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The role of TRAF6 in signal transduction and the immune response
Microbes and Infection 6 (2004) 1333–1338 www.elsevier.com/locate/micinf
Review
The role of TRAF6 in signal transduction and the immune response Takashi Kobayashi, Matthew C. Walsh, Yongwon Choi * Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 421 Curie Blvd., Room 308, Philadelphia, PA 19104, USA Available online 28 September 2004
Abstract Signals from the IL-1 receptor (IL-1R)/Toll-like receptor (TLR) family and TNF receptor (TNFR) superfamily are critical for regulating the function of antigen-presenting cells such as dendritic cells (DCs). It has been revealed that TNF receptor-associated factor 6 (TRAF6), a signaling adapter molecule common to the IL-1R/TLR family and TNFR superfamily, is important not only for DC maturation, cytokine production, and T cell stimulatory capacity of DCs in response to TLR ligands (e.g. lipopolysaccharide) or CD40 ligand, but also for the homeostasis of splenic DC subsets. © 2004 Elsevier SAS. All rights reserved. Keywords: TRAF6; Toll-like receptor; CD40; Dendritic cells; Innate immune response
1. Introduction Upon activation of the innate immune response during infection, antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), immediately recognize the invasion of microbes, such as bacteria or viruses, and then transmit the information to T cells to activate the adaptive immune response (Fig. 1) [1]. Immature DCs in the periphery uptake antigens efficiently but express low levels of MHC and costimulatory molecules, and thus possess a low level of T cell stimulatory capacity [1]. Upon maturation, DCs greatly augment their ability to stimulate naïve T cells by dramatically upregulating surface expression of antigen/ MHC complexes and various costimulatory molecules [1]. Activated DCs also secrete cytokines, including IL-12, which are critical for the differentiation of T cells into Th1 type effector cells [1]. DC maturation is not triggered by specific protein antigens, but rather by inflammatory cytokines like CD40L on activated T cells, or microbial components (e.g. lipopolysaccharide (LPS), double-stranded RNA (dsRNA), or CpG-DNA), which signal through CD40 and Toll-like receptors (TLRs), respectively, on the surface of the APC [2–4].
* Corresponding author. Tel.: +1-215-746-6404; fax: +1-215-573-0889. E-mail address: [email protected] (Y. Choi). 1286-4579/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.09.001
Fig. 1. TLRs function as sensors of infection in the innate immune system. During infection, immature DCs take up and digest the peptide antigens of pathogenic microbes, and simultaneously respond to specific molecular features of microbes via pattern-recognition receptors, so-called TLRs, by initiating an activation program. Upon activation, mature DCs dramatically upregulate surface expression of antigen/MHC complexes and various costimulatory molecules which potentiate stimulation of naïve T cells. Moreover, activated DCs secrete cytokines, such as IL-12, that drive T cell differentiation toward the Th1 effector phenotype. TRAF6 serves as a critical adapter molecule in transducing signals emanating from TLRs.
2. TLRs recognize pathogen-associated molecular patterns (PAMPs) Drosophila Toll was originally characterized as a crucial factor in regulating dorso-ventral axis formation during fly
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embryo development, but was later recognized as serving a second function as a regulator of antimicrobial (antifungal) peptides, such as drosomycin, in response to infection. In addition, another Drosophila Toll family member, 18wheeler, is known to induce the antibacterial peptide, attacin [2,5]. Soon after the Drosophila Toll family was identified, mammalian TLR family members and their cognate ligands were identified. To date, 11 mammalian TLRs (TLR1– TLR11), comprising a family of receptors that share homology to Drosophila Toll, have been cloned [5,6]. Interestingly, it has been demonstrated that the ligands of mammalian TLRs are components of pathogenic microbes called PAMPs. For example, TLR2 recognizes peptidoglycan (PGN; a component of Gram-positive bacteria), TLR3 recognizes dsRNA (a viral product), TLR4 recognizes LPS (a component of Gram-negative bacteria), TLR5 recognizes flagellin (protein subunits that make up bacterial flagella), and TLR9 recognizes unmethylated CpG-DNA motifs in bacterial DNA (Fig. 2) [5]. TLR2 functions by forming heterodimers with either TLR1 or TLR6, which are thought to generate distinct ligand–receptor specificities. It would appear that these various “first-alert” receptor sets have evolved to immediately signal the immune system at the first sign of a foreign invader capable of causing pathological damage [2]. While the extracellular domains of TLRs, which contain leucine-rich repeats (LRRs), and IL-1R, which bears an Ig-
like domain, are dissimilar, the cytoplasmic regions of each exhibit functional similarity, sharing a common motif, termed the TIR (TLR/IL-1R) domain [5]. Furthermore, various adapter molecules, including MyD88, IRAK and TRAF6 are associated with both signaling cascades [7,8]. 3. Signal transduction of IL-1R/TLRs and cytokine production The signal transduction pathway common to both IL-1R and TLRs was first defined by studying IL-1R signaling. IL-1 binds to IL-1R, which is associated with an accessory protein (IL-1RAcp), inducing the formation of a receptor complex that recruits a cytoplasmic adapter protein, MyD88 [2,5]. IL-1R and MyD88 interact through their TIR domains. This is followed by the recruitment of Ser/Thr kinases called IRAKs (IL-1 receptor-associated kinase; IRAK, IRAK-2, IRAK-M, and IRAK-4), and then by association with TRAF6, which mediates activation of downstream signaling via NF-jB transcription factors and MAP kinase (JNK, p38 and Erk) cascade-activated AP-1 transcription factors. The fundamental TLR signaling mechanisms are similar to those of IL-1R, specifically with respect to adapter molecules utilized in the proximal complex (MyD88, IRAKs and TRAF6), and downstream activation of both NF-jB and AP-1 (Fig. 2) [5]. DCs from MyD88-deficient mice fail to produce inflammatory cytokines (TNF-a, IL-6, IL-12) in response to IL-1 or
Fig. 2. TRAF6 is a common signaling adapter protein for the IL-1R/TLR family and TNFR superfamily. Signals from the IL-1R/TLR family are mediated by adapter molecules MyD88, IRAK and TRAF6, and lead to activation of transcription factors NF-jB and AP-1. TNFR superfamily members such as CD40 and RANK directly associate with TRAF6, which in turn activates the same transcription factors. TRAF6, a common signaling adapter molecule for both receptor families, is critical for DC maturation and inflammatory cytokine production.
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other TLR ligands, such as LPS or CpG-DNA, suggesting that MyD88 is indispensable for inflammatory cytokine production in IL-1R/TLRs signaling [3]. On the other hand, LPS (TLR4 ligand) or dsRNA (TLR3 ligand) do induce DC maturation (upregulation of MHC and costimulatory molecules), IFN-b production via activation of IRF-3, and IFN-inducible genes (RANTES, IP-10, GARG16, MCP-1) in MyD88deficient mice, suggesting the presence of a MyD88independent pathway in TLR3 and TLR4 signaling. A new TIR domain-containing protein, TIRAP/Mal (TIR domaincontaining adapter protein/MyD88-adapter-like), has been identified and shown, by examining TIRAP-deficient mice, to be involved in TLR2- and TLR4-mediated inflammatory cytokine production [9–12]. However, DC maturation and IFN-inducible gene transcription in response to LPS stimulation occurs normally in MyD88/TIRAP double knockout mice, suggesting that TIRAP is not the factor responsible for the TLR4-specific MyD88-independent pathway [10]. More recently, a third TIR-containing protein, TRIF (TIR domaincontaining adapter inducing IFN-b) was identified and knockout mice generated [13]. It has been reported that the production of inflammatory cytokine induced by LPS or dsRNA stimulation is absent, and upregulation of IFNinducible genes severely impaired in TRIF-deficient mice [14]. Moreover, LPS failed entirely to upregulate IFNinducible genes or to activate NF-jB in TRIF/MyD88 double knockout mice, demonstrating the involvement of TRIF in the MyD88-independent pathway [14]. It has also been shown that TRIF can associate with TRAF6 and TBK1 (TANK-binding kinase 1) in activating two distinct transcription factors: NF-kB and IRF3, respectively [15]. Production of inflammatory cytokines, such as TNF-a, IL-6 and IL-12, in response to TLR ligands appears to be dependent on MyD88, TIRAP and TRIF [5,14]. LPS (TLR4 ligand) stimulation, in particular, exhibits a nonredundant requirement for each adapter, whereas CpG-DNA (TLR9 ligand) appears to depend on MyD88 alone [4]. By contrast, LPS may utilize either MyD88 or TRIF for IFN-b production and upregulation of IFN-inducible genes, meaning that MyD88 and TRIF can compensate for the lack of one or the other in mediating LPS-induced IFN-b production, but not LPS-induced inflammatory cytokine production [5,14,16]. Although all proximal adapters are required for TLRinduced production of inflammatory cytokine, experimental evidence suggests that a degree of flexibility exists with regard to the downstream factors required to mediate the same process. Specifically, while there is no defect in production of LPS- or CD40L-induced inflammatory cytokines in the absence of a single NF-jB transcription factor, p50 or cRel, as evidenced by experiments using single knockout mice, production is severely impaired in p50/cRel double knockout mice [17]. These findings suggest that p50 and cRel, unlike upstream adapter molecules, MyD88 and TRIF, appear capable of compensating each other to produce inflammatory cytokine.
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4. TRAF6 is a common signaling adapter molecule among the IL-1R/TLR family and TNFR superfamily TNF receptor-associated factor (TRAF) family members are intracellular proteins that transmit signals from the intracellular portion of TNFR superfamily members. Currently, there are seven known mammalian TRAFs (TRAF1– TRAF7) [18,19]. TRAF family proteins comprise an N-terminal zinc-binding domain (containing a RING finger followed by several zinc fingers) and a C-terminal TRAF domain (consisting of a coiled-coil domain known as the TRAF-N domain and a highly conserved TRAF-C domain) (Fig. 3) [18,20]. It has been revealed that the N-terminal domain is essential for the activation of downstream signaling cascades, and the TRAF domain permits self-association and interactions with receptors and other signaling proteins [18]. Two groups independently cloned TRAF6. One searched a DNA database for TRAF2-like sequences followed by cDNA library screening [8], and the other performed a yeast two-hybrid screening using CD40 as bait [21]. TRAF6 is unique among TRAF family members with regard to the manner in which it interacts with receptors. For example, TNFR family members CD40 and RANK (receptor activator of NF-jB, also known as TRANCE receptor) associate with TRAFs 1–3, 5 and 6 via their cytoplasmic tails. While TRAFs 1–3 and 5 share common binding sites on CD40 and RANK, the binding sites of TRAF6 are distinct. Studies of crystal structures of TRAF6 in complex with CD40 and RANK peptides revealed remarkable differences between receptor recognition by TRAF6 and TRAF2 [22]. Not surprisingly, in both gene structure and sequence homology of the TRAF-C domain, TRAF6 appears to be one of the most divergent mammalian TRAFs, further highlighting its uniqueness as an adapter molecule [18]. An additional unique and important trait of TRAF6, in the context of TRAF biology, is the capacity to mediate signals emanating from
Fig. 3. TRAF family proteins. TRAF family members are signaling adapter proteins for the TNFR superfamily. TRAF family proteins comprise an N-terminal zinc-binding domain (containing a RING finger followed by several zinc fingers) and a C-terminal TRAF domain, which consists of a coiled-coil domain, known as the TRAF-N domain, and a highly conserved TRAF-C domain.
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both TNFR superfamily and IL-1R/TLR family members, interacting directly with receptors in the former case and with receptor-associated proteins like MyD88 and IRAKs in the latter case. These unique and diverse molecular characteristics are reflected in the physiologic functions of TRAF6. For instance, the RANK signal regulating osteoclast development and function is entirely dependent on TRAF6 (Fig. 2) [23,24]. Mice lacking the TNF family member TNF-related activation-induced cytokine (TRANCE, also known as RANKL) [25], and its cognate receptor, RANK [26], exhibit osteopetrosis (increased bone mass) due to defective development and function of osteoclasts. Among TRAF knockout mice (so far TRAF1–TRAF6 genes have been deleted in mice), only TRAF6 knockout mice showed osteopetrosis, even though other TRAFs, including TRAFs 1–3 and 5, can also associate with RANK. These findings imply that the TRANCE–RANK–TRAF6 axis regulates osteoclast development and function. It has been reported that TRAF6 is also a major regulator of CD40 signaling, which controls IL-6 secretion, Ig secretion, upregulation of costimulatory molecules, affinity maturation and generation of long-lived plasma cells [27,28]. 5. TRAF6 is required for optimal DC maturation and activation in response to TLR ligands and CD40L DCs function as sensors of infection of the innate immune system. DCs recognize the invasion of pathogenic microbes
via the binding of PAMPs to TLRs expressed on the cell surface [1]. It has been shown that TRAF6 is involved in the signaling cascade downstream of the IL-1R/TLR family; however, little is known about the specific physiologic role of TRAF6 in DCs. Thus, we investigated TRAF6 function in DCs using TRAF6 knockout mice. While wild-type DCs activated by various TLR ligands or CD40L produce a substantial amount of the inflammatory cytokines IL-6 and IL-12, DCs generated from TRAF6 knockout mice exhibit a severe cytokine production defect (Fig. 4A) [29]. These results suggest that TRAF6, like the proximal signaling adapter MyD88, is required for cytokine production induced by signals from various TLRs. In addition, TRAF6 is necessary for cytokine production induced upon CD40 ligation [29]. Moreover, whereas TLR ligands or CD40L induce upregulation of surface molecules like MHC class II and costimulatory molecules on wild-type DCs in vitro, upregulation is severely impaired in TRAF6-deficient DCs (Fig. 4B). Similarly, administration of LPS or anti-CD40 antibody in vivo induces DC maturation in wild-type mice but not in TRAF6 knockout mice (Fig. 4C) [29]. It would appear, therefore, that both MyD88-dependent and -independent pathways of the LPS-TLR4 signaling cascade, which together regulate DC maturation and activation, are dependent on TRAF6. Interestingly though, in vivo LPS treatment of fetal liver cell chimeras, in which only the hematopoietic compartment is TRAF6-deficient, results in normal upregulation of CD80, CD86, and MHC class II by TRAF6-
Fig. 4. Production of inflammatory cytokines and upregulation of surface molecules are severely impaired in TRAF6-deficient DCs. DCs generated in vitro from wild-type or TRAF6 knockout (KO) mice were stimulated with various TLR ligands or CD40L. (A) Culture supernatant IL-12 (p40) concentration was measured by ELISA. (B) Surface MHC class II expression levels were analyzed by flow cytometry. The gray filled histogram represents the untreated condition, and the red lined histogram represents the treated condition. LTA: lipoteichoic acid (C). Wild-type and TRAF6 knockout mice were injected with LPS or anti-CD40 antibody, and surface MHC class II expression levels on splenic DCs examined as in (B) [29].
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Fig. 5. TRAF6 deficiency results in a severe reduction in the CD4+ splenic DC subset. Analysis of spleens harvested from BM chimeras reconstituted with fetal liver cells from either TRAF6 knockout or wild-type littermate fetuses revealed a striking reduction in the number of CD11chigh DCs in knockout chimeras (left panels), as well as a near complete absence of the CD4+ DC subset, the majority subset, in wild-type spleen (right panels). Numbers denote the ratio of each population (%) [29].
deficient DCs (Walsh and Kobayashi, unpublished data). This observation suggests that non-hematopoietic cell types may be capable of responding to LPS by producing factors that induce DC maturation in a TRAF6-independent manner. Further evidence that TRAF6 plays a critical role in DC function is provided by the observation that LPS treatment of TRAF6-deficient DCs fails to augment their T cell stimulatory capacity [29]. TRAF6-deficient DCs treated with LPS do, however, exhibit normal induction of IFN-inducible genes, as well as activation, though attenuated, of IjBa, Akt and MAP kinases p38 and Erk [29]. These observations indicate that there are TRAF6-independent pathways operating downstream of TLR4 (Walsh and Kobayashi, unpublished data). The molecular mechanisms and physiologic significance of these TRAF6-independent pathways remain to be solved.
6. TRAF6 is essential for the development of a DC subset It has been demonstrated that TRAF6 is essential for the development of multiple tissues. The most prominent example is the osteopetrotic phenotype of TRAF6 knockout mice, which is due to defective development and function of osteoclasts [23,24]. TRAF6 knockout mice also lack lymph nodes, and it has been reported that TRAF6 deficiency increases the incidence of CNS developmental defects, such as exencephaly, due to failed neural tube closure [30]. Moreover, TRAF6 knockout mice exhibit hypohidrotic ectodermal dysplasia as a result of defective development of epidermal appendices [31]. Interestingly, we have found that TRAF6 is also essential for the development of a DC subset in the spleen (Fig. 5). It has been described that DCs in the mouse spleen can be
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assigned to one of three distinct subsets according to differential expression of CD4 or CD8a, namely CD4+CD8a–, CD4–CD8a+ or CD4–CD8a–, in conjunction with high expression of the pan-DC marker CD11c [32]. The number and frequency of CD11chigh DCs are strikingly reduced in the spleens of 2-week-old TRAF6 knockout mice compared to those of age-matched wild-type mice [29]. Among the three DC subsets, CD4+ DCs predominate in the wild-type spleen, while the CD4+ DC population is almost completely absent in TRAF6 knockout spleen [29]. However, analysis of TRAF6 knockout mice is problematic, because the mice exhibit growth retardation and osteopetrosis, and die between 2 and 3 weeks of age. These factors may indirectly affect the development of hematopoietic cell lineages, confounding analysis of DC homeostasis in vivo. To exclude such a possibility, we generated bone marrow (BM) chimeras reconstituted with fetal liver cells from TRAF6 knockout or wild-type littermate fetuses. As in straight TRAF6 knockout mice, the number and frequency of DCs were found to be remarkably diminished and the CD4+ DC subset almost completely absent in the TRAF6 knockout fetal liver cell chimeras, suggesting that the defective development of the CD4+ DC subset results from a direct effect of TRAF6 deficiency in the hematopoietic compartment (Fig. 5) [29]. This phenotype bears similarity to that of knockout mice deficient for the NF-jB subunit RelB, a transcription factor which may be positioned downstream of TRAF6 activation, suggesting that a TRAF6-RelB signaling pathway could control the development of the CD4+ DC subset in the spleen (Fig. 2). Future efforts should focus on further understanding the physiologic role of each DC subset, in addition to identifying the responsible receptor–ligand pair (or pairs) and concomitant signaling mechanisms that regulate DC development.
7. Conclusions and future directions In this review we discussed the function of TRAF6, an adapter molecule common to both the IL-1R/TLR family and TNFR superfamily and a critical factor for signaling in DCs and regulation of the innate immune system. Gaining further understanding of the physiologic functions of TRAF6 is a fascinating and important undertaking, as this adaptor protein functions not only in immune cells, but in osteoclasts and various other cell types, as well. However, because TRAF6 deficiency causes multiple disorders in various organ systems, leading to early death, analysis of knockout mice is difficult. To understand the role of TRAF6 in different physiologic contexts, powerful analytical tools, such as tissue-specific TRAF6 knockout mouse models utilizing the Cre/loxP system will be required. Another important area of study will be in determining what role TRAF6 plays in assigning specificity to signals emanating from different TLRs, such that the downstream outcomes are appropriate for combating different pathogenic
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threats. TRAF6 is positioned at a point where signals from various TLRs and TNFR superfamily members converge to exert common effects on DC maturation and activation. However, TRAF6 must also be considered in the context of other adapters, including those involved in the TRAF6independent pathway downstream of TLR3 and TLR4, for which the molecular mechanisms and physiologic significance remain unclear. Understanding the divergent signaling cascades underlying the TLR family will provide clues toward clarifying specific functions of individual TLRs. References [1]
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Review
The role of TRAF6 in signal transduction and the immune response Takashi Kobayashi, Matthew C. Walsh, Yongwon Choi * Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 421 Curie Blvd., Room 308, Philadelphia, PA 19104, USA Available online 28 September 2004
Abstract Signals from the IL-1 receptor (IL-1R)/Toll-like receptor (TLR) family and TNF receptor (TNFR) superfamily are critical for regulating the function of antigen-presenting cells such as dendritic cells (DCs). It has been revealed that TNF receptor-associated factor 6 (TRAF6), a signaling adapter molecule common to the IL-1R/TLR family and TNFR superfamily, is important not only for DC maturation, cytokine production, and T cell stimulatory capacity of DCs in response to TLR ligands (e.g. lipopolysaccharide) or CD40 ligand, but also for the homeostasis of splenic DC subsets. © 2004 Elsevier SAS. All rights reserved. Keywords: TRAF6; Toll-like receptor; CD40; Dendritic cells; Innate immune response
1. Introduction Upon activation of the innate immune response during infection, antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), immediately recognize the invasion of microbes, such as bacteria or viruses, and then transmit the information to T cells to activate the adaptive immune response (Fig. 1) [1]. Immature DCs in the periphery uptake antigens efficiently but express low levels of MHC and costimulatory molecules, and thus possess a low level of T cell stimulatory capacity [1]. Upon maturation, DCs greatly augment their ability to stimulate naïve T cells by dramatically upregulating surface expression of antigen/ MHC complexes and various costimulatory molecules [1]. Activated DCs also secrete cytokines, including IL-12, which are critical for the differentiation of T cells into Th1 type effector cells [1]. DC maturation is not triggered by specific protein antigens, but rather by inflammatory cytokines like CD40L on activated T cells, or microbial components (e.g. lipopolysaccharide (LPS), double-stranded RNA (dsRNA), or CpG-DNA), which signal through CD40 and Toll-like receptors (TLRs), respectively, on the surface of the APC [2–4].
* Corresponding author. Tel.: +1-215-746-6404; fax: +1-215-573-0889. E-mail address: [email protected] (Y. Choi). 1286-4579/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.09.001
Fig. 1. TLRs function as sensors of infection in the innate immune system. During infection, immature DCs take up and digest the peptide antigens of pathogenic microbes, and simultaneously respond to specific molecular features of microbes via pattern-recognition receptors, so-called TLRs, by initiating an activation program. Upon activation, mature DCs dramatically upregulate surface expression of antigen/MHC complexes and various costimulatory molecules which potentiate stimulation of naïve T cells. Moreover, activated DCs secrete cytokines, such as IL-12, that drive T cell differentiation toward the Th1 effector phenotype. TRAF6 serves as a critical adapter molecule in transducing signals emanating from TLRs.
2. TLRs recognize pathogen-associated molecular patterns (PAMPs) Drosophila Toll was originally characterized as a crucial factor in regulating dorso-ventral axis formation during fly
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embryo development, but was later recognized as serving a second function as a regulator of antimicrobial (antifungal) peptides, such as drosomycin, in response to infection. In addition, another Drosophila Toll family member, 18wheeler, is known to induce the antibacterial peptide, attacin [2,5]. Soon after the Drosophila Toll family was identified, mammalian TLR family members and their cognate ligands were identified. To date, 11 mammalian TLRs (TLR1– TLR11), comprising a family of receptors that share homology to Drosophila Toll, have been cloned [5,6]. Interestingly, it has been demonstrated that the ligands of mammalian TLRs are components of pathogenic microbes called PAMPs. For example, TLR2 recognizes peptidoglycan (PGN; a component of Gram-positive bacteria), TLR3 recognizes dsRNA (a viral product), TLR4 recognizes LPS (a component of Gram-negative bacteria), TLR5 recognizes flagellin (protein subunits that make up bacterial flagella), and TLR9 recognizes unmethylated CpG-DNA motifs in bacterial DNA (Fig. 2) [5]. TLR2 functions by forming heterodimers with either TLR1 or TLR6, which are thought to generate distinct ligand–receptor specificities. It would appear that these various “first-alert” receptor sets have evolved to immediately signal the immune system at the first sign of a foreign invader capable of causing pathological damage [2]. While the extracellular domains of TLRs, which contain leucine-rich repeats (LRRs), and IL-1R, which bears an Ig-
like domain, are dissimilar, the cytoplasmic regions of each exhibit functional similarity, sharing a common motif, termed the TIR (TLR/IL-1R) domain [5]. Furthermore, various adapter molecules, including MyD88, IRAK and TRAF6 are associated with both signaling cascades [7,8]. 3. Signal transduction of IL-1R/TLRs and cytokine production The signal transduction pathway common to both IL-1R and TLRs was first defined by studying IL-1R signaling. IL-1 binds to IL-1R, which is associated with an accessory protein (IL-1RAcp), inducing the formation of a receptor complex that recruits a cytoplasmic adapter protein, MyD88 [2,5]. IL-1R and MyD88 interact through their TIR domains. This is followed by the recruitment of Ser/Thr kinases called IRAKs (IL-1 receptor-associated kinase; IRAK, IRAK-2, IRAK-M, and IRAK-4), and then by association with TRAF6, which mediates activation of downstream signaling via NF-jB transcription factors and MAP kinase (JNK, p38 and Erk) cascade-activated AP-1 transcription factors. The fundamental TLR signaling mechanisms are similar to those of IL-1R, specifically with respect to adapter molecules utilized in the proximal complex (MyD88, IRAKs and TRAF6), and downstream activation of both NF-jB and AP-1 (Fig. 2) [5]. DCs from MyD88-deficient mice fail to produce inflammatory cytokines (TNF-a, IL-6, IL-12) in response to IL-1 or
Fig. 2. TRAF6 is a common signaling adapter protein for the IL-1R/TLR family and TNFR superfamily. Signals from the IL-1R/TLR family are mediated by adapter molecules MyD88, IRAK and TRAF6, and lead to activation of transcription factors NF-jB and AP-1. TNFR superfamily members such as CD40 and RANK directly associate with TRAF6, which in turn activates the same transcription factors. TRAF6, a common signaling adapter molecule for both receptor families, is critical for DC maturation and inflammatory cytokine production.
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other TLR ligands, such as LPS or CpG-DNA, suggesting that MyD88 is indispensable for inflammatory cytokine production in IL-1R/TLRs signaling [3]. On the other hand, LPS (TLR4 ligand) or dsRNA (TLR3 ligand) do induce DC maturation (upregulation of MHC and costimulatory molecules), IFN-b production via activation of IRF-3, and IFN-inducible genes (RANTES, IP-10, GARG16, MCP-1) in MyD88deficient mice, suggesting the presence of a MyD88independent pathway in TLR3 and TLR4 signaling. A new TIR domain-containing protein, TIRAP/Mal (TIR domaincontaining adapter protein/MyD88-adapter-like), has been identified and shown, by examining TIRAP-deficient mice, to be involved in TLR2- and TLR4-mediated inflammatory cytokine production [9–12]. However, DC maturation and IFN-inducible gene transcription in response to LPS stimulation occurs normally in MyD88/TIRAP double knockout mice, suggesting that TIRAP is not the factor responsible for the TLR4-specific MyD88-independent pathway [10]. More recently, a third TIR-containing protein, TRIF (TIR domaincontaining adapter inducing IFN-b) was identified and knockout mice generated [13]. It has been reported that the production of inflammatory cytokine induced by LPS or dsRNA stimulation is absent, and upregulation of IFNinducible genes severely impaired in TRIF-deficient mice [14]. Moreover, LPS failed entirely to upregulate IFNinducible genes or to activate NF-jB in TRIF/MyD88 double knockout mice, demonstrating the involvement of TRIF in the MyD88-independent pathway [14]. It has also been shown that TRIF can associate with TRAF6 and TBK1 (TANK-binding kinase 1) in activating two distinct transcription factors: NF-kB and IRF3, respectively [15]. Production of inflammatory cytokines, such as TNF-a, IL-6 and IL-12, in response to TLR ligands appears to be dependent on MyD88, TIRAP and TRIF [5,14]. LPS (TLR4 ligand) stimulation, in particular, exhibits a nonredundant requirement for each adapter, whereas CpG-DNA (TLR9 ligand) appears to depend on MyD88 alone [4]. By contrast, LPS may utilize either MyD88 or TRIF for IFN-b production and upregulation of IFN-inducible genes, meaning that MyD88 and TRIF can compensate for the lack of one or the other in mediating LPS-induced IFN-b production, but not LPS-induced inflammatory cytokine production [5,14,16]. Although all proximal adapters are required for TLRinduced production of inflammatory cytokine, experimental evidence suggests that a degree of flexibility exists with regard to the downstream factors required to mediate the same process. Specifically, while there is no defect in production of LPS- or CD40L-induced inflammatory cytokines in the absence of a single NF-jB transcription factor, p50 or cRel, as evidenced by experiments using single knockout mice, production is severely impaired in p50/cRel double knockout mice [17]. These findings suggest that p50 and cRel, unlike upstream adapter molecules, MyD88 and TRIF, appear capable of compensating each other to produce inflammatory cytokine.
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4. TRAF6 is a common signaling adapter molecule among the IL-1R/TLR family and TNFR superfamily TNF receptor-associated factor (TRAF) family members are intracellular proteins that transmit signals from the intracellular portion of TNFR superfamily members. Currently, there are seven known mammalian TRAFs (TRAF1– TRAF7) [18,19]. TRAF family proteins comprise an N-terminal zinc-binding domain (containing a RING finger followed by several zinc fingers) and a C-terminal TRAF domain (consisting of a coiled-coil domain known as the TRAF-N domain and a highly conserved TRAF-C domain) (Fig. 3) [18,20]. It has been revealed that the N-terminal domain is essential for the activation of downstream signaling cascades, and the TRAF domain permits self-association and interactions with receptors and other signaling proteins [18]. Two groups independently cloned TRAF6. One searched a DNA database for TRAF2-like sequences followed by cDNA library screening [8], and the other performed a yeast two-hybrid screening using CD40 as bait [21]. TRAF6 is unique among TRAF family members with regard to the manner in which it interacts with receptors. For example, TNFR family members CD40 and RANK (receptor activator of NF-jB, also known as TRANCE receptor) associate with TRAFs 1–3, 5 and 6 via their cytoplasmic tails. While TRAFs 1–3 and 5 share common binding sites on CD40 and RANK, the binding sites of TRAF6 are distinct. Studies of crystal structures of TRAF6 in complex with CD40 and RANK peptides revealed remarkable differences between receptor recognition by TRAF6 and TRAF2 [22]. Not surprisingly, in both gene structure and sequence homology of the TRAF-C domain, TRAF6 appears to be one of the most divergent mammalian TRAFs, further highlighting its uniqueness as an adapter molecule [18]. An additional unique and important trait of TRAF6, in the context of TRAF biology, is the capacity to mediate signals emanating from
Fig. 3. TRAF family proteins. TRAF family members are signaling adapter proteins for the TNFR superfamily. TRAF family proteins comprise an N-terminal zinc-binding domain (containing a RING finger followed by several zinc fingers) and a C-terminal TRAF domain, which consists of a coiled-coil domain, known as the TRAF-N domain, and a highly conserved TRAF-C domain.
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both TNFR superfamily and IL-1R/TLR family members, interacting directly with receptors in the former case and with receptor-associated proteins like MyD88 and IRAKs in the latter case. These unique and diverse molecular characteristics are reflected in the physiologic functions of TRAF6. For instance, the RANK signal regulating osteoclast development and function is entirely dependent on TRAF6 (Fig. 2) [23,24]. Mice lacking the TNF family member TNF-related activation-induced cytokine (TRANCE, also known as RANKL) [25], and its cognate receptor, RANK [26], exhibit osteopetrosis (increased bone mass) due to defective development and function of osteoclasts. Among TRAF knockout mice (so far TRAF1–TRAF6 genes have been deleted in mice), only TRAF6 knockout mice showed osteopetrosis, even though other TRAFs, including TRAFs 1–3 and 5, can also associate with RANK. These findings imply that the TRANCE–RANK–TRAF6 axis regulates osteoclast development and function. It has been reported that TRAF6 is also a major regulator of CD40 signaling, which controls IL-6 secretion, Ig secretion, upregulation of costimulatory molecules, affinity maturation and generation of long-lived plasma cells [27,28]. 5. TRAF6 is required for optimal DC maturation and activation in response to TLR ligands and CD40L DCs function as sensors of infection of the innate immune system. DCs recognize the invasion of pathogenic microbes
via the binding of PAMPs to TLRs expressed on the cell surface [1]. It has been shown that TRAF6 is involved in the signaling cascade downstream of the IL-1R/TLR family; however, little is known about the specific physiologic role of TRAF6 in DCs. Thus, we investigated TRAF6 function in DCs using TRAF6 knockout mice. While wild-type DCs activated by various TLR ligands or CD40L produce a substantial amount of the inflammatory cytokines IL-6 and IL-12, DCs generated from TRAF6 knockout mice exhibit a severe cytokine production defect (Fig. 4A) [29]. These results suggest that TRAF6, like the proximal signaling adapter MyD88, is required for cytokine production induced by signals from various TLRs. In addition, TRAF6 is necessary for cytokine production induced upon CD40 ligation [29]. Moreover, whereas TLR ligands or CD40L induce upregulation of surface molecules like MHC class II and costimulatory molecules on wild-type DCs in vitro, upregulation is severely impaired in TRAF6-deficient DCs (Fig. 4B). Similarly, administration of LPS or anti-CD40 antibody in vivo induces DC maturation in wild-type mice but not in TRAF6 knockout mice (Fig. 4C) [29]. It would appear, therefore, that both MyD88-dependent and -independent pathways of the LPS-TLR4 signaling cascade, which together regulate DC maturation and activation, are dependent on TRAF6. Interestingly though, in vivo LPS treatment of fetal liver cell chimeras, in which only the hematopoietic compartment is TRAF6-deficient, results in normal upregulation of CD80, CD86, and MHC class II by TRAF6-
Fig. 4. Production of inflammatory cytokines and upregulation of surface molecules are severely impaired in TRAF6-deficient DCs. DCs generated in vitro from wild-type or TRAF6 knockout (KO) mice were stimulated with various TLR ligands or CD40L. (A) Culture supernatant IL-12 (p40) concentration was measured by ELISA. (B) Surface MHC class II expression levels were analyzed by flow cytometry. The gray filled histogram represents the untreated condition, and the red lined histogram represents the treated condition. LTA: lipoteichoic acid (C). Wild-type and TRAF6 knockout mice were injected with LPS or anti-CD40 antibody, and surface MHC class II expression levels on splenic DCs examined as in (B) [29].
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Fig. 5. TRAF6 deficiency results in a severe reduction in the CD4+ splenic DC subset. Analysis of spleens harvested from BM chimeras reconstituted with fetal liver cells from either TRAF6 knockout or wild-type littermate fetuses revealed a striking reduction in the number of CD11chigh DCs in knockout chimeras (left panels), as well as a near complete absence of the CD4+ DC subset, the majority subset, in wild-type spleen (right panels). Numbers denote the ratio of each population (%) [29].
deficient DCs (Walsh and Kobayashi, unpublished data). This observation suggests that non-hematopoietic cell types may be capable of responding to LPS by producing factors that induce DC maturation in a TRAF6-independent manner. Further evidence that TRAF6 plays a critical role in DC function is provided by the observation that LPS treatment of TRAF6-deficient DCs fails to augment their T cell stimulatory capacity [29]. TRAF6-deficient DCs treated with LPS do, however, exhibit normal induction of IFN-inducible genes, as well as activation, though attenuated, of IjBa, Akt and MAP kinases p38 and Erk [29]. These observations indicate that there are TRAF6-independent pathways operating downstream of TLR4 (Walsh and Kobayashi, unpublished data). The molecular mechanisms and physiologic significance of these TRAF6-independent pathways remain to be solved.
6. TRAF6 is essential for the development of a DC subset It has been demonstrated that TRAF6 is essential for the development of multiple tissues. The most prominent example is the osteopetrotic phenotype of TRAF6 knockout mice, which is due to defective development and function of osteoclasts [23,24]. TRAF6 knockout mice also lack lymph nodes, and it has been reported that TRAF6 deficiency increases the incidence of CNS developmental defects, such as exencephaly, due to failed neural tube closure [30]. Moreover, TRAF6 knockout mice exhibit hypohidrotic ectodermal dysplasia as a result of defective development of epidermal appendices [31]. Interestingly, we have found that TRAF6 is also essential for the development of a DC subset in the spleen (Fig. 5). It has been described that DCs in the mouse spleen can be
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assigned to one of three distinct subsets according to differential expression of CD4 or CD8a, namely CD4+CD8a–, CD4–CD8a+ or CD4–CD8a–, in conjunction with high expression of the pan-DC marker CD11c [32]. The number and frequency of CD11chigh DCs are strikingly reduced in the spleens of 2-week-old TRAF6 knockout mice compared to those of age-matched wild-type mice [29]. Among the three DC subsets, CD4+ DCs predominate in the wild-type spleen, while the CD4+ DC population is almost completely absent in TRAF6 knockout spleen [29]. However, analysis of TRAF6 knockout mice is problematic, because the mice exhibit growth retardation and osteopetrosis, and die between 2 and 3 weeks of age. These factors may indirectly affect the development of hematopoietic cell lineages, confounding analysis of DC homeostasis in vivo. To exclude such a possibility, we generated bone marrow (BM) chimeras reconstituted with fetal liver cells from TRAF6 knockout or wild-type littermate fetuses. As in straight TRAF6 knockout mice, the number and frequency of DCs were found to be remarkably diminished and the CD4+ DC subset almost completely absent in the TRAF6 knockout fetal liver cell chimeras, suggesting that the defective development of the CD4+ DC subset results from a direct effect of TRAF6 deficiency in the hematopoietic compartment (Fig. 5) [29]. This phenotype bears similarity to that of knockout mice deficient for the NF-jB subunit RelB, a transcription factor which may be positioned downstream of TRAF6 activation, suggesting that a TRAF6-RelB signaling pathway could control the development of the CD4+ DC subset in the spleen (Fig. 2). Future efforts should focus on further understanding the physiologic role of each DC subset, in addition to identifying the responsible receptor–ligand pair (or pairs) and concomitant signaling mechanisms that regulate DC development.
7. Conclusions and future directions In this review we discussed the function of TRAF6, an adapter molecule common to both the IL-1R/TLR family and TNFR superfamily and a critical factor for signaling in DCs and regulation of the innate immune system. Gaining further understanding of the physiologic functions of TRAF6 is a fascinating and important undertaking, as this adaptor protein functions not only in immune cells, but in osteoclasts and various other cell types, as well. However, because TRAF6 deficiency causes multiple disorders in various organ systems, leading to early death, analysis of knockout mice is difficult. To understand the role of TRAF6 in different physiologic contexts, powerful analytical tools, such as tissue-specific TRAF6 knockout mouse models utilizing the Cre/loxP system will be required. Another important area of study will be in determining what role TRAF6 plays in assigning specificity to signals emanating from different TLRs, such that the downstream outcomes are appropriate for combating different pathogenic
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threats. TRAF6 is positioned at a point where signals from various TLRs and TNFR superfamily members converge to exert common effects on DC maturation and activation. However, TRAF6 must also be considered in the context of other adapters, including those involved in the TRAF6independent pathway downstream of TLR3 and TLR4, for which the molecular mechanisms and physiologic significance remain unclear. Understanding the divergent signaling cascades underlying the TLR family will provide clues toward clarifying specific functions of individual TLRs. References [1]
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