Download Pathogen recognition in the innate immune response

Biochem. J. (2009) 420, 1–16 (Printed in Great Britain)
1
doi:10.1042/BJ20090272
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 receptors 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.
INTRODUCTION
T-cells, which provide pathogen specific immunity to the host
through somatic rearrangement of antigen receptor genes. Bcells 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
The innate immune system is the first line of the defence system
against microbial pathogens such as Gram-positive and Gramnegative 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 production 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
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).
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 inhibitor 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-mdiaminopimelic 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, mitogenactivated 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, nucleotidebinding 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, receptorinteracting 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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H. Kumar, T. Kawai and S. Akira
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 families
of receptors are collectively known as PRRs (pattern-recognition
receptors) [14], which recognize the specific molecular structures
of pathogens known as PAMPs (pathogen-associated molecular
patterns) in various compartments of cells, such as the plasma
membrane, the endolysosome and the cytoplasm.
In the present review we will focus on recent advances in the
study of recognition and signalling mediated by TLRs, RLRs and
NLRs.
TOLL-LIKE RECEPTORS
The Toll protein was originally identified in fruitflies (Drosophila)
and is involved in dorsoventral polarity during embryonic
development [6]. Further studies have shown that the Toll protein
plays an essential role in mounting an effective immune response
against the fungus Aspergillus fumigatus [6]. These studies led
to the identification of homologues of Toll proteins in humans
and mice through database searches and which are referred to
as TLRs [7]. To date, 10 and 13 TLR members have been
identified in humans and mice respectively [1]. The TLRs are
type I membrane glycoproteins and consist of an extracellular
LRR (leucine-rich repeat) domain, a transmembrane domain and
a cytoplasmic TIR ]Toll/IL (interleukin)-1 receptor] domain [15].
The LRR domain of TLRs consists of 16–28 tandem repeats
of the LRR motif [16] and is involved in the recognition of
ligands 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 lipopeptides
from various pathogens), derivatives of lipid (lipoarabinomannan)
from mycobacteria) and a complex derivative of protein or
peptides, sugar and lipid (diacyl lipopeptides from mycoplasma)
[1]. The TIR domain of TLRs consists of approx. 150 amino
acids and shows homology with the cytoplasmic region of the
IL-1 receptor. Therefore, it is termed the TIR domain [15,17].
The TIR domain interacts with TIR-domain-containing adaptors
such as MyD88 (myeloid differentiation primary response gene
88), TIRAP [TIR-containing adaptor protein, also known as MAL
(MyD88-adaptor-like], TRIF [TIR-containing adaptor-inducing
IFN (interferon)-β, also known as TICAM1 (TIR-domaincontaining adaptor molecule 1] and TRAM (TRIF-related adaptor
molecule, also known as TICAM2). In turn, the downstream
signalling pathways activate MAPKs (mitogen-activated protein
kinases) and transcription factors such as NF-κB (nuclear factor
κB) and IRFs (IFN regulatory factors) to induce production of
inflammatory cytokines and type I IFNs.
The TLR family members are expressed on various immune and
non-immune cells such as B-cells, NK (natural killer) cells, DCs,
macrophages, fibroblast cells, epithelial cells and endothelial
cells. However, TLRs are differentially localized within the cells.
TLR1, TLR2, TLR4, TLR5 and TLR6 are expressed on the cell
surface, whereas TLR3, TLR7, TLR8 and TLR9 are expressed in
the endosomes (Figure 1).
Recently, co-crystallization of TLR1–TLR2, TLR3 and TLR4
and their ligand-recognition properties has been reported. The
heterodimer or homodimer of the LRR domain of TLR1–TLR2,
TLR4 and TLR3 show a horseshoe-like structure or m-shaped
framework and consists of both a concave and a convex surface.
These surfaces are responsible for ligand binding and ligandinduced TLR dimerization [18–21].
c The Authors Journal compilation c 2009 Biochemical Society
Pathogen recognition by the extracellular cell-surface TLRs
TLR1, TLR2 and TLR6
TLR2 recognizes a structurally diverse range of PAMPs that
include proteins such as V-antigen (LcrV) from Yersinia,
haemagglutinin protein from measles virus, glycolipids, LTA
from Staphylococcus aureus and Streptococcus pneumoniae
[22], lipopeptides or lipoproteins such as MALP (macrophageactivating lipopeptide)-2 and R-MALP from Mycoplasma species
[23–25], lipoproteins from Escherichia coli [26], Borrelia
burgdorferi [27], Mycoplasma species [28] and Mycobacterium
tuberculosis [29], peptidoglycan from Staph. aureus, Strep.
pneumoniae and Strep. pyogenes [30–32] and polysaccharides
known as zymosan from Saccharomyces cerevisiae [33,34]. The
ligands 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], viruses
such as HSV (herpes simplex virus) [37] and varicella-zoster virus
[38] (Figure 1). The diversity of ligand recognition by TLR2 is
possible because TLR2 can recognize the ligands in association
with structurally related TLRs such as TLR1 and TLR6. The
TLR2–TLR1 and TLR2–TLR6 heterodimers recognize triacyl
lipopeptide derived from Gram-negative bacteria and diacyl lipopeptide derived from mycoplasma respectively [39,40]. TLR2
also recognizes zymosan (β-1,3-glucan and β-1,6-glucan) in
association with the structurally unrelated C-type lectin family
known as dectin-1 [41]. Furthermore, the class II scavenger
receptor CD36 has been shown to be involved in phagocytosis
and cytokine production in response to Staph. aureus and its cellwall components such as LTA and MALP-2, suggesting that CD36
functions as a co-receptor of TLR2/6 [25].
TLR4
TLR4 recognizes LPS from Gram-negative bacteria [8,9],
glycoinositolphospholipids from Trypanosoma [42], the fusion
protein from RSV (respiratory syncytial virus) [43] and the
envelope protein from MMTV (mouse mammary tumour virus)
[44]. TLR4 also recognizes diterpene (taxol) purified from the
bark of Taxus brevifolia (the Pacific yew) [45,46] (Figure 1). In
addition, TLR4 directly or indirectly recognizes endogenous molecules such as heat-shock proteins, fibrinogen, hyaluronic acid,
β-defensin and extracellular domain A in fibronectin [47]. The
recognition of LPS is triggered by a complex that contains TLR4,
a recognition subunit MD2 (myeloid differentiation protein-2) and
membrane-bound GPI (glycosylphosphatidylinositol)-anchored
CD14. The activation of TLR4 is further supported by another
protein known as LBP (LPS-binding protein) [48]. The studies
show that the lipid A (an active component of LPS) binds to
MD2 and forms a complex. The lipid A–MD2 complex interacts
with TLR4 and activates signalling, suggesting that MD2 is more
important in the recognition of lipid A [18].
TLR5
TLR5 recognizes a monomer of flagellin [49], an important
structural protein of pathogenic and non-pathogenic motile
bacteria. It is also important for adhesion and invasion at the
luminal surface of the epithelial cells covering the mucosal tissues
during infection [50]. Flagellin from Salmonella typhimurium
contains 494 amino acids and consists of two functional domains.
The 140 amino acids of the N-terminal domain and 90 amino acids
of the C-terminal domain of this protein are highly conserved and
essential for the polymerization and motility of flagellin [51]. The
central domain of the protein is highly variable among Salmonella
Pathogen recognition in the innate immune response
Figure 1
3
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 indicated
ligands. 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 recruit
MyD88, whereas TLR3 recruits TRIF.
serovars and bacterial species, and this region is exposed at the
outer surface of the flagellum. Amino acids 89–96 are essential
for TLR5 activation, but this region is located deep inside the
tertiary structure of the flagellin protein and becomes accessible
only when flagellin is present in monomer form [52,53]. However,
it is not clear how flagellated bacteria deliver monomeric flagellin
under physiological conditions. TLR5 is mainly expressed on the
luminal surface of the epithelial cells covering the mucosal tissues
and trachea, and the bronchi and the alveoli of the respiratory tract
[54–58] (Figure 1). Upon activation with flagellin, epithelial cells
induce cytokines and chemokines, and neutrophil recruitment.
Recently, we have shown that TLR5+ small-intestinal LPDCs
(lamina propria DCs) are important for the induction of humoral
and cellular immunity in the intestine. These LPDCs can induce
retinoic acid and are involved in the generation of IgA+ plasma
cells, as well as the differentiation of both Th1 (T-helper 17) and
Th1 cells in the intestine in a TLR5-dependent manner [59].
TLR11
TLR11 recognizes profilins from Toxoplasma gondii, an
obligate intracellular protozoan parasite [60]. It also recognizes
uropathogenic E. coli (Figure 1). Mice deficient in TLR11 show
increased 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 predicted
mRNA has at least one stop codon [62].
Pathogen recognition by intracellular TLRs
TLR3
TLR3 recognizes viral ds (double-stranded) RNA originating
from dsRNA viruses such as reovirus [63]. TLR3 also recognizes
dsRNA produced during replication of ss (single-stranded) RNA
viruses, such as WNV (West Nile virus) [64], RSV [65] and
EMCV (encephalomyocarditis virus) [66]. In addition, TLR3
recognizes 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-presenting
cell that induces various cytokines after stimulation with ligands),
macrophages, B-cells, NK cells and non-immune cells, including
epithelial cells. However, TLR3 is not expressed on pDCs
(plasmacytoid DCs – a type of DC that produces high amounts of
type I IFNs). In addition, TLR3 is highly expressed in the brain
[67]. TLR3-deficient mice infected with various RNA viruses such
as MCMV (murine cytomegalovirus), VSV (vesicular-stomatitis
virus), LCMV (lymphocytic choriomeningitis virus), RSV or
c The Authors Journal compilation c 2009 Biochemical Society
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H. Kumar, T. Kawai and S. Akira
reovirus show comparable susceptibility with WT (wild-type)
mice, suggesting that TLR3 is dispensable for protection against
these viruses [68]. However, TLR3-deficient mice infected with
lethal doses of WNV show resistance to the WNV infection,
suggesting that TLR3-mediated inflammatory responses induce
death of the mice [69]. Therefore, the role of TLR3 in viral
infection is unclear.
TLR7, TLR8 and TLR9
TLR7, TLR8 and TLR9 are located in the intracellular endosomal
compartment, where they sense microbial nucleic acids such
as RNA and DNA. TLR7 and TLR8 (but not mouse TLR8)
respond to synthetic antiviral imidazoquinoline compounds such
as R848, loxoribine and imiquimod and ssRNAs rich in guanosine
or uridine derived from viruses [70–72] (Figure 1). Generally,
these viruses gain entry into cells through receptor-mediated
endocytosis and reach the phagolysosome, where the virus-coat
protein is hydrolysed to expose the viral RNA to the TLRs. In
contrast, the host ssRNA does not reach the endocytic vesicles
because it is degraded by RNase.
TLR9 recognizes unmethylated CpG motifs of ssDNA and
induces inflammatory cytokines and type I IFNs. These sequences
are commonly present in the genomes of bacteria and viruses
[73–77]. However, in the host, these sequences are highly
methylated at the cytosine base and, therefore, the host CpG motifs
stimulate poorly. Synthetic ssDNA-containing CpG dinucleotides
motifs can also induce the production of inflammatory cytokines
and type I IFNs through TLR9. There are two structurally distinct
types of CpG DNAs known, namely the A-type (D-type) and
the B-type (K-type). A-type CpG ODNs (oligodeoxynucleotides)
stimulate pDCs to induce a robust amount of IFN-α and a little
IL-12 [78]. By contrast, B-type CpG ODN is a potent inducer of
inflammatory cytokines such as IL-6, IL-12 and TNF-α (tumour
necrosis factor-α) and up-regulates co-stimulatory molecules such
as CD80, CD86 and MHC class II in pDCs and, to lesser extent, in
B-cells [79]. DNA viruses such as MCMV, HSV (herpes simplex
virus)-1 and HSV-2 induce inflammatory cytokines and type I
IFNs through TLR9 [73–77]. It has been shown that TLR9deficient mice are susceptible to MCMV infection [76]. HSV
recognition by pDCs does not require viral replication, because
UV-inactivated virus still induces IFN-α [75]. In addition to these
ligands, TLR9 also recognizes the malarial pigment known as
haemozoin [80]. Recently, it was shown that cleavage of TLR9
by lysosomal cathepsins is involved in the activation of signalling
[81–83].
Signalling through TLR
Upon recognition, TLRs recruit various TIR-domain-containing
adaptors to the TIR domain of TLRs. TLR5, TLR7, TLR9 and
TLR11 only use MyD88. TLR1, TLR2, TLR4 and TLR6 use
TIRAP in addition to MyD88, which links TLR to MyD88. TLR3
only uses TRIF. TLR4 uses TRIF and TRAM, and TRAM links
TLR4 with TRIF. Taken together, TLR signalling can be broadly
divided into two signalling pathways: the MyD88-dependent and
TRIF-dependent pathways [1,17,47,84,85] (Figures 1 and 2).
In the MyD88-dependent signalling pathway, the IRAK (IL-1
receptor-associated kinase) family members such as IRAK4,
IRAK1 and IRAK2 are recruited to the MyD88. IRAK4 is initially
activated, and IRAK1 and IRAK2 are sequentially activated [86].
The activated IRAK family proteins associate with TRAF6 (TNF
receptor-associated factor 6), an E3 ubiquitin ligase, which forms
a complex with E2 ubiquitin-conjugating enzymes such as UBC13
(ubiquitin C 13) and UEV1A (ubiquitin-conjugating enzyme variant 1A). This complex polyubiquitinates TRAF6 itself and IKKγ
c The Authors Journal compilation c 2009 Biochemical Society
[IκB kinase γ , also known as NEMO (NF-κB essential modifier)]
through K63 (Lys63 ) linkage [87,88]. The polyubiquitinated
TRAF6 activates the protein kinase TAK (transforming growth
factor-β-activated kinase 1) and TABs (TAK1-binding proteins)
such as TAB1, TAB2 and TAB3, which subsequently activate
transcription factors such as NF-κB and AP-1 (activator protein1) through the canonical IKK complex and the MAPK [ERK
(extracellular-signal-regulated kinase), JNK (c-jun N-terminal
kinase) and p38] pathway respectively for the transcription of
inflammatory cytokine genes [87]. TAK1-deficient cells show
reduced inflammatory cytokine levels and impaired NFκB and
MAPK activation after stimulation with various TLR ligands
[89,90]. TLR4 also activates NF-κB via TRIF by two distinct
signalling pathways [91]. The N-terminus of TRIF interacts with
TRAF6 through the TRAF6-binding motifs [92] and the C-terminus of TRIF interacts with RIP1 (receptor-interacting protein
1), both of which co-operate to activate NF-κB [93] (Figure 2).
Recently, we showed that the TRIF-dependent signalling pathway
is 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 immune
regulation [94].
Stimulation with TLR4 and TLR3 ligands activates the
TRIF-dependent signalling pathway and induces inflammatory
cytokines in addition to type I IFNs and IFN-inducible genes
in 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-associated
kinase) or NAK (NF-κB activating kinase)] and IKKi (also
known 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, which
subsequently translocates into the nucleus and binds to the ISREs
(IFN-stimulated response elements) to induce type I IFNs and
IFN-inducible genes [98]. TRAF3 has been proposed to link
TRIF to TBK1, because TRAF3 interacts with these proteins and
the production of IFN-β is abrogated in TRAF3-deficient cells
[99,100] (Figure 2).
In pDCs, TLR7 and TLR9 are highly expressed and induce
a huge amount of type I IFNs, particularly IFN-α, after virus
infection. Upon stimulation, MyD88 forms a complex with IRF7
[101,102] (which is highly expressed in pDCs) and TRAF6 to
induce the production of type I IFNs [100]. IRAK1 interacts
with MyD88 and can phosphorylate IRF7 [103]. IRAK1-deficient
pDCs consistently have defects in type I IFN production, but
show intact inflammatory cytokine production. Similarly, the
production of type I IFNs is decreased in IKKα-deficient mice,
and IKKα can bind to, and phosphorylate, IRF7 [104]. These
findings suggest that IRAK1 and IKKα act as IRF7 kinases.
By contrast, pDCs lacking MyD88 or IRAK4 do not induce
type I IFNs and inflammatory cytokines. Taken together, these
observations suggest that the TLR7– or TLR9–MyD88–TRAF6–
IRAK4–IRAK1–IKKα–IRF7 signalling pathway is active in
pDCs for the robust production of type I IFNs after virus infection
(Figure 3). Recently, the involvement of the serine/threonine
protein kinase mTOR (mammalian target of rapamycin) and its
downstream signalling kinases, such as the p70 ribosomal S6
protein kinases p70S6K1 and p70S6K2, has been shown to play
roles in pDC for the production of type I IFNs. Inhibition of
mTOR and its downstream kinases blocks the interaction between
TLR9 and MyD88 and inhibits further activation of IRF7
[105].
Pathogen recognition in the innate immune response
Figure 2
5
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 complex
consisting of IKKα, IKKβ and NEMO/IKKγ , and phosphorylates IκBs. Phosphorylated IκBs are ubiquitinated and undergo proteasome-mediated degradation, and NF-κB subunits, which consist of
p50 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 RIP1
and 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 phosphorylate
IRF3 and IRF7. The phosphorylated IRF3 and IRF7 are translocated to the nucleus for the transcription of type I IFNs.
The ER (endoplasmic reticulum)-localized transmembrane
protein known as UNC93B1 was shown to be essential for
the production of inflammatory cytokines after stimulation with
TLR3, TLR7 and TLR9 ligands [106]. In addition, UNC93B1
plays a crucial role in the cross-presentation of an exogenous
antigen via MHC Class I and Class II. Moreover, UNC93B1 is
required for the translocation of TLR7 and TLR9 from the ER to
the endolysosome [107–109].
TLR and human diseases
Genetic variations or a deficiency of genes encoding TLR and
TLR signalling proteins have been implicated in the predisposition
to innate immune diseases [110]. Recently, autosomal recessive
MyD88-deficient pediatric patients have been reported. These
patients show susceptibility to pyrogenic bacterial infections such
as Strep. pneumoniae and Staph. aureus, and severe complications
have been reported in several patients in early childhood.
Otherwise, these patients are healthy and have normal immunity
to other microbes, and their clinical severity improves with age.
These observations suggest that MyD88-dependent signalling is
essential for protective immunity against a few types of pyrogenic
bacteria, but is dispensable for the host defence to the majority of
other infections in humans [111].
IRAK4 deficiency has also been reported in humans. The
IRAK4 gene is located on an autosomal chromosome and 28
individuals with a recessive gene have been reported so far.
These patients show increased susceptibility to infection by Grampositive and Gram-negative bacteria in early childhood [112].
Similar to MyD88 deficiency or mutants, IRAK4-deficient people
also show an improvement in symptoms with advancing age.
These patients also show impaired induction of type I IFNs, but
do not show any susceptibility to viral infection or HSE (herpes
simplex virus encephalitis). Furthermore, these individuals do
not show susceptibility to any parasitic or fungal diseases. Patients with TLR3 deficiency have also been reported. These
patients show increased susceptibility to HSV-1 [113]. These observations suggest that TLRs play an important role in the
pathogenesis of some diseases. UNC93B deficiency has also
been documented in some patients. Cells from these patients
do not respond to TLR3, TLR7, TLR8 or TLR9. However,
similar to IRAK4-deficient patients, UNC93B patients do not
show susceptibility to viral diseases such as HSE [114].
RIG-I-LIKE RECEPTORS
Much progress has been made in TLR-independent recognition of
viral nucleic acids, particularly RNA recognition by intracellular
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Figure 3
H. Kumar, T. Kawai and S. Akira
TLR signalling in pDCs
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. MyD88
interacts 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 for
the detection of RNA derived from RNA viruses and replicating
DNA viruses. The recognition of RNA by intracellular sensors
subsequently activates innate antiviral responses, mainly through
the induction of type I IFNs and inflammatory cytokines in most
cell types.
In this regard, DExD/H-box RNA helicase, known RIG-I, was
found to have a role in the cytoplasmic recognition of dsRNA
and activation of IRFs and NF-κB [119]. Two members of
this family known as Mda5 (melanoma differentiation-associated
gene 5) and Lgp2 (Laboratory of Genetics and Physiology 2)
were subsequently identified [120]. These proteins are collectively
known as RLRs. RIG-I and Mda5 contain two tandem repeats
of the CARD (caspase recruitment domain) at their N-terminus,
which are important for activating downstream signalling. The
intermediate portion of these proteins contains the helicase
domain, which is similar to other members of the DExD/H-box
RNA helicase family. For RIG-I, this domain contains an ATPbinding region, which is essential for RIG-I function. However,
an ATP-binding region was not found in Mda5. In addition, RIG
c The Authors Journal compilation c 2009 Biochemical Society
I contains an RD (repressor domain) at the C-terminus, which
represses the activity of RIG-I [121,122]. Repression of Mda5 in
the steady state is not known, but it has been postulated that MdA5
might be negatively regulated by other proteins such as DAK
(dihydroacetone kinase) [123]. Lgp2 contains the RNA helicase
domain, but is devoid of the CARD. This protein was considered
to be a negative regulator for RIG-I and Mda5. However, a study
of Lgp2-deficient mice revealed that Lgp2 acts as both a negative
and positive regulator depending on the virus [124]. Recently, a
homologue of RIG-I helicase (DExD/H-box helicase), Dicer-2,
was identified in Drosophila and which controls the expression
of an antiviral protein known as Vago after virus infection,
suggesting an evolutionarily conserved role of RLR in antiviral
responses [125].
Pathogen recognition by RLRs
Studies of RIG-I- and Mda5-deficient mice revealed that these
sensors recognize different classes of RNA viruses. RIG-Ideficient cells infected with NDV (Newcastle-disease virus),
Pathogen recognition in the innate immune response
VSV, SeV (Sendai virus) and IV (influenza virus) show impaired
type 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) are
also recognized by RIG-I [128,129]. However, dengue virus and
WNV, which belong to the flavivirus family, do not require RIG-I.
By contrast, Mda5-deficient mice show normal production of
type I IFNs and inflammatory cytokines against NDV, VSV, SeV,
IV and JEV. However, they show impaired ability to produce type I
IFNs and inflammatory cytokines against picornaviruses such as
EMCV, Theiler’s virus and Mengo virus [128]. These observations
suggest that these two sensors induce antiviral responses against a
wide spectrum of RNA viruses with different specificity. However,
RLRs are not sufficient for the protection against other RNA
viruses such as IV, RSV and LCMV in vivo. In other words, in vivo
infection of LCMV induces the production of type I IFNs and the
promotion of virus-specific CD8 T-cells through the TLR7 rather
than the RLR signalling pathway [130]. For IV infection, the RLR
signalling pathway is essential for the induction of cytokines in
fibroblasts, alveolar macrophages, and cDCs, whereas TLR7 acts
in pDCs to induce cytokines [131,132]. However, the production
of virus-specific antibodies is dependent on TLR7 rather than
RLR. Similar observations were reported for RSV infection
[133], suggesting that TLR and RLR signalling pathways together
induce the host defence against these viruses. Moreover, cooperative activation of TLR and RLR is also required for the
adjuvant effects of poly(I-C) [134].
RIG-I and Mda5 recognize in-vitro-synthesized dsRNA and
a synthetic analogue of dsRNA poly(I-C) respectively [128].
Further studies revealed that the 5 -triphosphate moiety of RNA
is essential for RIG-I recognition [135] and it is independent of
the strand property (single or double) of RNA [136]. A recent
biochemical study revealed that the 5 -triphosphate moiety of
RNA is recognized by the CTD (C-terminal domain) of RIG-I
[122]. Host-cell RNA is not recognized by RIG-I because, during
synthesis of cellular RNA, the 5 -ends are either modified by
the addition of a 7-methylguanosine cap or the 5 -triphosphate
is removed before transportation to the cytoplasm. Thus RIG-I
can discriminate between viral and host RNA. RIG-I can bind to
a 25-bp dsRNA to efficiently induce type I IFNs and the RNA
end structures (blunt end, 5 -overhang and 3 -overhang), and the
nucleotide sequences are not critical for binding to RIG-I [137].
Furthermore, it has been shown, using NMR of the CTD, that
RIG-I recognizes two distinct viral RNA patterns, including ds
and 5 -triphosphate ssRNA, and gel-filtration analysis revealed
that a dimer of RIG-I CTD mediates the recognition of 5 triphosphate RNA [118]. The length of dsRNA is critical for
differential recognition by RIG-I and Mda5; RNA viruses have a
shorter RNA length (approx. 1.2–1.4 kbp) and are recognized by
RIG-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 Mda5
associate with the CARD-containing adaptor protein known
as IPS-1 {IFN promoter stimulator-1, also known as MAVS
(mitochondrial antiviral signalling), Cardif (CARD adaptor inducing IFN-β) and VISA (virus-induced signalling adaptor [139–
142]} to induce inflammatory cytokines and type I IFNs. Ectopic
expression of IPS-1 in cells activates NF-κB and IFN promoters.
In addition, IPS-1-deficient cells show a complete abrogation of
inflammatory cytokines and type I IFNs after virus infection.
Furthermore, IPS-1-deficient mice infected with various RNA
viruses recognized by RIG-I and Mda5 show enhanced motility
7
compared with that in WT mice [143,144]. These observations
collectively suggest that IPS-1 is the sole adaptor for RIGI/Mda5 and plays an essential role in host defence against
various RNA viruses. It has been shown that IPS-1 is localized
in the outer membrane of mitochondria [140]. NS3/4 (nonstructural 3/4) protease from HCV cleaves and dislodges IPS-1
from the mitochondria to block IFN production, suggesting that
mitochondrial localization is essential for IPS-1 function with
respect to antiviral responses [141]. The NLR family protein
known as NLRX1 was recently reported to be localized in the outer
membrane of mitochondria and to inhibit IPS-1-mediated type I
IFN induction in response to virus infection [145]. Signalling
through RIG-I is further regulated by ubiquitination. It was shown
that TRIM25 (tripartite motif 25), a ubiquitin E3 ligase which
contains an RNF (RING-finger) domain, a B-box/coiled-coil
domain and a SPRY (SPla/RYanodine) domain, interacts with the
N-terminal CARD of RIG-I. This interaction leads to the Lys63 linked ubiquitination of RIG-I. Furthermore, TRIM25-deficient
cells show abrogation of type I IFNs production, suggesting that
ubiquitination of RIG-I is essential for activation of the signalling
pathway[146]. By contrast, RIG-I is inhibited by another ubiquitin
ligase, RNF125, which induces ubiquitination and proteasomal
degradation of RIG-I. These observations suggest that the
ubiquitination is an additional regulatory mechanism for the RIGI-mediated signalling pathway [147] (Figure 4).
TRAF3, an E3 ubiquitin ligase that polyubiquitinates through
its C-terminal TRAF domain, was shown to interact with
IPS-1 and activates TBK1 and IKKi [99,100,148]. Recently,
a de-ubiquitinase enzymatic protein known as DUBA (deubiquitinating enzyme A) was reported to deubiquitinate TRAF3
and inhibit the RLR-mediated signalling pathway [149].
RLR-mediated NF-κB activation is achieved via the FADD
(Fas-associated death domain) protein, which interacts with
caspase 8 and caspase 10 and forms a complex with IPS-1
[139,150]. The TNFR-I (TNF receptor-I) signalling adaptor
TRADD (TNF-receptor-associated DD), an adaptor in the
TNFR-I signalling pathway, has been suggested to be involved
in RLR signalling. The engagement of RLRs by viruses leads to
the formation of a molecular complex that consists of TRADD,
FADD and RIP1. This complex interacts with IPS-1 and activates
IRF3 and NF-κB [151].
The ER-localized STING [stimulator of interferon genes, also
known as MITA (mediator of IRF3 activation)] protein was
recently identified and shown to be involved in host defence
against RNA virus. Knockdown of STING impaired IFN-β
production by poly(I-C) transfection. The replication of RNA
viruses is higher in STING-deficient fibroblast cells than in WT
cells. In addition, the production of IFN-β was also reduced
in STING-deficient fibroblast cells. However, STING-deficient
bone-marrow-derived macrophages and DCs show replication of
virus comparable with that shown by WT cells, suggesting a celltype-specific role of STING [152,153].
Autophagy is an essential biological process that maintains
cellular homoeostasis, development, differentiation and tissue
remodelling. Recent studies have highlighted the importance of
autophagy in innate immunity. Recently it was shown that IPS1 and RIG-I were associated with the Atg5–Atg12 complex,
which is an essential component for autophagy. Mouse embryonic
fibroblasts deficient in Atg5 and Atg12 show increased levels
of type I IFN production in response to VSV infection [154].
This observation suggests that autophagy is important in the
negative regulation of the RLR signalling pathway. However, it
has been shown that autophagy plays an opposite role in pDCs.
In pDCs, TLR7 recognizes ssRNA in the endolysosome, but
it also recognizes replicating VSV. Atg5-deficient pDCs show
c The Authors Journal compilation c 2009 Biochemical Society
8
Figure 4
H. Kumar, T. Kawai and S. Akira
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 and
ubiquitinated 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 forms
a 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 the
transcription 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 the
autophagosome is formed after VSV infection and contains viral
RNA, and this autophagosome traffics to the lysosome, where it
forms a complex with the autophagolysosome after fusion with
the lysosome. In the autophagolysosome, TLR7 recognizes viral
RNA for the production of IFN-α [155].
RECOGNITION OF DNA BY CYTOSOLIC RECEPTOR
Bacterial and viral dsDNA are immunostimulatory components
that activate various cell types to induce type I IFNs
and inflammatory cytokines through cytosolic DNA sensors.
Cytosolic recognition of dsDNA induces TBK1/IKKi-dependent
type I IFNs and NF-κB-dependent inflammatory cytokines
[156,157] (Figure 5). DAI (DNA-dependent activator of IRFs) has
been 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 consists
of plasmid DNA and has inherited properties of adjuvant induces
innate and adaptive immunity, such as antigen-specific antibody
production and CD4 and CD8 T-cell responses, through TBK1 but
not TLR9, MyD88 and DAI. This suggests that the DNA-induced
adjuvant effects are TLR- and DAI-independent phenomena and
c The Authors Journal compilation c 2009 Biochemical Society
these effects may depend on an unknown DNA sensor that signals
through TBK1 [159].
Recently, STING has been shown to be involved in the
recognition of dsDNA. STING was shown to play a pivotal role
in type I IFN production by dsDNA when introduced into the
cytosol. Furthermore, infection with a DNA virus such as HSV1, and Listeria monocytogenes, organisms which are known to
introduce DNA into the cytosol during infection, shows a reduced
type I IFN production in STING-deficient cells. This suggests
that STING plays an important role in DNA sensor signalling
pathways [152,153].
NOD-LIKE RECEPTORS
The NLR family of proteins are cytosolic, intracellular PRRs
that recognize PAMPs and endogenous ligands. The recognition
of ligands induces a signalling cascade leading to activation of
NF-κB, or a cytoplasmic multiprotein complex known as
the inflammasome, to produce inflammatory cytokines [1,11–
13,160,161]. In addition, NLRs are also involved in the signalling
for cell death after microbial infection [162]. The NLR family
comprises 23 proteins in humans and 34 proteins in mice
[163]. However, the function of most of the NLR proteins is
poorly understood. These proteins have a trimodular structure
Pathogen recognition in the innate immune response
Figure 5
9
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 and
IRFs. 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 into
active 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 domain
consists of tandem repeats of LRR, which are essential for
sensing or recognition of the microbial components. A centrally
located nucleotide binding NOD domain is essential for selfoligomerization and formation of a complex for the activation
and recruitment of downstream signalling proteins. The variable
N-terminal domains are defined by CARD, DED (death effector
domain), PYD (pyrin domain) or BIR (baculovirus inhibitor
of apoptosis protein repeat) domain [163]. These domains are
essential for downstream signal transduction through homotypic
protein–protein interactions. Mutations in these proteins were
reported to be associated with chronic inflammatory diseases
such as familial cold autoinflammatory syndromes (familial
cold 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 substructure
of PGN (peptidoglycan), namely iE-DAP (γ -D-glutamyl-m-
diaminopimelic acid, a dipeptide), which is present in Gramnegative and Gram-positive bacteria such as Bacillus subtilis and
L. monocytogenes [167,168]. NOD2 (CARD15) recognizes MDP
(muramyl dipeptide), the largest component of PGN motif, which
is also present in Gram-negative and Gram-positive bacteria [169].
Upon stimulation with these ligands, NOD1 and NOD2 interact
with a CARD domain-containing serine/threonine kinase known
as CARDIAK [CARD-containing IL-1β-converting-enzymeassociated kinase, also known as RICK (RIP-like interacting
caspase-like apoptosis-regulatory protein kinase) and RIP2]
[170,171] and induces antimicrobial peptides [172] and inflammatory cytokines through activation of MAPKs [173] and
NF-κB. MAPKs are activated through another CARD-containing
protein known as CARD9 [174] (Figure 6). Ex vivo studies
showed that various pathogenic bacteria, such as E. coli
[175], Shigella flexneri [168], Pseudomonas aeruginosa [176],
Chlamydia species [177–179], Campylobacter jejuni [180] and
Haemophilus 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 NOD1
and NOD2 [184,185]. In vivo, it has been shown that NOD1 and
c The Authors Journal compilation c 2009 Biochemical Society
10
H. Kumar, T. Kawai and S. Akira
NALP3 inflammasome
Figure 6
NLR signalling pathway
NOD1 and NOD2 sense various PAMPs of pathogenic bacteria in the cytoplasm or PAMPs that
are 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 the
association of CARDIAK, which in turn activates NF-κB. NOD1 and NOD2 also activate
the 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 of
various inflammatory cytokines. The IL-1 family of cytokines,
including IL-1β, IL-18 and IL-33, are key cytokines that regulate
various components of innate and adaptive immunity. The
production of these cytokines is regulated by two signals in
the innate immune cells (e.g. macrophages). The first signal is
transcriptional and translational up-regulation of pro-forms of the
cytokine (pro-IL-1β, pro-IL-18) in response to various TLR, NLR
and RLR agonists. The second signal is the processing of the proform of these cytokines to the mature, secretary form of the cytokines by the inflammasome. Depending on the NLR proteins, the
inflammasome is categorized into three types such as NALP3
[NACHT (NTPase-domain named after NAIP, CIITA, HETE and TP1)–LRR-PYD–containing protein 3, also known as
NLRP3, CIAS1 (cold-induced autoinflammatory syndrome 1) and
cryopyrin] inflammasome, the IPAF [IL-1β-converting-enzyme
protease-activating factor, also known as the NLRC4 (NLR
family, CARD domain containing 4)] and CLAN (CARD LRRand NACHT-domain-containing protein) inflammasome and the
NALP1 (also known as NLRP1) inflammasome (Figure 7).
c The Authors Journal compilation c 2009 Biochemical Society
The NALP3 inflammasome is activated by various exogenous and
host endogenous ligands. Exogenous ligands include microbial
ligands such as MDP, bacterial and viral RNA, toxins such as
nigericin, maitotoxin, environmental pollutants such as asbestos
and silica [187] and vaccine adjuvant alum (aluminium salts)
[188,189]. Host endogenous ligands include MSU (monosodium
urate), calcium pyrophosphate dehydrate, amyloid-β fibrillar
peptide and ATP. In addition, the NALP3 inflammasome is also
activated by UV light and skin irritants such as picryl chloride and
2,4-dinitrofluorobenzene. The NALP3 inflammasome consists
of NALP3, CARDINAL (CARD inhibitor of NF-κB-activating
ligands), ASC (apoptosis-associated speck-like protein containing
a CARD) and caspase 1, which process cytosolic pro-IL-1β to
bioactive secretory IL-1β [187].
Activation of the inflammasome leads to the production of IL1β, which may play an important role in clearing pathogens
from the host. A recent study has shown that the NALP3
inflammasome is essential for alum-induced adjuvant effects
such as antibody production to antigens and Th2-mediated
inflammation [189]. However, there is another study showing that
the NALP3 inflammasome is dispensable for the adjuvant effects,
but indispensible for IL-1β production [190]. There are several
possible differences among these studies, such as preparation
of mixture of antigen and alum before immunization, the dose of
antigen or adjuvant, the purity of antigen (some antigen has
impurity of immunomodulatory substances), and the route and
protocol of immunization. Therefore it is almost impossible to
evaluate the data from two different laboratories. Furthermore,
the differences in results could be due to multiple mechanisms
of action of alum. Taken together, further studies are needed to
conclude the involvement of NALP3 inflammasome in adjuvant
effects of alum. It has been suggested that activation of the
inflammasome by the host ligand plays an important role in
the pathogenesis of arthritic diseases such as gout, pseudogout
[187] and Alzheimer’s disease [191]. The exogenous ligand silica
induces NALP3-inflammasome-dependent silicosis [192,193].
These observations suggest that the NALP3 inflammasome plays
a pivotal role in the activation of innate immune responses for host
defence and is a mediator for the pathogenesis of various diseases.
Recently, the introduction of bacterial, viral and host dsDNA
into the cytosol of cells has been shown to induce the production
of IL-1β, which requires the inflammasome components ASC
and caspase 1, but not NALP3. However, the introduction of
adenoviral DNA showed that IL-1β production depends on
NALP3, ASC and caspase 1. AIM2 (absent in melanoma 2)
has been shown to recognize dsDNA. AIM2 is a member of the
IFI20X (interferon-inducible protein 20X)/IFI16 or PYHIN [PYD
and HIN (haematopoietic interferon-inducible) nuclear protein
domain family member 1]. It has been shown that the HIN200
domain of AIM2 binds to dsDNA, whereas PYD associates with
ASC [194–197] (Figure 5).
The NALP3 inflammasome is activated by numerous ligands;
however, it is unknown whether these ligands bind directly to
NALP3. Pore-forming toxins such as staphylococcal α-toxin and
pneumolysin, K+ ionophores and a low concentration of detergent
induce an efflux of K+ to activate the NALP3 inflammasome.
In addition, ATP, maitotoxin and nigericin activate hemichannel
pannexin-1 and the purinergic receptor P2X7 to induce efflux
of K+ and activate the NALP3 inflammasome. Furthermore,
ROS (reactive oxygen species) are also reported to activate the
NALP3 inflammasome [187]. Two different mechanisms for ROS
production have been proposed, including efflux of K+ -induced
stress to the mitochondria and NADPH oxidase-mediated
Pathogen recognition in the innate immune response
Figure 7
11
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 and
RLR 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. In
addition to ASC, the NALP3 inflammasome contains CARDINAL protein (the domain arrangement of each component is shown in the box labelled ‘Components of inflammasome’. The inflammasome
converts 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. The
NALP1 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 also
been proposed recently. Phagocytosis of silica crystal and fibrous
particles of amyloid-β induce lysosomal destabilization and
permeabilization which leads to the release of cathepsin B into the
cytosol [191,198]. The released cathepsin B activates the NALP3
inflammasome.
IPAF inflammasome
The IPAF inflammasome is activated by intracellular pathogens
such as Salmonella typhimurium [199] and Legionella
pneumophila [200]. It was suggested that S. typhimurium and
L. pneumophila deliver flagellin to the cytosol via the type III
and type IV secretion systems respectively [199,200]. The introduction of recombinant purified flagellin protein into the cytosol also promotes IPAF-dependent caspase 1 activation [201].
Flagellin of Ps. aeruginosa also induces IPAF- and caspase1-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 of
L. pneumophila [203,204]. NAIP5 consists of three consecutive
repeats of the BIR domain at the N-terminus. Previous studies
have indicated that NAIP5 regulates the replication of L.
pneumophila via caspase-1-dependent cell death. It has been
reported that 35 amino acids from the C-terminus of flagellin
and infection with L. pneumophila is sufficient to activate the
inflammasome, but is not activated in the absence of NAIP5. In
addition, full-length flagellin activates NAIP5-independent, but
IPAF-dependent, cell death [205]. These findings suggest that
the IPAF and NAIP5 proteins are essential for the flagellininduced immune response.
NALP1 inflammasome
Anthrax LT (lethal toxin) is a potent toxin of Bacillus anthracis
that induces cell death [206]. LT is composed of a protective
antigen and a lethal factor. The protective antigen is a receptorbinding protein that creates a pore to deliver the lethal factor
c The Authors Journal compilation c 2009 Biochemical Society
12
H. Kumar, T. Kawai and S. Akira
into the cytosol of infected cells and induce cell death. It has
been shown that the polymorphic gene known as Nlrp1b is
responsible for susceptibility to LT [207]. It was also shown
that LT-induced macrophage death requires caspase 1, which is
activated in susceptible macrophages [207]. Furthermore, the LT
of B. anthracis was shown to induce IL-1β, which depends on
the NALP1 inflammasome. In addition, it was shown that NOD2
is also required for IL-1β production and NOD2–NALP1 forms
a molecular complex during infection [208].
NALP1 is implicated in the association with anti-apoptotic
proteins Bcl2 (B-cell lymphoma-2) and Bcl-X(L), which suppress
caspase-1 activation, indicating a role of Bcl-2 proteins in the
inhibition of LT-induced cytotoxicity [209].
CONCLUSIONS AND FUTURE DIRECTIONS
Although numerous studies have greatly enhanced our
understanding of innate immune recognition by TLRs, RLRs,
NLRs and unknown PRRs, we still know relatively little about
the recognition of the vast array of micro-organisms and the
cross-talk among these receptors. Although the cytosolic receptor
for DNA, which induces IL-1β production, has been reported,
we have not yet characterized the sensors for DNA or DNA
viruses that activate IRFs. The function of most of the members
of the NLR family in host defence and their ligands are also
unidentified. Characterization of these PRRs in innate immunity
will further improve our understanding of the complexities of
innate immune regulation. In addition, structural studies of PRR–
ligand complexes will improve our understanding of innate
immunity and facilitate the design of novel drugs that target
PRRs.
ACKNOWLEDGEMENTS
We thank the members of our laboratory for insightful discussions and apologize
to those authors whose work could not be included in this review owing to space
limitations.
FUNDING
This work was supported by the Japan Society for the Promotion of Science [grant number
PO8123 (postdoctoral fellowship to H. K.)].
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