ARTICLES
Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-jB activation through Raf-1 and Syk © 2009 Nature America, Inc. All rights reserved.
Sonja I Gringhuis1,2, Jeroen den Dunnen1,2, Manja Litjens1, Michiel van der Vlist1, Brigitte Wevers1, Sven C M Bruijns1 & Teunis B H Geijtenbeek1 The C-type lectin dectin-1 activates the transcription factor NF-jB through a Syk kinase–dependent signaling pathway to induce antifungal immunity. Here we show that dectin-1 expressed on human dendritic cells activates not only the Syk-dependent canonical NF-jB subunits p65 and c-Rel, but also the noncanonical NF-jB subunit RelB. Dectin-1, when stimulated by the b-glucan curdlan or by Candida albicans, induced a second signaling pathway mediated by the serine-threonine kinase Raf-1, which integrated with the Syk pathway at the point of NF-jB activation. Raf-1 antagonized Syk-induced RelB activation by promoting sequestration of RelB into inactive p65-RelB dimers, thereby altering T helper cell differentiation. Thus, dectin-1 activates two independent signaling pathways, one through Syk and one through Raf-1, to induce immune responses.
Dendritic cells (DCs) are essential to initiate adaptive immune responses against various pathogens, such as bacteria, viruses and fungi1. The molecular mechanisms leading from pathogen recognition to adaptive immune responses remain poorly understood. DCs express pattern recognition receptors (PRRs) to sense pathogens and induce innate signaling pathways that lead to DC activation and cytokine responses. These PRRs include the archetypal Toll-like receptors (TLRs) and non-TLRs such as C-type lectins and intracellular nucleotide binding oligomerization domain proteins2–4. After pathogen binding, TLRs elicit signaling pathways through the MyD88 or TRIF adaptor proteins, leading to activation of NF-kB and other transcription factors5. TLR signaling can be modulated by other PRRs, such as the C-type lectins DC-SIGN and dectin-1, to tailor immune responses6–8. Dectin-1 is a unique C-type lectin that induces NF-kB activation after ligand binding9. Dectin-1 recognizes b-glucan carbohydrates on various fungi, including C. albicans, and dectin-1 activation induces both T helper type 1 (TH1) and interleukin (IL)-17-producing T helper (TH-17) cell immune responses by DCs that are essential to the defense against fungi10–14. Engagement of dectin-1 by fungal b-glucans leads to phosphorylation of the immunoreceptor tyrosine-based activation motif–like sequence within the cytoplasmic domain of dectin-1 (refs. 6,15). Subsequent association of the spleen tyrosine kinase Syk (A000040) with the phosphorylated receptor induces the assembly of a scaffold consisting of the caspase recruitment domain 9 (CARD9) protein and the adaptor proteins Bcl-10 and MALT1 (refs. 6,9,15). This
CARD9–Bcl-10–MALT1 scaffold couples dectin-1 to the canonical NF-kB pathway by activation of the IkB kinase (IKK) complex, leading to nuclear translocation of NF-kB subunit p65 (ref. 9). Notably, dectin-1 cross-talk with TLR2 is Syk independent15, suggesting that dectin-1 triggers a second signaling pathway to modulate TLR signaling. Moreover, little is known about how dectin-1-induced Sykdependent NF-kB activation leads to specific cytokine responses essential to T helper cell differentiation. Here we show that dectin-1-induced Syk signaling in human DCs activates not only the canonical NF-kB subunits p65 (A001645) and c-Rel (A002052), but also the noncanonical NFkB subunit RelB16, making dectin-1 the only known PRR to induce the noncanonical NF-kB pathway. Dectin-1 induced a second signaling pathway through the kinase Raf-1 (A002008) that was independent of the Syk pathway but integrated with it at the level of NF-kB activation. The dectin-1-induced Raf-1 signaling pathway repressed Syk-induced RelB activity and increased p65 transcriptional activity to induce TH1- and TH-17-polarizing cytokines in response to both curdlan and C. albicans. Moreover, dectin-1 cross-talk with various TLRs was dependent on Raf-1 signaling. Thus, we have identified a previously unknown innate signaling pathway induced by dectin-1 that is essential to protective inflammatory responses to fungi and might be involved in the pathogenesis of fungal infections. Modulation of this pathway in vaccination strategies might enhance specific cytokine responses to combat infections and inflammatory diseases.
1Department of Molecular Cell Biology and Immunology, Vrije University Medical Center, 1007 MC Amsterdam, The Netherlands. 2These authors contributed equally to this work. Correspondence should be addressed to T.B.H.G. (
[email protected]).
Received 18 August 2008; accepted 18 November 2008; published online 4 January 2009; doi:10.1038/ni.1692
NATURE IMMUNOLOGY
VOLUME 10
NUMBER 2
FEBRUARY 2009
203
ARTICLES RESULTS Dectin-1 modulates TLR-induced cytokine responses via Raf-1 Dectin-1 enhances TLR2-induced cytokine responses in a Sykindependent manner15, but little is known about the mechanisms involved and whether other TLRs are also modulated by dectin-1. Given that another C-type lectin, DC-SIGN, has been shown to enhance TLR signaling through Raf-1 (ref. 8), we investigated whether Raf-1 is also involved in dectin-1 cross-talk with TLRs. Human DCs expressed dectin-1 (Fig. 1a). We treated DCs with the TLR4 ligand lipopolysaccharide (LPS), alone or with the b-glucan curdlan11, which is a ligand for dectin-1 and induces Syk activation (Fig. 1b). LPSinduced cytokine responses were enhanced several fold by costimulation of dectin-1 with curdlan (Fig. 1c). Syk inhibition by piceatannol
3.0
60
2.5
Relative mRNA expression
% of max
c
80
40 20 0 100
101 102 103 Dectin-1 FI
104
Relative mRNA expression
% of max
20 0 100
101 102 103 104 Syk Y525-526P FI
e
2.0
Unstimulated
LPS
5.0
IL-12p40
4.0
1.5
3.0
1.0
2.0
0.5
1.0
Piceatannol
Unstimulated
LPS
IB α-S338P-Raf-1
0.0 LPS + curdlan Unstimulated
2.0
0.0
IP α-Raf-1
1.0
0.5
PP2
Relative mRNA expression
40 20
103
104
80 60 40 20
Control siRNA 1.2
IL-10
*
1.2
IL-12p35
*
1.2
IB α-Raf-1
f
60
101
IL-12p40
*
102
IB α-Y340-341P-Raf-1
Unstim Curdlan
80
0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 Raf-1 Y340-341P FI
LPS + curdlan
IL-23p19
Toxin B
g
LPS
0.0 Unstimulated LPS + curdlan
0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 Raf-1 S338P FI 100 α-Dectin1 Piceatannol PP2 Toxin B
% of max
d
3.0
Relative mRNA expression
% of max
100 α-Dectin1
Piceatannol GW5074
4.0
1.0
80
40
– α-Dectin 1 IL-12p35
1.5
2.5
60
5.0
IL-10
2.0
0.0
b 100
Relative mRNA expression
© 2009 Nature America, Inc. All rights reserved.
a 100
(Fig. 1b) did not abrogate the enhanced expression of IL-12p35 and IL-12p40 by dectin-1–TLR4 cross-talk, whereas expression of IL-10 and IL-23p19 was partially inhibited (Fig. 1c). As published reports have shown that cytokine responses induced by dectin-1 alone are completely abrogated by Syk inhibition9,15, this suggests that dectin-1 modulates TLR4-induced cytokine responses through a Syk-independent pathway. The Raf inhibitor GW5074 completely abrogated the curdlanmediated upregulation of IL-10 and IL-12p35 mRNA expression, and IL-12p40 expression was blocked below that obtained with LPS alone (Fig. 1c). IL-23p19 expression was twofold higher than that obtained with curdlan plus LPS (Fig. 1c). Similarly, Raf-1 activation by dectin-1 also modulated TLR2 signaling (Supplementary Fig. 1
103
104
LPS
LPS + curdlan –
GW5074 Piceatannol 1.2 IL-12p40 * 1.0 0.8 0.6 0.4 0.2 0.0 Unstimulated Curdlan
1.2 IL-10 * 1.0 0.8 0.6 0.4 0.2 0 Unstimulated Curdlan
1.2 IL-12p35 * 1.0 0.8 0.6 0.4 0.2 0 Unstimulated Curdlan
3.0 IL-23p19 * 2.5 2.0 1.5 1.0 0.5 0 Unstimulated Curdlan
1.2 IL-6 * 1.0 0.8 0.6 0.4 0.2 0 Unstimulated Curdlan
Syk siRNA Raf-1 siRNA 3.0 IL-23p19
*
1.2
IL-6
*
1.2
1.0
1.0
1.0
2.5
1.0
1.0
0.8
0.8
0.8
2.0
0.8
0.8
0.6
0.6
0.6
1.5
0.6
0.6
0.4
0.4
0.4
1.0
0.4
0.4
0.2
0.2
0.2
0.5
0.2
0.2
0
0
0
0
0
Unstimulated Curdlan
Unstimulated Curdlan
Unstimulated Curdlan
Unstimulated Curdlan
Unstimulated Curdlan
0
1.2 IL-1β * 1.0 0.8 0.6 0.4 0.2 0 Unstimulated Curdlan
IL-1β
*
Unstimulated Curdlan
Figure 1 Dectin-1-mediated signaling activates Raf-1 to control cytokine expression and modulate TLR-mediated signaling. (a) Flow cytometry analysis of dectin-1 expression (filled peak) and isotype control (open peak) on primary human immature DCs. FI, fluorescence intensity. (b) Syk phosphorylation at Tyr525 and Tyr526 in unstimulated (thin line) or curdlan-stimulated DCs in the absence (thick line) or presence (filled peak) of Syk inhibitor piceatannol. (c) Quantitative real-time PCR for indicated mRNAs in DCs after TLR4 triggering alone (LPS) or in combination with dectin-1 (+ curdlan) in the absence or presence of blocking dectin-1 antibodies, piceatannol or Raf inhibitor GW5074. Expression is normalized to GAPDH and set at 1 in LPS-stimulated cells. (d,e) Raf-1 phosphorylation at Ser338 or Tyr340 and Tyr341, determined by immunoblot (d) or flow cytometry (e) in unstimulated (thin line) or curdlanstimulated DCs in the absence (thick line) or presence (filled peak) of blocking dectin-1 antibodies, piceatannol, PP2 or toxin B. (f,g) Quantitative real-time PCR for indicated mRNAs in curdlan-stimulated DCs in the absence or presence of piceatannol or GW5074 (f) or after silencing of Syk or Raf-1 expression by RNAi (g). Expression is normalized to GAPDH and set at 1 in curdlan-stimulated cells. Data are representative of at least two (a,b,d) or three (e) independent experiments or presented as mean ± s.d. of at least three (g) or four (c,f) independent experiments. *, P o 0.01.
204
VOLUME 10
NUMBER 2
FEBRUARY 2009
NATURE IMMUNOLOGY
ARTICLES
IP α-p65 p65
c-Rel
p52
RelB
**
**
IB α-S276P-p65
ur dl an u G rd W la 50 n + 74
IB α-p65
Unstimulated
Unstimulated Curdlan
d
Curdlan
e
1.2 S536
Curdlan + GW5074
0.5 S276 0.4 0.3 0.2 0.1 0.0
0.8 0.4 0.0
*
IP α-RelB
IP α-p65
C ur dl an u G rd W la n 50 + 74
b
c
C
1.0 p50 0.8 0.6 0.4 0.2 0.0
Curdlan + piceatannol Curdlan + GW5074
C
IB α-p65
GW5074
f
Dimerization with p65 (OD450)
C
C
Control
IB α-RelB
RelB binding (OD450)
g
Hoechst
Merge
RelB
DNA binding (OD450)
h
1.0 p50 0.8
Hoechst
Merge
0.6 0.4
Curdlan + control siRNA
0.2
Curdlan + Raf-1 siRNA
0.0
1.2 1.0 0.8 0.6 0.4 0.2 0.0
RelB p50 Mock S276P-p65 blocking peptide
**
– NE + NE – NE + NE GST-p65 GST-p65-S276P
i
c-Rel DNA binding (OD450)
RelB
C
DNA binding (OD450)
Unstimulated Curdlan
Piceatannol
Curdlan + control siRNA Curdlan + p65 siRNA 1.0 p65 p52 0.8 0.6 0.4 0.2 0.0 – λ Ph – λ Ph + λ Ph 1.0 RelB 0.8 0.6 0.4 0.2 0.0 – λ Ph + λ Ph
NATURE IMMUNOLOGY
VOLUME 10
NUMBER 2
FEBRUARY 2009
DNA binding (OD450)
Figure 2 Syk signaling induces NF-kB activation, whereas Unstimulated 0.6 Raf-1 induces formation of inactive p65-RelB dimers. Curdlan 0.4 Curdlan + (a) DNA binding of NF-kB subunits in nuclear extracts of 0.2 p65 preclearing curdlan-stimulated DCs. OD450, optical density at 450 nm. 0.0 – λ Ph + λ Ph – λ Ph + λ Ph (b) Translocation of RelB (red) into the nuclei (blue Hoechst 1.0 p52 1.0 p65 staining) in curdlan-stimulated DCs. Colocalization (merge) RelB ** * 0.8 0.8 appears pink. (c,d) p65 phosphorylation at Ser276 or 0.6 0.6 Ser536 determined by immunoblot (c) or ELISA (d) in 0.4 0.4 nuclear extracts of curdlan-stimulated DCs. (e,f) Immunoblot 0.2 0.2 analysis (e) or ELISA (f) of the formation of dimers of p65 0.0 0.0 – λ Ph + λ Ph – λ Ph + λ Ph – λ Ph + λ Ph and either RelB or p50 in nuclear extracts of curdlanstimulated DCs in the absence or presence of GW5074 (e) or after Raf-1 silencing by RNAi (f). (g) ELISA of the formation of dimers of RelB with GST-p65, assessed after precipitation with Ser276-phosphorylated or nonphosphorylated GST-p65 from nuclear extracts (NE) of DCs in the absence or presence of a blocking peptide containing phosphorylated Ser276. (h) DNA binding of NF-kB subunits in nuclear extracts of curdlan-stimulated DCs. Effect of p65 phosphorylation on RelB and p52 DNA binding was investigated by dephosphorylating proteins with the dualspecificity l-phosphatase (+l Ph) or buffer only (–l Ph), before and after p65 preclearing. (i) p65, RelB and p52 DNA binding in nuclear extracts of curdlan-stimulated DCs after p65 silencing by RNAi. In a–e, curdlan-stimulated DCs were analyzed in the absence or presence of Syk inhibitor piceatannol or Raf inhibitor GW5074. Data are representative of two (b,d,e) independent experiments or presented as mean ± s.d. of three (a), four (d) or at least two (f–i) independent experiments. *, P o 0.05; **, P o 0.01. DNA binding (OD450)
© 2009 Nature America, Inc. All rights reserved.
a
ur dl an u G rd W la n 50 + 74
Dectin-1-induced cytokine expression requires Raf-1 We next investigated whether dectin-1 triggering activates Raf-1. Raf-1 kinase activity requires phosphorylation of Raf-1 on Ser338 and on Tyr340 and Tyr341 by Pak and Src kinases, respectively8,17. Curdlaninduced phosphorylation of Raf-1 Ser338, Tyr340 and Tyr341 was blocked by antibody to dectin-1 but not by the Syk inhibitor piceatannol (Fig. 1d,e). Phosphorylation of Ser338 and Tyr340 and Tyr341 was abrogated by toxin B (an inhibitor of the Rho family GTPases that are upstream effectors of Pak kinases) and PP2 (a Src kinase inhibitor), respectively (Fig. 1e). Thus, dectin-1 triggering results in activation of Raf-1, independent of Syk.
We then examined whether Raf-1 is involved in cytokine expression induced by dectin-1. Both Raf-1 inhibition with GW5074 and Raf-1 silencing by RNA-mediated interference (RNAi) strongly decreased curdlan-induced IL-10, IL-12p35, IL-12p40, IL-6 and IL-1b expression, whereas IL-23p19 expression was increased twofold compared to expression with curdlan alone (Fig. 1f,g). Identical results were obtained with three different small interfering RNAs (siRNAs) against Raf-1 (Supplementary Fig. 2 online), ensuring specificity of the RNAi and confirming specificity of the Raf inhibitor. We also obtained similar results when Raf-1 was inhibited indirectly through inhibition of the upstream Src and Pak kinases (Supplementary Fig. 3 online). Syk inhibition by piceatannol or Syk silencing by RNAi abrogated all cytokine responses (Fig. 1f,g). These data strongly suggest that dectin1 activates two independent signaling pathways, one through Syk and one through Raf-1, that lead to cytokine expression.
p65 phosphorylation (OD450)
online). These data strongly suggest that dectin-1 triggering activates a Raf-1-dependent signaling pathway that modulates both TLR2- and TLR4-induced cytokine expression.
205
Raf-1 signaling represses noncanonical RelB The NF-kB family consists of five distinct members that can form hetero- and homodimers: the transcriptionally active subunits p65, RelB (A002053) and c-Rel, and the subunits p50 (A002937) and p52 (A002936), which lack transactivation domains16,18. NF-kB dimers are normally retained inactive in the cytoplasm and translocate into the nucleus after activation by a variety of receptors. We investigated NF-kB activation after dectin-1 triggering by assessing the nuclear translocation and subsequent DNA binding of the different subunits. Curdlan stimulation of dectin-1 induced nuclear translocation and DNA binding of not only p65, but also c-Rel and the noncanonical RelB and p52 subunits (Fig. 2a). Further analysis strongly suggested that p65 and c-Rel formed dimers with p50, whereas RelB formed dimers with p52 to produce active dimers (data not shown; active dimers are associated with DNA binding and direct modulation of transcription—either activation or repression—as opposed to inactive dimers, which are incapable of DNA binding). Activation of all NF-kB subunits occurred in a Syk-dependent manner, as piceatannol abrogated their nuclear translocation (Fig. 2a,b). Raf-1 inhibition strongly increased DNA binding of both RelB and p52, whereas it did not significantly affect the DNA binding of p50, p65 or c-Rel (Fig. 2a). We determined that nuclear translocation of RelB was unaffected by Raf-1 activation (Fig. 2b), which strongly suggested that Raf-1 signaling represses DNA binding activity of most of the RelB
1.2 IL-6 ** * 1.2 IL-1β * 1.0 1.0 0.8 0.8 1.5 0.6 0.6 1.0 0.4 0.4 0.5 0.2 0.2 0.0 0.0 0.0 Unstimulated Curdlan Unstimulated Curdlan Unstimulated Curdlan 2.5 IL-23p19 2.0
b
*
*
Unstimulated
1.0 p65 0.8
p52
0.6
Control siRNA
0.4 0.2 0.0
– λ Ph – λ Ph + λ Ph
1.0 RelB 0.8 0.6 0.4 0.2 0.0
1.5 IL-23p19
1.2 IL-6 1.5 IL-1β 1.0 0.8 1.0 0.6 0.5 0.4 0.5 0.2 0.0 0.0 0.0 Unstimulated Curdlan Unstimulated Curdlan Unstimulated Curdlan 1.0
e
– λ Ph + λ Ph
Control siRNA
Curdlan
Control siRNA
Merge
RelB
Hoechst
Merge
Relative mRNA expression
Hoechst
NIK siRNA
7 CCL17 * 6 ** 5 4 3 2 1 0 Unstimulated Curdlan Control siRNA RelB siRNA
f
RelB
NIK siRNA
* 2.0 IL-12p40 1.2 IL-10 1.2 IL-12p35 1.0 1.0 1.5 0.8 0.8 1.0 0.6 0.6 0.4 0.4 0.5 0.2 0.2 0.0 0.0 0.0 Unstimulated Curdlan Unstimulated Curdlan Unstimulated Curdlan
Relative mRNA expression
1.2 IL-12p35 * * 2.5 IL-12p40 * * 1.0 * 2.0 0.8 1.5 0.6 1.0 0.4 0.5 0.2 0.0 0.0 Unstimulated Curdlan Unstimulated Curdlan
d Curdlan + control siRNA Curdlan + NIK siRNA
Relative mRNA expression
1.2 IL-10 * * 1.0 0.8 0.6 0.4 0.2 0.0 Unstimulated Curdlan
c
RelB siRNA RelB siRNA + GW5074
Relative mRNA expression
Control siRNA Control siRNA + GW5074
DNA binding (OD450) DNA binding (OD450)
Relative mRNA expression
Relative mRNA expression
a
present in the nucleus. Notably, not all RelB DNA binding activity was repressed by Raf-1 activation (see below). A recent study showed that Ser276 phosphorylation of p65 represses RelB activity by sequestering active RelB into inactive p65-RelB dimers that do not bind DNA19. As Raf-1 activation through DC-SIGN has been shown to induce Ser276 phosphorylation of p65 (ref. 8), we investigated whether dectin-1 triggering leads to phosphorylation of p65 at Ser276. Indeed, curdlan induced phosphorylation of p65 at Ser276, which was abrogated by both Raf-1 inhibition and Raf-1 silencing (Fig. 2c,d and Supplementary Fig. 4 online). Phosphorylation of p65 at Ser536, a prerequisite for p65-induced transactivation20, occurred in a Raf-1-independent manner (Fig. 2d). We next examined whether Raf-1-induced Ser276 phosphorylation of p65 leads to the formation of inactive p65-RelB dimers. We coimmunoprecipitated p65 and RelB from nuclear extracts of curdlan-treated DCs using a RelB-specific antibody or a p65-specific antibody (Fig. 2e). Raf-1 inhibition completely abrogated the coimmunoprecipitation of RelB and p65 (Fig. 2e). These data were confirmed with an enzyme-linked immunosorbent assay (ELISA) in which p65-RelB dimers were captured from nuclear extracts of curdlan-treated DCs with an antibody to p65. Again, the formation of p65-RelB dimers was abrogated by Raf-1 silencing, consequently resulting in enhanced formation of p65-p50 dimers (Fig. 2f). We also conducted a precipitation assay with a recombinant
NIK siRNA
© 2009 Nature America, Inc. All rights reserved.
ARTICLES
5 CCL17 4
* *
3 2
Raf-1 siRNA
4 CCL22 3
* **
2 1 0 Unstimulated Curdlan
Control siRNA + GW5074 RelB siRNA + GW5074 3 CCL22
* *
2 1
1 0 0 Unstimulated Curdlan Unstimulated Curdlan
Figure 3 Raf-1 counteracts RelB activation to induce IL-12p40 and IL-1b expression and to limit CCL17 and CCL22 expression. (a) Quantitative real-time PCR for indicated mRNAs in curdlan-stimulated DCs after RelB silencing by RNAi in the absence or presence of Raf inhibitor GW5074. Expression is normalized to GAPDH and set at 1 in curdlan-stimulated cells. (b) Translocation of RelB (red) into the nuclei (blue) of curdlan-stimulated DCs after NIK silencing by RNAi. Colocalization (merge) appears pink. (c) p65, RelB and p52 DNA binding in nuclear extracts of curdlan-stimulated DCs after NIK silencing by RNAi. Cells were treated with l-phosphatase (+l Ph) or buffer only (–l Ph) as described in Figure 2h. (d–f) Quantitative real-time PCR for indicated mRNAs in curdlan-stimulated DCs after silencing of NIK (d,e), Raf-1 (e) or RelB (f) expression by RNAi in the absence or presence of GW5074 (f). Expression is normalized to GAPDH and set at 1 in curdlan-stimulated cells. Data are representative of two (b,c) independent experiments or presented as mean ± s.d. of at least two (a,d–f) independent experiments. *, P o 0.05; **, P o 0.01.
206
VOLUME 10
NUMBER 2
FEBRUARY 2009
NATURE IMMUNOLOGY
ARTICLES
© 2009 Nature America, Inc. All rights reserved.
% input DNA
Relative mRNA expression
% input DNA
% input DNA
% input DNA
Unstimulated Curdlan + glutathione-S-transferase (GST)-p65 fusion Curdlan GW5074 a b protein to investigate whether Ser276 NF-κB IL12B promoter NF-κB IL1B promoter +1 IL12B promoter +1 IL1B promoter phosphorylation is indeed essential to the ** 3.5 3.5 ** 4.0 4.0 * * ** ** * 3.0 3.0 formation of dimers. In contrast to nonpho3.0 3.0 2.5 2.5 2.0 2.0 sphorylated GST-p65, Ser276-phosphory2.0 2.0 1.5 1.5 1.0 1.0 lated GST-p65 was able to precipitate RelB 1.0 1.0 0.5 0.5 0.0 0.0 0.0 from nuclear extracts of DCs (Fig. 2g). 0.0 IP: RNAPII IgG IP: p65 Ac-p65 c-Rel RelB IgG IP: RNAPII IgG IP: p65 Ac-p65 c-Rel RelB IgG Notably, the association between Ser276c NF-κB CCL22 promoter NF-κB CCL17 promoter +1 CCL17 promoter d +1 CCL22 promoter phosphorylated GST-p65 and RelB was 3.5 3.5 3.5 4.0 * ** * ** 3.0 3.0 3.0 prevented by the presence of a p65-derived 3.0 2.5 2.5 2.5 2.0 2.0 2.0 peptide containing the phosphorylated 2.0 1.5 1.5 1.5 Ser276 sequence (Fig. 2g). These data 1.0 1.0 1.0 1.0 0.5 0.5 0.5 indicate that Raf-1-induced Ser276 phos0.0 0.0 0.0 0.0 IP: p65 Ac-p65 c-Rel RelB IgG IP: RNAPII IgG IP: RNAPII IgG IP: p65 Ac-p65 c-Rel RelB IgG phorylation of p65 induces the formation of e 1.2 IL-12p40 * 1.2 IL-1β * 7 CCL17 * 4 CCL22 p65-RelB dimers after dectin-1 triggering. * Control siRNA 6 1.0 1.0 p65 siRNA 3 5 We next treated nuclear extracts from 0.8 0.8 4 0.6 2 0.6 curdlan-treated DCs with the dual-specificity 3 0.4 0.4 2 1 l-phosphatase to investigate whether dephos0.2 0.2 1 0.0 0.0 0 0 phorylation of p65 abolishes its formation of Unstimulated Curdlan Unstimulated Curdlan Unstimulated Curdlan Unstimulated Curdlan dimers with RelB and thereby increases the formation of RelB dimers with other NF-kB Figure 4 RelB repression affects NF-kB and RNA polymerase II recruitment differently at the IL12B, proteins. Indeed, treatment with l-phospha- IL1B, CCL17 and CCL22 promoters. (a–d) ChIP assays of the binding of p65, acetylated p65 (Ac-p65), c-Rel and RelB to NF-kB binding motifs or recruitment of RNA polymerase II to the transcription tase resulted in a strong increase in DNA initiation site (+1) of the IL12B (a), IL1B (b), CCL17 (c) and CCL22 (d) promoters. Protein-DNA binding of both RelB and p52 (Fig. 2h), to an complexes were immunoprecipitated from sheared chromatin isolated from paraformaldehyde-fixed amount similar to that obtained after chemi- DCs stimulated with curdlan in the absence or presence of Raf inhibitor GW5074. Immunoprecipitation cal inhibition of Raf-1 (Fig. 2a), which sug- with mouse IgG served as a negative control. Quantitative real-time PCR was done for various regions. gests that dephosphorylation of p65 prevents Results are normalized to that of ‘input DNA’ that had not undergone immunoprecipitation and are the formation of p65-RelB dimers and allows presented as the % input DNA. (e) Quantitative real-time PCR for indicated mRNAs in curdlanRelB and p52 to form functional dimers. stimulated DCs after p65 silencing by RNAi. Expression is normalized to GAPDH and set at 1 in curdlan-stimulated cells. Data are presented as mean ± s.d. of at least two independent experiments. Notably, DNA binding of the other subunits *, P o 0.05; **, P o 0.01. was unaffected by l-phosphatase treatment (Fig. 2h). To exclude the possibility that other mechanisms, such as expression: repression of RelB by the formation of p65-RelB dimers, direct phosphorylation of RelB, are involved in Raf-1-mediated which depends on Raf-1-induced phosphorylation of p65, and Raf-1repression of RelB, we precleared nuclear extracts of curdlan-treated induced activation that is independent of RelB. Concerning the latter, DCs with antibody to p65 to remove p65-RelB dimers and then our recent findings on the C-type lectin receptor DC-SIGN8 indicate treated the extracts with l-phosphatase. As anticipated, l-phosphatase that, independent of its induction of p65-RelB inactive dimers, Raf-1 treatment did not increase RelB and p52 DNA binding in extracts positively regulates p65 activity through acetylation of p65 (this aspect precleared of p65-RelB dimers but also did not abolish it (Fig. 2h), as of control will be discussed further below). Notably, Raf-1 inhibition our earlier results (Fig. 2a) showed that residual amounts of RelB and completely inhibited IL-1b mRNA expression by DCs in response to p52 DNA binding activities were present in curdlan-stimulated cells in curdlan stimulation, whereas Raf-1 inhibition in combination with which RelB-p65 dimers were present. Conversely, p65 silencing in DCs RelB silencing completely restored IL-1b mRNA expression (Fig. 3a), by RNAi resulted in a strong increase in DNA binding of p52 and suggesting that RelB is responsible for abrogating IL1B transcription in RelB, which was freed from sequestration by p65, and this DNA the absence of Raf-1 activation. These data indicate that the increase in binding was not further enhanced by l-phosphatase treatment functional RelB-containing dimers as a result of Raf-1 inhibition leads (Fig. 2i). These data strongly suggest that Raf-1 represses RelB by to repression of both IL12B and IL1B transcription. sequestering active RelB into inactive p65-RelB dimers after Ser276 Only a few members of the tumor necrosis factor receptor phosphorylation of p65. superfamily are known to activate noncanonical RelB-p52, which depends on NF-kB-inducing kinase (NIK) to activate IKKa16,23. Repression of RelB induces IL-12p40 and IL-1b IKKa-mediated phosphorylation and subsequent proteasomal To investigate the effect of RelB sequestration in inactive p65-RelB processing of p100 to p52 leads to nuclear translocation of RelB-p52 dimers on curdlan-induced cytokine responses, we silenced RelB complexes16,23. Indeed, we found that dectin-1 triggering induced expression in DCs by RNAi and inhibited Raf-1 by GW5074 treat- activation of RelB in a NIK-dependent manner, as NIK silencing by ment. RelB silencing had no effect on expression of IL-10, RNAi abrogated nuclear translocation of RelB and p52 after curdlan IL-12p35, IL-23p19, IL-6 or IL-1b (Fig. 3a). In contrast, RelB silencing treatment, whereas nuclear translocation and DNA binding of p50, increased IL-12p40 mRNA (encoded by IL12B; Fig. 3a), as it pre- p65 and c-Rel were unaffected (Fig. 3b,c and data not shown). vented repression of IL12B transcription by residual amounts of RelB Consistent with the above results for silencing of RelB, silencing of and p52 (Fig. 2a); this is in agreement with the known repressive NIK increased IL-12p40 mRNA after curdlan treatment (Fig. 3d), function of RelB in IL12B transcription21,22. Moreover, RelB silencing which suggests that a functional amount of active RelB-p52 dimers is in DCs treated with GW5074 resulted in partially restored IL12B present to bind to the IL12B promoter and limit transcription. transcription compared to that in control-silenced DCs (Fig. 3a). Expression of the other cytokines was unaffected by NIK silencing These data indicate that two regulatory components control IL-12p40 (Fig. 3d), again similar to RelB silencing.
NATURE IMMUNOLOGY
VOLUME 10
NUMBER 2
FEBRUARY 2009
207
ARTICLES
208
b
Unstimulated Curdlan 0.3
0.6
0.6
0.4
0.4
0.4
0.2
0.2
0.2
0.0
0.0
0.0
0.0 1.4 CCL22 1.2 1.0 0.8 0.6 0.4 0.2 0.0
CCL17
ed at tim ul
FEBRUARY 2009
an
0.8
0.6
0.2
dl
1.0
0.8
0.4
ed
1.0
NUMBER 2
1.0 0.6
0.8
VOLUME 10
AA
**
0.8
1.0 0.5 0.0 1.2
IL-1β
– IL-6
ur
*
1.2
at
0.0 1.2
2.5 2.0 1.5
**
ns
0.0 1.2 IL-12p40 1.0
IL-23p19
3.0
U
0.2
**
an
0.2
0.0
dl
0.4
0.6
0.1 0.0
1.2 IL-12p35 1.0 0.8 0.6 0.4
0.8
0.2
0.1
ur
**
*
0.3
C
1.2 IL-10 1.0
Ac
0.4
0.2
ns
c
0.5
*
C
IB α-p65
K310
Curdlan + GW5074
tim ul
IP α-p65 IB α-K310Ac-p65
U
Figure 6 Raf-1-mediated acetylation of p65 regulates IL-10, IL-12p35, IL-23p19, IL-6 and IL-12p40 expression. (a,b) p65 acetylation at Lys310 or overall p65 acetylation (Ac) determined by immunoblot (a) or ELISA (b) in nuclear extracts of curdlan-stimulated DCs in the absence or presence of Raf inhibitor GW5074. (c) Quantitative real-time PCR for indicated mRNAs in DCs after stimulation with curdlan in the absence or presence of CBP and p300 HAT activity inhibitor anacardic acid (AA). Expression is normalized to GAPDH and set at 1 in curdlan-stimulated cells. Data are representative of two (a,b) independent experiments or presented as mean ± s.d. of at least four (c) independent experiments. *, P o 0.05; **, P o 0.01.
a
p65 acetylation (OD450)
Differential recruitment of NF-jB and RNA polymerase II As shown above, RelB shows either repressive or stimulatory transactivation properties depending on the genes involved19,22. We therefore used chromatin immunoprecipitation (ChIP) assays to investigate the effect of Raf-1-induced RelB repression on binding of the NF-kB subunits to various cytokine promoters and enhancers (Fig. 4a–d). As mentioned above, transcription of IL12B and IL1B are believed to be repressed by RelB21,22,24. In agreement with this, we found that the NF-kB binding site within the IL12B promoter22 was bound by acetylated p65 and, to a lesser extent, by RelB after curdlan stimulation (Fig. 4a). Raf-1 inhibition abrogated p65 binding, whereas RelB now occupied most IL12B promoter NF-kB sites (Fig. 4a). This suggested that the increase in functionally repressive RelB resulting from the release of RelB from p65 dimers after Raf-1 inhibition increased
an
Although RelB functions as a negative regulator of transcription for IL12B transcription, it can also function as positive regulator of transcription of genes such as CCL17 and CCL22 (ref. 21,22). Consistent with this, curdlan-induced RelB-p52 was essential for expression of CCL17 and CCL22, as NIK or RelB silencing completely abrogated the expression of these chemokines after dectin-1 triggering (Fig. 3e,f). As expected, Raf-1 inhibition (through RNAi or GW5074 treatment) strongly increased the expression of these chemokines (Fig. 3e,f), which is consistent with our model that functional RelB, no longer sequestered in inactive p65-RelB dimers, positively regulates these chemokines. The above data show that dectin-1 induces RelB activation and DNA binding, which leads to transcriptional repression for some genes (IL12B) and transcriptional activation for others (CCL17 and CCL22), in a Syk- and NIK-dependent manner. Notably, RelB activation by Syk is largely counteracted by dectin-1-induced Raf-1 signaling and formation of inactive p65-RelB dimers. Thus, opposite functions of the Syk and Raf-1 signaling pathways differentially regulate cytokine expression.
dl
IgG
ur
RelB
ed
p65 Ac-p65 c-Rel
C
IgG
at
RelB
tim ul
p65 Ac-p65 c-Rel
ns
0.0 IP:
U
0.5
binding of RelB to the IL12B promoter. This result is consistent with our finding that Raf-1 signaling induces the formation of p65RelB dimers through phosphorylation of p65 on Ser276 (Fig. 2c–i). RelB binding to the promoter is thought to repress IL12B transcription by interfering with recruitment of RNA polymerase II (RNAPII) to the transcription preinitiation complex22. Consistent with that model, we found that RNAPII was efficiently recruited to the IL12B transcription initiation site (+1) after curdlan stimulation, whereas Raf-1 inhibition almost completely impaired RNAPII recruitment (Fig. 4a); this corresponded with a strong decrease in IL-12p40 expression by Raf-1 inhibition after dectin-1 stimulation (Fig. 1e,f). The importance of p65 in RelB repression was further underscored by our data showing that p65 silencing in DCs by RNAi decreased IL12B expression (Fig. 4e) similar to Raf-1 inhibition (Fig. 1e,f). We also evaluated DNA binding of the NF-kB subunits to the IL1B promoter. The IL1B promoter was specifically bound by c-Rel after curdlan stimulation, and Raf-1 inhibition resulted in complete displacement of c-Rel by RelB (Fig. 4b), which we interpreted as being caused by increased DNA binding of RelB-containing dimers, not altered c-Rel activity (Fig. 2a). This suggested that Raf-1 inhibition results in much less IL1B transcription as a result of the binding of repressive RelB dimers (Fig. 3). To further show this, we measured IL1B transcription in p65-silenced DCs after curdlan treatment. As expected, IL1B transcription was completely abrogated when p65 was silenced (Fig. 4e), strongly suggesting that the increase in functionally repressive RelB after p65 silencing displaced c-Rel to block IL1B transcription.
C ur dl an C u r G d W la 50 n + 74
1.0
**
an
1.5
**
dl
2.0
3.0 2.5 2.0 1.5 1.0 0.5 0.0 IP:
IgG
ur
d 3.5
*
RelB
ed
*
p65 Ac-p65 c-Rel NF-κB IL6 promoter
C
**
2.5
*
at
IgG
**
tim ul
RelB
NF-κB IL12A promoter
**
ns
p65 Ac-p65 c-Rel
3.0 2.5 2.0 1.5 1.0 0.5 0.0 IP:
Figure 5 Raf-1 signaling affects binding of canonical NF-kB subunits to transcriptional regulatory elements of the IL10, IL12A, IL23A and IL6 genes. (a–d) ChIP assays of the binding of p65, acetylated p65 (Ac-p65), c-Rel and RelB to NF-kB binding motifs of the IL10 enhancer (a) and the IL23A (b), IL12A (c) and IL6 (d) promoters of curdlan-stimulated DCs in the absence or presence of Raf inhibitor GW5074, as described in Figure 4. Chromatin DNA that had not undergone immunoprecipitation was used to normalize for DNA input. Data are presented as mean ± s.d. of three independent experiments. *, P o 0.05; **, P o 0.01.
Relative mRNA expression
1.0
c 3.0 % input DNA
% input DNA
2.0
0.0 IP:
© 2009 Nature America, Inc. All rights reserved.
*
3.0
% input DNA
% input DNA
*
b 3.5 NF-κB site 1 IL23A promoter
Relative mRNA expression
Curdlan + GW5074
U
Curdlan
a 4.0 NF-κB IL10 enhancer
NATURE IMMUNOLOGY
ARTICLES
100 50 0
ed
at
ns
U
16 12 8 4 0
an
dl
ur
*
IL-6
ed
C
ns
U
an
20 0 120 100 80 60 40 20 0
ur
IL-1β
**
ed
C
ns
2.5 0
ur
150 100 50 0
an
ed
at
ul tim
C
U
*
200 IL-23
dl
at
ul tim
5.0
ns
U
4 IL-12p40 3 2 1 0 50
30 20 10 0
ns
a C.
*
IL-6
40
ed
at
ca
i lb
*
ul tim
ns
U
500
a C.
c
TH1
*
TH2 Curdlan Curdlan + GW5074
20 10 0 10 20 30 40 % positive cells IFN-γ
IL-4
**
IL-1β
dT1
400
TH2
H
300
C. albicans
200
C. albicans + GW5074
100 0
ns
ca
i lb
GW5074
70 IL-12p70 60 50 40 30 20 10 0
Expression (pg/ml)
40
dl
at
ul tim
60
*
Expression (ng/ml)
0
20
– 7.5 IL-10
Expression (pg/ml)
150
1
b
Expression (ng/ml)
*
2
*
Expression (ng/ml)
0 250 IL-23 200
3
GW5074
80 IL-12p70
Expression (pg/ml)
100
*
Expression (pg/ml)
200
4 IL-12p40
Expression (pg/ml)
300
ul tim
© 2009 Nature America, Inc. All rights reserved.
*
Expression (ng/ml)
– 500 IL-10 400
Expression (ng/ml)
Expression (pg/ml)
Expression (pg/ml)
a
ed
at
ul tim
ns
U
ns
ca
bi
al C.
15
10 5 0 5 % positive cells IFN-γ
10
IL-4
Figure 7 Raf-1 signaling controls cytokine production and skews T helper cell differentiation toward TH1. (a,b) Cytokine production was determined by ELISA in supernatants of DCs stimulated with curdlan (a) or C. albicans (b) in the absence or presence of Raf inhibitor GW5074. (c,d) T helper cell polarization was assessed by flow cytometry by staining for intracellular IFN-g (TH1) and IL-4 (TH2) in individual T cells. DCs, stimulated with curdlan (c) or C. albicans (d), were added to naive CD4+ T cells and analyzed after 13 d. Data are presented as mean ± s.d. of duplicate samples representative of at least two (a,b) independent experiments or representative of three (c,d) independent experiments. *, P o 0.05; **, P o 0.01.
Similar to what has been reported for monocytes25, we found that IL1B in DCs is transcribed from a ‘poised’ promoter architecture, as RNAPII was recruited to the transcription initiation site before curdlan stimulation (Fig. 4b). Raf-1 inhibition induced RelB binding that resulted in an almost complete loss of association of RNAPII with the IL1B promoter (Fig. 4b), which corresponded with the block in IL-1b expression after Raf-1 inhibition that we noted earlier (Fig. 1e,f). Thus, Raf-1 signaling by dectin-1 is crucial for promoting p65 phosphorylation and the formation of p65-RelB dimers, which leads to IL-12p40 and IL-1b induction. In contrast, as we showed above, RelB is essential to the expression of CCL17 and CCL22 after curdlan stimulation (Fig. 3e,f). Consistent with this, we found that RelB almost exclusively occupied the NF-kB binding sites within the CCL17 and CCL22 promoters, and this corresponded with recruitment of RNAPII for transcription (Fig. 4c,d). After Raf-1 inhibition, RelB and RNAPII recruitment to the chemokine promoters increased (Fig. 4c,d), which corresponded with enhanced transcription. Consistent with those results, silencing of p65 increased chemokine expression similar to that obtained after Raf-1 inhibition (Fig. 4e), again resulting from the release of RelB from inactive p65-RelB dimers. These data show that Raf-1 signaling, through regulation of RelB, is crucial to IL12B and IL1B transcription by antagonizing Sykinduced RelB activation through the formation of p65-RelB dimers. However, not all RelB is antagonized by Raf-1 signaling, allowing some formation of RelB-p52 dimers, which function as repressors of IL12B expression and activators of CCL17 and CCL22 expression by recruiting RNAPII. Acetylation of p65 modulates cytokine expression In contrast to IL-12p40 and IL-1b, the expression of IL-10, IL-12p35, IL-23p19 and IL-6 after dectin-1 triggering was not dependent on RelB (Fig. 3a). Consistent with this, ChIP assays showed that only p65 and c-Rel were bound to the respective enhancer and promoter elements of IL10, IL12A, IL23A and IL6 (Fig. 5). Nonetheless, all of those cytokines are regulated by Raf-1 (Fig. 1f,g), suggesting a different, RelBindependent mode of regulation. Besides regulating the formation of p65-RelB dimers through Ser276 phosphorylation of p65, Raf-1 also affects the transactivation capacity of p65 itself, as Ser276 phosphorylation of p65 is a
NATURE IMMUNOLOGY
VOLUME 10
NUMBER 2
FEBRUARY 2009
prerequisite for acetylation of p65 by the histone acetyltransferases (HATs) CBP and p300 (refs. 23,26). As p65 acetylation increases its DNA affinity and transactivation activity and prolongs its nuclear localization18,27, we speculated that this modification of p65 is involved in modulation of cytokine expression by dectin-1, analogous to the function of acetylated p65 in DC-SIGN signaling8. Indeed, curdlan induced acetylation of p65 at Lys310 in a Raf-1-dependent manner (Fig. 6a,b and Supplementary Fig. 5 online). Preclearing the nuclear extracts of curdlan-treated DCs with antibody to Lys310acetylated p65 removed all detectable p65, suggesting that most active p65-containing dimers, such as p50-p65 and p65-p65 dimers, become acetylated after curdlan treatment (Supplementary Fig. 5). We next investigated whether acetylation of p65 is involved in cytokine expression by treating DCs with anacardic acid, an inhibitor of transcriptional coactivators CBP and p300 (ref. 28). Anacardic acid blocked IL-10, IL-12p35 and IL-6 and increased IL-23p19 expression, similar to Raf-1 inhibition (Fig. 6c); IL-12p40 expression was blocked, though significantly less than after Raf-1 inhibition (0.61 ± 0.08 versus 0.34 ± 0.05, respectively; P o 0.02; Fig. 1e,f). As anticipated, IL-1b, CCL17 and CCL22 expression was not inhibited (Fig. 6c), because p65 is not involved in IL1B, CCL17 or CCL22 transcriptional regulation (Fig. 4b–d). These data strongly suggest that Raf-1-induced acetylation of p65 is essential to expression of some, but not all, cytokines induced by dectin-1. To support that conclusion, we used ChIP assays to investigate the effect of Raf-1-induced p65 acetylation on binding of the NF-kB subunits to the transcriptional regulatory elements of cytokine genes. After dectin-1 triggering, the NF-kB binding motif in the enhancer region of IL10 (ref. 29) was bound by p65, whereas Raf-1 inhibition slightly but significantly decreased p65 binding (Fig. 5a). Further analysis with antibody to Lys310-acetylated p65 showed that most p65 bound to the IL10 enhancer in curdlan-stimulated cells was acetylated (Fig. 5a). These data strongly suggest that IL10 transcription depends on acetylated p65, whereas loss of acetylation of p65 after Raf-1 inhibition decreases binding of p65. The relative decrease in IL10 transcription after inhibition of either Raf-1 activation or p65 acetylation (Figs. 1e,f and 6c) was associated with the loss of acetylated p65 bound to the IL10 enhancer (Fig. 5a), which is consistent with the fact that acetylation enhances and prolongs transcriptional activity of p65 (refs. 8,18). We also found that after dectin-1 triggering, the IL23A
209
© 2009 Nature America, Inc. All rights reserved.
ARTICLES promoter was associated with approximately equal amounts of NF-kB dimers containing acetylated p65 or c-Rel (Fig. 5b). However, Raf-1 inhibition completely abolished p65 binding to the IL23A promoter but resulted in increased binding of c-Rel (Fig. 5b). These data, in combination with the data showing that Raf-1 inhibition results in increased IL23A expression (Fig. 1e,f), imply that c-Rel is a stronger activator of transcription when bound to the IL23A promoter than is acetylated p65, accounting for the increase in IL-23p19 expression. The IL12A promoter was mainly bound by acetylated p65–containing NF-kB dimers after curdlan triggering, although some c-Rel was associated as well (Fig. 5c). Raf-1 inhibition strongly enhanced the association of c-Rel with the IL12A promoter, whereas only a small amount of nonacetylated p65 was detected (Fig. 5c). The decrease in binding of acetylated p65–containing NF-kB after Raf-1 inhibition and the decrease in IL-12p35 expression after inhibition of either Raf1 or p65 acetylation (Figs. 1e,f and 6c) are consistent with the conclusion that p65 is a stronger transactivator of IL12A transcription than is c-Rel. The IL6 promoter was bound by both acetylated p65 and c-Rel after curdlan stimulation, whereas Raf-1 inhibition again abolished binding of acetylated p65, though some nonacetylated p65 did bind (Fig. 5d) The presence of nonacetylated p65 corresponded with the observed decrease in IL-6 expression when either Raf-1 or p65 acetylation were inhibited (Figs. 1e,f and 6c). Thus, Raf-1-induced p65 acetylation controls cytokine expression by enhanced DNA binding and transactivation activity. Finally, acetylated p65 was also bound to the IL12B promoter after curdlan stimulation (Fig. 4a), which was consistent with the significant decrease in IL-12p40 expression after inhibition of p65 acetylation by anacardic acid compared to the decrease after inhibition of Raf-1 (0.61 ± 0.08 versus 0.34 ± 0.05; P o 0.02; Figs. 1e,f and 6c). These data are consistent with our model in which IL12B transcription is regulated by Raf-1 not only through repression of RelB activity, but also through induction of p65 acetylation. Dectin-1 directs T helper cell differentiation via Raf-1 IL-12p70 expression is required for TH1 cell differentiation, whereas IL-1b, IL-6 and IL-23 are necessary for TH-17 induction and proliferation30–33. Because we have shown that that curdlan stimulation of dectin-1 induces production of cytokines involved in TH1 and TH-17 cell differentiation, we evaluated this more formally. Inhibition of Raf-1 completely abrogated IL-12p40, IL-12p70, IL-23 and IL-1b production, whereas IL-10 and IL-6 production were decreased (Fig. 7a). Thus, because Raf-1 activation is crucial for directly modulating the expression of IL-12p40 (through formation of p65RelB dimers), the amounts of IL-23 and IL-12p70 were increased indirectly, as IL-12p40 and IL-12p35 form bioactive IL-12p70 and IL-12p40 and IL-23p19 form bioactive IL-23 (ref. 34). We next investigated whether Raf-1 signaling is involved in cytokine expression after dectin-1 triggering by C. albicans. Similar to treatment with curdlan, treatment of DCs with C. albicans resulted in Raf-1 activation that induced a cytokine profile in a Syk-dependent manner (Fig. 7b and Supplementary Fig. 6 online). Although Syk activation was exclusively induced by dectin-1, C. albicans induced Raf-1 activation through both dectin-1 and DC-SIGN (Supplementary Fig. 6). These data indicate that Raf-1 signaling by dectin-1 is a crucial determinant in the induction of TH1- and TH-17-polarizing cytokines by curdlan as well as C. albicans. As IL-12p70 expression by DCs is a key factor in driving TH1 polarization30, we also investigated whether the Raf-1 signaling pathway induced by dectin-1 in response to curdlan and C. albicans affects the differentiation of T helper cells. As expected, given its crucial function in IL-12p70 expression, Raf-1
210
signaling was required for T cell differentiation toward a TH1 response, whereas Raf-1 inhibition led to a strong TH2 response after curdlan or C. albicans stimulation (Fig. 7c,d). Thus, by regulating cytokine expression essential to T helper cell differentiation, Raf-1 signaling serves as the crucial determinant in the induction of adaptive immunity to C. albicans by DCs (Supplementary Fig. 7 online). DISCUSSION Dectin-1 is an important innate receptor on DCs that dictates antifungal immunity35 by inducing NF-kB activation through Sykdependent CARD9–Bcl-10–MALT1 signaling9,15. Here we showed that the Syk signaling pathway activated not only the canonical NF-kB subunits p65 and c-Rel but also the noncanonical NF-kB subunit RelB. We also showed that dectin-1 induced a second signaling pathway, independent of Syk, that activated a Raf-1-dependent signaling pathway that integrated with the Syk-dependent CARD9–Bcl-10– MALT1 pathway at the level of NF-kB activation and regulation. Notably, dectin-1-induced Raf-1 signaling repressed Syk-induced RelB activity and increased p65 transactivation activity to induce cytokine expression. Raf-1 signaling induced the phosphorylation of p65 at Ser276, which regulates cytokine expression at two distinct levels: RelB inactivation and p65 acetylation. The Syk and Raf-1 signaling pathways triggered by dectin-1 are both essential to induce TH1and TH-17-polarizing cytokines in response to curdlan and C. albicans. Thus, dectin-1 induces two independent signaling pathways, one through Syk and one through Raf-1, which converge at the level of NF-kB activation to control adaptive immunity to fungi. To date, only a few members of the tumor necrosis factor receptor superfamily have been shown to induce the NIK-dependent noncanonical NF-kB pathway36. We found that dectin-1 induced NIKdependent RelB activation through Syk, further emphasizing the unique function of dectin-1 as a PRR. LPS has been reported to activate RelB after 24 h (ref. 22), suggesting that TLR4 indirectly activates the noncanonical NF-kB pathway. Remarkably, the kinetics of p100 processing by dectin-1-induced NIK activation is faster than has been reported for the tumor necrosis factor receptor family members37. Notably, Syk-induced RelB by dectin-1 was mostly inactivated by the Raf-1 signaling pathway. Raf-1 induced p65 phosphorylation at Ser276, which was required for p65 and RelB association and formation of inactive p65-RelB dimers. The antagonistic effect of Syk and Raf-1 signaling on RelB activity was essential to dectin-1-induced cytokine responses, as Raf-1-induced inactivation of RelB (through the formation of p65-RelB dimers) was essential for induction of IL-12p40 and IL-1b. Consistent with those results, inhibition of Raf-1 led to a strong increase in active RelB that blocked transcription of IL12B and IL1B by preventing RNAPII recruitment. A previous study reported that formation of inactive p65-RelB dimers represses both RelB and p65 activity38. However, our data showed no increase in p65dependent transcription of IL10, IL12A, IL6 and IL23A after RelB silencing, indicating that RelB, but not p65, is inactivated by p65-RelB sequestering after dectin-1 signaling. Notably, IL-12p40 induction by Raf-1 signaling is also essential to the formation of bioactive IL-12p70 and IL-23. Indeed, the decreased expression of IL-12p40 after Raf-1 inhibition prevented the formation of bioactive IL-23 protein, even though IL-23p19 mRNA was abundantly present. We found that the Raf-1 signaling pathway did not antagonize Syk-induced RelB activation absolutely, as some residual RelB remained unbound by p65 dimers, and this RelB was sufficient to activate CCL17 and CCL22 and to weakly repress IL-12p40 expression. Because dectin-1 is the only known PRR to induce RelB, we hypothesize that Raf-1 activation by this receptor is essential to prevent complete repression of IL-12p40,
VOLUME 10
NUMBER 2
FEBRUARY 2009
NATURE IMMUNOLOGY
© 2009 Nature America, Inc. All rights reserved.
ARTICLES which would abrogate TH1- and TH-17-mediated protection against fungi. Thus, the sequestering of RelB by p65 seems to be a mechanism that is essential to decrease RelB repression of TH1- and TH-17inducing cytokines and to moderate expression of TH2-attracting chemokines such as CCL17 and CCL22. Our data also strongly suggested that IL10, IL12A and IL6 transcription is increased by acetylation of p65, as the promoter or enhancer regions were occupied by acetylated p65, and inhibition of either Raf-1 or CBP- and p300-specific HAT activity decreased IL-10, IL-12p35 and IL-6. In contrast, IL-23p19 expression was increased twofold after inhibition. Recent data showed that the mouse IL23A promoter is preferentially transcribed by c-Rel39; likewise, our data suggest that human IL23A is preferentially transcribed by c-Rel. We also showed that IL-12p40 expression depends not only on Raf-1mediated RelB sequestering in inactive p65-RelB dimers, but also partially on Raf-1-induced acetylation, and thereby activation, of p65. This was further supported by our finding that RelB silencing did not completely restore IL12B transcription after Raf-1 inhibition to the amount in RelB-silenced DCs treated with curdlan. Several studies have shown that TLR signaling is enhanced by dectin-1 (refs. 9,15,40), and we showed here that the Raf-1 signaling pathway is essential to this cross-talk. Our data strongly suggest that the Raf-1 signaling pathway induced by dectin-1 modulates TLRinduced cytokine expression by inducing p65 acetylation. TLR4 triggering alone activates p65 (ref. 8) and induced expression of IL-10, IL-12p35 and IL-12p40, which were increased by dectin-1 signaling in a Raf-1-dependent manner. Our data indicated that these genes were positively regulated by p65 acetylation, strongly suggesting that Raf-1 signaling enhanced TLR4-induced expression by acetylation of p65. Notably, the Syk signaling pathway induced by dectin-1 is also involved in cross-talk with TLR4 signaling by activating both c-Rel and RelB. However, the contribution of the Syk pathway in TLR–dectin-1 cross-talk was dependent on the TLR involved, as dectin-1-induced Syk signaling was more important for TLR2- than TLR4-mediated cytokines. Thus, the Raf-1 signaling pathway induced by dectin-1 is essential in dectin-1–TLR cross-talk by inducing p65 acetylation and by repressing Syk-induced RelB. Dectin-1 is involved in the recognition of a broad range of fungal pathogens, including C. albicans, Aspergillus fumigatus and Pneumocystis carinii35, and protective antifungal immunity requires TH1 and TH-17 responses10–13,41. IL-12p70 is a key factor in the induction of TH1 immunity30. Here we showed that Raf-1 signaling is a crucial determinant for both IL-12p35 and IL-12p40 mRNA, and hence IL-12p70 protein expression, in human DCs after interaction of curdlan or C. albicans with dectin-1, thereby triggering TH1 responses. Dectin-1 triggering on mouse DCs by C. albicans leads to both TH1 and TH-17 differentiation11. In addition, IL-6, IL-1b and IL-23 are involved in promoting the proliferation of TH-17 cells in humans31–33. Our data show that Raf-1 signaling by dectin-1 is essential to IL-1b and IL-6 expression, as well as the expression of bioactive IL-23. Further studies will be necessary to specifically investigate the function of the Raf-1 signaling pathway in TH-17 differentiation. Curdlan and C. albicans binding to dectin-1 induce both Syk- and Raf-1-dependent signaling, but as both pathways are regulated independently, we cannot exclude the possibility that some pathogens can induce Syk-dependent signaling through dectin-1 without activation of Raf-1, thereby inducing a very specific cytokine program without IL-12p70, IL-23 and IL-1b but instead with expression of RelBregulated genes. Further research into the specific regulation of Raf1 signaling by dectin-1 and its ligands will shed light on this. The high homology between human and mouse dectin-1 suggest that Raf-1
NATURE IMMUNOLOGY
VOLUME 10
NUMBER 2
FEBRUARY 2009
signaling is also involved in mouse dectin-1 signaling. Considering the ability of Raf-1 signaling to modulate immune responses mediated by Syk-CARD9–Bcl-10–MALT1–, TLR-, and other NF-kB-related signaling, this pathway might serve a central function in directing immune responses against numerous pathogens. From a therapeutic point of view, targeting of this pathway might be a useful tool to steer the immune response to clear pathogenic infections more efficiently. METHODS Cells, stimulation and inhibition. Immature DCs were cultured as described7 and used for experiments on day 6 or 7. This study was done in accordance with the ethical guidelines of the VU University Medical Center. Final concentrations of stimuli were 10 mg/ml curdlan from Alcaligenes faecalis (Sigma-Aldrich), 10 ng/ml LPS from Salmonella typhosa (Sigma), 50 mg/ml zymosan from Saccharomyces cerevisiae (Sigma) and heat-killed C. albicans (multiplicity of infection of 10). Final concentrations of inhibitors were 10 mg/ml antibody to dectin-1 (MAB1859; R&D Systems), 20 mg/ml antibody to DC-SIGN (AZN-D2; ‘in house’)42, 1 mM GW5074 (Raf inhibitor43; Calbiochem), 20 ng/ml toxin B (inhibitor of Rho-family GTPases44; Calbiochem), 30 mM anacardic acid (p300 and CBP HAT inhibitor28; Calbiochem), 40 mM piceatannol (Syk inhibitor45; LC Laboratories) and 10 mM PP2 (Src inhibitor46; LC Laboratories). RNA interference. DCs were transfected with 50 nM siRNA with transfection reagent DF4 (Dharmacon) according to the manufacturer’s protocol. Silencing was confirmed at the mRNA and protein levels by quantitative real-time PCR and immunofluorescence cytospin or flow cytometry staining, respectively (Supplementary Fig. 8 online). Further details are given in Supplementary Methods online. Quantitative real-time PCR and enzyme-linked immunosorbent assay. DCs were lysed after 6 h of stimulation, and real-time PCR was done as described8 (see Supplementary Methods for additional details). Cytokines were measured in culture supernatants collected after 24 h of stimulation by ELISA (Invitrogen, except IL-23 ELISA from eBioscience). Phosphorylation of Syk and Raf-1. Phosphorylated Syk and Raf-1 were measured by flow cytometry after 5 or 15 min of stimulation, respectively. Phosphorylated Raf-1 was detected by immunoblotting after immunoprecipitating Raf-1 from whole cell extracts with antibody to Raf-1 (07-396; Upstate Biotechnology) after 15 min of stimulation. Further details are described in Supplementary Methods. NF-jB DNA binding and k-phosphatase treatment. Nuclear extracts of DCs were prepared after 30 min of stimulation with a NucBuster protein extraction kit (Novagen). NF-kB DNA binding was determined with a TransAM NF-kB family kit (Active Motif). Activation of the different subunits occurred rapidly and remained unchanged between 30 min and 2 h (data not shown). Nuclear extracts were precleared by incubating them with antibody to p65 (3034; Cell Signaling) coated on protein A/G-PLUS agarose beads (Santa Cruz Biotechnology) to remove all p65 and p65-associated proteins. Nuclear extracts were treated with l-phosphatase (New England Biolabs) for 1 h at 30 1C before DNA binding to dephosphorylate serine, threonine and tyrosine residues. p65 phosphorylation and acetylation. Phosphorylated or acetylated p65 was measured by ELISA or detected by immunoblotting. Nuclear extracts were precleared by incubating with antibody to Lys310-acetylated NF-kB p65 (3045; Cell Signaling) coated on protein A/G-PLUS agarose beads (Santa Cruz Biotechnology) to remove all Lys310-acetylated p65. Further details are provided in Supplementary Methods. Formation of p65-RelB dimers and GST precipitation. p65-RelB was detected by ELISA or by immunoblot analysis after immunoprecipitation of RelB or p65 from nuclear extracts with antibodies to RelB or p65 (both from Cell Signaling). Associated RelB, p65 and p50 were detected with antibodies to RelB (4954), p65 (3035) or p50 (all from Cell Signaling). Association of RelB with nonphosphorylated or Ser276-phosphorylated GST-p65 was detected by ELISA. Further details are given in Supplementary Methods.
211
ARTICLES
© 2009 Nature America, Inc. All rights reserved.
Chromatin immunoprecipitation assay. A ChIP-IT enzymatic kit (Active Motif) was used for ChIP assays. Protein-DNA complexes were immunoprecipitated from sheared chromatin isolated from fixed cells after 2 h of stimulation with salmon sperm–treated protein G–agarose beads and antibodies to p65, Lys310-acetylated p65, c-Rel or RelB (all from Cell Signaling); monoclonal antibody to RNAPII (included in the ChIP-IT kit; Active Motif); or negative control mouse IgG (included in the ChIP-IT kit; Active Motif). Real-time PCR was then used to quantify NF-kB binding or transcription initiation (+1) sites of IL10 (ref. 29), IL12A (refs. 22,47), IL12B (ref. 22), IL23A (ref. 39), IL6 (ref. 48), IL1B (ref. 49), CCL17 (ref. 50) and CCL22 (ref. 50). To normalize for DNA input, a sample for each condition was taken that had not undergone immunoprecipitation with a specific antibody (‘input DNA’); the results are presented as the percentage of input DNA. Further details are provided in Supplementary Methods. T cell differentiation assay. DCs were preincubated for 2 h with inhibitors as indicated and then stimulated for 48 h with curdlan. DCs were washed extensively, and naive CD4+ T cells were added to stimulated DCs. On days 6 and 9 of coculture, cells were stimulated with IL-2 (100 U/ml). On day 13, T cells were restimulated with the phorbol ester PMA (30 ng/ml) and ionomycin (1 mg/ml) in the presence of brefeldin A (10 mg/ml), stained for intracellular IL-4 and IFN-g with phycoerythrin- and fluorescein isothiocyanate–labeled antibodies (Becton Dickinson), respectively, and analyzed on a FACSCalibur (Becton Dickinson). Statistical analysis. Data are presented as means ± s.d. derived from at least three independent experiments, unless otherwise stated. Student t test for paired observations was used for statistical analyses. Statistical significance was set at P o 0.05. Accession codes. UCSD-Nature Signaling Gateway (http://www.signalinggateway.org/): A002053, A002937, A002936, A002008, A002052, A001645 and A000040. Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS We thank Y. van Kooyk and R. Mebius for critically reading the manuscript, M. Oudhoff (Academic Centre for Dentistry Amsterdam) for C. albicans and K. Kristiansen (University of Southern Denmark) for the GST-p65 expression plasmid. This work was supported by the Netherlands Organisation for Scientific Research (NWO 917-46-367 to M.L. and NWO 912-04-025 to J.d.D.), the AIDS Foundation (2007036 to M.v.d.V.) and the Dutch Asthma Foundation (3.2.03.39 to S.I.G.). AUTHOR CONTRIBUTIONS S.I.G. and J.d.D. executed most experiments and prepared the manuscript. M.L., M.v.d.V. and B.W. participated in the GST-p65 precipitation, nuclear extract isolation and dectin-1 expression experiments, respectively. S.C.M.B. helped with the T helper cell differentiation assay. S.I.G. and T.B.H.G. designed and interpreted most experiments. T.B.H.G. supervised all aspects of this study, including execution and manuscript preparation. Published online at http://www.nature.com/natureimmunology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Banchereau, J. & Steinman, R.M. Dendritic cells and the control of immunity. Nature 392, 245–252 (1998). 2. Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature 449, 819–826 (2007). 3. Robinson, M.J., Sancho, D., Slack, E.C., LeibundGut-Landmann, S. & Sousa, C.R.E. Myeloid C-type lectins in innate immunity. Nat. Immunol. 7, 1258–1265 (2006). 4. van Vliet, S.J., den Dunnen, J., Gringhuis, S.I., Geijtenbeek, T.B. & van Kooyk, Y. Innate signaling and regulation of dendritic cell immunity. Curr. Opin. Immunol. 19, 435–440 (2007). 5. Kawai, T. & Akira, S. TLR signaling. Cell Death Differ. 13, 816–825 (2006). 6. Underhill, D.M., Rossnagle, E., Lowell, C.A. & Simmons, R.M. Dectin-1 activates Syk tyrosine kinase in a dynamic subset of macrophages for reactive oxygen production. Blood 106, 2543–2550 (2005). 7. Geijtenbeek, T.B.H. et al. Mycobacteria target DC-SIGN to suppress dendritic cell function. J. Exp. Med. 197, 7–17 (2003).
212
8. Gringhuis, S.I. et al. C-type lectin DC-SIGN modulates toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappa B. Immunity 26, 605–616 (2007). 9. Gross, O. et al. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature 442, 651–656 (2006). 10. Romani, L. Immunity to fungal infections. Nat. Rev. Immunol. 4, 11–23 (2004). 11. LeibundGut-Landmann, S. et al. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17. Nat. Immunol. 8, 630–638 (2007). 12. Hohl, T.M., Rivera, A. & Pamer, E.G. Immunity to fungi. Curr. Opin. Immunol. 18, 465–472 (2006). 13. Acosta-Rodriguez, E.V. et al. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat. Immunol. 8, 639–646 (2007). 14. Ouaaz, F., Arron, J., Zheng, Y., Choi, Y. & Beg, A.A. Dendritic cell development and survival require distinct NF-kB subunits. Immunity 16, 257–270 (2002). 15. Rogers, N.C. et al. Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity 22, 507–517 (2005). 16. Hayden, M.S. & Ghosh, S. Signaling to NF-kB. Genes Dev. 18, 2195–2224 (2004). 17. Wellbrock, C., Karasarides, M. & Marais, R. NF-kB and the immune response. Nat. Rev. Mol. Cell Biol. 5, 875–885 (2004). 18. Chen, L.-F. & Greene, W.C. Shaping the nuclear action of NF-kB. Nat. Rev. Mol. Cell Biol. 5, 392–401 (2004). 19. Jacque, E., Tchenio, T., Piton, G., Romeo, P.-H. & Baud, V. RelA repression of RelB activity induces selective gene activation downstream of TNF receptors. Proc. Natl. Acad. Sci. USA 102, 14635–14640 (2005). 20. Yang, F., Tang, E., Guan, K.L. & Wang, C.Y. IKK beta plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J. Immunol. 170, 5630–5635 (2003). 21. Tas, S.W. et al. Noncanonical NF-\{kappa\}B signaling in dendritic cells is required for indoleamine 2,3-dioxygenase (IDO) induction and immune regulation. Blood 110, 1540–1549 (2007). 22. Saccani, S., Pantano, S. & Natoli, G. Modulation of NF-kappaB activity by exchange of dimers. Mol. Cell 11, 1563–1574 (2003). 23. Neumann, M. & Naumann, M. Beyond I\{kappa\}Bs: alternative regulation of NF-{kappa}B activity. FASEB J. 21, 2642–2654 (2007). 24. Yoza, B.K., Hu, J.Y.Q., Cousart, S.L., Forrest, L.M. & McCall, C.E. Induction of RelB participates in endotoxin tolerance. J. Immunol. 177, 4080–4085 (2006). 25. Liang, M.D., Zhang, Y., McDevit, D., Marecki, S. & Nikolajczyk, B.S. The Interleukin1beta gene is transcribed from a poised promoter architecture in monocytes. J. Biol. Chem. 281, 9227–9237 (2006). 26. Zhong, H.H., May, M.J., Jimi, E. & Ghosh, S. The phosphorylation status of nuclear NFkappa B determines its association with CBP/p300 or HDAC-1. Mol. Cell 9, 625–636 (2002). 27. Chen, L.F. & Greene, W.C. Regulation of distinct biological activities of the NF-kappa B transcription factor complex by acetylation. J. Mol. Med. 81, 549–557 (2003). 28. Sun, Y., Jiang, X., Chen, S. & Price, B.D. Inhibition of histone acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation. FEBS Lett. 580, 4353–4356 (2006). 29. Saraiva, M. et al. Identification of a macrophage-specific chromatin signature in the IL-10 locus. J. Immunol. 175, 1041–1046 (2005). 30. Murphy, K.M. & Reiner, S.L. The lineage decisions of helper T cells. Nat. Rev. Immunol. 2, 933–944 (2002). 31. Manel, N., Unutmaz, D. & Littman, D.R. The differentiation of human TH-17 cells requires transforming growth factor-[beta] and induction of the nuclear receptor ROR[gamma]t. Nat. Immunol. 9, 641–649 (2008). 32. Volpe, E. et al. A critical function for transforming growth factor-[beta], interleukin 23 and proinflammatory cytokines in driving and modulating human TH-17 responses. Nat. Immunol. 9, 650–657 (2008). 33. Yang, L. et al. IL-21 and TGF-[bgr] are required for differentiation of human TH17 cells. Nature 454, 350–352 (2008). 34. Hunter, C.A. New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions. Nat. Rev. Immunol. 5, 521–531 (2005). 35. Dennehy, K.M. & Brown, G.D. The role of the \{beta\}-glucan receptor Dectin-1 in control of fungal infection. J. Leukoc. Biol. 82, 253–258 (2007). 36. Doyle, S.L. & O’Neill, L.A. Toll-like receptors: from the discovery of NFkappaB to new insights into transcriptional regulations in innate immunity. Biochem. Pharmacol. 72, 1102–1113 (2006). 37. Zarnegar, B.J. et al. Noncanonical NF-[kappa]B activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK. Nat. Immunol. 9, 1371–1378 (2008). 38. Marienfeld, R. et al. RelB forms transcriptionally inactive complexes with RelA/p65. J. Biol. Chem. 278, 19852–19860 (2003). 39. Carmody, R.J., Ruan, Q.G., Liou, H.C. & Chen, Y.H.H. Essential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells. J. Immunol. 178, 186–191 (2007). 40. Gantner, B.N., Simmons, R.M., Canavera, S.J., Akira, S. & Underhill, D.M. Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J. Exp. Med. 197, 1107–1117 (2003). 41. Huffnagle, G.B. & Deepe, G.S. Innate and adaptive determinants of host susceptibility to medically important fungi. Curr. Opin. Microbiol. 6, 344–350 (2003).
VOLUME 10
NUMBER 2
FEBRUARY 2009
NATURE IMMUNOLOGY
ARTICLES 47. Liu, J., Guan, X., Tamura, T., Ozato, K. & Ma, X. Synergistic activation of interleukin-12 p35 gene transcription by interferon regulatory factor-1 and interferon consensus sequence-binding protein. J. Biol. Chem. 279, 55609–55617 (2004). 48. Faggioli, L., Costanzo, C., Donadelli, M. & Palmieri, M. Activation of the Interleukin-6 promoter by a dominant negative mutant of c-Jun. Biochim. Biophys. Acta 1692, 17–24 (2004). 49. Chan, C., Li, L., McCall, C.E. & Yoza, B.K. Endotoxin tolerance disrupts chromatin remodeling and NF-\{kappa\}B transactivation at the IL-1b promoter. J. Immunol. 175, 461–468 (2005). 50. Nakayama, T. et al. Selective induction of Th2-attracting chemokines CCL17 and CCL22 in human B cells by Latent Membrane Protein 1 of Epstein-Barr Virus. J. Virol. 78, 1665–1674 (2004).
© 2009 Nature America, Inc. All rights reserved.
42. Geijtenbeek, T.B. et al. Identification of DC-SIGN, a novel dendritic cell-specific ICAM3 receptor that supports primary immune responses. Cell 100, 575–585 (2000). 43. Lackey, K. et al. The discovery of potent cRaf1 kinase inhibitors. Bioorg. Med. Chem. Lett. 10, 223–226 (2000). 44. Just, I. et al. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375, 500–503 (1995). 45. Geahlen, R.L. & McLaughlin, J.L. Piceatannol (3,4,3¢,5¢-tetrahydroxy-trans-stilbene) is a naturally occurring protein-tyrosine kinase inhibitor. Biochem. Biophys. Res. Commun. 165, 241–245 (1989). 46. Hanke, J.H. et al. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J. Biol. Chem. 271, 695–701 (1996).
NATURE IMMUNOLOGY
VOLUME 10
NUMBER 2
FEBRUARY 2009
213