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Seminars in Immunology 19 (2007) 24–32

Review

TLR signaling

Taro Kawai a,b, Shizuo Akira a,b,∗a Department of Host Defense, Research Institute for Microbial Diseases, Osaka University,

3-1 Yamada-oka, Suita, Osaka 565-0871, Japanb Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Research Institute for Microbial Diseases,

Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan

bstract

The TLR family senses the molecular signatures of microbial pathogens, and plays a fundamental role in innate immune responses. TLRs signalia a common pathway that leads to the expression of diverse inflammatory genes. In addition, each TLR elicits specific cellular responses to

athogens owing to differential usage of intracellular adapter proteins. Recent studies have revealed the importance of the subcellular localizationf TLRs in pathogen recognition and signaling. TLR signaling pathways is negatively regulated by a number of cellular proteins to attenuatenflammation. Here, we describe recent advances in our understanding of the regulation of TLR-mediated signaling.

2007 Elsevier Ltd. All rights reserved.

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eywords: Innate immunity; Signal transduction; Toll-like receptor

. Introduction

The innate immune system is the first line of the defen-ive mechanisms that protect hosts from invading microbialathogens. Host cells express various pattern recognition recep-ors (PRRs) that sense diverse pathogen-associated molecularatterns (PAMPs), ranging from lipids, lipoproteins, proteinsnd nucleic acids [1]. Recognition of PAMPs by PRRs activatesntracellular signaling pathways that culminate in the inductionf inflammatory cytokines, chemokines, interferons (IFNs) andpregulation of co-stimulatory molecules. In mammals, the fam-ly of Toll-like receptors (TLR) expressed on antigen presentingells such as dendritic cells (DC) and macrophages serves asey PRRs with central roles in induction of innate immuneesponses as well as the subsequent development of adaptivemmune responses [1]. TLRs are type I membrane proteins char-cterized by an ectodomain composed of leucine rich repeatsLRR) that are responsible for recognition of PAMPs and a

ytoplasmic domain homologous to the cytoplasmic region ofhe IL-1 receptor, known as the TIR domain, which is requiredor downstream signaling. To date, 11 human TLRs and 13

∗ Corresponding author at: Department of Host Defense, Research Institute foricrobial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871,

apan. Tel.: +81 6 6879 8303; fax: +81 6 6879 8305.E-mail address: sakira@biken.osaka-u.ac.jp (S. Akira).

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044-5323/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.smim.2006.12.004

ouse TLRs have been identified, and each TLR appears to rec-gnize distinct PAMPs derived from various microorganisms,ncluding bacteria, viruses, protozoa and fungi [1]. TLRs arelassified into several groups based on the types of PAMPshey recognize. TLR1, 2, 4 and 6 recognize lipids. For exam-le, TLR4, together with its extracellular components such asD-2 and CD14, recognizes lipopolysaccharide (LPS) fromram-negative bacteria, which causes septic shock [1]. TLR2

orms heterodimers with TLR1, TLR6 and non-TLRs suchs CD36 to discriminate a wide variety of PAMPs, includingeptidoglycan, lipopeptides and lipoproteins of Gram-positiveacteria, mycoplasma lipopeptides and fungal zymosan [1]. Inarticular, TLR1/2 and TLR2/6 can discriminate triacyl- andiacyl-lipopeptide, respectively [1]. In addition, human TLR10s thought to heterodimerize with TLR2 and TLR1, although

ligand for these heterodimers remains unknown [1]. TLR5nd 11 recognize protein ligands. TLR5 is expressed abun-antly in intestinal CD11c-positive lamina propria cells wheret senses bacterial flagellin [1,2]. Mouse TLR11 recognizess yet unknown components of uropathogenic bacteria, andprofilin-like molecule of the protozoan parasite Toxoplasma

ondii. The third class of TLRs includes TLR3, 7, 8 and 9,hich are localized intracellularly where they detect nucleic

cids derived from viruses and bacteria. TLR3 was shown toecognize double stranded RNA (dsRNA), which is producedy many viruses during replication. TLR7 recognizes syntheticmidazoquinoline-like molecules, guanosine analogs such as

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oxoribine, single stranded RNA (ssRNA) derived from variousiruses and small interfering RNA [1]. Human TLR8, which hashe highest homology to TLR7, participates in the recognition ofmidazoquinolines and ssRNA. It has been reported that humanLR8 is expressed on regulatory T cells (Treg) and directly

ecognizes polyG-containing DNA oligonucleotides [1]. Thisecognition can reverse the suppressive function of Treg withoutrequirement for DC activation. In mice, however, the func-

ion and ligands of TLR8 remain unknown. TLR9 recognizespG DNA motifs present in bacterial and viral genomes asell as non-nucleic acids such as hemozoin from the malariaarasite [1].

. TIR domain-containing adapters

After recognizing PAMPs, TLRs activate intracellular signal-ng pathways that lead to the induction of inflammatory cytokineenes such as TNF�, IL-6, IL-1� and IL-12. Signaling fromLRs also elicits the upregulation of co-stimulatory moleculesn DCs, a step that is critical for the induction of pathogen-pecific adaptive immune responses. Furthermore, several TLRsre capable of inducing type I IFN (multiple IFN� and singleFN�) to elicit antiviral responses.

Recognition of PAMPs by TLRs stimulates the recruitmentf a set of intracellular TIR-domain-containing adaptors, includ-ng MyD88, TIRAP (also known as MAL), Trif (also known asICAM1) and TRAM (also known as TICAM2) via TIR–TIR

nteractions (Fig. 1) [3]. MyD88 is a universal adapter that acti-ates inflammatory pathways; it is shared by all TLRs with thexception of TLR3. Recruitment of MyD88 leads to the acti-ation of MAP kinases (MAPKs) (ERK, JNK, p38) and theranscription factor NF-�B to control the expression of inflam-

atory cytokine genes. TIRAP mediates the activation of ayD88-dependent pathway downstream of TLR2 and TLR4.

RIF is recruited to TLR3 and TLR4, and activates an alterna-ive pathway (TRIF-dependent pathway) that culminates in thectivation of NF-�B, MAPKs and the transcription factor IRF3.ctivation of IRF3 is pivotal for induction of type I IFN, par-

icularly IFN�. TRAM selectively participates in the activationf the TRIF-dependent pathway downstream of TLR4, but notLR3. More recently, an additional adapter, SARM, has beenhown to inhibit the TRIF-dependent pathway in human cellines; however, the physiological function of SARM in miceemains unknown [4]. Collectively, each TLR recruits a spe-ific combination of adapters to activate different transcriptionactors, giving rise to appropriate and effective responses toathogens.

. MyD88-dependent pathway

The association of TLRs and MyD88 stimulates the recruit-ent of members of the IRAK family, including IRAK1,

RAK2, IRAK4 and IRAK-M. In particular, IRAK4 is indis-

ensable for activation of the MyD88-dependent pathway. Oncehosphorylated, IRAKs dissociate from MyD88 and interactith TRAF6, a member of the TRAF family. TRAF6, an E3

igase, forms a complex with Ubc13 and Uev1A to promote

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munology 19 (2007) 24–32 25

he synthesis of lysine 63-linked polyubiquitin chains, whichn turn activate TAK1, a MAPKKK [5]. TAK1, in combina-ion with TAB1, TAB2 and TAB3, activates two downstreamathways involving the IKK complex and the MAPK family.he IKK complex, composed of the catalytic subunits IKK�nd IKK� and a regulatory subunit IKK�/NEMO, catalyzes thehosphorylation of I�B proteins. This phosphorylation is nec-ssary for the degradation of I�Bs and the subsequent nuclearranslocation of the transcription factor NF-�B, which controlshe expression of various inflammatory cytokine genes (Fig. 1).

embers of the MAPK family phosphorylate and activate theranscription factor AP-1, a dimer of basic region leucine zipperbZIP) proteins from the Jun, Fos, ATF and the Maf subfamilies6]. Among them, c-Jun is implicated in having a central role innflammatory responses in TLR signaling (Fig. 1). Cells derivedrom TAK1-deficient mice consistently display reduced inflam-atory cytokine production and impaired NF-�B and MAPK

ctivation in response to multiple TLR ligands, indicating anssential and non-redundant role of TAK1 in the activation ofF-�B and MAPK pathways [7]. However, substrates of TAK1,hich are responsible for activation of the IKK complex andAPK, remain unknown.Although in vitro analyses have implicated Ubc13 in the

ctivation of NF-�B and MAPK, via ubiquitination of TAK1,tudies on mice with a conditionally deleted Ubc13 gene in var-ous tissues have shown that it has a dispensable role in NF-�Bctivation [8]. Macrophages deficient for Ubc13 show defectivenflammatory cytokine induction after treatment with multipleLR ligands. They display impaired MAPK activation, but nor-al NF-�B activation, indicating that Ubc13 controls MAPK

ather than NF-�B. Importantly, TAK1 activation and TRAF6biquitination are normally observed in the absence of Ubc13.iven that TAK1 and TRAF6 are essential for optimal MAPK

nd NF-�B activation in TLR signaling, it is possible that Ubc13ctivates the MAPK pathway independently of TAK1/TRAF6r that it is located downstream of TAK1/TRAF6. Alternatively,ther E2 enzymes such as Ubc5 might compensate for the lossf Ubc13 function by activating the TAK1/TRAF6-dependentF-�B pathway in vivo. Notably, in Ubc13-deficient cells,

here is a reduction in the stimulus-dependent ubiquitinationf IKK�/NEMO, suggesting a link between Ubc13-dependentKK�/NEMO ubiquitination and MAPK activation (Fig. 1) [8].

In addition to TAK1, several other MAPKKKs are impli-ated in TLR4 signaling. Macrophages deficient for MEKK3how defective JNK and p38 activation and IL-6 productionfter LPS treatment [9]. In contrast, LPS-stimulated ERK activa-ion and TNF� production is severely impaired in Tpl2-deficient

acrophages, whereas activation of JNK and p38 is normal [10].t has been reported that ABIN2 (A20-binding inhibitor of NF-B 2) increases the stability of Tpl2 to facilitate ERK activation11]. ASK1-deficient splenocytes and DC have defects in LPS-timulated p38 activation as well as IL-6 and TNF� production12]. LPS-induced ROS production is implicated in facili-

ating the TRAF6-ASK1-p38 signaling axis. Taken together,hese observations suggest that activation of distinct MAPKKKs

ight determine the nature and magnitude of TLR4-mediatednflammatory responses.

26 T. Kawai, S. Akira / Seminars in Immunology 19 (2007) 24–32

Fig. 1. TLR signaling. Following stimulation, all TLRs except for TLR3 recruit MyD88, IRAKs and TRAF6 to activate the Ubc13/TAK1 pathway. The TAK1complex then activates the IKK complex composed of IKK�, IKK� and IKK�/NEMO, which catalyzes phosphorylation of I�B proteins. Phosphorylated I�Bproteins are degraded by a proteasome-dependent pathway, allowing NF-�B to translocate to the nucleus. TAK1 also activates the MAPK pathway, which mediatesAP-1 activation. IRF5 is recruited to the MyD88-IRAK4-TRAF6 complex, phosphorylated and translocated to the nucleus. NF-�B, AP-1 and IRF5 control theexpression of genes encoding inflammatory cytokines. TIRAP is recruited to TLR4, TLR1/2 and TLR2/6, activating the MyD88-dependent pathway. TRIF isrecruited to TLR3 and TLR4, and interacts with TBK1 and IKKi, which mediate phosphorylation of IRF3. Phosphorylated IRF3 dimerizes and is translocated tothe nucleus to induce expression of type I IFN and IFN-inducible genes. TRAF3 forms a complex with TBK1 and IKKi. TRIF interacts with TRAF6 and RIP1,which mediate NF-�B activation. TLR4, but not TLR3, utilizes TRAM for activation of the TRIF-dependent pathway. In pDC, a signaling complex consistingof MyD88-TRAF6-IRAK4-IRAK1-IRF7 is formed. In this complex, IRF7 is directly phosphorylated by IRAK1, and then translocated to the nucleus to inducee re alsI tment

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xpression of type I IFN and IFN-inducible genes. OPN-i, TRAF3 and IKK� aRF7, similar to IRAK1. TLR3, TLR7 and TLR9 locate in intracellular compar

. TRIF-dependent pathway

MyD88-deficient mice show a failure to activate NF-�B andAPK and produce inflammatory cytokines in response to lig-

nds specific for TLR2, 5, 7 and 9. Although MyD88-deficientacrophages also fail to produce inflammatory cytokines in

esponse to LPS, they appear to activate NF-�B and MAPK,lbeit with delayed kinetics [13]. Furthermore, activation ofRF3 and subsequent induction of IFN� after treatment withLR3 and TLR4 ligands is normal in MyD88-deficient mice

14]. These observations strongly suggest the presence of ayD88-independent pathway in TLR3 and TLR4 signaling. In

hese regards, TRIF was identified as an essential adapter ofhe MyD88-independent pathway [15,16]. TRIF, overexpression

f which activates IRF3 and NF-�B, is recruited to TLR3 andLR4. TRIF-deficient mice consistently show defective IRF3ctivation and IFN� induction after LPS and poly IC stimula-ion [17,18]. Moreover, late phase NF-�B and MAPK activation

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o involved in this complex. Among these, IKK� is capable of phosphorylatings such as endosomes.

fter LPS stimulation is totally abolished in MyD88 and TRIFouble-deficient mice, indicating that TRIF-dependent signal-ng contributes to late phase activation of NF-�B and MAPKFig. 1) [17].

The N-terminal and the C-terminal regions of TRIF haveistinct functions with regards to the recruitment of signal-ng molecules. The N-terminal region of TRIF activates bothFN� and NF-�B promoters, whereas the C-terminal regionctivates NF-�B but not the IFN� promoter. The N-terminalegion recruits non-canonical IKKs, TBK1 (T2K, NAK) andKKi (IKK�), which phosphorylate serine/threonine clustersresent in the C-terminal region of IRF3 [19,20]. Phosphory-ated IRF3 forms a dimer, which translocates from cytoplasm tohe nucleus to induce expression of target genes including IFN�

Fig. 1). Cells lacking both TBK1 and IKKi show a loss of IRF3ctivation and IFN� induction in response to TLR3 and TLR4igands, whereas activation of NF-�B and MAPK and inflam-

atory cytokine induction is unaffected in these cells [21–23].

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oreover, the N-terminal region of TRIF recruits TRAF6 viaRAF6-binding motifs [24]. Dominant-negative TRAF6 pre-ents TRIF-induced NF-�B activation and mutations in theRAF6-binding motifs of TRIF abrogate NF-�B activation,

ndicating the importance of TRAF6 in TRIF-dependent NF-B activation. The C-terminal region of TRIF contains a RHIMRip homotypic interaction motif), which mediates its interac-ion with RIP1, a member of the RIP family involved in TNFeceptor-mediated NF-�B activation [25]. TLR3- and TLR4-ediated NF-�B activation and the subsequent induction of

arget genes are impaired in the absence of RIP1, indicating thatIP1 participates in TRIF-dependent NF-�B activation. Further-ore, RIP1 is reportedly polyubiquitinated and forms a complexith TRAF6 and TAK1 [26]. Together, the recruitment by TRIFf RIP1 and TRAF6 might facilitate TAK1 activation, result-ng in activation of NF-�B and MAPK (Fig. 1). It is currentlyncertain whether Ubc13 is a component of the complex thatediates RIP1 polyubiqutination.Recent reports have suggested that TRIF-dependent IRF3

ctivation is tightly linked to late phase NF-�B activation27,28]. LPS-induced late phase NF-�B activation seems toequire de novo synthesis of TNF�. Indeed, a reduction inRF3 expression by siRNA results in the inhibition of latehase NF-�B activation. Thus, it is possible that IRF3, activatedy the TRIF-dependent pathway, binds the promoter regionf the TNF� gene to promote the synthesis of TNF�, whichubsequently binds TNF receptor to initiate late phase NF-�Bctivation in an autocrine manner; however, detailed analyses ofRF3-deficient mice will be required to support these findings.

oreover, given that RIP1 is critically involved in TNF receptor-ediated NF-�B activation, it is possible that the defects inRIF-dependent NF-�B activation observed in cells from RIP1-eficient mice are due to defective TNF receptor signaling inhose cells.

. IRFs in TLR signaling

There are nine members of the IRF family (IRF1 to IRF9),nd several IRFs, in addition to IRF3, are critically involvedn TLR-signaling [29]. IRF7, which is also present in the cyto-lasm and translocates to the nucleus after phosphorylation, istructurally the most similar to IRF3. IRF7 potently activateshe promoter of IFN� and various IFN� genes. Unlike IRF3,hich is ubiquitously expressed, IRF7 expression is weak innstimulated condition, but rapidly upregulated in response toiral infection or TLR-ligands in most cell types, suggesting aositive feedback regulation of type I IFN induction [29]. How-ver, IRF7 is constitutively expressed in plasmacytoid DC (pDC)also known as type I IFN-producing cells), which have the abil-ty to rapidly produce vast amounts of type I IFN in responseo a wide variety of viruses [30,31]. TLR7 and TLR9 are abun-antly expressed on pDC. Notably, TLR7- and TLR9-mediatedype I IFN induction is elicited by the MyD88-dependent path-

ay, but not by the TRIF-dependent pathway [32,33]. In these

ontexts, it has been shown that IRF7 forms a complex withyD88, IRAK1, IRAK4 and TRAF6 that translocates into the

ucleus in response to CpG DNA in pDC [34,35]. Although

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munology 19 (2007) 24–32 27

ice deficient for MyD88, IRAK4 or TRAF6 have defects inoth IRF7 and NF-�B activation, with a concomitant impair-ent in the induction of type I IFN and inflammatory cytokines

n response to CpG DNA, IRAK1 deficiency results in the lossf IRF7 activation without affecting NF-�B activity [36]. More-ver, IRAK1, but not IRAK4, is capable of phosphorylatingRF7. Together, IRAK1 is most likely a kinase that phospho-ylates IRF7 in pDC [36]. Recently, IKK� has been reportedo have a similar role to IRAK1, having the ability to bind andhosphorylate IRF7 [37]. Although the functional relationshipsetween IRAK1 and IKK� remain unclear, it is possible that theyunction as a heterodimer to potentiate IRF7 activation. Alter-atively, they might phosphorylate different residues of IRF7,oth of which are required for the activation. IRF7 also partic-pates in type I IFN induction activated by the cytosolic RNAelicases RIG-I and Mda5, which recognize viral dsRNA, inonventional DC, macrophages and fibroblast cells [38,39]. Inhese pathways, IRF7 is phosphorylated by TBK1/IKKi but notRAK1/IKK�. Thus, unlike TLR7 and TLR9, which requireRAK1 and IKK� for optimal IRF7 phosphorylation, RIG-I and

da5 use TBK1/IKKi to activate both IRF3 and IRF7. Collec-ively, IRF7 is the master regulator for type I IFN induction inoth TLR-dependent and TLR-independent cytosolic pathways.

Additional components involved in the MyD88-IRF7 com-lex in pDC have been recently identified. TRAF3, a member ofhe TRAF family, is critical for type I IFN induction in TLR7-nd TLR9-signaling [40,41]. TRAF3 binds MyD88, IRAK1,nd perhaps IKK�, modulating IRF7 activation. Furthermore,RAF3 is also required for TBK1/IKKi-dependent IRF7 and

RF3 activation in TLR3 and RIG-I/Mda5-signaling, indicat-ng that TRAF3 is an integral component in the activation ofRF7 and IRF3 by multiple pathways. Notably, TRAF3 is alsoecessary for the induction of anti-inflammatory cytokine IL-0, but not pro-inflammatory cytokines, in response to ligandspecific for TLR3, 4, 7 and 9. Furthermore, IRAK1-deficientplenocytes show a remarkable reduction in IL-10 after LPSreatment [42]. Thus, the IRAK1-TRAF3-dependent pathway

ight control the release of IL-10 as well. Osteopontin (OPN)s a secreted phosphoprotein implicated in diverse cellular func-ions, including bone resorption, vascularization, inflammationnd Th1 polarization. In pDC, OPN expression is upregulated byLR9 ligand through T-bet, a master transcription factor drivingh1 differentiation. pDC isolated from either T-bet- or OPN-eficient mice have defects in the induction of type I IFN inesponse to TLR9-ligand, but they can produce NF-�B-drivenL-6 as usual [43]. A previous report showing that a precursorf OPN (OPN-i) is retained in the cytoplasm suggests a possibleunction of OPN-i as an intracellular signaling molecule. In thisontext, it was shown that expression of a mutant OPN lack-ng the signal sequence stimulates IFN� production by pDC,ut not by conventional DC, and that OPN-i interacts and colo-alizes with MyD88 [43]. Furthermore, nuclear translocation ofRF7 in response to a TLR9 ligand is impaired in OPN-deficient

DC. These observations suggest that intracellular OPN is aomponent of the MyD88-IRF7 complex in pDC.

IRF1 also participates in TLR9 signaling [44]. IRF1 directlyinds the central region of MyD88 and is released into the

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ucleus in response to stimulation. Conventional DC derivedrom IRF1-deficient mice display impaired induction of IFN�,nducible nitric-oxide synthase and IL-12 p35, in response to

TLR9 ligand. In contrast, pDC derived from IRF1-deficientice show normal induction of IFN� and IFN�, suggesting a

ell type-specific involvement of IRF1 in MyD88-dependent sig-aling. It is well known that cytokine induction is enhancedy pretreatment with IFN� in response to TLR ligands. AsFN� stimulation induces IRF1 expression, it is possible thatRF1 is involved in IFN�-mediated enhancement of TLR signal-ng. Consistently, this enhancement is ablated in IRF1-deficient

ice. Thus, IRF1 is recruited to MyD88 when it is induced byFN� stimulation, and translocates into the nucleus in responseo TLR stimulation to induce a set of genes including IFN� inonventional DC.

In addition to IRF3, IRF7 and IRF1, IRF5 is also involvedn TLR signaling [45]. IRF5-deficient conventional DC and

acrophages exhibit impaired inflammatory cytokine produc-ion against multiple TLR ligands, whereas they exhibit normalecretion of type I IFN by pDC. IRF5 binds MyD88 andRAF6 and moves to the nucleus after phosphorylation. In theucleus, IRF5 binds ISRE motifs found in the promoter region ofenes encoding inflammatory cytokines to cause their expres-ion, presumably via collaborative activation with NF-�B. Asnflammatory cytokine induction after TLR ligation occurs inde-endently of TBK1/IKKi and IRAK1, other protein kinases suchs TAK1, IRAK4 and canonical IKKs, which mediate MyD88-ependent inflammatory cytokine induction, might participaten IRF5 phosphorylation. In humans, increased expression of

ultiple unique isoforms of IRF5 due to mutations is associatedith the pathogenesis of systemic lupus erythematosus (SLE), a

ystemic autoimmune disease exhibiting elevated levels of typeIFN [46].

IRF8 is implicated in TLR9-mediated responses. pDCerived from IRF8-deficient mice show a loss of TLR9-mediatednduction of type I IFN and inflammatory cytokines. These cellslso show a severe impairment in the DNA-binding activity ofF-�B in response to TLR9-ligand [47]. These data strongly

uggest the possibility that IRF8 facilitates NF-�B DNA-bindingctivity or is an activator for the IKK complex.

. TLR localization and signaling

TLRs can be classified into two groups based on subcellu-ar localization. The first group includes TLR1, 2, 4, 5 and 6,hich are all present at the plasma membrane [1]. The secondroup includes TLR3, 7, 8 and 9, which localize to intracellularompartments such as endosomes [1]. Intracellular TLRs senseiral and bacterial nucleic acids in particular. Viral particlesre endocytosed and degraded in late endosomes or lysosomes,nd this degradation causes the release of viral DNA and RNA,llowing TLRs to contact them. Moreover, it has been suggestedhat the intracellular localization of these TLRs is important to

nable discrimination of viral DNA and self-DNA. When theransmembrane and cytoplasmic region of TLR9 is replaced tohat of TLR4, the chimeric protein (TLR9N4C) is traffickedo the plasma membrane [48]. Whereas TLR9N4C responds to

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munology 19 (2007) 24–32

pG DNA, it has no ability to sense viral DNA. More impor-antly, when TLR9N4C is expressed on the cell surface of

acrophages, these cells respond to self-DNA. As abnormalecognition of self-DNA is associated with the pathogenesis ofutoimmune diseases, intracellular localization of TLR9 mighte the safeguards against contact with self-DNA.

In pDC, TLR9 acts as a sensor of viral infection, which leadso the transcription of type I IFN, particularly IFN�, throughhe MyD88-IRF7 pathway. In other cells, such as conventionalC or macrophages, TLR9 ligands poorly induce type I IFN but

hey can still induce inflammatory cytokines. One mechanismo explain these differences is that pDC express IRF7, but otherells do not. However, the spatiotemporal regulation of TLR9ignaling has been recently proposed as an alternative expla-ation for the ability of pDC to robustly produce IFN� [49]./D-type CpG DNA (CpG-A), a TLR9 ligand that strongly

nduces IFN� production from pDC, co-localizes with TLR9,yD88 and IRF7 in endosomes in pDC. By contrast, CpG-A is

apidly transferred and degraded in the lysosome in conventionalC and macrophages. However, when CpG-A is relocalized

o the endosome in conventional DC using a cationic lipid,hese cells can produce IFN� as a consequence of activationf the MyD88-IRF7 pathway. B/K-type CpG DNA (CpG-B), aonventional TLR9 ligand that poorly induces type I IFN, butnduces inflammatory cytokines, also elicits IFN� induction ift is manipulated to stay longer in the endosome of conventionalC [49]. These findings suggest that retention of the CpG DNA-LR9 complex in the endosome might cause the induction of

obust IFN� production.Signaling via intracellular TLR3, 7 and 9 is abrogated in

D mice, which have a single missense mutation in the genencoding UNC-93B, a twelve membrane spanning protein thatesides in the endoplasmic reticulum (ER) [50]. These micehow defective TNF� production as well as reduced upregu-ation of co-stimulatory molecules in response to TLR3, 7 and 9igands. Furthermore, these mice have no ability to present pep-ides derived from exogeneous antigens. Although it remainsnclear how ER-resident UNC-93B controls the function ofLR3, 7 and 9, it is possible that it regulates communicationetween the ER and the endosome.

In contrast to intracellular TLRs, which utilize MyD88 orRIF, plasma membrane-localized TLRs, such as TLR1, 2, 4nd 6, use TIRAP and/or TRAM as additional adapters, sug-esting a link between adapter usage and TLR localization.RAM localizes to the plasma membrane and Golgi appara-

us, where it colocalizes with TLR4, but not TLR3 [51]. TRAMossessing a mutation in a putative myristoylation site at the N-erminus distributes to the cytoplasm and abrogates activation ofF-�B and IRF3, indicating the importance of its plasma mem-rane localization. Myristoylated proteins are phosphorylatedy PKC resulting in dissociation from the plasma membrane;ccordingly, TRAM is phosphorylated at serine 16 by PKC� inesponse to LPS [52]. This phosphorylation is required for acti-

ation of NF-�B and MAPK in LPS-stimulated cells as well asor clearing infectious bacteria. Collectively, phosphorylationf TRAM by PKC� at the plasma membrane and subsequentissociation from the membrane is a prerequisite to activating

T. Kawai, S. Akira / Seminars in Immunology 19 (2007) 24–32 29

Fig. 2. Negative regulators for TLR signaling. TLR signaling is negatively regulated by multiple pathways. SIGIRR, ST2L and RP105 antagonize TLR signaling.T AK1d d �-ao ly reg

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riad3 downregulates the expression of TLR4 and TLR9. IRAK-M, MyD88c, IRownregulate the expression of TIRAP and TRIF, respectively. TRAF4, A20 anf IRF5 to MyD88. PIN1 mediates the degradation of IRF3, and ATF3 negative

he TRIF-dependent pathway. It is currently unclear how LPSctivates PKC� in TLR4 signaling. TIRAP contains a phos-hatidylinositol 4,5-bisphosphate (PIP2) binding domain, whichargets TIRAP to the plasma membrane and facilitates interac-ions with TLR4 [53]. Mutations in this PIP2 binding domainbrogate the ability of TIRAP to localize to the plasma mem-rane and to induce inflammatory cytokines in response to LPS,uggesting that PIP2-dependent recruitment of TIRAP to thelasma membrane is important for TLR4 signaling. Integrinignaling regulates the production of PIP2 through the activa-ion of ARF6 GTPase and PI5K. Macrophages lacking integrinD11b show a loss of TIRAP localization to the plasma mem-rane and a hypo-responsiveness to LPS. These findings suggesthat TLR4 recruits TIRAP to the plasma membrane as a resultf CD11b-mediated production of PIP2, which subsequentlyecruits MyD88 after which the signaling complex is assembledo initiate the MyD88-dependent signaling.

. Negative regulators

Negative regulation of TLR signaling is essential for limitingnflammation and a number of molecules have been identifiedhat serve to negatively regulate TLR signaling. Some of these

olecules downregulate TLR expression, whereas others neg-

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c and IRAK2c/d inhibit signaling downstream of MyD88. SOCS1 and TRAF1rrestins (�-arr) bind and block TRAF6 function. IRF4 prevents the recruitmentulates NF-�B activation.

tively regulate TLR signaling via sequestration of signalingolecules, blockade of their recruitment, degradation of target

roteins and inhibition of transcription (Fig. 2). Furthermore,ost of these proteins are inducible, suggesting a negative feed-

ack regulation of TLR-dependent innate immune responses.RP105, originally identified as a B cell-specific surface pro-

ein that triggers proliferation, contains extracellular LRR withomology to TLR4 and a short cytoplasmic tail [54]. The LRRf RP105 associate with MD-1 to form a complex on the cellurface [55]. RP105 is also expressed on non-B cells, and theP105-MD-1 complex interacts with a TLR4-MD-2 complex

o prevent LPS binding to TLR4-MD-2. RP105-deficient miceisplay hyper-production of inflammatory cytokines in responseo LPS, but respond normally to CpG DNA, indicating thatP105-MD-1 specifically inhibits TLR4-responses [56]. ST2Lelongs to the IL-1 receptor family, comprised of an extracellu-ar immunogloblin-like domain and an intracellular TIR domain.T2L-deficient mice show enhanced production of inflamma-

ory cytokines in response to LPS, and impaired induction of LPSolerance [57]. ST2L sequesters MyD88 and TIRAP to inhibit

he recruitment of these adapters to TLR4. SIGIRR also belongso the IL-1 receptor family, but lacks two amino acid residuesssential for signaling [58]. Kidney and intestinal epithelial cellsnd splenocytes derived from SIGIRR-deficient mice are hyper-

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esponsive to LPS and CpG DNA with respect to increasedctivation of NF-�B and JNK. SIGIRR interacts with IRAKsnd TRAF6 to block TLR signaling. Triad3A is an E3 ligasehat binds the TIR domain of TLR4 and 9, but not that of TLR2.riad3 overexpression promotes degradation of TLR4 and 9ia a proteasome-dependent pathway; conversely, a reduction inriad3A expression by siRNA increases the expression of theseLRs with a concomitant activation of signaling molecules [59].hese data suggest that Triad3A represents a negative regulatorf TLR signaling that downregulates the expression of TLR4nd 9.

Several intracellular proteins have been identified that neg-tively regulate the function of TIR domain-containing adapterroteins. Among IRAK family members, IRAK-M lacks intrin-ic kinase activity, and overexpression of IRAK-M results inrevention of the dissociation of IRAK4 and IRAK1 fromyD88 [60]. Accordingly, IRAK-M-deficient mice secreteore inflammatory cytokines in response to various TLR ligands

han wild type mice do. The finding that IRAK-M is LPS-nducible suggests a negative feedback function of IRAK-M inLR signaling. Splicing variants of IRAK1 (IRAK1c), IRAK2

IRAK2c, IRAK2d) and MyD88 (MyD88s) have been isolatednd implicated in antagonizing TLR signaling [61,62]. IRAK1cacks kinase activity and, therefore, interferes with TLR sig-aling. IRAK2c and IRAK2d lack a death domain, which isequired for interaction with MyD88, to prevent the recruit-ent of MyD88. MyD88s forms a heterodimer with MyD88.he MyD88s-MyD88 heterodimer is unable to recruit IRAK4,

hereby inhibiting IRAK1 phosphorylation. SOCS1, an E3 ligasenitially identified to suppress JAK-STAT pathways, also sup-resses signaling downstream of TLR2 and TLR4 [63]. TIRAPs phosphorylated by a tyrosine kinase, Btk, in response toLR2 and TLR4 ligands, and interacts with SOCS1, whichubsequently targets TIRAP for ubiquitination and degradation63].

Whereas TRAF3 and TRAF6 positively regulate TLR signal-ng, TRAF1 and TRAF4 are implicated in negatively regulatingLR signaling. TRAF1 interacts with TRIF and overexpressionf TRIF causes Caspase 8-dependent cleavage of TRAF1 [64].he cleaved fragment of TRAF1 can inhibit TRIF-dependentctivation of NF-�B and IRF3, suggesting that TRIF-inducedleavage of TRAF1 is responsible for shutdown of the TRIF-ependent pathway. TRAF4 interacts with TRIF and TRAF6,nd its overexpression results in inhibition of NF-�B activa-ion induced by TRIF and TRAF6 [65]. TRAF4 also suppresses

yD88-dependent NF-�B activation. Therefore, TRAF4 mayerve to antagonize TRAF6 function by preventing recruit-ent of TRAF6 to the adapter complex. Additionally, the

unction of TRAF6 is negatively regulated by several indepen-ent mechanisms. In response to LPS, �-arrestins interact withRAF6 to prevent its oligomerization, resulting in inhibition ofRAF6 polyubiquitination and subsequent activation of NF-�Bnd MAPK [66]. A20, an inducible de-ubiqutination enzyme,

emoves ubiquitin moieties from TRAF6 to terminate TLR sig-aling [67]. A20-deficient mice consistently show enhancedF-�B activation in response to multiple TLR ligands, includingLR2, 4 and 9 [67].

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munology 19 (2007) 24–32

The activation of transcription factors is also negatively reg-lated. Phosphorylation of the C-terminal region of IRF3 byBK1/IKKi is essential for transcriptional activity. IRF3 is alsohosphorylated at Ser339, and this phosphorylation is linkedo IRF3 destabilization rather than activation. The cytoplasmiceptidyl-prolyl-isomerase Pin1, which catalyzes the cis-transsomerization of peptide-bonds located N-terminal to prolineo modulate substrate function, binds IRF3 when it is phos-horylated at Ser339, triggering ubiquitination and subsequentegradation of IRF3 by a proteasome-dependent pathway toerminate IFN responses [68]. LPS stimulation leads to thexpression of ATF3, a member of the ATF/CREB family ofranscription factors. ATF3-deficient macrophages produce highmounts of IL-6 and IL-12. Mechanistically, ATF3 recruits his-one deacetylases, which alter chromatin structure to restrictccess of NF-�B and AP-1 to the promoter region of genesncoding IL-6 and IL12b, thereby repressing expression [69].RF4 is reported to bind MyD88 [70]. Whereas IRF7 binds-terminal MyD88, IRF5 and IRF4 interact with the same

entral region of MyD88. Macrophages derived from IRF4-eficient mice show markedly high expression of inflammatoryytokine genes, which depend on IRF5 [70]. As expressionf the IRF4 gene is upregulated after exposure with TLRigands, the induced IRF4 might associate with MyD88 to pre-ent recruitment of IRF5, thereby attenuating IRF5-dependentnflammatory responses.

. Future perspectives

Recent progress has revealed that TLR responses are tightlyontrolled by multiple mechanisms to induce appropriateesponses against diverse microbial pathogens. First, TLRs uti-ize different combinations of adapter molecules, which activateifferent transcription factors to elicit specific innate immuneesponses. Second, several TLRs are expressed in the intracellu-ar compartments to detect nucleic acids derived from microbialathogens, and the intracellular localization of TLRs is likelyo be important for discrimination between self- and non-selfucleic acids. Cellular localization of TIR domain-containingdapters is also important to determine the specificity of sig-aling and responses. Third, TLR expression and signaling areightly controlled by a number of negative regulators to termi-ate immune and inflammatory responses and prevent excessivenflammation. Despite their beneficial roles in host defense,berrant activation of TLR signaling and abnormal cellular local-zation of TLRs are suggested to contribute to the pathogenesisf autoimmune diseases such as SLE.

Accumulating evidence has shown that cells express addi-ional PRRs in the cytoplasm. These include the RNA helicasesIG-I and Mda5, which recognize cytoplasmic dsRNA, proteinsf the NLR (NACHT-LRR) family, which sense intracellu-ar bacterial PAMPs and even host-derived molecules such asonosodium urate crystal and calcium pyrophosphate dihy-

rate, and an as-yet-unidentified cytosolic receptor(s) thatecognizes cytosolic double-stranded DNA released from DNAiruses, bacteria and host damaged cells [1,71,72]. Thus, annderstanding of the precise mechanisms of how PRRs activate

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nnate immune cells, and how they discriminate between hostnd pathogens, will be required to improve therapeutic strategieshat control infectious, inflammatory and autoimmune diseases.

cknowledgement

We thank members of our lab for helpful discussions.

eferences

[1] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immu-nity. Cell 2006;124:783–801.

[2] Uematsu S, Jang MH, Chevrier N, Guo Z, Kumagai Y, Yamamoto M, etal. Detection of pathogenic intestinal bacteria by Toll-like receptor 5 onintestinal CD11c+ lamina propria cells. Nat Immunol 2006;7:868–74.

[3] Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol2004;4:499–511.

[4] Carty M, Goodbody R, Schroder M, Stack J, Moynagh PN, Bowie AG.The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling. Nat Immunol 2006;7:1074–81.

[5] Chen ZJ. Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol2005;7:758–65.

[6] Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat CellBiol 2002;4:131–6.

[7] Sato S, Sanjo H, Takeda K, Ninomiya-Tsuji J, Yamamoto M, Kawai T, etal. Essential function for the kinase TAK1 in innate and adaptive immuneresponses. Nat Immunol 2005;6:1087–95.

[8] Yamamoto M, Okamoto T, Takeda K, Sato S, Sanjo H, Uematsu S, et al.Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immunereceptor signaling. Nat Immunol 2006;7:962–70.

[9] Huang Q, Yang J, Lin Y, Walker C, Cheng J, Liu ZG, et al. Differential regu-lation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3.Nat Immunol 2004;5:98–103.

10] Dumitru CD, Ceci JD, Tsatsanis C, Kontoyiannis D, Stamatakis K, Lin JH,et al. TNF-alpha induction by LPS is regulated posttranscriptionally via aTpl2/ERK-dependent pathway. Cell 2000;103:1071–83.

11] Papoutsopoulou S, Symons A, Tharmalingham T, Belich MP, Kaiser F,Kioussis D, et al. ABIN-2 is required for optimal activation of Erk MAPkinase in innate immune responses. Nat Immunol 2006;7:606–15.

12] Matsuzawa A, Saegusa K, Noguchi T, Sadamitsu C, Nishitoh H, NagaiS, et al. ROS-dependent activation of the TRAF6-ASK1-p38 pathway isselectively required for TLR4-mediated innate immunity. Nat Immunol2005;6:587–92.

13] Kawai T, Adachi O, Ogawa T, Takeda K, Akira S. Unresponsiveness ofMyD88-deficient mice to endotoxin. Immunity 1999;11:115–22.

14] Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S, etal. Lipopolysaccharide stimulates the MyD88-independent pathway andresults in activation of IFN-regulatory factor 3 and the expression of a subsetof lipopolysaccharide-inducible genes. J Immunol 2001;167:5887–94.

15] Yamamoto M, Sato S, Mori K, Hoshino K, Takeuchi O, Takeda K, et al. Anovel Toll/IL-1 receptor domain-containing adapter that preferentially acti-vates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol2002;169:6668–72.

16] Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediatedinterferon-beta induction. Nat Immunol 2003;4:161–7.

17] Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, et al. Roleof adaptor TRIF in the MyD88-independent toll-like receptor signalingpathway. Science 2003;301:640–3.

18] Hoebe K, Du X, Georgel P, Janssen E, Tabeta K, Kim SO, et al. Identifi-

cation of Lps2 as a key transducer of MyD88-independent TIR signalling.Nature 2003;424:743–8.

19] Sharma S, tenOever BR, Grandvaux N, Zhou GP, Lin R, Hiscott J. Trig-gering the interferon antiviral response through an IKK-related pathway.Science 2003;300:1148–51.

[

[

munology 19 (2007) 24–32 31

20] Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, GolenbockDT, et al. IKKepsilon and TBK1 are essential components of the IRF3signaling pathway. Nat Immunol 2003;4:491–6.

21] McWhirter SM, Fitzgerald KA, Rosains J, Rowe DC, Golenbock DT, Mani-atis T. IFN-regulatory factor 3-dependent gene expression is defective inTbk1-deficient mouse embryonic fibroblasts. Proc Natl Acad Sci USA2004;101:233–8.

22] Hemmi H, Takeuchi O, Sato S, Yamamoto M, Kaisho T, Sanjo H, etal. The roles of two IkappaB Kinase-related kinases in lipopolysaccha-ride and double stranded RNA signaling and viral infection. J Exp Med2004;199:1641–50.

23] Perry AK, Chow EK, Goodnough JB, Yeh WC, Cheng G. Differ-ential requirement for TANK-binding kinase-1 in type I interferonresponses to Toll-like receptor activation and viral infection. J Exp Med2004;199:1651–8.

24] Sato S, Sugiyama M, Yamamoto M, Watanabe Y, Kawai T, Takeda K, et al.Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)associates with TNF receptor-associated factor 6 and TANK-bindingkinase 1, and activates two distinct transcription factors, NF-kappa B andIFN-regulatory factor-3, in the Toll-like receptor signaling. J Immunol2003;171:4304–10.

25] Meylan E, Burns K, Hofmann K, Blancheteau V, Martinon F, Kelliher M, etal. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappaBactivation. Nat Immunol 2004;5:503–7.

26] Cusson-Hermance N, Lee TH, Fitzgerald KA, Kelliher MA. Rip1 mediatesthe Trif-dependent toll-like receptor 3 and 4-induced NF-kappa B activa-tion but does not contribute to IRF-3 activation. J Biol Chem 2005;280:36560–6.

27] Covert MW, Leung TH, Gaston JE, Baltimore D. Achieving sta-bility of lipopolysaccharide-induced NF-kappaB activation. Science2005;309:1854–7.

28] Werner SL, Barken D, Hoffmann A. Stimulus specificity of gene expres-sion programs determined by temporal control of IKK activity. Science2005;309:1857–61.

29] Honda K, Taniguchi T. IRFs: master regulators of signalling by Toll-likereceptors and cytosolic pattern-recognition receptors. Nat Rev Immunol2006;6:644–58.

30] Colonna M, Trinchieri G, Liu YJ. Plasmacytoid dendritic cells in immunity.Nat Immunol 2004;5:1219–26.

31] Liu YJ. IPC: professional Type 1 interferon-producing cells and plas-macytoid dendritic cell precursors. Annu Rev Immunol 2005;23:275–306.

32] Hoshino K, Kaisho T, Iwabe T, Takeuchi O, Akira S. Differential involve-ment of IFN-beta in Toll-like receptor-stimulated dendritic cell activation.Int Immunol 2002;14:1225–31.

33] Hemmi H, Kaisho T, Takeda K, Akira S. The roles of Toll-like receptor9, MyD88, and DNA-dependent protein kinase catalytic subunit in theeffects of two distinct CpG DNAs on dendritic cell subsets. J Immunol2003;170:3059–64.

34] Kawai T, Sato S, Ishii KJ, Coban C, Hemmi H, Yamamoto M, etal. Interferon-alpha induction through Toll-like receptors involves adirect interaction of IRF7 with MyD88 and TRAF6. Nat Immunol2004;5:1061–8.

35] Honda K, Yanai H, Mizutani T, Negishi H, Shimada N, Suzuki N, etal. Role of a transductional-transcriptional processor complex involvingMyD88 and IRF-7 in Toll-like receptor signalling. Proc Natl Acad SciUSA 2004;101:15416–21.

36] Uematsu S, Sato S, Yamamoto M, Hirotani T, Kato H, Takeshita F, et al.Interleukin-1 Receptor-Associated Kinase-1 (IRAK-1) plays an essentialrole for TLR7- and TLR9-mediated interferon-a induction. J Exp Med2005;201:915–23.

37] Hoshino K, Sugiyama T, Matsumoto M, Tanaka T, Saito M, Hemmi H, etal. IkappaB kinase-alpha is critical for interferon-alpha production induced

by Toll-like receptors 7 and 9. Nature 2006;440:949–53.

38] Meylan E, Tschopp J, Karin M. Intracellular pattern recognition receptorsin the host response. Nature 2006;442:39–44.

39] Meylan E, Tschopp J. Toll-like receptors and RNA helicases: two parallelways to trigger antiviral responses. Mol Cell 2006;22:561–9.

3 s in Im

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

2 T. Kawai, S. Akira / Seminar

40] Hacker H, Redecke V, Blagoev B, Kratchmarova I, Hsu LC, Wang GG,et al. Specificity in Toll-like receptor signalling through distinct effectorfunctions of TRAF3 and TRAF6. Nature 2006;439:204–7.

41] Oganesyan G, Saha SK, Guo B, He JQ, Shahangian A, Zarnegar B, et al.Critical role of TRAF3 in the Toll-like receptor-dependent and -independentantiviral response. Nature 2006;439:208–11.

42] Huang Y, Li T, Sane DC, Li L. IRAK1 serves as a novel regulator essen-tial for lipopolysaccharide-induced interleukin-10 gene expression. J BiolChem 2004;279:51697–703.

43] Shinohara ML, Lu L, Bu J, Werneck MB, Kobayashi KS, Glimcher LH, etal. Osteopontin expression is essential for interferon-alpha production byplasmacytoid dendritic cells. Nat Immunol 2006;7:498–506.

44] Negishi H, Fujita Y, Yanai H, Sakaguchi S, Ouyang X, Shinohara M, et al.Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 tran-scription factor by MyD88 in Toll-like receptor-dependent gene inductionprogram. Proc Natl Acad Sci USA 2006;103:15136–41.

45] Takaoka A, Yanai H, Kondo S, Duncan G, Negishi H, Mizutani T, et al.Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 2005;434:243–9.

46] Graham RR, Kozyrev SV, Baechler EC, Reddy MV, Plenge RM, BauerJW, et al. A common haplotype of interferon regulatory factor 5 (IRF5)regulates splicing and expression and is associated with increased risk ofsystemic lupus erythematosus. Nat Genet 2006;38:550–5.

47] Tsujimura H, Tamura T, Kong HJ, Nishiyama A, Ishii KJ, Klinman DM, etal. Toll-like receptor 9 signaling activates NF-kappaB through IFN regula-tory factor-8/IFN consensus sequence binding protein in dendritic cells. JImmunol 2004;172:6820–7.

48] Barton GM, Kagan JC, Medzhitov R. Intracellular localization of Toll-likereceptor 9 prevents recognition of self-DNA but facilitates access to viralDNA. Nat Immunol 2006;7:49–56.

49] Honda K, Ohba Y, Yanai H, Negishi H, Mizutani T, Takaoka A, et al.Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-Iinterferon induction. Nature 2005;434:1035–40.

50] Tabeta K, Hoebe K, Janssen EM, Du X, Georgel P, Crozat K, et al. TheUnc93b1 mutation 3d disrupts exogenous antigen presentation and signal-ing via Toll-like receptors 3, 7 and 9. Nat Immunol 2006;7:156–64.

51] Rowe DC, McGettrick AF, Latz E, Monks BG, Gay NJ, Yamamoto M,et al. The myristoylation of TRIF-related adaptor molecule is essentialfor Toll-like receptor 4 signal transduction. Proc Natl Acad Sci USA2006;103:6299–304.

52] McGettrick AF, Brint EK, Palsson-McDermott EM, Rowe DC, GolenbockDT, Gay NJ, et al. Trif-related adapter molecule is phosphorylated by PKC{epsilon} during Toll-like receptor 4 signaling. Proc Natl Acad Sci USA2006;103:9196–201.

53] Kagan JC, Medzhitov R. Phosphoinositide-mediated adaptor recruitmentcontrols Toll-like receptor signaling. Cell 2006;125:943–55.

54] Miyake K, Yamashita Y, Ogata M, Sudo T, Kimoto M. RP105, a novel B

cell surface molecule implicated in B cell activation, is a member of theleucine-rich repeat protein family. J Immunol 1995;154:3333–40.

55] Miyake K, Shimazu R, Kondo J, Niki T, Akashi S, Ogata H, et al. MouseMD-1, a molecule that is physically associated with RP105 and positivelyregulates its expression. J Immunol 1998;161:1348–53.

[

[

munology 19 (2007) 24–32

56] Divanovic S, Trompette A, Atabani SF, Madan R, Golenbock DT, VisintinA, et al. Negative regulation of Toll-like receptor 4 signaling by the Toll-likereceptor homolog RP105. Nat Immunol 2005;6:571–8.

57] Brint EK, Xu D, Liu H, Dunne A, McKenzie AN, O’Neill LA, etal. ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor4 signaling and maintains endotoxin tolerance. Nat Immunol 2004;5:373–9.

58] Wald D, Qin J, Zhao Z, Qian Y, Naramura M, Tian L, et al. SIGIRR, anegative regulator of Toll-like receptor-interleukin 1 receptor signaling.Nat Immunol 2003;4:920–7.

59] Chuang TH, Ulevitch RJ. Triad3A, an E3 ubiquitin-protein ligase regulat-ing Toll-like receptors. Nat Immunol 2004;5:495–502.

60] Kobayashi K, Hernandez LD, Galan JE, Janeway CAJ, Medzhitov R,Flavell R. IRAK-M is a negative regulator of Toll-like receptor signaling.Cell 2002;110:191–202.

61] Janssens S, Burns K, Vercammen E, Tschopp J, Beyaert R. MyD88S, asplice variant of MyD88, differentially modulates NF-kappaB- and AP-1-dependent gene expression. FEBS Lett 2003;548:103–7.

62] Hardy MP, O’Neill LA. The murine IRAK2 gene encodes four alter-natively spliced isoforms, two of which are inhibitory. J Biol Chem2004;279:27699–708.

63] Mansell A, Smith R, Doyle SL, Gray P, Fenner JE, Crack PJ, et al. Suppres-sor of cytokine signaling 1 negatively regulates Toll-like receptor signalingby mediating Mal degradation. Nat Immunol 2006;7:148–55.

64] Su X, Li S, Meng M, Qian W, Xie W, Chen D, et al. TNF receptor-associated factor-1 (TRAF1) negatively regulates Toll/IL-1 receptordomain-containing adaptor inducing IFN-beta (TRIF)-mediated signaling.Eur J Immunol 2006;36:199–206.

65] Takeshita F, Ishii KJ, Kobiyama K, Kojima Y, Coban C, Sasaki S, et al.TRAF4 acts as a silencer in TLR-mediated signaling through the associa-tion with TRAF6 and TRIF. Eur J Immunol 2005;35:2477–85.

66] Wang Y, Tang Y, Teng L, Wu Y, Zhao X, Pei G. Association of beta-arrestinand TRAF6 negatively regulates Toll-like receptor-interleukin 1 receptorsignaling. Nat Immunol 2006;7:139–47.

67] Boone DL, Turer EE, Lee EG, Ahmad RC, Wheeler MT, Tsui C, et al. Theubiquitin-modifying enzyme A20 is required for termination of Toll-likereceptor responses. Nat Immunol 2004;5:1052–60.

68] Saitoh T, Tun-Kyi A, Ryo A, Yamamoto M, Finn G, Fujita T, et al.Negative regulation of interferon-regulatory factor 3-dependent innateantiviral response by the prolyl isomerase Pin1. Nat Immunol 2006;7:598–605.

69] Gilchrist M, Thorsson V, Li B, Rust AG, Korb M, Kennedy K, et al. Sys-tems biology approaches identify ATF3 as a negative regulator of Toll-likereceptor 4. Nature 2006;441:173–8.

70] Negishi H, Ohba Y, Yanai H, Takaoka A, Honma K, Yui K, et al. Negativeregulation of Toll-like-receptor signaling by IRF-4. Proc Natl Acad SciUSA 2005;102:15989–94.

71] Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature2006;440:237–41.

72] Ishii KJ, Akira S. Innate immune recognition of, and regulation by, DNA.Trends Immunol 2006;27:525–32.