Abstract
Signaling pathways from TLRs are mediated by the Toll/IL-1R (TIR) domain-containing adaptor molecules. TNF receptor-associated factor (TRAF) 6 is thought to activate NF-κB and MAPKs downstream of these TIR domain-containing proteins to induce production of inflammatory cytokines. However, the precise role of TRAF6 in signaling from individual TLRs has not been appropriately addressed. We analyzed macrophages from TRAF6-deficient mice and made the following observations. In the absence of TRAF6, 1) ligands for TLR2, TLR5, TLR7, and TLR9 failed to induce activation of NF-κB and MAPKs or production of inflammatory cytokines; 2) TLR4 ligand-induced cytokine production was remarkably reduced and activation of NF-κB and MAPKs was observed, albeit with delayed kinetics; and 3) in contrast with previously reported findings, TLR3 signaling was not affected. These results indicate that TRAF6 is essential for MyD88-dependent signaling but is not required for TIR domain-containing adaptor-inducing IFN-β (TRIF)-dependent signaling.
Toll-like receptors (TLRs) recognize specific structural motifs of various pathogens, known as pathogen-associated microbial patterns, and are critical in provoking innate immune responses (1). In addition to the specificity for pathogen-associated microbial pattern recognition, the intracellular signaling pathways utilized by various TLRs differ and this may provide a molecular basis for differences in the expression profile of immune genes induced by distinct TLRs.
The cytoplasmic regions of TLRs share a protein motif known as the Toll/IL-1R (TIR)3 domain, which meditates homo- and heteromeric associations between TLRs and TIR-containing adaptors upon ligand binding. Five such adaptors have been identified: MyD88, MyD88 adaptor like (Mal)/TIR domain-containing adaptor protein (TIRAP), TIR domain-containing adaptor-inducing IFN-β (TRIF)/TIR containing adaptor molecule-1 (TICAM-1), TRIF-related adaptor molecule (TRAM), and sterile α and HEAT-Armadillo motifs (SARM) (2). MyD88 is essential for production of inflammatory cytokines, including TNF-α, IL-1, and IL-6, induced by TLR2, TLR4, TLR5, TLR7, and TLR9, whereas Mal/TIRAP is required for cytokine production induced by TLR2 and TLR4 (1, 3, 4). In MyD88-deficient mice, stimulation of TLR3 or TLR4 can lead not only to activation of NF-κB but also to expression of IFN-β- and IFN-inducible genes, including IFN-γ-inducible protein 10 (IP10), MCP-1, and RANTES (1, 5). This MyD88-independent pathway is mediated by TRIF, which is also essential for TLR3- and TLR4-induced inflammatory cytokine production (6, 7, 8). TRAM-deficient mice showed defects in cytokine production and IFN-β induction only in response to TLR4 (9). Although the physiological role of SARM is not known, the genetic data enable us to classify the TLR family into at least four groups on the basis of differential 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 both MyD88 and Mal/TIRAP. The third group, comprising TLR3, has a pathway mediated only by TRIF. Involvement of MyD88 in TLR3 signaling is still controversial (7, 10). The fourth group, which contains TLR4, uses both MyD88-Mal/TIRAP-mediated and TRAM-TRIF-mediated pathways.
Although there is increasing information about TIR-containing adaptors, the pathways downstream of these adaptors remain to be elucidated. In the IL-1R signaling, which is MyD88 dependent, members of the IL-1R-associated kinase (IRAK) family are recruited to the receptor complex along with MyD88 upon stimulation and are then activated (11). Activated IRAK1 and IRAK4 are then released from the complex and associate with TNFR-associated factor 6 (TRAF6), which in turn acts as an E3 enzyme to catalyze lysine-63-linked polyubiquitination of TRAF6 and IκB kinase γ, leading to activation of the IκB kinase complex (11, 12). By analogy with IL-1 signaling, it has been thought that all TLR pathways are mediated by TRAF6 without genetic evidence. In addition, two groups reported that TRAF6 binds to TRIF and is involved in TRIF-mediated activation of NFκB (13, 14). These findings suggest that TRAF6 may be involved in both the MyD88-dependent and -independent pathways. However, there are discrepancies between findings in these studies, and the roles of TRAF6 in TLR signaling under physiological conditions were not adequately addressed. In this study, we report differential roles of TRAF6 in TLR signaling determined with TRAF6-deficient macrophages.
Materials and Methods
Reagents and Abs
Macrophage-activating lipopeptide 2 (MALP-2) was purchased from EMC Microcollections (Tuebingen, Germany). Flagellin was obtained from Calbiochem (San Diego, CA). LPS and R848 were purchased from Sigma-Aldrich (St. Louis, MO). Poly(I:C) was obtained from Amersham Pharmacia (Piscataway, NJ). Phosphorothioate-stabilized CpG DNA (5′-TCCATGACGTTCCTGATGCT-3′) 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-IκBα and anti-p38 Abs 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-κB and MAPK activation in macrophages from TRAF6-deficient mice
Spleen cells from 2-wk-old TRAF6+/− and TRAF6−/− mice (15) were incubated in 10% FBS α-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 TRAF6−/− and TRAF6+/− macrophages. Primer pairs for amplification of TLRs were described previously (16). For amplification of MyD88, TRAF6, and hypoxanthine guanine phosphoribosyl transferase (HPRT), the following sets of primers were used. MyD88, 5′-CCAGAGTGGAAAGCAGTGTC-3′ and 5′-GTCCTTCTTCATCGCCTTGT-3′; TRAF6, 5′-CTGCAAAGCCTGCATCATCA-3′ and 5′-GAGGACAGCTTTGATCATGG-3′; and HPRT, 5′-GTTGGATACAGGCCAGACTTTGTTG-3′ and 5′-GAAGGGTAGGCTGGCCTATAGGCT-3′. 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 μg of total RNA was separated on 1% agarose gels containing formaldehyde and transferred to nylon membranes (New England Nuclear, Boston, MA). The membranes were incubated with a 32P-labeled mouse IP10 probe (nt 101–719) or a mouse GAPDH probe (nt 168–585).
Results and Discussion
To elucidate the physiological role of TRAF6 in TLR signaling, macrophages were generated from splenocytes of TRAF6−/− and TRAF6+/− mice, because the number of bone marrow cells was reduced significantly in TRAF6−/− mice due to severe osteopetrosis (15). After 6 days of culture with M-CSF, almost equal numbers of adherent cells, which displayed typical macrophage morphology, were obtained from both TRAF6−/− and TRAF6+/− splenocytes. More than 95% of the adherent cells from both TRAF6+/− and TRAF6−/− mice expressed CD11b and F4/80 (data not shown), indicating that TRAF6 deficiency does not affect macrophage differentiation.
To determine whether TRAF6 is involved in MyD88-mediated signaling pathways, we first examined the effects of TRAF6 deficiency on activation of NFκB and MAPKs by TLR2, TLR5, TLR7, and TLR9, whose signals are dependent on MyD88 (1). MALP-2, a ligand for the TLR2/TLR6 heterodimer (17), induced degradation of IκBα and phosphorylation of JNK, p38, and ERK in TRAF6+/− macrophages (Fig. 1⇓A). However, TRAF6−/− macrophages showed a severe defect in activation of NFκB and MAPKs in response to MALP-2 (Fig. 1⇓A). Bacterial lipopeptide, a ligand of the TLR1/TLR2 heterodimer (18), also did not induce IκBα degradation or MAPK phosphorylation in TRAF6−/− macrophages (data not shown). Flagellin, imidazoquinoline (R848), and CpG DNA, which are ligands for TLR5, TLR7, and TLR9, respectively (1), did not induce IκBα degradation or MAPK phosphorylation in TRAF6−/− macrophages (Fig. 1⇓A). We then analyzed production of inflammatory cytokines in response to these TLR ligands, which occurs in a MyD88-dependent manner. TRAF6+/− macrophages produced TNF-α or IL-6 in response to MALP-2, R848, and CpG DNA, whereas TRAF6−/− macrophages did not produce these cytokines in response to any of these ligands (Fig. 1⇓B). Expression of TLR1, TLR2, TLR5, TLR6, TLR7, TLR9, and MyD88 was not significantly affected in the absence of TRAF6 (Fig. 1⇓C). These results indicate that TRAF6 is essential for activation of NFκB and MAPKs and cytokine production via TLR2, TLR5, TLR7, and TLR9.
TRAF6 is required for TLR2, TLR5, TLR7, and TLR9 signalings. A, TRAF6+/− and TRAF6−/− macrophages were stimulated for the indicated periods with 50 ng/ml MALP-2, 1 μg/ml flagellin, 1 μg/ml R848, or 1 μM CpG DNA. Total lysates were used for immunoblotting. Total p38 proteins were detected as a loading control. B, TRAF6+/− (□) and TRAF6−/− (▪) macrophages were stimulated for the indicated periods with 50 ng/ml MALP-2, 1 μg/ml R848, or 10 μM CpG DNA. TNF-α and IL-6 in the supernatants were quantified by ELISA. Values are TNF-α or IL-6 concentration (picograms per milliliter) per 5 μg of cellular protein. Error bars indicate SD of the mean of three independent experiments. ND, Not detectable. C, Analysis of TLR expression. cDNA was amplified by semiquantitative PCR using sets of primers specific for the murine TLR1, 2, 5, 6, 7, and 9, MyD88, TRAF6, and HPRT.
We next examined whether TRAF6 is involved in TLR3 signaling, which is dependent on TRIF instead of MyD88 or TIRAP/Mal. Poly(I:C), a ligand of TLR3 (10), induced degradation of IκBα and phosphorylation of MAPKs to a similar extent and with similar kinetics in both TRAF6+/− and TRAF6−/− macrophages (Fig. 2⇓A). In addition, there was no significant difference in the production of TNF-α and IL-6 in response to poly(I:C) between TRAF6+/− and TRAF6−/− macrophages (Fig. 2⇓B). TLR3 induces expression of IFN-β and several IFN-inducible genes, including IP10, MCP-1, and RANTES, via the TRIF-TANK-binding kinase 1-IFN regulatory factor 3 pathway (6, 7). In TRAF6+/− macrophages, expression of IP10 mRNA was detected at 1 h after poly(I:C) stimulation and was significantly increased at 4 h (Fig. 2⇓C). Induction of IP10 expression was also observed in response to poly(I:C) in TRAF6−/− macrophages, and there was no significant difference in extent or kinetics of the induction between TRAF6+/− and TRAF6−/− macrophages (Fig. 2⇓C). Although TLR3-dependent and -independent pathways leading to up-regulation of CD86 (B7.2) expression are present (19), up-regulation of CD86 in response to poly(I:C) stimulation was not affected by TRAF6 deficiency (Fig. 2⇓D). These findings indicate that TRAF6 is not necessary for TLR3 signaling in macrophages.
TRAF6 is not necessary for TLR3 signaling. A, TRAF6+/− and TRAF6−/− macrophages were stimulated for the indicated periods with 10 μg/ml poly(I:C). Total lysates were used for immunoblotting. Total p38 proteins were detected as a loading control. B, TRAF6+/− (□) and TRAF6−/− (▪) macrophages were stimulated for the indicated periods with 10 μg/ml poly(I:C). TNF-α and IL-6 in the supernatants were quantified by ELISA. Values are TNF-α or IL-6 concentration (picograms per milliliter) per 5 μg of cellular protein. Error bars indicate SD of the mean of three independent experiments. ND, Not detectable. C, TRAF6+/− and TRAF6−/− macrophages were stimulated with 10 μg/ml poly(I:C). Total RNA was prepared and analyzed by Northern blotting with an IP10-specific probe. GAPDH mRNA was detected as a loading control. Numbers below columns represent IP10 levels normalized to those of GAPDH. D, Macrophages were cultured for 24 h with 10 μg/ml poly(I:C). Cells were then harvested and stained with FITC-conjugated CD86 Ab. Expression of CD86 on the cell surface was analyzed by flow cytometry. Shaded and open histograms represent cells cultured in the absence and presence of poly(I:C), respectively.
We next examined the effects of TRAF6 deficiency on TLR4 signaling, which has both MyD88-dependent and TRIF-dependent pathways (1, 6). When TRAF6−/− macrophages were stimulated with LPS, a ligand of TLR4, degradation of IκBα and phosphorylation of MAPKs were observed, but the kinetics were somewhat slower than those in TRAF6+/− macrophages (Fig. 3⇓A). It has been shown that early-phase NFκB activation is dependent on MyD88, whereas late-phase NFκB activation is dependent on TRIF and TRAM and that both phases of NFκB activation are required for cytokine production (6, 9). LPS did not induce production of TNF-α or IL-6 by TRAF6−/− macrophages (Fig. 3⇓B). These results indicate that TRAF6 is involved in MyD88-mediated NFκB activation but not TRIF-mediated NFκB activation. In addition, LPS induced expression of IP10 and enhanced expression of CD86, which is TRIF-dependent (19), in both TRAF6+/− and TRAF6−/− macrophages (Fig. 3⇓, C and D), indicating that theTLR4-TRIF-IRF3 pathway is independent of TRAF6. Thus, TRAF6 is required for the MyD88-dependent pathway but not the TRIF-dependent pathway of TLR4.
TRAF6 is required for TLR4-induced cytokine production but not IFN-β induction. A, TRAF6+/− and TRAF6−/− macrophages were stimulated for the indicated periods with 1 μg/ml LPS. Total lysates were used for immunoblotting. Total p38 proteins were detected as a loading control. B, TRAF6+/− (□) and TRAF6−/− (▪) macrophages were stimulated for the indicated periods with 1 μg/ml LPS. TNF-α and IL-6 in the supernatants were quantified by ELISA. Values are TNF-α or IL-6 concentration (picograms per milliliter) per 5 μg of cellular protein. Error bars indicate SD of the mean of three independent experiments. ND, Not detectable. C, TRAF6+/− and TRAF6−/− macrophages were stimulated with 1 μg/ml LPS. Total RNA was prepared and analyzed by Northern blotting with an IP10-specific probe. GAPDH mRNA was detected as a loading control. Numbers below columns represent IP10 levels normalized to those of GAPDH. D, Macrophages were cultured for 24 h with 1 μg/ml LPS. Cells were then harvested and stained with FITC-conjugated CD86 Ab. Expression of CD86 on the cell surface was analyzed by flow cytometry. Shaded and open histograms represent cells cultured in the absence and presence of LPS, respectively.
In the present study, we examined the physiological role of TRAF6 in the signal transduction pathways used by members of the TLR family. All MyD88-dependent pathways of the TLR family, which lead to activation of NFκB and MAPKs and production of cytokines, require TRAF6, irrespective of involvement of Mal/TIRAP. In contrast, TRAF6 is not necessary for signaling via the TRIF-dependent pathways of TLR3 and TLR4, which induce production of cytokines and secretion of IFN-β.
Several recent reports have suggested that TRAF6 is involved in the TRIF-dependent pathways for TLR3 and TLR4 (13, 14, 20). Both human and mouse TRIF contain three putative TRAF6 binding sites that match the consensus TRAF6-binding motif (X-X-P-X-E-X-X-acidic or aromatic) deduced from other known TRAF6-binding proteins (21). Transient transfection experiments have revealed that TRIF associates with TRAF6 (13, 14). It was theorized that substitution of Ala for Glu at the fifth position of the TRAF6 binding site would abolish binding to TRAF6 (21, 22); various TRIF mutants in which one or all of the TRAF6 binding sites were mutated were generated and the abilities of these mutants to activate NFκB were analyzed. Sato et al. (13) demonstrated that mutation of all three TRAF6 binding sites in TRIF abolished binding to TRAF6, although activation of NFκB by this mutant was only 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 TRIF, resulted in complete abrogation of binding to TRAF6 and activation of NFκB. The inconsistencies in the findings of the two groups may be attributable to differences in experimental conditions and strongly suggest that the function of TRAF6 in TLR3 signaling should be examined under physiological conditions. In this context, the association between endogenous TRIF and TRAF6 and enhancement of this association after poly(I:C) stimulation as reported by Sato et al. (13) is intriguing. However, it is possible that the interaction between TRIF and TRAF6 is indirect, although they could be in the same complex, such as signalosome. Jiang et al. (14) transiently transfected a TLR3 expression plasmid and an NFκB-driven luciferase reporter construct into embryonic fibroblasts from wild-type or TRAF6−/− mice and found that poly(I:C) stimulation-dependent activation of NFκB requires TRAF6. The reason for the discrepancy between their result and our present results is unclear; however, it may be due to differences in experimental systems or cell type-specific roles of TRAF6 in TLR3 signaling. It has been reported that TRAF6-deficient dendritic cells (DCs) do not mature after stimulation with poly(I:C) or other TLR ligands, including peptidoglycan, LPS, and CpG DNA (20). However, in our hands, few CD11c+ DCs were generated from TRAF6−/− splenocytes in the presence of GM-CSF, whereas with identical culture conditions, a large number of functional CD11c+ DCs were differentiated from splenocytes of control littermates (data not shown). Therefore, we could not address whether TRAF6 is required for TLR3 signaling in DCs. However, it is unlikely that intracellular signal transduction pathways for TLR3 differ between DCs and macrophages in terms of TRAF6 dependency.
Our experiments showed that signaling pathways that lead to activation of NFκB and MAPKs, cytokine production, induction of IFN-β-inducible genes, and up-regulation of surface CD86 (B7.2) in response to poly(I:C) are normal in macrophages in the absence of TRAF6. However, we cannot rule out the possibility that under physiological conditions TRIF may interact with and signal through TRAF6, but that in TRAF6−/− macrophages another factor(s) may substitute. Mutational analysis of TRIF revealed that the N-terminal portion of TRIF bears a dual role to activate NFκB and the IFN-β promoter while the C-terminal portion activates NFκB but failed to induce the IFN-β promoter (13). It has recently been reported that receptor-interacting protein 1 (RIP-1), which binds the C-terminal tail of TRIF, plays a crucial role in TLR3-induced NFκB activation (23), suggesting that RIP-1 may be one such factor. However, RIP-1 deficiency does not affect activation of JNK and induction of IFN-β, suggesting that another factor(s) or TRAF6 may compensate. Precise studies of the TRIF-dependent pathways will greatly improve our understanding of the molecular mechanisms underlying TLR-mediated transduction of specific signals.
Acknowledgments
We thank Drs. H. Hayashi and K. Onozaki for providing bacterial lipopeptide. We also thank Drs. S. Akira, S. Sato, and M. Yamamoto for helpful discussions.
Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 This work was supported by Grants-in-Aid for Special Coordination Funds for Promoting Science and Technology and Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government and by grants from the Mitsubishi Foundation.
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↵2 Address correspondence and reprint requests to Dr. Jun-ichiro Inoue, Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Shirokane-dai, Minato-ku, Tokyo, 108-8639, Japan. E-mail address: jun-i{at}ims.u-tokyo.ac.jp
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↵3 Abbreviations used in this paper: TIR, Toll/IL-1R; TRIF, TIR domain-containing adaptor-inducing IFN-β; TRAF, TNFR-associated factor; Mal, MyD88 adaptor-like; TIRAP, TIR domain-containing adaptor protein; TRAM, TRIF-related adaptor molecule; IRAK, IL-1R-associated kinase; HPRT, hypoxanthine guanine phosphoribosyltransferase; IP10, IFN-γ-inducible protein 10; DC, dendritic cell; RIP-1, receptor-interacting protein 1; MALP-2, macrophage-activating lipopeptide 2.
- Received May 12, 2004.
- Accepted July 7, 2004.
- Copyright © 2004 by The American Association of Immunologists
References
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