuppressive effects of HIV-1 have been found consistently in AIDS patients (Redpath et al., 2001). Notably, a recent study demonstrated that AIDS patients also have elevated amounts of microbial LPS in their serum (Brenchley et al., 2006). Our data strongly suggest that the concerted effect of LPS and HIV-1 in these patients will modulate DC functions through the Raf-1-acetylation-dependent pathway resulting in the elevated IL-10 amounts observed in AIDS patients (Redpath et al., 2001). We have shown that DC-SIGN signaling converges with TLR signaling at the level of NF-kB to modulate pathogenspecific cytokine responses by DCs. Our data have identified an important role for Raf-1 in the differentiation of immune responses against pathogens, and the Raf-1dependent acetylation of p65 might be a general mechanism by which DC-SIGN, and maybe other PRRs, modulate the various NF-kB-dependent immune responses. The modulatory effect of DC-SIGN on NF-kB coupled with its well-reported binding to a wide array of pathogens (van Kooyk and Geijtenbeek, 2003) further confirms the important role of DC-SIGN in the regulation of DC-specific immune responses to pathogens. These data suggest that this signaling pathway might be a promising target in combating immune suppression induced by pathogens. EXPERIMENTAL PROCEDURES Cells, Stimulation, and Inhibition Immature DCs were cultured as described before (Geijtenbeek et al., 2003). In brief, human blood monocytes were isolated from buffy coats (Sanquin bloodbank Amsterdam, the Netherlands) by use of a Ficoll gradient and a subsequent CD14 selection step with a MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Purified monocytes were differentiated into immature DCs in the presence of 500 U/ml interleukin-4 and 800 U/ml granulocyte-macrophage colony-stimulating factor (Schering-Plough, Brussels, Belgium). DCs were used for experiments at day 6 and 7. This study was performed in accordance with the ethical guidelines of the VU University Medical Center. Jurkat cells expressing DC-SIGN mutants were generated by retroviral transduction with lentiviral vectors as described (Arrighi et al., 2004). Cells were stimulated with different TLR ligands and pathogens in the presence or absence of signaling inhibitors as described in the Supplemental Experimental Procedures. The Ras inhibitor Tat-RasN17 (a gift from D. Hall, University of Wisconsin, Madison, WI) and the control peptide Tat-HA (a gift from S. Dowdy, UCSD School of Medicine, La Jolla, CA) were expressed and purified as described previously (Remans

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et al., 2004) and used at concentrations of 25 mg/ml. Cells were incubated with Tat proteins for 1 hr prior to stimulation. IL-10 Production For the detection of IL-10 protein, culture supernatants were harvested after 24 hr of stimulation and analyzed for IL-10 by ELISA (Biosource International, Camarillo, CA). Quantitative Real-Time PCR and mRNA Half-Life Determination mRNA was specifically isolated with the mRNA capture kit (Roche, Basel, Switzerland), and cDNA was synthesized with the Reverse transcriptase kit (Promega, Madison, WI). For the determination of mRNA half-life, DCs were stimulated for 3 hr prior to the addition of 10 mg/ml actinomycin D (Sigma) to block transcription; mRNA was then isolated at 20 min time periods. For real-time PCR analysis, PCR amplification was performed with the SYBR green method as described (Garcia-Vallejo et al., 2004). Details are described in Supplemental Experimental Procedures. RNA Interference DCs were transfected with 100 nM siRNA with transfection reagens DF4 (Dharmacon, Lafayette, CO), according to the manufacturer’s protocol. The siRNAs used were: Raf-1 SMARTpool (M-003601-00); nontargeting siRNA pool (D-001206-13), as a control (Dharmacon). This protocol resulted in nearly 100% transfection efficiency as determined by flow cytometry of cells transfected with siGLO-RISC free-siRNA (D-001600-01). At 72 hr after transfection, cells were used for experiments. Silenced expression of Raf-1 was confirmed at the mRNA level by quantitative real-time PCR. Ras Pull-Down Assay Active GTP-bound Ras was precipitated from cell lysates with Raf-RBD-GST (Upstate Biotechnology, Lake Placid, NY) as previously described (Remans et al., 2004), resolved by SDS-PAGE, and detected by immunoblotting with anti-Ras mAbs (BD Transduction Laboratories, Lexington, KY). Total Ras was detected in lysates to confirm equal amounts. Kinase Activity Assays for Raf-1, B-Raf, Syk, and ERK B-Raf or Raf-1 was isolated from cell lysates by specific antibodies (Upstate). The immuno-complexes were incubated with 0.5 mg inactive MEK1 (Santa Cruz Biotechnology, Santa Cruz, CA) as a substrate at 30 C. After 20 min, 0.4 mg inactive ERK2 (Upstate) and 0.4 mg biotinMBP (Chemicon, Temecula, CA) were added as substrate for activated MEK1, and after 30 min at 30 C, biotin-MBP was captured in streptavidin-coated wells and phosphorylation was determined with mouse anti-phospho-MBP (Chemicon), which is a measure for Raf-1 activity. Further details are described in the Supplemental Experimental Procedures. Phosphorylation of Raf-1 and Syk Phosphorylation of Raf-1 was measured by flow cytometry with rabbit anti-phospho-c-Raf (Ser338) mAb (Cell Signaling, Danvers, MA) and rabbit anti-c-Raf (pTyr340, Tyr341) pAb (Calbiochem). Syk phosphorylation was detected by rabbit anti-phospho Syk (Tyr525/526) mAb (Cell Signaling). Details are described in the Supplemental Experimental Procedures. DNA Binding, Acetylation, and Phosphorylation of NF-kB DC nuclear extracts were prepared with the NucBuster protein extraction kit (Novagen, Madison, WI), and 5 mg of nuclear extract was used to determine the specific subunits within the DNA binding NF-kB dimers with the TransAM NF-kB family kit (Active Motif, Carlsbad, CA), according to the manufacturer’s protocol. p65 from cell lysates was captured with the Pathscan phosphoNF-kB p65 (Ser536) sandwich ELISA kit (Cell Signaling), according to the manufacturer’s protocol. Total p65 was detected with rabbit antip65 pAbs (Active Motif), p65 phosphorylated at Ser276 or Ser536

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was detected with rabbit anti-phospho-p65(Ser276) or -p65(Ser536) pAbs (Cell Signaling), and acetylated p65 was detected with rabbit anti-acetyl-lysine pAbs (Upstate). Nuclear Run-on Nuclei were isolated from DCs. Transcription was initiated by the addition of transcription buffer containing 16-biotin-UTP (Roche) and allowed to proceed at 26 C for 20 min. Nuclei were lysed, and newly synthesized biotin-containing RNA was captured in streptavidincoated tubes with the mRNA capture kit (Roche). cDNA synthesis and real-time PCR analysis was performed as described above. Details are described in Supplemental Experimental Procedures. Statistical Analysis Data are presented as the means ± standard deviations derived from at least three independent experiments unless otherwise stated. Statistical analyses were performed on the data via the Student’s t test for paired observations. Statistical significance of the data was set at p < 0.05. Supplemental Data Three figures and Experimental Procedures are available at http:// www.immunity.com/cgi/content/full/26/5/605/DC1/. ACKNOWLEDGMENTS We would like to thank L. Colledge for critically reading the manuscript. ManLAM and Mycobacterium tuberculosis were a kind gift from J. Belisle, Colorado State University, as part of the NIH, NIAID Contract No. HHSN266200400091C, entitled ‘‘Tuberculosis Vaccine Testing and Research Materials.’’ M. leprae was a kind gift from J.P. Brennan, Colorado State University, as part of NIH, NIAID Contract NO1 AI-25469, entitled ‘‘Leprosy Research Support.’’ Candida albicans was a kind gift from M.J. Oudhoff (VU Amsterdam). J. den D. and M.L. are supported by grants from the Dutch Scientific Research program (M.L., NWO 917-46-367; J. den D., NWO 912-04-025); S.I.G. is supported by the Dutch Asthma Foundation (3.2.03.39). The authors declare that they have no competing financial interests. Received: October 16, 2006 Revised: February 12, 2007 Accepted: March 26, 2007 Published online: April 26, 2007 REFERENCES Abel, B., Thieblemont, N., Quesniaux, V.J., Brown, N., Mpagi, J., Miyake, K., Bihl, F., and Ryffel, B. (2002). Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice. J. Immunol. 169, 3155–3162. Arrighi, J.-F., Rebsamen, M., Rousset, F., Kindler, V., and Hauser, C. (2001). A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-a, and contact sensitizers. J. Immunol. 166, 3837–3845. Arrighi, J.-F., Pion, M., Wiznerowicz, M., Geijtenbeek, T.B., Garcia, E., Abraham, S., Leuba, F., Dutoit, V., Ducrey-Rundquist, O., van Kooyk, Y., et al. (2004). Lentivirus-mediated RNA interference of DC-SIGN expression inhibits human immunodeficiency virus transmission from dendritic cells to T cells. J. Virol. 78, 10848–10855. Baccarini, M. (2005). Second nature: biological functions of the Raf-1 ‘‘kinase’’. FEBS Lett. 579, 3271–3277. Banchereau, J., and Steinman, R.M. (1998). Dendritic cells and the control of immunity. Nature 392, 245–252. Baumann, B., Weber, C.K., Troppmair, J., Whiteside, S., Israel, A., Rapp, U.R., and Wirth, T. (2000). Raf induces NF-kappa B by


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