REVIEW

Toll-like receptors and innate immunity

Satoshi Uematsu & Shizuo Akira

Received: 10 April 2006 /Accepted: 8 June 2006 / Published online: 10 August 2006# Springer-Verlag 2006

Abstract The innate immune system is an evolutionallyconserved host defense mechanism against pathogens.Innate immune responses are initiated by pattern recogni-tion receptors (PRRs), which recognize specific structuresof microorganisms. Among them, Toll-like receptors(TLRs) are capable of sensing organisms ranging frombacteria to fungi, protozoa, and viruses, and play a majorrole in innate immunity. However, TLRs recognize patho-gens either on the cell surface or in the lysosome/endosomecompartment. Recently, cytoplasmic PRRs have beenidentified to detect pathogens that have invaded cytosols.In this review, we focus on the functions of PRRs in innateimmunity and their downstream signaling cascades.

Keywords TLR . NLR . RIG-I

Introduction

The innate immune response is the first line of defenseagainst microbial infections. Since the discovery of theDrosophila protein Toll, which induces effective immuneresponses to Aspergillus fumigatus [1], accumulatingevidence showed that the innate immune system specificallyrecognizes invading microorganisms. The targets of innate

immune recognition are the conserved molecular patterns(pathogen-associated molecular patterns, PAMPs) of micro-organisms. Receptors in innate immunity are therefore calledpattern recognition receptors (PRRs) [2]. PAMPs aregenerated by microbes and not by the host, suggesting thatPAMPs are good targets for innate immunity to discriminatebetween self- and nonself. Furthermore, PAMPs are essentialfor microbial survival and are conserved structures amongmany pathogens, which allows innate immunity to recognize

J Mol Med (2006) 84:712–725DOI 10.1007/s00109-006-0084-y

S. Uematsu : S. AkiraDepartment of Host Defense,Research Institute for Microbial Diseases, Osaka University,3-1 Yamada-oka,Suita, Osaka 565-0851, Japan

S. Akira (*)ERATO, Japan Science and Technology Corporation,3-1 Yamada-oka,Suita, Osaka 565-0871, Japane-mail: sakira@biken.osaka-u.ac.jp

Dr. SATOSHI UEMATSU

received his M.D. in 1997 fromOsaka University medicalschool, Osaka City, Japan. Heobtained his Ph.D. in 2004 atOsaka University. He iscurrently an assistant professorof the Department of HostDefense at the Research Institutefor Microbial Diseases, OsakaUniversity, Japan. His researchfocuses on innate immunity,especially the defensemechanism of macrophages anddendritic cells in intracellularbacterial infection.

Dr. SHIZUO AKIRA

received his M.D. and Ph.D.degree from the OsakaUniversity, Japan. He iscurrently a professor in theDepartment of Host Defense atthe Research Institute forMicrobial Diseases, OsakaUniversity. His research interestsfocus on pathogen recognitionand signaling pathways ofToll-like receptors andTLR-independent pathogenrecognition in innate immunity.

microorganisms with limited numbers of PRRs. AmongPRRs, Toll-like receptors (TLRs), mammalian homologs ofToll, were highlighted as the key recognition structures ofthe innate immune system in the past few years [3]. TLRsare capable of sensing organisms ranging from bacteria tofungi, protozoa, and viruses. However, host recognition andresponse is not restricted to TLRs. Some members of theNACHT (domain present in NAIP, CIITA, HET-E, andTP1)-LRR (leucine-rich repeat) (NLR) family, whichincludes both nucleotide-binding oligomerization domain(NOD) proteins and NACHT-LRR- and pyrin-domain-containing proteins (NALPs), recognize microbial compo-nents in the cytosol [4]. Furthermore, the RNA helicase,retinoic acid inducible gene-I (RIG-I), was identified as acytosolic receptor for intracellular double-stranded (dsRNA)[5] and induces type I interferons (IFNs) in response todsRNA viruses in a TLR-independent manner [6]. Therecent understanding of these PRRs in innate immunity andtheir signaling pathways is the focus of this review.

TLR

A mammalian homolog of a Toll receptor (now termedTLR4) was identified through database searches. The TLRfamily now consists of 13 mammalian members: with eachTLR having its intrinsic signaling pathway and inducingspecific biological responses against microorganisms. Rec-ognition of microbial components by TLRs triggers theactivation of signal transduction pathways, which theninduces dendritic cell (DC) maturation and cytokine produc-tion, resulting in development of adaptive immunity [3].

Structure

The cytoplasmic portion of a TLR is similar to that of theinterleukin (IL)-1 receptor family. It is therefore called theToll/IL-1 receptor (TIR) domain. A TIR domain is requiredfor initiating intracellular signaling. The extracellular regionof TLRs and IL-1R are markedly different. Whereas IL-1Rpossesses an Ig-like domain, TLRs contain LRRs in theextracellular domain. LRRs are responsible for the recog-nition of PAMPs [3] (Fig. 1).

Pathogen recognition

Bacteria

Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria, a strong immunostimulant (Table 1). LPSis composed of lipid A (endotoxin), core oligosaccharide,and O-antigen. TLR4 recognizes lipid A of LPS; this occursthrough the TLR4/MD2/CD14 complex, which is present on

various cells such as macrophages and DCs [7]. LPS forms acomplex with an accessory protein, LPS-binding protein inserum, which converts oligomeric micelles of LPS to amonomer for delivery to CD14, a glycosylphosphatidylino-sitol (GPI)-anchored, high-affinity membrane protein thatcan also circulate in a soluble form. CD14 concentrates LPSfor binding to the TLR4/MD2 complex [8].

TLR2 recognizes various microbial components, such aslipoproteins/lipopeptides and peptidoglycans from gram-pos-itive and gram-negative bacteria, and lipoteichoic acid fromgram-positive bacteria, a phenol-soluble modulin from Staph-ylococcus aureus, and glycolipids from Treponema maltophi-lum [3, 9]. TLR2 is also reported to be involved in therecognition of atypical LPS from nonenterobacteria, whosestructures are different from typical LPS of gram-negativebacteria [3, 10]. However, a recent report has indicated thatlipoproteins contaminated in a LPS preparation from Por-phyromonas gingivalis stimulated TLR2 and that LPS from P.gingivalis itself had poor activity for TLR4 stimulation [11].There are also controversial reports regarding peptidoglycanrecognition by TLR2. More careful analyses will be needed toexclude any possibility of contaminates remaining.

TLR1 and TLR6 are structurally related to TLR2 [12].TLR2 and TLR1 or TLR6 form a heterodimer, which isinvolved in the discrimination between the molecularstructure of diacyl and triacyl lipopeptides [13–15]. CD36,a member of the class II scavenger family of proteins wasshown to serve as a facilitator or coreceptor for diacyllipopeptide recognition through the TLR2/6 complex [16].

TLR5 is responsible for the recognition of flagellin, themajor portion of bacterial flagella [17]. Gewirtz et al. [18]reported that TLR5 is expressed on the basolateral surface,but not the apical side of intestinal epithelial cells,suggesting that TLR5 serves as the sensor for thepathogenic bacteria that invade across the epithelium. Acommon stop codon polymorphism in the ligand-bindingdomain of TLR5 (TLR5 392STOP SNP) is unable tomediate flagellin signaling and is associated with suscep-tibility to pneumonia caused by Legionella pneumophila[19]. However, researchers in Vietnam have reported thatthe TLR5 392STOP SNP is not associated with suscepti-bility to typhoid fever [20]. A recent work has reported thatsome commensal bacteria, such as a and e Proteobacteria,change the TLR5 recognition site of flagellin without losingflagellar motility [21]. This modification may contribute tothe persistence of these bacteria on mucosal surfaces.

Bacterial DNA, which contains unmethylated CpGmotifs, is a potent stimulator of the host immune response.In vertebrates, the frequency of CpG motifs is severelyreduced and the cysteine residues of CpG motifs are highlymethylated, which leads to abrogation of the immunostim-ulatory activity. Analysis of TLR9-deficient mice showedthat TLR9 is a receptor for CpG DNA [22].

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Mouse TLR11, a relative of TLR5, is expressed abun-dantly in the kidney and bladder. TLR11-deficient mice aresusceptible to uropathogenic bacterial infections, indicatingthat TLR11 senses the component of uropathogenic bacteria.However, TLR11 is thought to be nonfunctional in humanbecause of the presence of stop codon [23].

Fungi

TLRs are involved in the recognition of fungal pathogenssuch as Candida albicans, A. fumigatus, Cryptococcusneoformans, and Pneumocystis carinii [3, 24] (Table 1).

Several components located in the cell wall or cell surfaceof fungi were identified as potential ligands for TLRs. Yeastzymosan activates TLR2/TLR6 heterodimers, whereasmannan, derived from Saccharomyces cerevisiae and C.albicans is detected by TLR4. Phospholipomannan,present on the cell surface of C. albicans, is recognizedby TLR2, while TLR4 mainly interacts with glucuronox-ylomannan, the major capsular polysaccharide of C.neoformans [24].

Dectin-1 is a lectin family receptor for the fungal cellwall component, b-glucan [25]. Dectin-1 functionallycollaborates with TLR2 and induces a strong immune

Fig. 1 TLR signaling pathways. TLR signaling pathways originatefrom the cytoplasmic TIR domain. A TIR domain-containing adaptor,MyD88, associates with the cytoplasmic TIR domain of TLRs andrecruits IRAKs to the receptor upon ligand binding. IRAKs thenactivates TRAF6, leading to the activation of TAK1. TAK1 activatesthe IKK complex consisting of IKKα, IKKβ, and NEMO/IKKγ. TheIKK complex phosphorylates IκB, resulting in nuclear translocation ofNF-kB, which induces expression of inflammatory cytokines. Amongthe TLR family members, TLR4 on cell surfaces and TLR3, 7, 8, and9 on endosomal membrane can induce type I IFNs. Stimulation ofmacrophages and DCs with LPS and dsRNA results in the activationof IRF3 and subsequent induction of IFN-β and IFN-inducible genes.A TIR domain-containing adapter, TRIF is responsible for TLR3- and

TLR4-mediated IRF3 activation. In addition, TLR4 also requiresanother adaptor, TRAM, for IRF3 activation. TRIF interacts withTBK1, IKKi, RIP1, and TRAF6. IRF3 is activated by TBK1 andIKKi, which catalyze IRF3 phosphorylation and stimulate its nucleartranslocation and DNA binding. pDCs express TLR7 (also TLR8 inhuman) and TLR9 and secrete large amounts of IFN-αs and IFN-β inresponse to ssRNA and CpG DNA. This induction by TLR7 andTLR9 depends entirely on MyD88. After stimulation, IRF7 trans-locates into the nucleus to induce type I IFNs. IRF7 is activatedthrough forming a signaling complex with MyD88, IRAKs, andTRAF6 in the cytoplasm. IRAK-1 but not IRAK-4 is an essentialenzyme for phosphorylation and activation of IRF7 in TLR7- andTLR9-mediated type I IFNs induction

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response to yeast via recruitment of the tyrosine kinase, Syk[26–28]. TLR2 recognizes a variety of microbial productsthrough functional cooperation with several proteins, someof which are structurally related to TLR2 whereas othersare not.

Protozoa

TLRs also sense the components of protozoa includingTrypanosoma cruzi, Trypanosoma brucei, Toxoplasmagondii, Leishmania major, and Plasmodium falciparum(Table 1). GPI anchors, glycoinositolphospholipids, andgenomic DNA-derived from T. cruzi are recognized byTLR2, TLR4, and TLR9, respectively [29]. Murine TLR11senses the profilin-like molecule of T. gondii [30]. Malariaparasites digest host hemoglobin into a hydrophobic hemepolymer known as hemozoin. TLR9 recognizes hemozoinfrom P. falciparum [31]. However, the precise mechanismof how TLR9 recognizes both DNA and non-DNA crystalsremains unclear.

Virus

TLR4 recognizes not only bacterial components but alsoviral envelope proteins (Table 1). The fusion protein fromrespiratory syncytial virus (RSV) is recognized by TLR4[32]. TLR4-mutated C3H/HeJ mice are sensitive to RSVinfection [33]. The envelope protein of mouse mammarytumor virus directly activates B cells via TLR4 [34].

TLR2 is also involved in the recognition of viralcomponents such as measles virus, human cytomegalovirus,and herpes simplex virus type 1 (HSV-1) [35–37].

During viral replication, dsRNA is generated, and TLR3is involved in the recognition of a synthetic analog ofdsRNA, polyinosine–deoxycytidylic acid (poly I:C), apotent inducer of type I IFNs [38, 39]. TLR3-deficientmice are susceptible to mouse cytomegalovirus [40]. On theother hand, TLR3-deficient mice show more resistance toWest Nile virus (WNV) infection. This single-strandedRNA (ssRNA) flavivirus triggers inflammatory responsesvia TLR3, which leads a disruption of the blood brainbarrier, followed by enhanced brain infection [41]. Thesefindings suggested that WNV utilizes TLR3 to efficientlyenter the brain.

Mouse splenic DCs are divided into CD11c high B220−and CD11c dull B220+ cells. The latter contain plasmacytoidDCs (pDCs), which induce large amounts of IFN-α duringviral infection. CpG DNA motifs are also found in genomesof DNA viruses, such as HSV-1, HSV-2, and murinecytomegalovirus (MCMV). Mouse pDCs recognize CpGDNA of HSV-2 via TLR9 and produce IFN-α [42]. TLR9-deficient mice are susceptible to MCMV infection, suggest-ing that TLR9 induces antiviral responses by sensing CpGDNA of DNA virus [40, 43, 44].

TLR7 and TLR8 are structurally highly conservedproteins [12]. The synthetic imidazoquinoline-like mole-cules imiquimod (R-837) and resiquimod (R848) havepotent antiviral activities and are used for clinical treatmentof viral infections. Analysis of TLR7-deficient miceshowed that TLR7 recognizes these synthetic compounds[45]. Human TLR7 and TLR8, but not murine TLR8,recognize imidazoquinoline compounds [46]. Furthermore,murine TLR7 recognizes guanosine analogs such asloxoribine, which has antiviral and antitumor activities[47]. All these compounds are structurally similar toribonucleic acids. After a short while, TLR7 and humanTLR8 also were shown to recognize guanosine- or uridine-rich ssRNA from viruses such as human immunodeficiencyvirus, vesicular stomatitis virus (VSV), and influenza virus[48, 49].

Signaling pathways

The activation of TLR signaling pathways originatesfrom the cytoplasmic TIR domains. Four TIR domain-containing adaptors (MyD88, TIRAP/MAL, TRIF, andTRAM) were shown to play an important role inTLR signaling pathway. These adaptors are associatedwith TLRs through homophilic interaction of TIRdomains. Each TLR mediates distinctive responses inassociation with a different combination of theseadapters (Fig. 1).

Table 1 Pathogen recognition of TLRs

TLRs Ligands

TLR1 Triacyl lipopeptides (bacteria)TLR2 Peptidoglycan, lipoprotein, lipopeptides, atypical

LPS (bacteria)Zymosan, phospholipomannan (fungi)GPI anchor (protozoa)Envelope protein (virus)

TLR3 Poly (I:C), dsRNA (virus)TLR4 LPS (bacteria)

Mannan, glucuronoxylomannan (fungi)Glycoinositolphospholipids (protozoa)RSV fusion protein (virus)

TLR5 Flagellin (bacteria)TLR6 Diacyl lipopeptides (bacteria)TLR7/TLR8 Synthetic imidazoquinoline-like molecules,

ssRNA (virus)TLR9 CpG DNA (bacteria, protozoa, virus)

Hemozoin (protozoa)TLR11 Component of uropathogenic bacteria (bacteria)

Profilin like molecule (protozoa)

TLRs recognize molecular patterns associated with a broad range ofpathogens including bacteria, fungi, protozoa, and viruses

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MyD88-dependent pathway

TLR family members use many of the same signalingcomponents as IL-1R because both have a conservedcytoplasmic domain, the TIR domain. The TIR domain-containing adapter MyD88 is utilized by all TLRs with theexception of TLR3. MyD88 possesses a TIR domain in itsC terminus and a death domain in its N terminus. Uponstimulation, MyD88 recruits IL-1 receptor kinase (IRAK) toTLRs through the interaction of both molecules’ deathdomains. Two members of the IRAK family, IL-1 receptor-associated kinase (IRAK-1) and IRAK-4 are activated byphosphorylation, and associate with TRAF6. TRAF6 formsa complex with ubiquitin-conjugating enzymes to activateTAK1, leading to the activation of nuclear factor (NF)-κB(IκB) and activator protein-1 through the canonical IκBkinase (IKK) complex and the mitogen-activated proteinkinase pathway, respectively. The IKK complex phosphor-ylates the inhibitor of IκB, which sequesters the transcrip-tion factor NF-κB to induce target genes. This signalingpathway is called the “MyD88-dependent pathway” and isessential for the expression of inflammatory cytokinegenes, including TNF-α, IL-6, IL-12, and IL-1β, andcostimulatory molecules [3]. In the case of the TLR2 andTLR4 signaling pathways, TIRAP/MAL is also required forthe activation of the MyD88-dependent pathway [50, 51].

MyD88-independent pathway

In addition to proinflammatory signals, some members ofthe TLR family trigger the induction of type I IFNs. Inparticular, TLR3 and TLR4 have the ability to induce IFN-β and IFN-inducible genes in a MyD88-independentmanner [52]. Thus, this signaling pathway is called theMyD88-independent pathway. A third adapter, TRIF [53,54], is essential for the TLR3- and TLR4-mediatedMyD88-independent pathway [39, 55]. TRIF is alsorequired for the induction of inflammatory cytokines inthe TLR4 signaling pathway.

A fourth adapter, TRAM is essential for the TLR4-mediated MyD88-independent pathway [56–58]. The Nterminus of TRAM has a myristoylation site, the mutationof which alters its normal membrane localization andabolishes the TLR4 signaling. TRAM acts as a bridgingadapter between TLR4 and TRIF [58].

TLR3- and TLR4-mediated signaling activates a memberof the IRF family of transcription factors, IRF3 to induceIFN-β and IFN-inducible genes. Two noncanonical IKKs,inducible IKK (IKKi)/IKKɛ and TRAF family member-associated NF-κB activator-binding kinase 1 (TBK1)/NF-κB-activating kinase (NAK)/TRAF2-associated kinase(T2K), were shown to be responsible for TLR3- andTLR4-mediated IRF-3 activation [59–63]. Upon stimula-

tion, TRIF interacts with TBK1 and IKKi, which phos-phorylates IRF3, allowing IRF3 to translocate into thenucleus and activate the IFN-β promoter.

TRAF6 is reported to bind TRIF and cooperativelyactivate NF-κB [64]. However, cells doubly deficient inTRAF6 and MyD88 still partially activated NF-κB inresponse to LPS, suggesting that TRIF activates NF-κBthrough both TRAF6-dependent and TRAF6-independentpathways in TLR4 signaling [65, 66].

Receptor-interacting protein-1 (RIP1) was implicated inthe TLR3-mediated NF-κB response to dsRNA. RIP1 bindsthe C terminus of TRIF via a RIP homotypic interactionmotif (RHIM). In cells lacking RIP1, TLR3-mediated NF-κB activation and the subsequent induction of intercellularadhesion molecule 1 is severely impaired, whereas IFN-βinduction is intact. By contrast, responses to LPS werenormal in the absence of RIP1. However, LPS fails tostimulate NF-κB activation in rip−/−MyD88−/− cells,revealing that RIP1 is also required for the TRIF-dependentTLR4-induced NF-κB pathway. In the TNF-α pathway,RIP1 interacts with the E3 ubiquitin ligase TRAF2 and ismodified by a polyubiquitin chain. Upon TLR3 activation,RIP1 is also modified by polyubiquitin chains and isrecruited to TLR3 along with TRAF6 and TAK1, suggest-ing that RIP1 uses a similar ubiquitin-dependent mecha-nism to activate IKK-β in response to TNF-α and TLR3ligands [67].

Specific pathway in pDCs

pDCs are specialized for producing large amounts of type IIFNs during viral infection [68]. pDCs highly expressTLR7 and TLR9 and produce high levels of IFN-α inresponse to TLR7 and TLR9 ligands [47]. It appears thatIFN-α induction by TLR7 and TLR9 depends entirely onMyD88 [69]. IRF7 is a transcriptional factor structurallyrelated to IRF3, which is expressed constitutively in pDCs.Overexpression of IRF7 activates IFN-α- and IFN-β-dependent promoters. IRF7 forms a signaling complex withMyD88 and TRAF6 in the cytoplasm [70, 71]. After ligandstimulation, IRF7 translocates into the nucleus to induceIFN-αs [70, 71]. Similar to IRF3, IRF7 is activated by itsphosphorylation. However, in TBK1-deficient cells, TLR9-mediated IFN-α production is still observed [70]. MousepDCs lacking IRAK-4 fails to produce both inflammatorycytokines and IFN-αs [71]. Human TLR7-, TLR8-, andTLR9-mediated induction of IFN-α/β and -λ was alsoIRAK-4 dependent [72]. Recently, IRAK-1 was shown toserve as an IRF7 kinase. In IRAK-1-deficient mice, TLR7-and TLR9-induced IFN-α production was completelyabolished, although inflammatory cytokines are normallyproduced. Furthermore, IRF7 activation by TLR9 ligand isimpaired in IRAK-1-deficient mice, in spite of normal

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NF-κB activation. IRAK-1 but not IRAK-4 can directlybind and phosphorylate IRF7; thus, IRAK-1 specificallymediates IFN-α induction downstream of MyD88 andIRAK-4 [73].

IRF8 is involved in TLR9-mediated responses becausepDCs lacking IRF8 fails to produce proinflammatorycytokines and IFN-α in response to TLR9 ligand; inIRF8-deficient cells, TLR9-stimulated NF-κB activation isunexpectedly impaired, suggesting that IRF8 mediatesNF-κB activation in TLR9 signaling [74].

NLR

Structure

Mammalian NLR family members are also thought torecognize PAMPs. NLR proteins contain three characteristicdomains. The first is a LRR domain, which is involved inligand recognition. The second is a centrally located NOD(also known as a NACHT domain), which acts as an ATP-dependent dimerization domain. The last N-teminal domain iscomposed of protein–protein interaction cassettes. NLRs aredivided into subgroups according to the N-terminal domain:caspase-recruitment domain (CARD), pyrin domain (PYD),baculoviral inhibitory repeat (BIR)-like, and unclassified.NODs, NALPs, and IPAFs contain CARDs, PYDs, and BIR,respectively. All these domains are involved in the regulationof proapoptotic and proinflammatory signaling [75].

Pathogen recognition and signaling pathway

Recent reports showed that some members of the NLRfamily are involved in intracellular recognition of microbialproducts. Although initial studies identified LPS as aNOD2 ligand [76], it is well established now that NOD1and NOD2 recognize peptidoglycan-derived peptides, γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) [77] andmuramyl dipeptide (MDP) [78, 79], respectively. Recogni-tion of ligands through the LRR domains activates NODproteins, which then recruit the serine–threonine kinaseRICK (also known as RIP2 or CARDIACK) through aCARD–CARD interaction [80]. In the NOD2 signalingpathway, activation of RICK leads to K63 (Lys63)-linkedpolyubiquitylation of NEMO/IKKγ, which is followed byphosphorylation of IKKβ and the phosphorylation of IκB,leading to the translocation of NF-κB to the nucleus [81,82]. CARD12 (also known as IPAF or CLAN) binds bothNOD1 and NOD2 through NOD–NOD interactions [83]and inhibits NOD1- and NOD2-mediated activation ofNF-κB in vitro [84]. However, recent reports show thatinvasive bacteria lead to the activation of CARD12 and theinduction of expression of IL-1β [85]. CARD6 was shown

to physically interact with NOD1 and RICK (but not withNOD2) and suppress NOD1/RICK-mediated NF-κB activa-tion [86]. In addition to NF-κB activation and NOD1 andNOD2 signaling give rise to the activation of the mitogen-activated protein kinase (MAPK) pathway although theprecise mechanism remains unknown [4] (Fig. 2).

Inflammasome is a multiprotein complex and is respon-sible for the activation of caspase 1 and 5, leading to theprocessing and secretion of the proinflammatory cytokinesIL-1β and IL-18 [87]. Two types of the inflammasomewere reported to date: NALP1 inflammasome and NALP2/3 inflammasome (Fig. 3). Whereas NALP1 inflammasomeis composed of NALP1, ASC, caspase 1, and caspase 5,NALP2/3 inflammasome contains NALP2/3, ASC, caspase1, and Cardinal. NALPs are the chief platforms forinflammasome [88]. ASC is an essential adapter moleculethat connects the NALPs to caspase 1. Because NALP2/3do not have FIND domain and CARD domains in the Cterminus, the NALP2/3 inflammasome contains Cardinal,which has the same structural organizations as the C-terminal region of NALP1 [88]. All of the inflammasomecomponents reside in the cytoplasm. Activation of inflam-masome is thought to occur through the recognition ofPAMPS by NALPs. However, the precise activationmechanism remains unknown. After activation of inflam-masome, activated caspases cleave the proinflammatorycytokines, IL-1β and IL-18. Processing leads to thesecretion of mature IL-1β and IL-18 and induces inflam-matory responses. IL-1β production is tightly controlled toavoid excessive inflammation. Caspase-1 activation isinhibited by CARD containing ICEBERG, COP, andDASC (POP1) and possibly by human caspase-12. Acti-vated caspase-1 is blocked by PI-9 and IL-1β activity isinhibited by IL1Ra [89].

The NALP3/cryopyrin/CIAS1 inflammasome is activatedby MDP. Recent reports showed that NALP3/cryopyrin/CIAS1 is involved in the recognition of bacterial RNA, ATP,and uric acid crystals, and is essential for caspase-1activation and IL-1β and IL-18 production in response tothese ligands [90–92] (Fig. 3).

Another CARD-containing NOD-LRR protein, Ipaf isresponsible for Salmonella typhimurium-induced, but notTLR-induced, caspase-1 activation. ASC but not RICK/RIP2 is also essential for S. typhimurium-induced caspase-1activation [85] (Fig. 3).

The Lgn1 locus was found to control the intracellularreplication of L. pneumophila in murine macrophages.NAIP5/Birc1e, a NLR protein with BIR domains, wasidentified to be responsible for the Lgn1 effect [93].NAIP5/Birc1e is assumed to function as a cytoplasmicsensor of L.pneumophila and is responsible for L.pneumo-phila-mediated caspase-1 activation. The pathway used forcaspase-1-dependent processing and secretion of IL-1 was

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distinct from the pathway used for caspase-1-dependentinhibition of L. pneumophila growth. It is interesting to notethat the adapter molecule ASC is essential for the secretionof IL-1β after L. pneumophila infection but not for thecontrol of L. pneumophila replication. These data suggestthat Ipaf and Asc have separate and distinct functions incontrolling innate immune responses to L. pneumophila [94](Fig. 3).

Diseases

The importance of NLRs was highlighted by findings thatmutations affecting the function of these proteins areassociated with the occurrence of autoimmune diseases.NOD2 is associated with two autoinflammatory disorders.A missense point mutation in the human NOD2 gene iscorrelated with susceptibility to Crohn’s disease [4]. Blausyndrome, an inheritable disorder characterized by granu-

Fig. 3 Bacterial recognition ofNLRs and their signaling. NLRsrecognize bacterial proteins inthe cytoplasm and trigger signal-ing pathways. MDP, RNA, ATP,and uric acid crystal are recog-nized by NALP3, which formsan inflammasome comprised ofASC, CARDINAL, and caspase-1. NALP1 also forms an inflam-masome composed of ASC,caspase-1, and caspase-5. Littleis known about the naturalstimuli that lead to the activationof NALP1 inflammasome.Activated caspase-1 cleavespro-IL-1β for the maturation ofIL-1β. Another NOD-LRRprotein, IPAF, is activated by S.typhimurium and inducesmaturation of IL-1β associatingwith ASC. Naip5/Birc1e is acti-vated by L. pneumophila andinduces caspase-1 activation inan IPAF/ASC-dependent manner

Fig. 2 Signaling pathways ofNOD1 and NOD2. NOD1 andNOD2 recognize iE-DAP andMDP, respectively, and activateNF-κB via RIP2/RICK. NOD1and NOD2 signaling gives riseto the activation of MAPKs byunknown mechanism. CARD12negatively regulates RICK/RIP2-mediated NF-κBactivation by both NOD1 andNOD2. CARD6 also negativelyregulates RICK/RIP2-mediatedNF-κB activation by NOD1

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lomatous polyarthritis, panuveitis, and exanthema, wasshown to share a mutated form of CARD15 (which encodesNOD2) [95].

NALP3/cryopyrin/CIAS1 is associated with three auto-inflammatory syndromes: familial-cold autoinflammatorysyndrome, Muckle–Wells syndrome, and neonatal-onsetmultisystem inflammatory disease [96, 97]. These diseasesare now referred as cryopyrin-associated periodic syndrome(CAPS). CAPS patients have systemic inflammation withincreased levels of C-reactive protein, serum amyloid A,and inflammatory cytokines. However, it is not wellunderstood how the mutated NALP3/cryopyrin/CIAS1contributes to the induction of systemic infection [75].

CIITA is a master transcription regulator of MHC classII genes [98]. Mutations in CIITA lead to MHC class IIdeficiency or a rare form of autosomal recessive hereditaryimmunodeficiency, group A bare lymphocyte syndrome[99, 100].

Familial Mediterranean fever (FMF) is an autosomalrecessive inherited disease that is characterized by recurrentepisodes of fever accompanied with topical signs ofinflammation. The gene, Mediterranean fever, whichencodes the pyrin (also known as marenostrin) protein ismutated in patients with FMF [75].

Antiviral RNA helicases

Whereas TLR2 and TLR4 recognize viral components atthe cell surface, TLR3, TLR7, TLR8, and TLR9 areexclusively expressed in endosomal compartments. Afterphagocytes internalize viruses or virus-infected apoptoticcells, viral nucleic acids are released in phagolysosomesand are recognized by these TLRs [3]. Recent reportsshowed that hosts also have a mechanism to detectreplicating viruses in the cytoplasm in a TLR-independentmanner [101].

RIG-I

RIG-I was identified as a cytoplasmic receptor for viraldsRNA. RIG-I is a DExD/H box RNA helicase, whichpossesses C-terminal helicase domain and two tandemcaspase-recruiting domains at the N terminus (Fig. 4).RIG-I binds to RNA with its C-terminal helicase domainand unwinds dsRNA in an ATPase dependent manner. Thebinding of the helicase domain and RNA is likely to induceconformational changes that exposes the N-terminalCARDs to recruit downstream signaling proteins, leadingto the activation of IRF3 and NF-κB [5].

The functional significance of RIG-I was shown in vitroand in vivo [5, 6]. Overexpression of RIG-I blocksreplication of VSV and encephalomyocarditis virus

(EMCV), and RNAi of RIG-I type I inhibited induction oftype I IFNs in response to Newcastle disease virus (NDV),Sendai virus, and EMCV [5]. Most RIG-I-deficient miceare embryonic lethal because of severe liver degeneration.Conventional DCs and fibroblasts derived from RIG-I-deficient mice are almost completely unable to producetype I IFN and inflammatory cytokines after infection withNDV, Sendai virus, or VSV. However, RIG-I-deficientpDCs produced type I IFNs normally in response to NDV.In pDCs, TLR systems are responsible for RNA virus-mediated type I IFN induction. Thus, RIG-I and the TLRsystem exert antiviral responses in a cell-type-specificmanner [6].

Other molecules

Melanoma differentiation-associated gene 5 (MDA5, alsocalled Helicard) was identified as the DExD/H box RNAhelicase and contains two CARD-like domains and a singlehelicase domain [102–105]. Like RIG-I, overexpression ofMDA5 enhanced NDV-induced type I IFN production andantiviral responses in response to VSV or EMCV. RNAi ofMDA5 blocks NDV-induced activation of type I IFNpromoters [105]. MDA5 was identified as a binding targetfor V proteins of paramyxoviruses [104]. Although theprecise mechanism is unclear, these V proteins inhibit thedsRNA-induced activation of the IFN-β gene throughMDA5. Virus-encoded proteins may specifically targetthese RNA helicases to escape from antiviral detection bythe host cells.

An RNA helicase Lgp2 is also related to RIG-I andMDA5, but lacks the CARD-like domains. Overexpressionof Lgp2 inhibits Sendai virus-induced activation of IFN-βpromoter, suggesting that Lgp2 is a negative regulator ofRIG-I and MDA5 [105, 106] (Fig. 4).

Signaling pathway of antiviral RNA helicases

The signaling pathway of RIG-I and MDA5 ultimatelyengages the IKK complex (IKKα/β/γ) and TBK1/IKKi,which leads to phosphorylation and activation of NF-κBand IRF3 and IRF7, respectively. IFN-β promoter stimula-tor 1 (IPS-1) was identified as a molecule that actsdownstream of RIG-I and MDA5. IPS-1 consists of twodomains characterized as an N-terminal CARD domain anda C-terminal effector domain. IPS-1 associates with RIG-Iand MDA5 with a CARD domain. Overexpression of IPS-1activates IFN-β, IFN-α4, IFN-α6, and NF-κB promotersand inhibits VSV replication. RNAi of IPS-1 blocks type IIFN production in response to intracellular administrationof dsRNA or infection with VSV or NDV. TBK1 and IKKiare required for IPS-1-mediated activation of the type I IFNpromoters, although it does not directly bind to these

J Mol Med (2006) 84:712–725 719

protein kinases. IPS-1 interacts with FADD and RIP1 via itseffector domain to facilitate NF-κB activation. Thus, IPS-1is an adaptor protein that mediates RIG-I- and MDA5-dependent antiviral responses [107].

IPS-1 was independently identified by three differentgroups: mitochondrial antiviral signaling protein (MAVS)[108], virus-induced signaling adaptor [109], and CARD-adaptor-inducing IFN-β (Cardif) [110]. The MAVS studyshows that MAVS localizes to the outer mitochondrialmembrane. The mitochondrial-targeting transmembranedomain is essential for MAVS signaling, implying theimportance of mitochondria in innate immunity [108](Fig. 4).

Viral modification of the host immune system

Viruses have various mechanisms to escape from hostimmunity. Vaccinia virus (VV) protein A52R was shown toblock the activation of NF-κB by multiple TLRs, inparticular, TLR3 [111, 112]. A52R associates with bothIRAK2 and TRAF6, and disrupts signaling complexescontaining these proteins. Furthermore, deletion of theA52R gene from VV reduced virus virulence [112]. Theother VV protein, A46R, has a TIR domain, and as such isthe only viral member of the IL-1R/TLR family identifiedto date [111]. Recent studies revealed that A46R inhibitedIL-1R/TLR, but not TNF-induced NF-κB activation, byassociating with TIR-domain containing adaptor molecules[111, 113]. A46R interacts with multiple TIR-containingadaptors and thereby inhibits the activation of IRF3 andNF-κB [113]. NS3-4A, a multifunctional protein of

hepatitis C virus that has serine protease activity essentialfor the production of mature viral proteins, was recentlyshown to block the activation of IRF3. NS3-4A counteractsnot only TLR3-dependent pathways by targeting Trif forproteolytic cleavage, but also counteracts TLR3-indepen-dent pathways by targeting an undefined protein of theRIG-I-dependent pathway [114, 115]. Meylan et al. [110]showed that Cardif is cleaved by wild-type but not by acatalytically inactive form of NS3-4A, which cleaves theC-terminal region of Cardif, causing disruption of IRF3 andNF-κB activation, probably by causing mislocalization ofcleaved Cardif from mitochondria. Also, the V proteins ofparamyxoviruses, such as simian virus 5, human para-influenza virus 2, mumps virus, Sendai virus, and Hendravirus, associate with MDA5 and block the downstreamsignaling of MDA5 [104, 105]. Thus, for their survivalmany viruses antagonize important components of the hostantiviral defense.

DNA recognition in cytoplasm

DNA, including host DNA, can be recognized indepen-dently of TLR9. DNase II, a chief DNase present in thephagosome of macrophages, clears DNA from phagocy-tosed apoptotic cells or debris [116]. In DNase II-deficientmice, macrophages begin to produce IFN-β, which lead tosubsequent TLR9-independent inflammatory events [117].Furthermore, dsDNA but not ssDNA derived from eitherpathogens or the host activates both immune and nonim-mune cells when introduced into the cytoplasm bytransfection [118, 119]. Double-stranded breaks form of

Fig. 4 Signaling pathways ofantiviral RNA helicases. Virusesproduce dsRNA duringreplication in cytoplasm. RIG-Iand Mda5 recognize dsRNAto initiate antiviral signaling.IPS-1 interacts with RIG-I andMda5 via the CARD-likedomain, followed by theactivation of IRF3 and IRF7 viaTBK1- and IKKi-dependentphosphorylation. IPS-1 alsoactivates NF-κB via FADD/RIP1-dependent pathways.Synthetic dsDNA also activatestype I IFN promoters, althougha receptor responsible forDNA recognition was notidentified

720 J Mol Med (2006) 84:712–725

DNA (B-DNA) but not Z-form DNA stimulated mouse andhuman stromal cells and DCs, resulting in the production oftype I IFN and chemokines. B-DNA activated IRF3 and theIFN-β promoter via both TBK1 and IKKi, whereas itactivated transcription factor NF-κB independently of bothTBK1 and IKKi. Both of these pathways required theadaptor molecule IPS-1 in vitro but not TLRs or RIG-I andMDA5. In addition, B-DNA activation of fibroblastsconferred resistance to viral infection, suggesting thatB-DNA-induced IFN genes might be important in antiviralinnate immune responses. Furthermore, B-DNA activationof the signaling pathways may be involved in the pathogen-esis of nucleic acid-dependent autoimmune diseases [120](Fig. 4). It is thus necessary to identify a cytoplasmicdetector of DNA.

Conclusion

Since the discovery of mammalian TLRs, there was a rapidclarification of the molecular mechanisms of pathogenrecognition. Research on TLRs has also revealed thatrecognition of intracellular pathogens is also mediated viaa TLR-independent mechanism. Newly described NLRfamily members, which recognize pathogens in the cyto-plasm, are genetically linked to several human immuno-logical disorders. Innate immune responses via RIG-I areessential for the control of viral replication in thecytoplasm. Furthermore, TLR system and RIG-I systemwere tactfully utilized against viral infection in a cell-type-specific manner. Thus, hosts have developed the skillfuland complicated innate immune system to counter thevarious pathogens with various modes of infection. Futurestudies on PRRs should provide a more comprehensiveunderstanding of innate immune responses.

Acknowledgements We thank M. Hashimoto for secretarial assis-tance. This work was supported by grants from Special CoordinationFunds, the Ministry of Education, Culture, Sports, Science andTechnology, and the Japan Research Foundation for ClinicalPharmacology.

References

1. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA(1996) The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophilaadults. Cell 86:973–983

2. Medzhitov R, Janeway CJ (1997) Innate immunity: the virtues ofa nonclonal system of recognition. Cell 91:295–298

3. Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognitionand innate immunity. Cell 124:783–801

4. Strober W, Murray PJ, Kitani A, Watanabe T (2006) Signallingpathways and molecular interactions of NOD1 and NOD2. NatRev Immunol 6:9–20

5. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T,Miyagishi M, Taira K, Akira S, Fujita T (2004) The RNA helicaseRIG-I has an essential function in double-stranded RNA-inducedinnate antiviral responses. Nat Immunol 5:730–737

6. Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, MatsuiK, Tsujimura T, Takeda K, Fujita T, Takeuchi O, Akira S (2005)Cell type-specific involvement of RIG-I in antiviral response.Immunity 23:19–28

7. Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, MiyakeK, Kimoto M (1999) MD-2, a molecule that confers lipopoly-saccharide responsiveness on Toll-like receptor 4. J Exp Med189:1777–1782

8. Takeda K, Kaisho T, Akira S (2003) Toll-like receptors. AnnuRev Immunol 21:335–376

9. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T,Takeda K, Akira S (1999) Differential roles of TLR2 and TLR4in recognition of gram-negative and gram-positive bacterial cellwall components. Immunity 11:443–451

10. Netea MG, van Deuren M, Kullberg BJ, Cavaillon JM, Van derMeer JW (2002) Does the shape of lipid A determine theinteraction of LPS with Toll-like receptors? Trends Immunol23:135–139

11. Hashimoto M, Asai Y, Ogawa T (2004) Separation and structuralanalysis of lipoprotein in a lipopolysaccharide preparation fromPorphyromonas gingivalis. Int Immunol 16:1431–1437

12. Akira S (2004) Toll receptor families: structure and function.Semin Immunol 16:1–2

13. Takeuchi O, Kawai T, Muhlradt PF, Morr M, Radolf JD,Zychlinsky A, Takeda K, Akira S (2001) Discrimination ofbacterial lipoproteins by Toll-like receptor 6. Int Immunol13:933–940

14. Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z,Modlin RL, Akira S (2002) Cutting edge: role of Toll-like receptor1 in mediating immune response to microbial lipoproteins.J Immunol 169:10–14

15. Alexopoulou L, Thomas V, Schnare M, Lobet Y, Anguita J,Schoen RT, Medzhitov R, Fikrig E, Flavell RA (2002)Hyporesponsiveness to vaccination with Borrelia burgdorferiOspA in humans and in TLR1- and TLR2-deficient mice. NatMed 8:878–884

16. Hoebe K, Georgel P, Rutschmann S, Du X, Mudd S, Crozat K,Sovath S, Shamel L, Hartung T, Zahringer U, Beutler B (2005)CD36 is a sensor of diacylglycerides. Nature 433:523–527

17. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, GoodlettDR, Eng JK, Akira S, Underhill DM, Aderem A (2001) Theinnate immune response to bacterial flagellin is mediated byToll-like receptor 5. Nature 410:1099–1103

18. Gewirtz AT, Navas TA, Lyons S, Godowski PJ, Madara JL(2001) Cutting edge: bacterial flagellin activates basolaterallyexpressed TLR5 to induce epithelial proinflammatory geneexpression. J Immunol 167:1882–1885

19. Hawn TR, Verbon A, Lettinga KD, Zhao LP, Li SS, Laws RJ,Skerrett SJ, Beutler B, Schroeder L, Nachman A, Ozinsky A,Smith KD, Aderem A (2003) A common dominant TLR5 stopcodon polymorphism abolishes flagellin signaling and isassociated with susceptibility to legionnaires’ disease. J ExpMed 198: 1563–1572

20. Dunstan SJ, Hawn TR, Hue NT, Parry CP, Ho VA, Vinh H, DiepTS, House D, Wain J, Aderem A, Hien TT, Farrar JJ (2005) Hostsusceptibility and clinical outcomes in toll-like receptor5-deficient patients with typhoid fever in Vietnam. J Infect Dis191:1068–1071

21. Andersen-Nissen E, Smith KD, Strobe KL, Barrett SL,Cookson BT, Logan SM, Aderem A (2005) Evasion of Toll-like receptor 5 by flagellated bacteria. Proc Natl Acad SciUSA 102:9247–9252

J Mol Med (2006) 84:712–725 721

22. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H,Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S (2000)A Toll-like receptor recognizes bacterial DNA. Nature 408:740–745

23. Zhang D, Zhang G, Hayden MS, Greenblatt MB, Bussey C,Flavell RA, Ghosh S (2004) A toll-like receptor that preventsinfection by uropathogenic bacteria. Science 303:1522–1526

24. Netea MG, Van der Graaf C, Van der Meer JW, Kullberg BJ(2004) Recognition of fungal pathogens by Toll-like receptors.Eur J Clin Microbiol Infect Dis 23:672–676

25. Brown GD, Taylor PR, Reid DM, Willment JA, Williams DL,Martinez-Pomares L, Wong SY, Gordon S (2002) Dectin-1 isa major beta-glucan receptor on macrophages. J Exp Med196:407–412

26. Gantner BN, Simmons RM, Canavera SJ, Akira S, UnderhillDM (2003) Collaborative induction of inflammatory responsesby dectin-1 and Toll-like receptor 2. J Exp Med 197:1107–1117

27. Rogers NC, Slack EC, Edwards AD, Nolte MA, Schulz O,Schweighoffer E, Williams DL, Gordon S, Tybulewicz VL,Brown GD, Reis ESC (2005) Syk-dependent cytokine inductionby Dectin-1 reveals a novel pattern recognition pathway for Ctype lectins. Immunity 22:507–517

28. Underhill DM, Rossnagle E, Lowell CA, Simmons RM(2005) Dectin-1 activates Syk tyrosine kinase in a dynamicsubset of macrophages for reactive oxygen production.Blood 106:2543–2550

29. Gazzinelli RT, Ropert C, Campos MA (2004) Role of the Toll/interleukin-1 receptor signaling pathway in host resistance andpathogenesis during infection with protozoan parasites. ImmunolRev 201:9–25

30. Yarovinsky F, Zhang D, Andersen JF, Bannenberg GL, SerhanCN, Hayden MS, Hieny S, Sutterwala FS, Flavell RA, Ghosh S,Sher A (2005) TLR11 activation of dendritic cells by a protozoanprofilin-like protein. Science 308(5728):1626–1629

31. Coban C, Ishii KJ, Kawai T, Hemmi H, Sato S, Uematsu S,Yamamoto M, Takeuchi O, Itagaki S, Kumar N, Horii T, Akira S(2005) Toll-like receptor 9 mediates innate immune activation bythe malaria pigment hemozoin. J Exp Med 201:19–25

32. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP,Tripp RA, Walsh EE, Freeman MW, Golenbock DT, AndersonLJ, Finberg RW (2000) Pattern recognition receptors TLR4 andCD14 mediate response to respiratory syncytial virus. NatImmunol 1:398–401

33. Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW,Anderson LJ, Tripp RA (2001) Involvement of toll-likereceptor 4 in innate immunity to respiratory syncytial virus.J Virol 75:10730–10737

34. Rassa JC, Meyers JL, Zhang Y, Kudaravalli R, Ross SR (2002)Murine retroviruses activate B cells via interaction with toll-likereceptor 4. Proc Natl Acad Sci USA 99:2281–2286

35. Bieback K, Lien E, Klagge IM, Avota E, Schneider-SchauliesJ, Duprex WP, Wagner H, Kirschning CJ, Ter Meulen V,Schneider-Schaulies S (2002) Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling. JVirol 76:8729–8736

36. Compton T, Kurt-Jones EA, Boehme KW, Belko J, Latz E,Golenbock DT, Finberg RW (2003) Human cytomegalovirusactivates inflammatory cytokine responses via CD14 andToll-like receptor 2. J Virol 77:4588–4596

37. Kurt-Jones EA, Chan M, Zhou S, Wang J, Reed G, Bronson R,Arnold MM, Knipe DM, Finberg RW (2004) Herpes simplexvirus 1 interaction with Toll-like receptor 2 contributes to lethalencephalitis. Proc Natl Acad Sci USA 101:1315–1320

38. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA (2001)Recognition of double-stranded RNA and activation ofNF-kappaB by Toll-like receptor 3. Nature 413:732–738

39. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, SanjoH, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S(2003) Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301:640–643

40. Tabeta K, Georgel P, Janssen E, Du X, Hoebe K, Crozat K,Mudd S, Shamel L, Sovath S, Goode J, Alexopoulou L, FlavellRA, Beutler B (2004) Toll-like receptors 9 and 3 as essentialcomponents of innate immune defense against mouse cytomeg-alovirus infection. Proc Natl Acad Sci USA 101:3516–3521

41. Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E,Flavell RA (2004) Toll-like receptor 3 mediates West Nilevirus entry into the brain causing lethal encephalitis. NatMed 10: 1366–1373

42. Lund J, Sato A, Akira S, Medzhitov R, Iwasaki A (2003) Toll-like receptor 9-mediated recognition of herpes simplex virus-2by plasmacytoid dendritic cells. J Exp Med 198:513–520

43. Krug A, French AR, Barchet W, Fischer JA, Dzionek A, PingelJT, Orihuela MM, Akira S, Yokoyama WM, Colonna M (2004)TLR9-dependent recognition of MCMV by IPC and DCgenerates coordinated cytokine responses that activate antiviralNK cell function. Immunity 21:107–119

44. Krug A, Luker GD, Barchet W, Leib DA, Akira S, Colonna M(2004) herpes simplex virus type 1 activates murine naturalinterferon-producing cells through toll-like receptor 9. Blood103:1433–1437

45. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K,Horiuchi T, Tomizawa H, Takeda K, Akira S (2002) Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 3:196–200

46. Ito T, Amakawa R, Kaisho T, Hemmi H, Tajima K, Uehira K,Ozaki Y, Tomizawa H, Akira S, Fukuhara S (2002) Interferon-alpha and interleukin-12 are induced differentially by Toll-likereceptor 7 ligands in human blood dendritic cell subsets. J ExpMed 195:1507–1512

47. Akira S, Hemmi H (2003) Recognition of pathogen-associatedmolecular patterns by TLR family. Immunol Lett 85:85–95

48. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis E, Sousa C(2004) Innate antiviral responses by means of TLR7-mediatedrecognition of single-stranded RNA. Science 303:1529–1531

49. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C,Akira S, Lipford G, Wagner H, Bauer S (2004) Species-specificrecognition of single-stranded RNA via toll-like receptor 7 and 8.Science 303:1526–1529

50. Horng T, Barton GM, Flavell RA, Medzhitov R (2002) Theadaptor molecule TIRAP provides signalling specificity forToll-like receptors. Nature 420:329–333

51. Yamamoto M, Sato S, Hemmi H, Sanjo H, Uematsu S,Kaisho T, Hoshino K, Takeuchi O, Kobayashi M, Fujita T,Takeda K, Akira S (2002) Essential role for TIRAP inactivation of the signalling cascade shared by TLR2 andTLR4. Nature 420:324–329

52. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S (1999)Unresponsiveness of MyD88-deficient mice to endotoxin.Immunity 11:115–122

53. Yamamoto M, Sato S, Mori K, Hoshino K, Takeuchi O, TakedaK, Akira S (2002) Cutting edge: a novel Toll/IL-1 receptordomain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol169:6668–6672

54. Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T(2003) TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat Immunol4:161–167

55. Hoebe K, Du X, Georgel P, Janssen E, Tabeta K, Kim SO,Goode J, Lin P, Mann N, Mudd S, Crozat K, Sovath S, Han J,

722 J Mol Med (2006) 84:712–725

Beutler B (2003) Identification of Lps2 as a key transducer ofMyD88-independent TIR signalling. Nature 424:743–748

56. Yamamoto M, Sato S, Hemmi H, Uematsu S, Hoshino K, KaishoT, Takeuchi O, Takeda K, Akira S (2003) TRAM is specificallyinvolved in the Toll-like receptor 4-mediated MyD88-indepen-dent signaling pathway. Nat Immunol 4:1144–1150

57. Bin LH, Xu LG, Shu HB (2003) TIRP, a novel Toll/interleukin-1receptor (TIR) domain-containing adapter protein involved inTIR signaling. J Biol Chem 278:24526–24532

58. Oshiumi H, Sasai M, Shida K, Fujita T, Matsumoto M, Seya T(2003) TIR-containing adapter molecule (TICAM)-2, a bridgingadapter recruiting to toll-like receptor 4 TICAM-1 that inducesinterferon-beta. J Biol Chem 278:49751–49762

59. Sharma S, tenOever BR, Grandvaux N, Zhou GP, Lin R, HiscottJ (2003) Triggering the interferon antiviral response through anIKK-related pathway. Science 300:1148–1151

60. Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E,Golenbock DT, Coyle AJ, Liao SM, Maniatis T (2003)IKKepsilon and TBK1 are essential components of the IRF3signaling pathway. Nat Immunol 4:491–496

61. McWhirter SM, Fitzgerald KA, Rosains J, Rowe DC, GolenbockDT, Maniatis T (2004) IFN-regulatory factor 3-dependent geneexpression is defective in Tbk1-deficient mouse embryonicfibroblasts. Proc Natl Acad Sci USA 101:233–238

62. Perry AK, Chow EK, Goodnough JB, Yeh WC, Cheng G (2004)Differential requirement for TANK-binding kinase-1 in type Iinterferon responses to toll-like receptor activation and viralinfection. J Exp Med 199:1651–1658

63. Hemmi H, Takeuchi O, Sato S, Yamamoto M, Kaisho T, SanjoH, Kawai T, Hoshino K, Takeda K, Akira S (2004) The roles oftwo IkappaB Kinase-related kinases in lipopolysaccharide anddouble stranded RNA signaling and viral infection. J Exp Med199:1641–1650

64. Sato S, Sugiyama M, Yamamoto M, Watanabe Y, Kawai T,Takeda K, Akira S (2003) Toll/IL-1 receptor domain-containingadaptor inducing IFN-beta (TRIF) associates with TNF receptor-associated factor 6 and TANK-binding kinase 1, and activatestwo distinct transcription factors, NF-kappa B and IFN-regula-tory factor-3, in the Toll-like receptor signaling. J Immunol171:4304–4310

65. Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S,Hoshino K, Akira S (2001) Lipopolysaccharide stimulates theMyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopoly-saccharide-inducible genes. J Immunol 167:5887–5894

66. Gohda J, Matsumura T, Inoue J (2004) Cutting edge: TNFR-associated factor (TRAF) 6 is essential for MyD88-dependentpathway but not toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent pathway in TLR signaling.J Immunol 173:2913–2917

67. Meylan E, Burns K, Hofmann K, Blancheteau V, Martinon F,Kelliher M, Tschopp J (2004) RIP1 is an essential mediator ofToll-like receptor 3-induced NF-kappaB activation. Nat Immunol5:503–507

68. Liu YJ (2005) IPC: professional type 1 interferon-producingcells and plasmacytoid dendritic cell precursors. Annu RevImmunol 23:275–306

69. Hemmi H, Kaisho T, Takeda K, Akira S (2003) The roles ofToll-like receptor 9, MyD88, and DNA-dependent protein kinasecatalytic subunit in the effects of two distinct CpG DNAs ondendritic cell subsets. J Immunol 170:3059–3064

70. Kawai T, Sato S, Ishii KJ, Coban C, Hemmi H, Yamamoto M,Terai K, Matsuda M, Inoue J, Uematsu S, Takeuchi O, Akira S(2004) Interferon-alpha induction through Toll-like receptorsinvolves a direct interaction of IRF7 with MyD88 and TRAF6.Nat Immunol 5:1061–1068

71. Honda K, Yanai H, Mizutani T, Negishi H, Shimada N, SuzukiN, Ohba Y, Takaoka A, Yeh WC, Taniguchi T (2004) Role of atransductional-transcriptional processor complex involvingMyD88 and IRF-7 in Toll-like receptor signaling. Proc NatlAcad Sci USA 101:15416–15421

72. Yang K, Puel A, Zhang S, Eidenschenk C, Ku CL, Casrouge A,Picard C, von Bernuth H, Senechal B, Plancoulaine S, Al-HajjarS, Al-Ghonaium A, Marodi L, Davidson D, Speert D, RoifmanC, Garty BZ, Ozinsky A, Barrat FJ, Coffman RL, Miller RL, LiX, Lebon P, Rodriguez-Gallego C, Chapel H, Geissmann F,Jouanguy E, Casanova JL (2005) Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4dependent and redundant for protective immunity to viruses.Immunity 23:465–478

73. Uematsu S, Sato S, Yamamoto M, Hirotani T, Kato H, TakeshitaF, Matsuda M, Coban C, Ishii KJ, Kawai T, Takeuchi O, Akira S(2005) Interleukin-1 receptor-associated kinase-1 plays anessential role for Toll-like receptor (TLR)7- and TLR9-mediatedinterferon-{alpha} induction. J Exp Med 201:915–923

74. Tsujimura H, Tamura T, Kong HJ, Nishiyama A, Ishii KJ,Klinman, DM, Ozato K (2004) Toll-like receptor 9 signalingactivates NF-kappaB through IFN regulatory factor-8/IFNconsensus sequence binding protein in dendritic cells. J Immunol172:6820–6827

75. Ting JP, Kastner DL, Hoffman HM (2006) CATERPILLERs,pyrin and hereditary immunological disorders. Nat Rev Immunol6:183–195

76. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, RamosR, Britton H, Moran T, Karaliuskas R, Duerr RH, Achkar JP,Brant SR, Bayless TM, Kirschner BS, Hanauer SB, Nunez G,Cho JH (2001) A frameshift mutation in NOD2 associated withsusceptibility to Crohn’s disease. Nature 411:603–606

77. Chamaillard M, Hashimoto M, Horie Y, Masumoto J, Qiu S,Saab L, Ogura Y, Kawasaki A, Fukase K, Kusumoto S, ValvanoMA, Foster SJ, Mak TW, Nunez G, Inohara N (2003) Anessential role for NOD1 in host recognition of bacterialpeptidoglycan containing diaminopimelic acid. Nat Immunol4:702–707

78. Inohara N, Ogura Y, Fontalba A, Gutierrez O, Pons F, CrespoJ, Fukase K, Inamura S, Kusumoto S, Hashimoto M, FosterSJ, Moran AP, Fernandez-Luna JL, Nunez G (2003) Hostrecognition of bacterial muramyl dipeptide mediated throughNOD2. Implications for Crohn’s disease. J Biol Chem 278:5509–5512

79. Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A,Thomas G, Philpott DJ, Sansonetti PJ (2003) Nod2 is a generalsensor of peptidoglycan through muramyl dipeptide (MDP)detection. J Biol Chem 278:8869–8872

80. Kobayashi K, Inohara, N, Hernandez LD, Galan JE, Nunez G,Janeway CA, Medzhitov R, Flavell RA (2002) RICK/Rip2/CARDIAK mediates signalling for receptors of the innate andadaptive immune systems. Nature 416:194–199

81. Abbott DW, Wilkins A, Asara JM, Cantley LC (2004) TheCrohn’s disease protein, NOD2, requires RIP2 in order to induceubiquitinylation of a novel site on NEMO. Curr Biol 14:2217–2227

82. Zhou H, Wertz I, O’Rourke K, Ultsch M, Seshagiri S, Eby M,Xiao W, Dixit VM (2004) Bcl10 activates the NF-kappaBpathway through ubiquitination of NEMO. Nature 427:167–171

83. Damiano JS, Stehlik C, Pio F, Godzik A, Reed JC (2001) CLAN,a novel human CED-4-like gene. Genomics 75:77–83

84. Damiano JS, Oliveira V, Welsh K, Reed JC (2004) Heterotypicinteractions among NACHT domains: implications for regulationof innate immune responses. Biochem J 381:213–219

85. Mariathasan S, Newton K, Monack DM, Vucic D, French DM,Lee WP, Roose-Girma M, Erickson S, Dixit VM (2004)

J Mol Med (2006) 84:712–725 723

Differential activation of the inflammasome by caspase-1adaptors ASC and Ipaf. Nature 430:213–218

86. Stehlik C, Hayashi H, Pio F, Godzik A, Reed JC (2003) CARD6is a modulator of NF-kappa B activation by Nod1- and Cardiak-mediated pathways. J Biol Chem 278:31941–31949

87. Martinon F, Burns K, Tschopp J (2002) The inflammasome:a molecular platform triggering activation of inflammatorycaspases and processing of proIL-beta. Mol Cell 10:417–426

88. Petrilli V, Papin S, Tschopp J (2005) The inflammasome. CurrBiol 15:R581

89. Martinon F, Tschopp J (2004) Inflammatory caspases: linking anintracellular innate immune system to autoinflammatory dis-eases. Cell 117:561–574

90. Kanneganti TD, Ozoren N, Body-Malapel M, Amer A, ParkJH, Franchi L, Whitfield J, Barchet W, Colonna M,Vandenabeele P, Bertin J, Coyle A, Grant EP, Akira S, NunezG (2006) Bacterial RNA and small antiviral compoundsactivate caspase-1 through cryopyrin/Nalp3. Nature 440:233–236

91. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K,Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM(2006) Cryopyrin activates the inflammasome in response totoxins and ATP. Nature 440:228–232

92. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J(2006) Gout-associated uric acid crystals activate the NALP3inflammasome. Nature 440:237–241

93. Diez E, Lee SH, Gauthier S, Yaraghi Z, Tremblay M, Vidal S,Gros P (2003) Birc1e is the gene within the Lgn1 locusassociated with resistance to Legionella pneumophila. Nat Genet33:55–60

94. Zamboni DS, Kobayashi KS, Kohlsdorf T, Ogura Y, LongEM, Vance RE, Kuida K, Mariathasan S, Dixit VM, FlavellRA, Dietrich WF, Roy CR (2006) The Birc1e cytosolicpattern-recognition receptor contributes to the detection andcontrol of Legionella pneumophila infection. Nat Immunol7:318–325

95. Miceli-Richard C, Lesage S, Rybojad M, Prieur AM, Manouvrier-Hanu S, Hafner R, Chamaillard M, Zouali H, Thomas G, HugotJP (2001) CARD15 mutations in Blau syndrome. Nat Genet29:19–20

96. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, KolodnerRD (2001) Mutation of a new gene encoding a putativepyrin-like protein causes familial cold autoinflammatorysyndrome and Muckle–Wells syndrome. Nat Genet 29:301–305

97. Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT,Hofmann SR, Stein L, Russo R, Goldsmith D, Dent P,Rosenberg HF, Austin F, Remmers EF, Balow JE Jr, RosenzweigS, Komarow H, Shoham NG, Wood G, Jones J, Mangra N,Carrero H, Adams BS, Moore TL, Schikler K, Hoffman H,Lovell DJ, Lipnick R, Barron K, O’Shea JJ, Kastner DL,Goldbach-Mansky R (2002) De novo CIAS1 mutations, cytokineactivation, and evidence for genetic heterogeneity in patientswith neonatal-onset multisystem inflammatory disease(NOMID): a new member of the expanding family ofpyrin-associated autoinflammatory diseases. Arthritis Rheum46:3340–3348

98. Harton JA, Linhoff MW, Zhang J, Ting JP (2002) Cutting edge:CATERPILLER: a large family of mammalian genes containingCARD, pyrin, nucleotide-binding, and leucine-rich repeatdomains. J Immunol 169:4088–4093

99. Ting JP, Davis BK (2005) CATERPILLER: a novel gene familyimportant in immunity, cell death, and diseases. Annu RevImmunol 23:387–414

100. Steimle V, Otten LA, Zufferey M, Mach B (1993) Complemen-tation cloning of an MHC class II transactivator mutated in

hereditary MHC class II deficiency (or bare lymphocytesyndrome). Cell 75:135–146

101. Kawai T, Akira S (2006) Innate immune recognition of viralinfection. Nat Immunol 7:131–137

102. Kang DC, Gopalkrishnan RV, Wu Q, Jankowsky E, Pyle AM,Fisher PB (2002) mda-5: An interferon-inducible putative RNAhelicase with double-stranded RNA-dependent ATPase activityand melanoma growth-suppressive properties. Proc Natl AcadSci USA 99:637–642

103. Kovacsovics M, Martinon F, Micheau O, Bodmer JL, HofmannK, Tschopp J (2002) Overexpression of Helicard, a CARD-containing helicase cleaved during apoptosis, accelerates DNAdegradation. Curr Biol 12:838–843

104. Andrejeva J, Childs KS, Young DF, Carlos TS, Stock N,Goodbourn S, Randall RE (2004) The V proteins ofparamyxoviruses bind the IFN-inducible RNA helicase,mda-5, and inhibit its activation of the IFN-beta promoter.Proc Natl Acad Sci USA 101:17264–17269

105. Yoneyama M, Kikuchi M, Matsumoto K, Imaizumi T, MiyagishiM, Taira K, Foy E, Loo YM, Gale M Jr, Akira S, Yonehara S,Kato A, Fujita T (2005) Shared and unique functions of theDExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviralinnate immunity. J Immunol 175:2851–2858

106. Rothenfusser S, Goutagny N, DiPerna G, Gong M, Monks BG,Schoenemeyer A, Yamamoto M, Akira S, Fitzgerald KA (2005)The RNA helicase Lgp2 inhibits TLR-independent sensing ofviral replication by retinoic acid-inducible gene-I. J Immunol175:5260–5268

107. Kawai T, Takahashi K, Sato S, Coban C, Kumar H, Kato H, IshiiKJ, Takeuchi O, Akira S (2005) IPS-1, an adaptor triggeringRIG-I- and Mda5-mediated type I interferon induction. NatImmunol 6:981–988

108. Seth RB, Sun L, Ea CK, Chen ZJ (2005) Identification andcharacterization of MAVS, a mitochondrial antiviral signalingprotein that activates NF-kappaB and IRF 3. Cell 122:669–682

109. Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, Shu HB (2005) VISAis an adapter protein required for virus-triggered IFN-betasignaling. Mol Cell 19:727–740

110. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M,Bartenschlager R, Tschopp J (2005) Cardif is an adaptor proteinin the RIG-I antiviral pathway and is targeted by hepatitis Cvirus. Nature 437:1167–1172

111. Bowie A, Kiss-Toth E, Symons JA, Smith GL, Dower SK,O’Neill LA (2000) A46R and A52R from vaccinia virus areantagonists of host IL-1 and toll-like receptor signaling. ProcNatl Acad Sci USA 97:10162–10167

112. Harte MT, Haga IR, Maloney G, Gray P, Reading PC, BartlettNW, Smith GL, Bowie A, O’Neill LA (2003) The poxvirusprotein A52R targets Toll-like receptor signaling complexes tosuppress host defense. J Exp Med 197:343–351

113. Stack J, Haga IR, Schroder M, Bartlett NW, Maloney G,Reading PC, Fitzgerald KA, Smith GL, Bowie AG (2005)Vaccinia virus protein A46R targets multiple Toll-like-interleu-kin-1 receptor adaptors and contributes to virulence. J Exp Med201:1007–1018

114. Foy E, Li K, Sumpter R Jr, Loo YM, Johnson CL, Wang C, FishPM, Yoneyama M, Fujita T, Lemon SM, Gale M Jr (2005)Control of antiviral defenses through hepatitis C virus disruptionof retinoic acid-inducible gene-I signaling. Proc Natl Acad SciUSA 102:2986–2991

115. Li XD, Sun L, Seth RB, Pineda G, Chen ZJ (2005) Hepatitis Cvirus protease NS3/4A cleaves mitochondrial antiviral signalingprotein off the mitochondria to evade innate immunity. Proc NatlAcad Sci USA 102:17717–17722

116. Kawane K, Fukuyama H, Yoshida H, Nagase H, Ohsawa Y,Uchiyama Y, Okada K, Iida T, Nagata S (2003) Impaired thymic

724 J Mol Med (2006) 84:712–725

development in mouse embryos deficient in apoptotic DNAdegradation. Nat Immunol 4:138–144

117. Yoshida H, Okabe Y, Kawane K, Fukuyama H, Nagata S (2005)Lethal anemia caused by interferon-beta produced in mouseembryos carrying undigested DNA. Nat Immunol 6:49–56

118. Suzuki K, Mori A, Ishii KJ, Saito J, Singer DS, Klinman DM,Krause PR, Kohn LD (1999) Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides. ProcNatl Acad Sci USA 96:2285–2290

119. Ishii KJ, Suzuki K, Coban C, Takeshita F, Itoh Y, Matoba H,Kohn LD, Klinman DM (2001) Genomic DNA released bydying cells induces the maturation of APCs. J Immunol167:2602–2607

120. Ishii KJ, Coban C, Kato H, Takahashi K, Torii Y, Takeshita F,Ludwig H, Sutter G, Suzuki K, Hemmi H, Sato S, Yamamoto M,Uematsu S, Kawai T, Takeuchi O, Akira S (2006) A Toll-likereceptor-independent antiviral response induced by double-stranded B-form DNA. Nat Immunol 7:40–48

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