Biochem. J. (2009) 420, 1–16 (Printed in Great Britain) doi:10.1042/BJ20090272 1 REVIEW ARTICLE Pathogen recognition in the innate immune response Himanshu KUMAR*†, Taro KAWAI*† and Shizuo AKIRA*† 1 *Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan, and †Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan Immunity against microbial pathogens primarily depends on the recognition of pathogen components by innate receptors expressed on immune and non-immune cells. Innate receptors are evolutionarily conserved germ-line-encoded proteins and include TLRs (Toll-like receptors), RLRs [RIG-I (retinoic acid-inducible gene-I)-like receptors] and NLRs (Nod-like receptors). These re- ceptors recognize pathogens or pathogen-derived products in different cellular compartments, such as the plasma membrane, the endosomes or the cytoplasm, and induce the expression of cytokines, chemokines and co-stimulatory molecules to eliminate pathogens and instruct pathogen-specific adaptive immune responses. In the present review, we will discuss the recent progress in the study of pathogen recognition by TLRs, RLRs and NLRs and their signalling pathways. Key words: innate immune response, Nod-like receptor (NLR), pathogen recognition, retinoic acid-inducible gene-I receptor (RIG-I-like receptor, RLR), Toll-like receptor (TLR). INTRODUCTION The innate immune system is the first line of the defence system against microbial pathogens such as Gram-positive and Gram- negative bacteria, fungi and viruses. Innate immune cells such as macrophages and DCs (dendritic cells) directly kill the pathogenic micro-organism through phagocytosis or induce the produc- tion of cytokines, which aid elimination of the pathogens [1– 4]. The responses of the innate immune system instruct the development of long-lasting pathogen-specific adaptive immune responses. The adaptive immune system consists of B- and T-cells, which provide pathogen specific immunity to the host through somatic rearrangement of antigen receptor genes. B- cells produce pathogen-specific antibodies to neutralize toxins produced by pathogens, whereas T-cells provide the cytokine milieu to clear pathogen-infected cells through their cytotoxic effects or via signals to B-cells [5]. The mechanisms for innate immune recognition of pathogens and signalling have received increasing research attention. These studies were initiated after the discovery of Toll protein, which plays an important role in the defence against fungal infection in Drosophila (fruitfly) [6]. Further studies led to the discovery Abbreviations used: AIM2, absent in melanoma 2; alum, aluminium salts; AP-1, activator protein-1; ASC, apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain); Atg, autophagy-related; Atg16L1, autophagy-related 16-like 1; Bcl, B-cell lymphoma; BIR, baculovirus inhibitor of apoptosis protein repeat; Birc1, BIR-containing 1; CARD, caspase recruitment domain; CARDIAK, CARD-containing IL (interleukin)-1β converting enzyme-associated kinase; Cardif, CARD adaptor inducing IFN-β; CARDINAL, CARD i nhibitor of nuclear factor-κB-activating ligands; cDC, conventional dendritic cell; CIAS, cold-induced autoinflammatory syndrome 1; CLAN, CARD LRR- and NACHT-domain-containing protein; CLR, C-type lectin receptor; CTD, C-terminal domain; DAI, DNA-dependent activator of IRFs; DAK, dihydroacetone kinase; DC, dendritic cell; DED, death effector domain; ds, double-stranded; DUBA, de-ubiquitinating enzyme A; DV, dengue virus; EDA, extracellular domain A; EMCV, encephalomyocarditis virus; ER, endoplasmic reticulum; ERK, extracellular-signal-regulated kinase; FADD, Fas-associated death-domain; GPI, glycosylphosphatidylinositol; HCV, hepatitis C virus; HIN, haematopoietic interferon-inducible; HSE, herpes simplex virus encephalitis; HSV, herpes simplex virus; iE-DAP, γ-D-glutamyl-m- diaminopimelic acid; IFI, interferon-inducible, IFN, interferon; IκB, inhibitor of κB; IKK, IκB kinase; IL, interleukin; iNOS, inducible NO synthase; IPAF, IL-1β converting enzyme protease activating factor; IPS-1, IFN-β promoter stimulator-1; IRAK, IL-1 receptor-associated kinase; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; IV, influenza virus; JEV, Japanese-encephalitis virus; JNK, c-jun N-terminal kinase; LBP, LPS-binding protein; LCMV, lymphocytic choriomeningitis virus; Lgp2, Laboratory of Genetics and Physiology 2; LPDC, lamina propria DC; LPS, lipopolysaccharide; LRR, leucine-rich repeat; LT, lethal toxin; LTA, lipoteichoic acid; MAL, MyD88-adaptor-like; MALP-2, macrophage-activating lipopeptide; MAPK, mitogen- activated protein kinase; MAVS, mitochondrial antiviral signalling; MCMV, murine cytomegalovirus; MD2, myeloid differentiation protein-2; Mda5, melanoma differentiation-associated gene 5; MDP, muramyl dipeptide; MITA, mediator of IRF3 activation; MMTV, mouse mammary tumour virus; MSU, monosodium urate; mTOR, mammalian target of rapamycin; MyD88, myeloid differentiation primary response gene 88; NACHT, NTPase-domain named after NAIP, CIITA, HET-E and TP1; NAIP, NLR family; apoptosis inhibitory protein; NAIP5, neuronal apoptosis inhibitor protein 5; NAK, NF-κB activating kinase; NALP, NACHT/LRR/PYD-containing protein; NAP, NAK-associated protein; NDV, Newcastle-disease virus; NEMO, NF-κB essential modifier; NF-κB, nuclear factor κB; NK, natural killer; NLR, Nod-like receptor; NLRC, NLR family CARD-domain-containing; NLRP, NLR family pyrin-domain-containing; NOD, nucleotide- binding oligomerization domain; ODN, oligodeoxynucleotide; PAMP, pathogen-associated molecular pattern; pDCs, plasmacytoid dendritic cells; PGN, peptidoglycan; Phox, phagocyte oxidase; PKR, protein kinase receptor; PRR, pattern-recognition receptor; PYCARD, PYD- and CARD-domain-containing; PYD, pyrin domain; PYHIN, PYD and HIN domain family member 1; PYPAF, PYD-containing APAF-1 (apoptotic peptidase activating factor 1)-like protein; RD, repressor domain; RICK, RIP-like interacting caspase-like apoptosis-regulatory protein kinase; RIG-I, retinoic acid-inducible gene-I; RIP, receptor- interacting protein; RLR, RIG-I-like receptor; RNF, ring finger; ROS, reactive oxygen species; RSV, respiratory syncytial virus; SeV, Sendai virus; SINTBAD, similar to NAP1 TBK1 adaptor; SNP, single-nucleotide polymorphism; ss, single-stranded; STING, stimulator of interferon genes; T2K, TRAF2-associated kinase; TAB, TAK1-binding protein; TAK1, transforming-growth-factor-β-activated kinase 1; TANK, TRAF family member associated NF-κB activator; TBK1, TANK-binding kinase 1; Th, T-helper; TICAM, TIR-domain-containing adaptor molecule; TIR, Toll/IL-1 receptor; TIRAP, TIR-containing adaptor protein; TLR, Toll-like receptor; TNF, tumour necrosis factor; TNFR, TNF receptor; TRADD, TNF-receptor-associated DD; TRAF, TNF-receptor-associated factor; TRAM, TRIF-related adaptor molecule; TRIF, TIR-containing adaptor inducing IFN-β; TRIM, tripartite motif; UBC, ubiquitin C; UEV, ubiquitin-conjugating enzyme variant; VISA, virus-induced signalling adaptor; VSV, vesicular-stomatitis virus; WNV, West Nile virus; WT, wild-type. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2009 Biochemical Society
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Biochem. J. (2009) 420, 1–16 (Printed in Great Britain) doi:10.1042/BJ20090272 1
REVIEW ARTICLEPathogen recognition in the innate immune responseHimanshu KUMAR*†, Taro KAWAI*† and Shizuo AKIRA*†1
*Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan, and †Department of Host Defense,Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Immunity against microbial pathogens primarily depends onthe recognition of pathogen components by innate receptorsexpressed on immune and non-immune cells. Innate receptors areevolutionarily conserved germ-line-encoded proteins and includeTLRs (Toll-like receptors), RLRs [RIG-I (retinoic acid-induciblegene-I)-like receptors] and NLRs (Nod-like receptors). These re-ceptors recognize pathogens or pathogen-derived products indifferent cellular compartments, such as the plasma membrane,the endosomes or the cytoplasm, and induce the expression
of cytokines, chemokines and co-stimulatory molecules toeliminate pathogens and instruct pathogen-specific adaptiveimmune responses. In the present review, we will discuss therecent progress in the study of pathogen recognition by TLRs,RLRs and NLRs and their signalling pathways.
The innate immune system is the first line of the defence systemagainst microbial pathogens such as Gram-positive and Gram-negative bacteria, fungi and viruses. Innate immune cells such asmacrophages and DCs (dendritic cells) directly kill the pathogenicmicro-organism through phagocytosis or induce the produc-tion of cytokines, which aid elimination of the pathogens [1–4]. The responses of the innate immune system instruct thedevelopment of long-lasting pathogen-specific adaptive immuneresponses. The adaptive immune system consists of B- and
T-cells, which provide pathogen specific immunity to the hostthrough somatic rearrangement of antigen receptor genes. B-cells produce pathogen-specific antibodies to neutralize toxinsproduced by pathogens, whereas T-cells provide the cytokinemilieu to clear pathogen-infected cells through their cytotoxiceffects or via signals to B-cells [5].
The mechanisms for innate immune recognition of pathogensand signalling have received increasing research attention. Thesestudies were initiated after the discovery of Toll protein, whichplays an important role in the defence against fungal infectionin Drosophila (fruitfly) [6]. Further studies led to the discovery
Abbreviations used: AIM2, absent in melanoma 2; alum, aluminium salts; AP-1, activator protein-1; ASC, apoptosis-associated speck-like proteincontaining a CARD (caspase recruitment domain); Atg, autophagy-related; Atg16L1, autophagy-related 16-like 1; Bcl, B-cell lymphoma; BIR, baculovirusinhibitor of apoptosis protein repeat; Birc1, BIR-containing 1; CARD, caspase recruitment domain; CARDIAK, CARD-containing IL (interleukin)-1β
of the TLR (Toll-like receptor) family of proteins in mammals[7–9]. Recently, other proteins families such as RLRs [RIG-I(retinoic acid-inducible gene-I)-like receptors] [1,10] and NLRs(Nod-like receptors) [1,11–13] were discovered. These familiesof receptors are collectively known as PRRs (pattern-recognitionreceptors) [14], which recognize the specific molecular structuresof pathogens known as PAMPs (pathogen-associated molecularpatterns) in various compartments of cells, such as the plasmamembrane, the endolysosome and the cytoplasm.
In the present review we will focus on recent advances in thestudy of recognition and signalling mediated by TLRs, RLRs andNLRs.
TOLL-LIKE RECEPTORS
The Toll protein was originally identified in fruitflies (Drosophila)and is involved in dorsoventral polarity during embryonicdevelopment [6]. Further studies have shown that the Toll proteinplays an essential role in mounting an effective immune responseagainst the fungus Aspergillus fumigatus [6]. These studies ledto the identification of homologues of Toll proteins in humansand mice through database searches and which are referred toas TLRs [7]. To date, 10 and 13 TLR members have beenidentified in humans and mice respectively [1]. The TLRs aretype I membrane glycoproteins and consist of an extracellularLRR (leucine-rich repeat) domain, a transmembrane domain anda cytoplasmic TIR ]Toll/IL (interleukin)-1 receptor] domain [15].The LRR domain of TLRs consists of 16–28 tandem repeatsof the LRR motif [16] and is involved in the recognition ofligands such as protein (e.g. flagellin and porin from bacteria),sugar (e.g. zymosan from fungi), lipid [LPS (lipopolysaccharide),lipid A and LTA (lipoteichoic acid) from bacteria], nucleic acid(CpG-containing DNA from bacteria and viruses and viral RNA),derivatives of protein or peptide (lipoprotein and lipopeptidesfrom various pathogens), derivatives of lipid (lipoarabinomannan)from mycobacteria) and a complex derivative of protein orpeptides, sugar and lipid (diacyl lipopeptides from mycoplasma)[1]. The TIR domain of TLRs consists of approx. 150 aminoacids and shows homology with the cytoplasmic region of theIL-1 receptor. Therefore, it is termed the TIR domain [15,17].The TIR domain interacts with TIR-domain-containing adaptorssuch as MyD88 (myeloid differentiation primary response gene88), TIRAP [TIR-containing adaptor protein, also known as MAL(MyD88-adaptor-like], TRIF [TIR-containing adaptor-inducingIFN (interferon)-β, also known as TICAM1 (TIR-domain-containing adaptor molecule 1] and TRAM (TRIF-related adaptormolecule, also known as TICAM2). In turn, the downstreamsignalling pathways activate MAPKs (mitogen-activated proteinkinases) and transcription factors such as NF-κB (nuclear factorκB) and IRFs (IFN regulatory factors) to induce production ofinflammatory cytokines and type I IFNs.
The TLR family members are expressed on various immune andnon-immune cells such as B-cells, NK (natural killer) cells, DCs,macrophages, fibroblast cells, epithelial cells and endothelialcells. However, TLRs are differentially localized within the cells.TLR1, TLR2, TLR4, TLR5 and TLR6 are expressed on the cellsurface, whereas TLR3, TLR7, TLR8 and TLR9 are expressed inthe endosomes (Figure 1).
Recently, co-crystallization of TLR1–TLR2, TLR3 and TLR4and their ligand-recognition properties has been reported. Theheterodimer or homodimer of the LRR domain of TLR1–TLR2,TLR4 and TLR3 show a horseshoe-like structure or m-shapedframework and consists of both a concave and a convex surface.These surfaces are responsible for ligand binding and ligand-induced TLR dimerization [18–21].
Pathogen recognition by the extracellular cell-surface TLRs
TLR1, TLR2 and TLR6
TLR2 recognizes a structurally diverse range of PAMPs thatinclude proteins such as V-antigen (LcrV) from Yersinia,haemagglutinin protein from measles virus, glycolipids, LTAfrom Staphylococcus aureus and Streptococcus pneumoniae[22], lipopeptides or lipoproteins such as MALP (macrophage-activating lipopeptide)-2 and R-MALP from Mycoplasma species[23–25], lipoproteins from Escherichia coli [26], Borreliaburgdorferi [27], Mycoplasma species [28] and Mycobacteriumtuberculosis [29], peptidoglycan from Staph. aureus, Strep.pneumoniae and Strep. pyogenes [30–32] and polysaccharidesknown as zymosan from Saccharomyces cerevisiae [33,34]. Theligands for TLR2 have been described in detail in a review article[35]. In addition, TLR2 also recognizes complete pathogens,including the species of the bacterium Chlamydia [36], virusessuch as HSV (herpes simplex virus) [37] and varicella-zoster virus[38] (Figure 1). The diversity of ligand recognition by TLR2 ispossible because TLR2 can recognize the ligands in associationwith structurally related TLRs such as TLR1 and TLR6. TheTLR2–TLR1 and TLR2–TLR6 heterodimers recognize triacyllipopeptide derived from Gram-negative bacteria and diacyl lipo-peptide derived from mycoplasma respectively [39,40]. TLR2also recognizes zymosan (β-1,3-glucan and β-1,6-glucan) inassociation with the structurally unrelated C-type lectin familyknown as dectin-1 [41]. Furthermore, the class II scavengerreceptor CD36 has been shown to be involved in phagocytosisand cytokine production in response to Staph. aureus and its cell-wall components such as LTA and MALP-2, suggesting that CD36functions as a co-receptor of TLR2/6 [25].
TLR4
TLR4 recognizes LPS from Gram-negative bacteria [8,9],glycoinositolphospholipids from Trypanosoma [42], the fusionprotein from RSV (respiratory syncytial virus) [43] and theenvelope protein from MMTV (mouse mammary tumour virus)[44]. TLR4 also recognizes diterpene (taxol) purified from thebark of Taxus brevifolia (the Pacific yew) [45,46] (Figure 1). Inaddition, TLR4 directly or indirectly recognizes endogenous mo-lecules such as heat-shock proteins, fibrinogen, hyaluronic acid,β-defensin and extracellular domain A in fibronectin [47]. Therecognition of LPS is triggered by a complex that contains TLR4,a recognition subunit MD2 (myeloid differentiation protein-2) andmembrane-bound GPI (glycosylphosphatidylinositol)-anchoredCD14. The activation of TLR4 is further supported by anotherprotein known as LBP (LPS-binding protein) [48]. The studiesshow that the lipid A (an active component of LPS) binds toMD2 and forms a complex. The lipid A–MD2 complex interactswith TLR4 and activates signalling, suggesting that MD2 is moreimportant in the recognition of lipid A [18].
TLR5
TLR5 recognizes a monomer of flagellin [49], an importantstructural protein of pathogenic and non-pathogenic motilebacteria. It is also important for adhesion and invasion at theluminal surface of the epithelial cells covering the mucosal tissuesduring infection [50]. Flagellin from Salmonella typhimuriumcontains 494 amino acids and consists of two functional domains.The 140 amino acids of the N-terminal domain and 90 amino acidsof the C-terminal domain of this protein are highly conserved andessential for the polymerization and motility of flagellin [51]. Thecentral domain of the protein is highly variable among Salmonella
Pathogen recognition in the innate immune response 3
Figure 1 PAMPs recognized by TLRs and their adaptors
Plasma-membrane-localized TLRs (TLR2, TLR4, TLR5, TLR11 alone and TLR2 in association with TLR1 or TLR6) and endosomally localized TLRs (TLR3, TLR7 and TLR9) recognize the indicatedligands. TLR1, TLR2, TLR4 and TLR6 recruit TIRAP and MyD88. MyD88 also contains the DD. In addition to TIRAP and MyD88, TLR4 recruits TRAM and TRIF. TLR5, TLR7, TLR9 and TLR11 recruitMyD88, whereas TLR3 recruits TRIF.
serovars and bacterial species, and this region is exposed at theouter surface of the flagellum. Amino acids 89–96 are essentialfor TLR5 activation, but this region is located deep inside thetertiary structure of the flagellin protein and becomes accessibleonly when flagellin is present in monomer form [52,53]. However,it is not clear how flagellated bacteria deliver monomeric flagellinunder physiological conditions. TLR5 is mainly expressed on theluminal surface of the epithelial cells covering the mucosal tissuesand trachea, and the bronchi and the alveoli of the respiratory tract[54–58] (Figure 1). Upon activation with flagellin, epithelial cellsinduce cytokines and chemokines, and neutrophil recruitment.Recently, we have shown that TLR5+ small-intestinal LPDCs(lamina propria DCs) are important for the induction of humoraland cellular immunity in the intestine. These LPDCs can induceretinoic acid and are involved in the generation of IgA+ plasmacells, as well as the differentiation of both Th1 (T-helper 17) andTh1 cells in the intestine in a TLR5-dependent manner [59].
TLR11
TLR11 recognizes profilins from Toxoplasma gondii, anobligate intracellular protozoan parasite [60]. It also recognizesuropathogenic E. coli (Figure 1). Mice deficient in TLR11 showincreased susceptibility to these pathogens [61]. TLR11 has been
shown to be expressed on epithelial cells of the bladder in mouse.However, TLR11 is not expressed in humans, as the predictedmRNA has at least one stop codon [62].
Pathogen recognition by intracellular TLRs
TLR3
TLR3 recognizes viral ds (double-stranded) RNA originatingfrom dsRNA viruses such as reovirus [63]. TLR3 also recognizesdsRNA produced during replication of ss (single-stranded) RNAviruses, such as WNV (West Nile virus) [64], RSV [65] andEMCV (encephalomyocarditis virus) [66]. In addition, TLR3recognizes a synthetic analogue of dsRNA known as poly(I-C)(Figure 1). TLR3 is expressed in the endosomes of immune cells,including cDCs (conventional DCs – a type of antigen-presentingcell that induces various cytokines after stimulation with ligands),macrophages, B-cells, NK cells and non-immune cells, includingepithelial cells. However, TLR3 is not expressed on pDCs(plasmacytoid DCs – a type of DC that produces high amounts oftype I IFNs). In addition, TLR3 is highly expressed in the brain[67]. TLR3-deficient mice infected with various RNA viruses suchas MCMV (murine cytomegalovirus), VSV (vesicular-stomatitisvirus), LCMV (lymphocytic choriomeningitis virus), RSV or
reovirus show comparable susceptibility with WT (wild-type)mice, suggesting that TLR3 is dispensable for protection againstthese viruses [68]. However, TLR3-deficient mice infected withlethal doses of WNV show resistance to the WNV infection,suggesting that TLR3-mediated inflammatory responses inducedeath of the mice [69]. Therefore, the role of TLR3 in viralinfection is unclear.
TLR7, TLR8 and TLR9
TLR7, TLR8 and TLR9 are located in the intracellular endosomalcompartment, where they sense microbial nucleic acids suchas RNA and DNA. TLR7 and TLR8 (but not mouse TLR8)respond to synthetic antiviral imidazoquinoline compounds suchas R848, loxoribine and imiquimod and ssRNAs rich in guanosineor uridine derived from viruses [70–72] (Figure 1). Generally,these viruses gain entry into cells through receptor-mediatedendocytosis and reach the phagolysosome, where the virus-coatprotein is hydrolysed to expose the viral RNA to the TLRs. Incontrast, the host ssRNA does not reach the endocytic vesiclesbecause it is degraded by RNase.
TLR9 recognizes unmethylated CpG motifs of ssDNA andinduces inflammatory cytokines and type I IFNs. These sequencesare commonly present in the genomes of bacteria and viruses[73–77]. However, in the host, these sequences are highlymethylated at the cytosine base and, therefore, the host CpG motifsstimulate poorly. Synthetic ssDNA-containing CpG dinucleotidesmotifs can also induce the production of inflammatory cytokinesand type I IFNs through TLR9. There are two structurally distincttypes of CpG DNAs known, namely the A-type (D-type) andthe B-type (K-type). A-type CpG ODNs (oligodeoxynucleotides)stimulate pDCs to induce a robust amount of IFN-α and a littleIL-12 [78]. By contrast, B-type CpG ODN is a potent inducer ofinflammatory cytokines such as IL-6, IL-12 and TNF-α (tumournecrosis factor-α) and up-regulates co-stimulatory molecules suchas CD80, CD86 and MHC class II in pDCs and, to lesser extent, inB-cells [79]. DNA viruses such as MCMV, HSV (herpes simplexvirus)-1 and HSV-2 induce inflammatory cytokines and type IIFNs through TLR9 [73–77]. It has been shown that TLR9-deficient mice are susceptible to MCMV infection [76]. HSVrecognition by pDCs does not require viral replication, becauseUV-inactivated virus still induces IFN-α [75]. In addition to theseligands, TLR9 also recognizes the malarial pigment known ashaemozoin [80]. Recently, it was shown that cleavage of TLR9by lysosomal cathepsins is involved in the activation of signalling[81–83].
Signalling through TLR
Upon recognition, TLRs recruit various TIR-domain-containingadaptors to the TIR domain of TLRs. TLR5, TLR7, TLR9 andTLR11 only use MyD88. TLR1, TLR2, TLR4 and TLR6 useTIRAP in addition to MyD88, which links TLR to MyD88. TLR3only uses TRIF. TLR4 uses TRIF and TRAM, and TRAM linksTLR4 with TRIF. Taken together, TLR signalling can be broadlydivided into two signalling pathways: the MyD88-dependent andTRIF-dependent pathways [1,17,47,84,85] (Figures 1 and 2).
In the MyD88-dependent signalling pathway, the IRAK (IL-1receptor-associated kinase) family members such as IRAK4,IRAK1 and IRAK2 are recruited to the MyD88. IRAK4 is initiallyactivated, and IRAK1 and IRAK2 are sequentially activated [86].The activated IRAK family proteins associate with TRAF6 (TNFreceptor-associated factor 6), an E3 ubiquitin ligase, which formsa complex with E2 ubiquitin-conjugating enzymes such as UBC13(ubiquitin C 13) and UEV1A (ubiquitin-conjugating enzyme vari-ant 1A). This complex polyubiquitinates TRAF6 itself and IKKγ
[IκB kinase γ , also known as NEMO (NF-κB essential modifier)]through K63 (Lys63) linkage [87,88]. The polyubiquitinatedTRAF6 activates the protein kinase TAK (transforming growthfactor-β-activated kinase 1) and TABs (TAK1-binding proteins)such as TAB1, TAB2 and TAB3, which subsequently activatetranscription factors such as NF-κB and AP-1 (activator protein-1) through the canonical IKK complex and the MAPK [ERK(extracellular-signal-regulated kinase), JNK (c-jun N-terminalkinase) and p38] pathway respectively for the transcription ofinflammatory cytokine genes [87]. TAK1-deficient cells showreduced inflammatory cytokine levels and impaired NFκB andMAPK activation after stimulation with various TLR ligands[89,90]. TLR4 also activates NF-κB via TRIF by two distinctsignalling pathways [91]. The N-terminus of TRIF interacts withTRAF6 through the TRAF6-binding motifs [92] and the C-ter-minus of TRIF interacts with RIP1 (receptor-interacting protein1), both of which co-operate to activate NF-κB [93] (Figure 2).Recently, we showed that the TRIF-dependent signalling pathwayis negatively regulated by Atg16L1 (autophagy-related 16-like 1).Mice lacking Atg16L1 show enhanced production of cytokine,suggesting an essential role for autophagy in innate immuneregulation [94].
Stimulation with TLR4 and TLR3 ligands activates theTRIF-dependent signalling pathway and induces inflammatorycytokines in addition to type I IFNs and IFN-inducible genesin DCs and macrophages, which depend on IRF3 and IRF7.The production of type I IFNs is absent in TRIF-deficient cells[95]. IRF3 and IRF7 are activated by IKK-related kinase TBK1[TANK binding kinase 1, also known as T2K (TRAF2-associatedkinase) or NAK (NF-κB activating kinase)] and IKKi (alsoknown as IKKε) [96,97]. TBK1 and IKKi interact with TANK(TRAF family member-associated NF-κB activator), NAP1(NAK-associated protein 1) and, similar to NAP1, SINTBAD(TBK1 adaptor), which then phosphorylates IRF3 and IRF7.Phosphorylated IRF3 and IRF7 form a homodimer, whichsubsequently translocates into the nucleus and binds to the ISREs(IFN-stimulated response elements) to induce type I IFNs andIFN-inducible genes [98]. TRAF3 has been proposed to linkTRIF to TBK1, because TRAF3 interacts with these proteins andthe production of IFN-β is abrogated in TRAF3-deficient cells[99,100] (Figure 2).
In pDCs, TLR7 and TLR9 are highly expressed and inducea huge amount of type I IFNs, particularly IFN-α, after virusinfection. Upon stimulation, MyD88 forms a complex with IRF7[101,102] (which is highly expressed in pDCs) and TRAF6 toinduce the production of type I IFNs [100]. IRAK1 interactswith MyD88 and can phosphorylate IRF7 [103]. IRAK1-deficientpDCs consistently have defects in type I IFN production, butshow intact inflammatory cytokine production. Similarly, theproduction of type I IFNs is decreased in IKKα-deficient mice,and IKKα can bind to, and phosphorylate, IRF7 [104]. Thesefindings suggest that IRAK1 and IKKα act as IRF7 kinases.By contrast, pDCs lacking MyD88 or IRAK4 do not inducetype I IFNs and inflammatory cytokines. Taken together, theseobservations suggest that the TLR7– or TLR9–MyD88–TRAF6–IRAK4–IRAK1–IKKα–IRF7 signalling pathway is active inpDCs for the robust production of type I IFNs after virus infection(Figure 3). Recently, the involvement of the serine/threonineprotein kinase mTOR (mammalian target of rapamycin) and itsdownstream signalling kinases, such as the p70 ribosomal S6protein kinases p70S6K1 and p70S6K2, has been shown to playroles in pDC for the production of type I IFNs. Inhibition ofmTOR and its downstream kinases blocks the interaction betweenTLR9 and MyD88 and inhibits further activation of IRF7[105].
Pathogen recognition in the innate immune response 5
Figure 2 TLR signalling in conventional DCs or macrophages
The engagement of TLRs with their respective ligands initiates signalling. MyD88 recruits the IRAK family of proteins and TRAF6. TRAF6 activates TAK1 which, in turn, activates the IKK complexconsisting of IKKα, IKKβ and NEMO/IKKγ , and phosphorylates IκBs. Phosphorylated IκBs are ubiquitinated and undergo proteasome-mediated degradation, and NF-κB subunits, which consist ofp50 and p65, translocate to the nucleus. TAK1 also activates the MAPK signalling pathway. The activated NF-κB and MAPK initiate the transcription of inflammatory cytokine genes. TRIF recruits RIP1and TRAF6. Activated TRAF6 and RIP1 activate NF-κB and MAPK to induce transcription of inflammatory cytokine genes. TRIF interacts with TRAF3 and activates TBK1/IKKi, which phosphorylateIRF3 and IRF7. The phosphorylated IRF3 and IRF7 are translocated to the nucleus for the transcription of type I IFNs.
The ER (endoplasmic reticulum)-localized transmembraneprotein known as UNC93B1 was shown to be essential forthe production of inflammatory cytokines after stimulation withTLR3, TLR7 and TLR9 ligands [106]. In addition, UNC93B1plays a crucial role in the cross-presentation of an exogenousantigen via MHC Class I and Class II. Moreover, UNC93B1 isrequired for the translocation of TLR7 and TLR9 from the ER tothe endolysosome [107–109].
TLR and human diseases
Genetic variations or a deficiency of genes encoding TLR andTLR signalling proteins have been implicated in the predispositionto innate immune diseases [110]. Recently, autosomal recessiveMyD88-deficient pediatric patients have been reported. Thesepatients show susceptibility to pyrogenic bacterial infections suchas Strep. pneumoniae and Staph. aureus, and severe complicationshave been reported in several patients in early childhood.Otherwise, these patients are healthy and have normal immunityto other microbes, and their clinical severity improves with age.These observations suggest that MyD88-dependent signalling isessential for protective immunity against a few types of pyrogenicbacteria, but is dispensable for the host defence to the majority ofother infections in humans [111].
IRAK4 deficiency has also been reported in humans. TheIRAK4 gene is located on an autosomal chromosome and 28individuals with a recessive gene have been reported so far.These patients show increased susceptibility to infection by Gram-positive and Gram-negative bacteria in early childhood [112].Similar to MyD88 deficiency or mutants, IRAK4-deficient peoplealso show an improvement in symptoms with advancing age.These patients also show impaired induction of type I IFNs, butdo not show any susceptibility to viral infection or HSE (herpessimplex virus encephalitis). Furthermore, these individuals donot show susceptibility to any parasitic or fungal diseases. Pat-ients with TLR3 deficiency have also been reported. Thesepatients show increased susceptibility to HSV-1 [113]. These ob-servations suggest that TLRs play an important role in thepathogenesis of some diseases. UNC93B deficiency has alsobeen documented in some patients. Cells from these patientsdo not respond to TLR3, TLR7, TLR8 or TLR9. However,similar to IRAK4-deficient patients, UNC93B patients do notshow susceptibility to viral diseases such as HSE [114].
RIG-I-LIKE RECEPTORS
Much progress has been made in TLR-independent recognition ofviral nucleic acids, particularly RNA recognition by intracellular
TLR7 and TLR9, which are associated with UNC-93B, are transported to the endolysosome. In the endolysosome, these TLRs interact with their respective ligands and recruit MyD88. MyD88interacts with TRAF6 through IRAK4 and activates TAK1. TAK1 activates NF-κB through the IKK complex. MyD88 also interacts with IRAK4 and IRAK1. The IRAK1 and IKKα phosphorylate IRF7.IRAK1 interacts with TRAF3 and phosphorylates IRF7. The phosphorylated IRF7 is translocated to the nucleus for the transcription of type I IFNs.
sensors [1,115–118]. The intracellular sensors are important forthe detection of RNA derived from RNA viruses and replicatingDNA viruses. The recognition of RNA by intracellular sensorssubsequently activates innate antiviral responses, mainly throughthe induction of type I IFNs and inflammatory cytokines in mostcell types.
In this regard, DExD/H-box RNA helicase, known RIG-I, wasfound to have a role in the cytoplasmic recognition of dsRNAand activation of IRFs and NF-κB [119]. Two members ofthis family known as Mda5 (melanoma differentiation-associatedgene 5) and Lgp2 (Laboratory of Genetics and Physiology 2)were subsequently identified [120]. These proteins are collectivelyknown as RLRs. RIG-I and Mda5 contain two tandem repeatsof the CARD (caspase recruitment domain) at their N-terminus,which are important for activating downstream signalling. Theintermediate portion of these proteins contains the helicasedomain, which is similar to other members of the DExD/H-boxRNA helicase family. For RIG-I, this domain contains an ATP-binding region, which is essential for RIG-I function. However,an ATP-binding region was not found in Mda5. In addition, RIG-
I contains an RD (repressor domain) at the C-terminus, whichrepresses the activity of RIG-I [121,122]. Repression of Mda5 inthe steady state is not known, but it has been postulated that MdA5might be negatively regulated by other proteins such as DAK(dihydroacetone kinase) [123]. Lgp2 contains the RNA helicasedomain, but is devoid of the CARD. This protein was consideredto be a negative regulator for RIG-I and Mda5. However, a studyof Lgp2-deficient mice revealed that Lgp2 acts as both a negativeand positive regulator depending on the virus [124]. Recently, ahomologue of RIG-I helicase (DExD/H-box helicase), Dicer-2,was identified in Drosophila and which controls the expressionof an antiviral protein known as Vago after virus infection,suggesting an evolutionarily conserved role of RLR in antiviralresponses [125].
Pathogen recognition by RLRs
Studies of RIG-I- and Mda5-deficient mice revealed that thesesensors recognize different classes of RNA viruses. RIG-I-deficient cells infected with NDV (Newcastle-disease virus),
Pathogen recognition in the innate immune response 7
VSV, SeV (Sendai virus) and IV (influenza virus) show impairedtype I IFN and inflammatory cytokine production [126,127].Furthermore, members of the flavivirus family, such as JEV(Japanese encephalitis virus) and HCV (hepatitis C virus) arealso recognized by RIG-I [128,129]. However, dengue virus andWNV, which belong to the flavivirus family, do not require RIG-I.By contrast, Mda5-deficient mice show normal production oftype I IFNs and inflammatory cytokines against NDV, VSV, SeV,IV and JEV. However, they show impaired ability to produce type IIFNs and inflammatory cytokines against picornaviruses such asEMCV, Theiler’s virus and Mengo virus [128]. These observationssuggest that these two sensors induce antiviral responses against awide spectrum of RNA viruses with different specificity. However,RLRs are not sufficient for the protection against other RNAviruses such as IV, RSV and LCMV in vivo. In other words, in vivoinfection of LCMV induces the production of type I IFNs and thepromotion of virus-specific CD8 T-cells through the TLR7 ratherthan the RLR signalling pathway [130]. For IV infection, the RLRsignalling pathway is essential for the induction of cytokines infibroblasts, alveolar macrophages, and cDCs, whereas TLR7 actsin pDCs to induce cytokines [131,132]. However, the productionof virus-specific antibodies is dependent on TLR7 rather thanRLR. Similar observations were reported for RSV infection[133], suggesting that TLR and RLR signalling pathways togetherinduce the host defence against these viruses. Moreover, co-operative activation of TLR and RLR is also required for theadjuvant effects of poly(I-C) [134].
RIG-I and Mda5 recognize in-vitro-synthesized dsRNA anda synthetic analogue of dsRNA poly(I-C) respectively [128].Further studies revealed that the 5′-triphosphate moiety of RNAis essential for RIG-I recognition [135] and it is independent ofthe strand property (single or double) of RNA [136]. A recentbiochemical study revealed that the 5′-triphosphate moiety ofRNA is recognized by the CTD (C-terminal domain) of RIG-I[122]. Host-cell RNA is not recognized by RIG-I because, duringsynthesis of cellular RNA, the 5′-ends are either modified bythe addition of a 7-methylguanosine cap or the 5′-triphosphateis removed before transportation to the cytoplasm. Thus RIG-Ican discriminate between viral and host RNA. RIG-I can bind toa 25-bp dsRNA to efficiently induce type I IFNs and the RNAend structures (blunt end, 5′-overhang and 3′-overhang), and thenucleotide sequences are not critical for binding to RIG-I [137].Furthermore, it has been shown, using NMR of the CTD, thatRIG-I recognizes two distinct viral RNA patterns, including dsand 5′-triphosphate ssRNA, and gel-filtration analysis revealedthat a dimer of RIG-I CTD mediates the recognition of 5′-triphosphate RNA [118]. The length of dsRNA is critical fordifferential recognition by RIG-I and Mda5; RNA viruses have ashorter RNA length (approx. 1.2–1.4 kbp) and are recognized byRIG-I, whereas viruses with longer dsRNA (longer than 3.4 kbp)are recognized by Mda5 [138].
SIGNALLING THROUGH RLRs FOR ANTIVIRAL RESPONSES
In response to viral infection, the CARDs of RIG-I and Mda5associate with the CARD-containing adaptor protein knownas IPS-1 {IFN promoter stimulator-1, also known as MAVS(mitochondrial antiviral signalling), Cardif (CARD adaptor in-ducing IFN-β) and VISA (virus-induced signalling adaptor [139–142]} to induce inflammatory cytokines and type I IFNs. Ectopicexpression of IPS-1 in cells activates NF-κB and IFN promoters.In addition, IPS-1-deficient cells show a complete abrogation ofinflammatory cytokines and type I IFNs after virus infection.Furthermore, IPS-1-deficient mice infected with various RNAviruses recognized by RIG-I and Mda5 show enhanced motility
compared with that in WT mice [143,144]. These observationscollectively suggest that IPS-1 is the sole adaptor for RIG-I/Mda5 and plays an essential role in host defence againstvarious RNA viruses. It has been shown that IPS-1 is localizedin the outer membrane of mitochondria [140]. NS3/4 (non-structural 3/4) protease from HCV cleaves and dislodges IPS-1from the mitochondria to block IFN production, suggesting thatmitochondrial localization is essential for IPS-1 function withrespect to antiviral responses [141]. The NLR family proteinknown as NLRX1 was recently reported to be localized in the outermembrane of mitochondria and to inhibit IPS-1-mediated type IIFN induction in response to virus infection [145]. Signallingthrough RIG-I is further regulated by ubiquitination. It was shownthat TRIM25 (tripartite motif 25), a ubiquitin E3 ligase whichcontains an RNF (RING-finger) domain, a B-box/coiled-coildomain and a SPRY (SPla/RYanodine) domain, interacts with theN-terminal CARD of RIG-I. This interaction leads to the Lys63-linked ubiquitination of RIG-I. Furthermore, TRIM25-deficientcells show abrogation of type I IFNs production, suggesting thatubiquitination of RIG-I is essential for activation of the signallingpathway[146]. By contrast, RIG-I is inhibited by another ubiquitinligase, RNF125, which induces ubiquitination and proteasomaldegradation of RIG-I. These observations suggest that theubiquitination is an additional regulatory mechanism for the RIG-I-mediated signalling pathway [147] (Figure 4).
TRAF3, an E3 ubiquitin ligase that polyubiquitinates throughits C-terminal TRAF domain, was shown to interact withIPS-1 and activates TBK1 and IKKi [99,100,148]. Recently,a de-ubiquitinase enzymatic protein known as DUBA (de-ubiquitinating enzyme A) was reported to deubiquitinate TRAF3and inhibit the RLR-mediated signalling pathway [149].
RLR-mediated NF-κB activation is achieved via the FADD(Fas-associated death domain) protein, which interacts withcaspase 8 and caspase 10 and forms a complex with IPS-1[139,150]. The TNFR-I (TNF receptor-I) signalling adaptorTRADD (TNF-receptor-associated DD), an adaptor in theTNFR-I signalling pathway, has been suggested to be involvedin RLR signalling. The engagement of RLRs by viruses leads tothe formation of a molecular complex that consists of TRADD,FADD and RIP1. This complex interacts with IPS-1 and activatesIRF3 and NF-κB [151].
The ER-localized STING [stimulator of interferon genes, alsoknown as MITA (mediator of IRF3 activation)] protein wasrecently identified and shown to be involved in host defenceagainst RNA virus. Knockdown of STING impaired IFN-βproduction by poly(I-C) transfection. The replication of RNAviruses is higher in STING-deficient fibroblast cells than in WTcells. In addition, the production of IFN-β was also reducedin STING-deficient fibroblast cells. However, STING-deficientbone-marrow-derived macrophages and DCs show replication ofvirus comparable with that shown by WT cells, suggesting a cell-type-specific role of STING [152,153].
Autophagy is an essential biological process that maintainscellular homoeostasis, development, differentiation and tissueremodelling. Recent studies have highlighted the importance ofautophagy in innate immunity. Recently it was shown that IPS-1 and RIG-I were associated with the Atg5–Atg12 complex,which is an essential component for autophagy. Mouse embryonicfibroblasts deficient in Atg5 and Atg12 show increased levelsof type I IFN production in response to VSV infection [154].This observation suggests that autophagy is important in thenegative regulation of the RLR signalling pathway. However, ithas been shown that autophagy plays an opposite role in pDCs.In pDCs, TLR7 recognizes ssRNA in the endolysosome, butit also recognizes replicating VSV. Atg5-deficient pDCs show
Figure 4 Recognition of RNA and RNA viruses by RLRs
Viral RNA, ssRNA, dsRNA and dsRNA analogue poly(I-C) are recognized in the cytoplasm of cDCs, macrophages and fibroblast cells by RIG-I and Mda5. Upon recognition, the CARD of Mda5 andubiquitinated CARD of RIG-I bind to IPS-1 (located on the outer membrane of mitochondria). The CARD of RIG-I undergoes ubiquitination by TRIM25. IPS-1 then interacts with TRADD and formsa complex with FADD and caspase 8/10 to activate NF-κB through the IKK complex. TRADD also interacts with TRAF3 and activates TBK1/IKKi, which phosphorylates IRF3 and IRF7 to induce thetranscription of type I IFNs. The mitochondrial NLRX1 inhibits IPS-1-mediated signalling. Mitochondrially localized STING interacts with RIG-I and IPS-1 and activates NF-κB and IRFs.
reduced IFN-α production after VSV infection, suggesting that theautophagosome is formed after VSV infection and contains viralRNA, and this autophagosome traffics to the lysosome, where itforms a complex with the autophagolysosome after fusion withthe lysosome. In the autophagolysosome, TLR7 recognizes viralRNA for the production of IFN-α [155].
RECOGNITION OF DNA BY CYTOSOLIC RECEPTOR
Bacterial and viral dsDNA are immunostimulatory componentsthat activate various cell types to induce type I IFNsand inflammatory cytokines through cytosolic DNA sensors.Cytosolic recognition of dsDNA induces TBK1/IKKi-dependenttype I IFNs and NF-κB-dependent inflammatory cytokines[156,157] (Figure 5). DAI (DNA-dependent activator of IRFs) hasbeen reported to be a cytosolic sensor for DNA [158]. However,DAI-deficient cells do not show impaired cytokine production,suggesting that DAI is not essential for the recognition of DNA[159]. Recently, we have shown that a DNA vaccine that consistsof plasmid DNA and has inherited properties of adjuvant inducesinnate and adaptive immunity, such as antigen-specific antibodyproduction and CD4 and CD8 T-cell responses, through TBK1 butnot TLR9, MyD88 and DAI. This suggests that the DNA-inducedadjuvant effects are TLR- and DAI-independent phenomena and
these effects may depend on an unknown DNA sensor that signalsthrough TBK1 [159].
Recently, STING has been shown to be involved in therecognition of dsDNA. STING was shown to play a pivotal rolein type I IFN production by dsDNA when introduced into thecytosol. Furthermore, infection with a DNA virus such as HSV-1, and Listeria monocytogenes, organisms which are known tointroduce DNA into the cytosol during infection, shows a reducedtype I IFN production in STING-deficient cells. This suggeststhat STING plays an important role in DNA sensor signallingpathways [152,153].
NOD-LIKE RECEPTORS
The NLR family of proteins are cytosolic, intracellular PRRsthat recognize PAMPs and endogenous ligands. The recognitionof ligands induces a signalling cascade leading to activation ofNF-κB, or a cytoplasmic multiprotein complex known asthe inflammasome, to produce inflammatory cytokines [1,11–13,160,161]. In addition, NLRs are also involved in the signallingfor cell death after microbial infection [162]. The NLR familycomprises 23 proteins in humans and 34 proteins in mice[163]. However, the function of most of the NLR proteins ispoorly understood. These proteins have a trimodular structure
Pathogen recognition in the innate immune response 9
Figure 5 Recognition of DNA and DNA viruses by DNA sensors
An unknown cytosolic DNA sensor or DAI activates NF-κB through activation of the IKK complex and IRF3 and IRF7 through TBK1/IKKi. ER-localized STING induces the activation of NF-κB andIRFs. DNA virus infection and introduction of dsDNA activates the inflammasome consisting of AIM2/ASC and NALP3/ASC respectively. The inflammasome converts inactive pro-caspase 1 intoactive caspase 1 for the maturation and production of IL-1β . Adenovirus infection directly or indirectly activates the NALP3 inflammasome.
and consist of the following domains. The C-terminal domainconsists of tandem repeats of LRR, which are essential forsensing or recognition of the microbial components. A centrallylocated nucleotide binding NOD domain is essential for self-oligomerization and formation of a complex for the activationand recruitment of downstream signalling proteins. The variableN-terminal domains are defined by CARD, DED (death effectordomain), PYD (pyrin domain) or BIR (baculovirus inhibitorof apoptosis protein repeat) domain [163]. These domains areessential for downstream signal transduction through homotypicprotein–protein interactions. Mutations in these proteins werereported to be associated with chronic inflammatory diseasessuch as familial cold autoinflammatory syndromes (familialcold urticaria) [164,165], Muckle-well (or urticaria–deafness–amyloidosis) syndrome and Crohn’s disease [166].
NOD1 AND NOD2
NOD1 (also known as CARD4) recognizes a distinct substructureof PGN (peptidoglycan), namely iE-DAP (γ -D-glutamyl-m-
diaminopimelic acid, a dipeptide), which is present in Gram-negative and Gram-positive bacteria such as Bacillus subtilis andL. monocytogenes [167,168]. NOD2 (CARD15) recognizes MDP(muramyl dipeptide), the largest component of PGN motif, whichis also present in Gram-negative and Gram-positive bacteria [169].
Upon stimulation with these ligands, NOD1 and NOD2 interactwith a CARD domain-containing serine/threonine kinase knownas CARDIAK [CARD-containing IL-1β-converting-enzyme-associated kinase, also known as RICK (RIP-like interactingcaspase-like apoptosis-regulatory protein kinase) and RIP2][170,171] and induces antimicrobial peptides [172] and in-flammatory cytokines through activation of MAPKs [173] andNF-κB. MAPKs are activated through another CARD-containingprotein known as CARD9 [174] (Figure 6). Ex vivo studiesshowed that various pathogenic bacteria, such as E. coli[175], Shigella flexneri [168], Pseudomonas aeruginosa [176],Chlamydia species [177–179], Campylobacter jejuni [180] andHaemophilus influenza [181] are sensed by NOD1 and Strep.pneumonia [182] and M. tuberculosis [183] are sensed by NOD2.L. monocytogenes has been reported to be sensed by both NOD1and NOD2 [184,185]. In vivo, it has been shown that NOD1 and
NOD1 and NOD2 sense various PAMPs of pathogenic bacteria in the cytoplasm or PAMPs thatare transported through the type III or type IV secretion systems. Upon recognition of PAMPs,these sensors undergo for self-oligomerization and formation of a complex that triggers theassociation of CARDIAK, which in turn activates NF-κB. NOD1 and NOD2 also activatethe MAPK signalling pathway through CARD9.
NOD2-deficient mice are susceptible to L. monocytogenes and H.pylori respectively [185,186].
The inflammasome
Pathogen infection into the host results in the production ofvarious inflammatory cytokines. The IL-1 family of cytokines,including IL-1β, IL-18 and IL-33, are key cytokines that regulatevarious components of innate and adaptive immunity. Theproduction of these cytokines is regulated by two signals inthe innate immune cells (e.g. macrophages). The first signal istranscriptional and translational up-regulation of pro-forms of thecytokine (pro-IL-1β, pro-IL-18) in response to various TLR, NLRand RLR agonists. The second signal is the processing of the pro-form of these cytokines to the mature, secretary form of the cyto-kines by the inflammasome. Depending on the NLR proteins, theinflammasome is categorized into three types such as NALP3[NACHT (NTPase-domain named after NAIP, CIITA, HET-E and TP1)–LRR-PYD–containing protein 3, also known asNLRP3, CIAS1 (cold-induced autoinflammatory syndrome 1) andcryopyrin] inflammasome, the IPAF [IL-1β-converting-enzymeprotease-activating factor, also known as the NLRC4 (NLRfamily, CARD domain containing 4)] and CLAN (CARD LRR-and NACHT-domain-containing protein) inflammasome and theNALP1 (also known as NLRP1) inflammasome (Figure 7).
NALP3 inflammasome
The NALP3 inflammasome is activated by various exogenous andhost endogenous ligands. Exogenous ligands include microbialligands such as MDP, bacterial and viral RNA, toxins such asnigericin, maitotoxin, environmental pollutants such as asbestosand silica [187] and vaccine adjuvant alum (aluminium salts)[188,189]. Host endogenous ligands include MSU (monosodiumurate), calcium pyrophosphate dehydrate, amyloid-β fibrillarpeptide and ATP. In addition, the NALP3 inflammasome is alsoactivated by UV light and skin irritants such as picryl chloride and2,4-dinitrofluorobenzene. The NALP3 inflammasome consistsof NALP3, CARDINAL (CARD inhibitor of NF-κB-activatingligands), ASC (apoptosis-associated speck-like protein containinga CARD) and caspase 1, which process cytosolic pro-IL-1β tobioactive secretory IL-1β [187].
Activation of the inflammasome leads to the production of IL-1β, which may play an important role in clearing pathogensfrom the host. A recent study has shown that the NALP3inflammasome is essential for alum-induced adjuvant effectssuch as antibody production to antigens and Th2-mediatedinflammation [189]. However, there is another study showing thatthe NALP3 inflammasome is dispensable for the adjuvant effects,but indispensible for IL-1β production [190]. There are severalpossible differences among these studies, such as preparationof mixture of antigen and alum before immunization, the dose ofantigen or adjuvant, the purity of antigen (some antigen hasimpurity of immunomodulatory substances), and the route andprotocol of immunization. Therefore it is almost impossible toevaluate the data from two different laboratories. Furthermore,the differences in results could be due to multiple mechanismsof action of alum. Taken together, further studies are needed toconclude the involvement of NALP3 inflammasome in adjuvanteffects of alum. It has been suggested that activation of theinflammasome by the host ligand plays an important role inthe pathogenesis of arthritic diseases such as gout, pseudogout[187] and Alzheimer’s disease [191]. The exogenous ligand silicainduces NALP3-inflammasome-dependent silicosis [192,193].These observations suggest that the NALP3 inflammasome playsa pivotal role in the activation of innate immune responses for hostdefence and is a mediator for the pathogenesis of various diseases.
Recently, the introduction of bacterial, viral and host dsDNAinto the cytosol of cells has been shown to induce the productionof IL-1β, which requires the inflammasome components ASCand caspase 1, but not NALP3. However, the introduction ofadenoviral DNA showed that IL-1β production depends onNALP3, ASC and caspase 1. AIM2 (absent in melanoma 2)has been shown to recognize dsDNA. AIM2 is a member of theIFI20X (interferon-inducible protein 20X)/IFI16 or PYHIN [PYDand HIN (haematopoietic interferon-inducible) nuclear proteindomain family member 1]. It has been shown that the HIN200domain of AIM2 binds to dsDNA, whereas PYD associates withASC [194–197] (Figure 5).
The NALP3 inflammasome is activated by numerous ligands;however, it is unknown whether these ligands bind directly toNALP3. Pore-forming toxins such as staphylococcal α-toxin andpneumolysin, K+ ionophores and a low concentration of detergentinduce an efflux of K+ to activate the NALP3 inflammasome.In addition, ATP, maitotoxin and nigericin activate hemichannelpannexin-1 and the purinergic receptor P2X7 to induce effluxof K+ and activate the NALP3 inflammasome. Furthermore,ROS (reactive oxygen species) are also reported to activate theNALP3 inflammasome [187]. Two different mechanisms for ROSproduction have been proposed, including efflux of K+-inducedstress to the mitochondria and NADPH oxidase-mediated
Pathogen recognition in the innate immune response 11
Figure 7 Activation of the inflammasome
The inflammasome is activated in two steps. The first step is transcriptional and translational up-regulation of pro-forms of the cytokine (pro-IL-1β , pro-IL-18) in response to various TLR, NLR andRLR agonists through activation of NF-κB. In the second step, the inactive form of the cytokines is converted into a biologically active form by the inflammasome. The ligands (described in the text)directly and indirectly (through an unknown sensor) induce the formation of various inflammasomes such as the NALP3 inflammasome, the IPAF inflammasome or the NALP1 inflammasome. Inaddition to ASC, the NALP3 inflammasome contains CARDINAL protein (the domain arrangement of each component is shown in the box labelled ‘Components of inflammasome’. The inflammasomeconverts inactive pro-caspase 1 into active caspase 1, which induces cell death (in the case of NALP1 inflammasome) and/or converts pro-cytokines (inactive) into bioactive secretory cytokines. TheNALP1 protein interacts with anti-apoptotic proteins such as Bcl-2 and Bcl-XL and inhibits caspase-1-dependent cell death and production of IL-1β and IL-18.
induction in the phagolysosome. Other mechanisms have alsobeen proposed recently. Phagocytosis of silica crystal and fibrousparticles of amyloid-β induce lysosomal destabilization andpermeabilization which leads to the release of cathepsin B into thecytosol [191,198]. The released cathepsin B activates the NALP3inflammasome.
IPAF inflammasome
The IPAF inflammasome is activated by intracellular pathogenssuch as Salmonella typhimurium [199] and Legionellapneumophila [200]. It was suggested that S. typhimurium andL. pneumophila deliver flagellin to the cytosol via the type IIIand type IV secretion systems respectively [199,200]. The intro-duction of recombinant purified flagellin protein into the cyto-sol also promotes IPAF-dependent caspase 1 activation [201].Flagellin of Ps. aeruginosa also induces IPAF- and caspase-1-dependent production of IL-1β [202]. In addition to IPAF,NAIP5 (apoptosis inhibitory protein 5, also known as Birc1 (BIR-
containing 1)] was reported to be involved in the recognition ofL. pneumophila [203,204]. NAIP5 consists of three consecutiverepeats of the BIR domain at the N-terminus. Previous studieshave indicated that NAIP5 regulates the replication of L.pneumophila via caspase-1-dependent cell death. It has beenreported that 35 amino acids from the C-terminus of flagellinand infection with L. pneumophila is sufficient to activate theinflammasome, but is not activated in the absence of NAIP5. Inaddition, full-length flagellin activates NAIP5-independent, butIPAF-dependent, cell death [205]. These findings suggest thatthe IPAF and NAIP5 proteins are essential for the flagellin-induced immune response.
NALP1 inflammasome
Anthrax LT (lethal toxin) is a potent toxin of Bacillus anthracisthat induces cell death [206]. LT is composed of a protectiveantigen and a lethal factor. The protective antigen is a receptor-binding protein that creates a pore to deliver the lethal factor
into the cytosol of infected cells and induce cell death. It hasbeen shown that the polymorphic gene known as Nlrp1b isresponsible for susceptibility to LT [207]. It was also shownthat LT-induced macrophage death requires caspase 1, which isactivated in susceptible macrophages [207]. Furthermore, the LTof B. anthracis was shown to induce IL-1β, which depends onthe NALP1 inflammasome. In addition, it was shown that NOD2is also required for IL-1β production and NOD2–NALP1 formsa molecular complex during infection [208].
NALP1 is implicated in the association with anti-apoptoticproteins Bcl2 (B-cell lymphoma-2) and Bcl-X(L), which suppresscaspase-1 activation, indicating a role of Bcl-2 proteins in theinhibition of LT-induced cytotoxicity [209].
CONCLUSIONS AND FUTURE DIRECTIONS
Although numerous studies have greatly enhanced ourunderstanding of innate immune recognition by TLRs, RLRs,NLRs and unknown PRRs, we still know relatively little aboutthe recognition of the vast array of micro-organisms and thecross-talk among these receptors. Although the cytosolic receptorfor DNA, which induces IL-1β production, has been reported,we have not yet characterized the sensors for DNA or DNAviruses that activate IRFs. The function of most of the membersof the NLR family in host defence and their ligands are alsounidentified. Characterization of these PRRs in innate immunitywill further improve our understanding of the complexities ofinnate immune regulation. In addition, structural studies of PRR–ligand complexes will improve our understanding of innateimmunity and facilitate the design of novel drugs that targetPRRs.
ACKNOWLEDGEMENTS
We thank the members of our laboratory for insightful discussions and apologizeto those authors whose work could not be included in this review owing to spacelimitations.
FUNDING
This work was supported by the Japan Society for the Promotion of Science [grant numberPO8123 (postdoctoral fellowship to H. K.)].
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Received 16 February 2009/5 March 2009; accepted 6 March 2009Published on the Internet 28 April 2009, doi:10.1042/BJ20090272