Pathway but Not Toll/IL-1 Receptor (TRAF) 6

Cutting Edge: TNFR-Associated Factor (TRAF) 6 Is Essential for MyD88-Dependent Pathway but Not Toll/IL-1 Receptor Domain-Containing Adaptor-Inducing IFN- β This information is current as (TRIF)-Dependent Pathway in TLR Signaling of September 27, 2021. Jin Gohda, Takayuki Matsumura and Jun-ichiro Inoue J Immunol 2004; 173:2913-2917; ; doi: 10.4049/jimmunol.173.5.2913 http://www.jimmunol.org/content/173/5/2913 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE

JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: TNFR-Associated Factor (TRAF) 6 Is Essential for MyD88-Dependent Pathway but Not Toll/ IL-1 Receptor Domain-Containing Adaptor-Inducing IFN-␤ (TRIF)-Dependent Pathway in TLR Signaling1 Jin Gohda, Takayuki Matsumura, and Jun-ichiro Inoue2

Signaling pathways from TLRs are mediated by the Toll/ have been identified: MyD88, MyD88 adaptor like (Mal)/TIR IL-1R (TIR) domain-containing adaptor molecules. TNF domain-containing adaptor protein (TIRAP), TIR domain- containing adaptor-inducing IFN-␤ (TRIF)/TIR containing

receptor-associated factor (TRAF) 6 is thought to activate Downloaded from NF-␬B and MAPKs downstream of these TIR domain- adaptor molecule-1 (TICAM-1), TRIF-related adaptor mole- ␣ containing proteins to induce production of inflammatory cule (TRAM), and sterile and HEAT-Armadillo motifs (SARM) (2). MyD88 is essential for production of inflamma- cytokines. However, the precise role of TRAF6 in signaling ␣ from individual TLRs has not been appropriately ad- tory cytokines, including TNF- , IL-1, and IL-6, induced by dressed. We analyzed macrophages from TRAF6-deficient TLR2, TLR4, TLR5, TLR7, and TLR9, whereas Mal/TIRAP

is required for cytokine production induced by TLR2 and http://www.jimmunol.org/ mice and made the following observations. In the absence of TLR4 (1, 3, 4). In MyD88-deficient mice, stimulation of TRAF6, 1) ligands for TLR2, TLR5, TLR7, and TLR9 ␬ ␬ TLR3 or TLR4 can lead not only to activation of NF- B but failed to induce activation of NF- B and MAPKs or produc- also to expression of IFN-␤- and IFN-inducible genes, includ- tion of inflammatory cytokines; 2) TLR4 ligand-induced cy- ing IFN-␥-inducible protein 10 (IP10), MCP-1, and RANTES tokine production was remarkably reduced and activation of (1, 5). This MyD88-independent pathway is mediated by NF-␬B and MAPKs was observed, albeit with delayed kinet- TRIF, which is also essential for TLR3- and TLR4-induced in- ics; and 3) in contrast with previously reported findings, flammatory cytokine production (6–8). TRAM-deficient mice TLR3 signaling was not affected. These results indicate that showed defects in cytokine production and IFN-␤ induction TRAF6 is essential for MyD88-dependent signaling but is only in response to TLR4 (9). Although the physiological role by guest on September 27, 2021 not required for TIR domain-containing adaptor-inducing of SARM is not known, the genetic data enable us to classify the IFN-␤ (TRIF)-dependent signaling. The Journal of Im- TLR family into at least four groups on the basis of differential munology, 2004, 173: 2913–2917. usage of TIR-containing adaptors. The first group is composed of TLR5, TLR7, and TLR9, which have only MyD88 in their pathways, whereas the second group, comprising TLR2, utilizes oll-like receptors (TLRs) recognize specific structural both MyD88 and Mal/TIRAP. The third group, comprising motifs of various pathogens, known as pathogen-asso- TLR3, has a pathway mediated only by TRIF. Involvement of T ciated microbial patterns, and are critical in provoking MyD88 in TLR3 signaling is still controversial (7, 10). The innate immune responses (1). In addition to the specificity for fourth group, which contains TLR4, uses both MyD88-Mal/ pathogen-associated microbial pattern recognition, the intra- TIRAP-mediated and TRAM-TRIF-mediated pathways. cellular signaling pathways utilized by various TLRs differ and Although there is increasing information about TIR-contain- this may provide a molecular basis for differences in the expres- ing adaptors, the pathways downstream of these adaptors re- sion profile of immune genes induced by distinct TLRs. main to be elucidated. In the IL-1R signaling, which is MyD88 The cytoplasmic regions of TLRs share a protein motif dependent, members of the IL-1R-associated kinase (IRAK) known as the Toll/IL-1R (TIR)3 domain, which meditates family are recruited to the receptor complex along with MyD88 homo- and heteromeric associations between TLRs and TIR- upon stimulation and are then activated (11). Activated IRAK1 containing adaptors upon ligand binding. Five such adaptors and IRAK4 are then released from the complex and associate

Division of Cellular and Molecular Biology, Institute of Medical Science, University of 2 Address correspondence and reprint requests to Dr. Jun-ichiro Inoue, Division of Cel- Tokyo, Tokyo, Japan lular and Molecular Biology, Institute of Medical Science, University of Tokyo, Shirokane- dai, Minato-ku, Tokyo, 108-8639, Japan. E-mail address: [email protected] Received for publication May 12, 2004. Accepted for publication July 7, 2004. 3 Abbreviations used in this paper: TIR, Toll/IL-1R; TRIF, TIR domain-containing adap- The costs of publication of this article were defrayed in part by the payment of page charges. tor-inducing IFN-␤; TRAF, TNFR-associated factor; Mal, MyD88 adaptor-like; TIRAP, This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. TIR domain-containing adaptor protein; TRAM, TRIF-related adaptor molecule; IRAK, Section 1734 solely to indicate this fact. IL-1R-associated kinase; HPRT, hypoxanthine guanine phosphoribosyltransferase; IP10, 1 This work was supported by Grants-in-Aid for Special Coordination Funds for Promot- IFN-␥-inducible protein 10; DC, dendritic cell; RIP-1, receptor-interacting protein 1; ing Science and Technology and Scientific Research on Priority Areas from the Ministry of MALP-2, macrophage-activating lipopeptide 2. Education, Culture, Sports, Science and Technology of the Japanese government and by grants from the Mitsubishi Foundation.

ϩ Ϫ with TNFR-associated factor 6 (TRAF6), which in turn acts as TRAF6 / splenocytes. More than 95% of the adherent cells ϩ Ϫ Ϫ Ϫ an E3 enzyme to catalyze lysine-63-linked polyubiquitination from both TRAF6 / and TRAF6 / mice expressed CD11b of TRAF6 and I␬B kinase ␥, leading to activation of the I␬B and F4/80 (data not shown), indicating that TRAF6 deficiency kinase complex (11, 12). By analogy with IL-1 signaling, it has does not affect macrophage differentiation. been thought that all TLR pathways are mediated by TRAF6 To determine whether TRAF6 is involved in MyD88-medi- without genetic evidence. In addition, two groups reported that ated signaling pathways, we first examined the effects of TRAF6 TRAF6 binds to TRIF and is involved in TRIF-mediated acti- deficiency on activation of NF␬B and MAPKs by TLR2, vation of NF␬B (13, 14). These findings suggest that TRAF6 TLR5, TLR7, and TLR9, whose signals are dependent on may be involved in both the MyD88-dependent and -indepen- MyD88 (1). MALP-2, a ligand for the TLR2/TLR6 het- dent pathways. However, there are discrepancies between find- erodimer (17), induced degradation of I␬B␣ and phosphoryla- ϩ Ϫ ings in these studies, and the roles of TRAF6 in TLR signaling tion of JNK, p38, and ERK in TRAF6 / macrophages (Fig. Ϫ Ϫ under physiological conditions were not adequately addressed. 1A). However, TRAF6 / macrophages showed a severe defect In this study, we report differential roles of TRAF6 in TLR sig- in activation of NF␬B and MAPKs in response to MALP-2 naling determined with TRAF6-deficient macrophages. (Fig. 1A). Bacterial lipopeptide, a ligand of the TLR1/TLR2 heterodimer (18), also did not induce I␬B␣ degradation or Ϫ Ϫ Materials and Methods MAPK phosphorylation in TRAF6 / macrophages (data not Reagents and Abs shown). Flagellin, imidazoquinoline (R848), and CpG DNA,

Macrophage-activating lipopeptide 2 (MALP-2) was purchased from EMC which are ligands for TLR5, TLR7, and TLR9, respectively (1), Downloaded from Microcollections (Tuebingen, Germany). Flagellin was obtained from Calbio- did not induce I␬B␣ degradation or MAPK phosphorylation in Ϫ Ϫ chem (San Diego, CA). LPS and R848 were purchased from Sigma-Aldrich (St. TRAF6 / macrophages (Fig. 1A). We then analyzed pro- Louis, MO). Poly(I:C) was obtained from Amersham Pharmacia (Piscataway, duction of inflammatory cytokines in response to these TLR NJ). Phosphorothioate-stabilized CpG DNA (5Ј-TCCATGACGTTCCT GATGCT-3Ј) was purchased from Japan Bioservice (Saitama, Japan). Anti- phospho-JNK, anti-phospho-p38, and anti-phospho-ERK Abs were purchased ␬ ␣ from Cell Signaling Technology (Beverly, MA). Anti-I B and anti-p38 Abs http://www.jimmunol.org/ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Human M- CSF and mouse GM-CSF were purchased from PeproTech (London, U.K.). Anti-CD16/32 and anti-CD11b Abs and streptavidin-CyChrome were obtained from BD Biosciences (Mountain View, CA). PE-conjugated anti-F4/80 Ab was purchased from Serotec Ltd. (Oxford, U.K.). FITC-conjugated anti- CD86 Ab was obtained from Immunotech (Marseille, France). Analyses of NF-␬B and MAPK activation in macrophages from TRAF6- deficient mice

ϩ/Ϫ Ϫ/Ϫ Spleen cells from 2-wk-old TRAF6 and TRAF6 mice (15) were incu- by guest on September 27, 2021 bated in 10% FBS ␣-MEM for 8 h, and nonadherent cells were cultured with 10 ng/ml M-CSF. Adherent cells obtained after 6 days of culture were used as macrophages. Macrophages were unstimulated or stimulated with various TLR ligands and then lysed. The lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were incubated with various first Abs and then incubated with anti- rabbit IgG Ab linked to HRP. Immunoreactive proteins were visualized with an ECL detection system (Amersham Pharmacia). PCR and Northern blotting Total cDNA was synthesized from total RNA extracted from TRAF6Ϫ/Ϫ and TRAF6ϩ/Ϫ macrophages. Primer pairs for amplification of TLRs were de- scribed previously (16). For amplification of MyD88, TRAF6, and hypoxanthine guanine phosphoribosyl transferase (HPRT), the following sets of primers were used. MyD88, 5Ј-CCAGAGTGGAAAGCAGTGTC-3Ј and 5Ј-GTC CTTCTTCATCGCCTTGT-3Ј; TRAF6, 5Ј-CTGCAAAGCCTGCATCA TCA-3Ј and 5Ј-GAGGACAGCTTTGATCATGG-3Ј; and HPRT, 5Ј-GTT GGATACAGGCCAGACTTTGTTG-3Ј and 5Ј-GAAGGGTAGGCTGGC CTATAGGCT-3Ј. Semiquantitative PCR was conducted and the data shown for each primer pair correspond to the cycle number at which the amplicons could be detected but were not yet at saturation. For Northern blotting, 3 ␮gof total RNA was separated on 1% agarose gels containing formaldehyde and transferred to nylon membranes (New England Nuclear, Boston, MA). The 32 membranes were incubated with a P-labeled mouse IP10 probe (nt 101–719) FIGURE 1. TRAF6 is required for TLR2, TLR5, TLR7, and TLR9 signal- or a mouse GAPDH probe (nt 168–585). ings. A, TRAF6ϩ/Ϫ and TRAF6Ϫ/Ϫ macrophages were stimulated for the indicated periods with 50 ng/ml MALP-2, 1 ␮g/ml flagellin, 1 ␮g/ml R848, or 1 Results and Discussion ␮M CpG DNA. Total lysates were used for immunoblotting. Total p38 pro- To elucidate the physiological role of TRAF6 in TLR signaling, teins were detected as a loading control. B, TRAF6ϩ/Ϫ (Ⅺ) and TRAF6Ϫ/Ϫ (f) Ϫ Ϫ macrophages were generated from splenocytes of TRAF6 / macrophages were stimulated for the indicated periods with 50 ng/ml MALP-2, ϩ Ϫ ␮ ␮ ␣ and TRAF6 / mice, because the number of bone marrow cells 1 g/ml R848, or 10 M CpG DNA. TNF- and IL-6 in the supernatants Ϫ/Ϫ were quantified by ELISA. Values are TNF-␣ or IL-6 concentration (picograms was reduced significantly in TRAF6 mice due to severe os- per milliliter) per 5 ␮g of cellular protein. Error bars indicate SD of the mean of teopetrosis (15). After 6 days of culture with M-CSF, almost three independent experiments. ND, Not detectable. C, Analysis of TLR ex- equal numbers of adherent cells, which displayed typical mac- pression. cDNA was amplified by semiquantitative PCR using sets of primers Ϫ Ϫ rophage morphology, were obtained from both TRAF6 / and specific for the murine TLR1, 2, 5, 6, 7, and 9, MyD88, TRAF6, and HPRT. The Journal of Immunology 2915

Ϫ Ϫ ligands, which occurs in a MyD88-dependent manner. poly(I:C) in TRAF6 / macrophages, and there was no signif- ϩ Ϫ TRAF6 / macrophages produced TNF-␣ or IL-6 in response icant difference in extent or kinetics of the induction between Ϫ Ϫ ϩ Ϫ Ϫ Ϫ to MALP-2, R848, and CpG DNA, whereas TRAF6 / mac- TRAF6 / and TRAF6 / macrophages (Fig. 2C). Although rophages did not produce these cytokines in response to any of TLR3-dependent and -independent pathways leading to up- these ligands (Fig. 1B). Expression of TLR1, TLR2, TLR5, regulation of CD86 (B7.2) expression are present (19), up-reg- TLR6, TLR7, TLR9, and MyD88 was not significantly af- ulation of CD86 in response to poly(I:C) stimulation was not fected in the absence of TRAF6 (Fig. 1C). These results indicate affected by TRAF6 deficiency (Fig. 2D). These findings indi- that TRAF6 is essential for activation of NF␬B and MAPKs cate that TRAF6 is not necessary for TLR3 signaling in and cytokine production via TLR2, TLR5, TLR7, and TLR9. macrophages. We next examined whether TRAF6 is involved in TLR3 We next examined the effects of TRAF6 deficiency on TLR4 signaling, which has both MyD88-dependent and TRIF-de- signaling, which is dependent on TRIF instead of MyD88 or Ϫ Ϫ pendent pathways (1, 6). When TRAF6 / macrophages were TIRAP/Mal. Poly(I:C), a ligand of TLR3 (10), induced degra- stimulated with LPS, a ligand of TLR4, degradation of I␬B␣ dation of I␬B␣ and phosphorylation of MAPKs to a similar ϩ/Ϫ and phosphorylation of MAPKs were observed, but the kinetics extent and with similar kinetics in both TRAF6 and ϩ/Ϫ Ϫ/Ϫ were somewhat slower than those in TRAF6 macrophages TRAF6 macrophages (Fig. 2A). In addition, there was no (Fig. 3A). It has been shown that early-phase NF␬B activation significant difference in the production of TNF-␣ and IL-6 in ␬ ϩ/Ϫ Ϫ/Ϫ is dependent on MyD88, whereas late-phase NF B activation response to poly(I:C) between TRAF6 and TRAF6 is dependent on TRIF and TRAM and that both phases of Downloaded from ␤ macrophages (Fig. 2B). TLR3 induces expression of IFN- and NF␬B activation are required for cytokine production (6, 9). several IFN-inducible genes, including IP10, MCP-1, and LPS did not induce production of TNF-␣ or IL-6 by RANTES, via the TRIF-TANK-binding kinase 1-IFN regula- Ϫ/Ϫ ϩ Ϫ TRAF6 macrophages (Fig. 3B). These results indicate that tory factor 3 pathway (6, 7). In TRAF6 / macrophages, ex- TRAF6 is involved in MyD88-mediated NF␬B activation but pression of IP10 mRNA was detected at 1 h after poly(I:C) not TRIF-mediated NF␬B activation. In addition, LPS in- stimulation and was significantly increased at 4 h (Fig. 2C). In- duced expression of IP10 and enhanced expression of CD86, http://www.jimmunol.org/ ϩ Ϫ duction of IP10 expression was also observed in response to which is TRIF-dependent (19), in both TRAF6 / and Ϫ Ϫ TRAF6 / macrophages (Fig. 3, C and D), indicating that the by guest on September 27, 2021

FIGURE 2. TRAF6 is not necessary for TLR3 signaling. A, TRAF6ϩ/Ϫ and FIGURE 3. TRAF6 is required for TLR4-induced cytokine production but TRAF6Ϫ/Ϫ macrophages were stimulated for the indicated periods with 10 not IFN-␤ induction. A, TRAF6ϩ/Ϫ and TRAF6Ϫ/Ϫ macrophages were stim- ␮g/ml poly(I:C). Total lysates were used for immunoblotting. Total p38 pro- ulated for the indicated periods with 1 ␮g/ml LPS. Total lysates were used for teins were detected as a loading control. B, TRAF6ϩ/Ϫ (Ⅺ) and TRAF6Ϫ/Ϫ (f) immunoblotting. Total p38 proteins were detected as a loading control. B, macrophages were stimulated for the indicated periods with 10 ␮g/ml poly(I: TRAF6ϩ/Ϫ (Ⅺ) and TRAF6Ϫ/Ϫ (f) macrophages were stimulated for the in- C). TNF-␣ and IL-6 in the supernatants were quantified by ELISA. Values are dicated periods with 1 ␮g/ml LPS. TNF-␣ and IL-6 in the supernatants were TNF-␣ or IL-6 concentration (picograms per milliliter) per 5 ␮g of cellular quantified by ELISA. Values are TNF-␣ or IL-6 concentration (picograms per protein. Error bars indicate SD of the mean of three independent experiments. milliliter) per 5 ␮g of cellular protein. Error bars indicate SD of the mean of ND, Not detectable. C, TRAF6ϩ/Ϫ and TRAF6Ϫ/Ϫ macrophages were stim- three independent experiments. ND, Not detectable. C, TRAF6ϩ/Ϫ and ulated with 10 ␮g/ml poly(I:C). Total RNA was prepared and analyzed by TRAF6Ϫ/Ϫ macrophages were stimulated with 1 ␮g/ml LPS. Total RNA was Northern blotting with an IP10-specific probe. GAPDH mRNA was detected prepared and analyzed by Northern blotting with an IP10-specific probe. as a loading control. Numbers below columns represent IP10 levels normalized GAPDH mRNA was detected as a loading control. Numbers below columns to those of GAPDH. D, Macrophages were cultured for 24 h with 10 ␮g/ml represent IP10 levels normalized to those of GAPDH. D, Macrophages were poly(I:C). Cells were then harvested and stained with FITC-conjugated CD86 cultured for 24 h with 1 ␮g/ml LPS. Cells were then harvested and stained with Ab. Expression of CD86 on the cell surface was analyzed by flow cytometry. FITC-conjugated CD86 Ab. Expression of CD86 on the cell surface was ana- Shaded and open histograms represent cells cultured in the absence and pres- lyzed by flow cytometry. Shaded and open histograms represent cells cultured ence of poly(I:C), respectively. in the absence and presence of LPS, respectively. 2916 CUTTING EDGE: DIFFERENTIAL ROLE OF TRAF6 IN TLR SIGNALING

TLR4-TRIF-IRF3 pathway is independent of TRAF6. Thus, lar signal transduction pathways for TLR3 differ between DCs TRAF6 is required for the MyD88-dependent pathway but not and macrophages in terms of TRAF6 dependency. the TRIF-dependent pathway of TLR4. Our experiments showed that signaling pathways that lead to In the present study, we examined the physiological role of activation of NF␬B and MAPKs, cytokine production, induc- TRAF6 in the signal transduction pathways used by members tion of IFN-␤-inducible genes, and up-regulation of surface of the TLR family. All MyD88-dependent pathways of the CD86 (B7.2) in response to poly(I:C) are normal in macro- TLR family, which lead to activation of NF␬B and MAPKs and phages in the absence of TRAF6. However, we cannot rule out production of cytokines, require TRAF6, irrespective of in- the possibility that under physiological conditions TRIF may interact with and signal through TRAF6, but that in volvement of Mal/TIRAP. In contrast, TRAF6 is not necessary Ϫ/Ϫ for signaling via the TRIF-dependent pathways of TLR3 and TRAF6 macrophages another factor(s) may substitute. Mu- tational analysis of TRIF revealed that the N-terminal portion TLR4, which induce production of cytokines and secretion of ␬ ␤ IFN-␤. of TRIF bears a dual role to activate NF B and the IFN- promoter while the C-terminal portion activates NF␬B but failed Several recent reports have suggested that TRAF6 is involved to induce the IFN-␤ promoter (13). It has recently been re- in the TRIF-dependent pathways for TLR3 and TLR4 (13, 14, ported that receptor-interacting protein 1 (RIP-1), which binds 20). Both human and mouse TRIF contain three putative the C-terminal tail of TRIF, plays a crucial role in TLR3-in- TRAF6 binding sites that match the consensus TRAF6-bind- duced NF␬B activation (23), suggesting that RIP-1 may be one ing motif (X-X-P-X-E-X-X-acidic or aromatic) deduced from such factor. However, RIP-1 deficiency does not affect activa- Downloaded from other known TRAF6-binding proteins (21). Transient trans- tion of JNK and induction of IFN-␤, suggesting that another fection experiments have revealed that TRIF associates with factor(s) or TRAF6 may compensate. Precise studies of the TRAF6 (13, 14). It was theorized that substitution of Ala for TRIF-dependent pathways will greatly improve our under- Glu at the fifth position of the TRAF6 binding site would abol- standing of the molecular mechanisms underlying TLR-medi- ish binding to TRAF6 (21, 22); various TRIF mutants in which ated transduction of specific signals. one or all of the TRAF6 binding sites were mutated were gen- http://www.jimmunol.org/ erated and the abilities of these mutants to activate NF␬B were analyzed. Sato et al. (13) demonstrated that mutation of all Acknowledgments three TRAF6 binding sites in TRIF abolished binding to We thank Drs. H. Hayashi and K. Onozaki for providing bacterial lipopeptide. TRAF6, although activation of NF␬B by this mutant was only We also thank Drs. S. Akira, S. Sato, and M. Yamamoto for helpful discussions. partially reduced in comparison to that by wild-type TRIF. In contrast, Jiang et al. (14) reported that a single mutation, substitution of Ala for Glu252 in the middle TRAF6 binding site of References

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