J Mol Med (2006) 84:712–725

DOI 10.1007/s00109-006-0084-y

REVIEW

Toll-like receptors and innate immunity

Satoshi Uematsu & Shizuo Akira

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

Abstract The innate immune system is an evolutionally

conserved host defense mechanism against pathogens.
Innate immune responses are initiated by pattern recogni-
tion receptors (PRRs), which recognize specific structures
of microorganisms. Among them, Toll-like receptors
(TLRs) are capable of sensing organisms ranging from
bacteria to fungi, protozoa, and viruses, and play a major
role in innate immunity. However, TLRs recognize patho-
gens either on the cell surface or in the lysosome/endosome
compartment. Recently, cytoplasmic PRRs have been
identified to detect pathogens that have invaded cytosols.
In this review, we focus on the functions of PRRs in innate
immunity and their downstream signaling cascades. Dr. SATOSHI UEMATSU Dr. SHIZUO AKIRA
received his M.D. in 1997 from received his M.D. and Ph.D.
Keywords TLR . NLR . RIG-I Osaka University medical degree from the Osaka
school, Osaka City, Japan. He University, Japan. He is
obtained his Ph.D. in 2004 at currently a professor in the
Osaka University. He is Department of Host Defense at
Introduction currently an assistant professor the Research Institute for
of the Department of Host Microbial Diseases, Osaka
Defense at the Research Institute University. His research interests
The innate immune response is the first line of defense for Microbial Diseases, Osaka focus on pathogen recognition
against microbial infections. Since the discovery of the University, Japan. His research and signaling pathways of
Drosophila protein Toll, which induces effective immune focuses on innate immunity, Toll-like receptors and
responses to Aspergillus fumigatus [1], accumulating especially the defense TLR-independent pathogen
mechanism of macrophages and recognition in innate immunity.
evidence showed that the innate immune system specifically dendritic cells in intracellular
recognizes invading microorganisms. The targets of innate bacterial infection.

S. Uematsu : S. Akira immune recognition are the conserved molecular patterns

Department of Host Defense, (pathogen-associated molecular patterns, PAMPs) of micro-
Research Institute for Microbial Diseases, Osaka University,
organisms. Receptors in innate immunity are therefore called
3-1 Yamada-oka,
Suita, Osaka 565-0851, Japan pattern recognition receptors (PRRs) [2]. PAMPs are
generated by microbes and not by the host, suggesting that
S. Akira (*) PAMPs are good targets for innate immunity to discriminate
ERATO, Japan Science and Technology Corporation,
between self- and nonself. Furthermore, PAMPs are essential
3-1 Yamada-oka,
Suita, Osaka 565-0871, Japan for microbial survival and are conserved structures among
e-mail: sakira@biken.osaka-u.ac.jp many pathogens, which allows innate immunity to recognize
J Mol Med (2006) 84:712–725
microorganisms with limited numbers of PRRs. Among
PRRs, Toll-like receptors (TLRs), mammalian homologs of
Toll, were highlighted as the key recognition structures of
the innate immune system in the past few years [3]. TLRs
are capable of sensing organisms ranging from bacteria to
fungi, protozoa, and viruses. However, host recognition and
response is not restricted to TLRs. Some members of the
NACHT (domain present in NAIP, CIITA, HET-E, and
TP1)-LRR (leucine-rich repeat) (NLR) family, which
includes both nucleotide-binding oligomerization domain
(NOD) proteins and NACHT-LRR- and pyrin-domain-
containing proteins (NALPs), recognize microbial compo-
nents in the cytosol [4]. Furthermore, the RNA helicase,
retinoic acid inducible gene-I (RIG-I), was identified as a
cytosolic receptor for intracellular double-stranded (dsRNA)
[5] and induces type I interferons (IFNs) in response to
dsRNA viruses in a TLR-independent manner [6]. The
recent understanding of these PRRs in innate immunity and
their signaling pathways is the focus of this review.
TLR
A mammalian homolog of a Toll receptor (now termed
TLR4) was identified through database searches. The TLR
family now consists of 13 mammalian members: with each
TLR having its intrinsic signaling pathway and inducing
specific biological responses against microorganisms. Rec-
ognition of microbial components by TLRs triggers the
activation of signal transduction pathways, which then
induces dendritic cell (DC) maturation and cytokine produc-
tion, resulting in development of adaptive immunity [3].
Structure
The cytoplasmic portion of a TLR is similar to that of the
interleukin (IL)-1 receptor family. It is therefore called the
Toll/IL-1 receptor (TIR) domain. A TIR domain is required
for initiating intracellular signaling. The extracellular region
of TLRs and IL-1R are markedly different. Whereas IL-1R
possesses an Ig-like domain, TLRs contain LRRs in the
extracellular domain. LRRs are responsible for the recog-
nition of PAMPs [3] (Fig. 1).
Pathogen recognition
Bacteria
Lipopolysaccharide (LPS) is a cell wall component of gram-
negative bacteria, a strong immunostimulant (Table 1). LPS
is composed of lipid A (endotoxin), core oligosaccharide,
and O-antigen. TLR4 recognizes lipid A of LPS; this occurs
through the TLR4/MD2/CD14 complex, which is present on
713
various cells such as macrophages and DCs [7]. LPS forms a
complex with an accessory protein, LPS-binding protein in
serum, which converts oligomeric micelles of LPS to a
monomer for delivery to CD14, a glycosylphosphatidylino-
sitol (GPI)-anchored, high-affinity membrane protein that
can also circulate in a soluble form. CD14 concentrates LPS
for binding to the TLR4/MD2 complex [8].
TLR2 recognizes various microbial components, such as
lipoproteins/lipopeptides and peptidoglycans from gram-pos-
itive and gram-negative bacteria, and lipoteichoic acid from
gram-positive bacteria, a phenol-soluble modulin from Staph-
ylococcus aureus, and glycolipids from Treponema maltophi-
lum [3, 9]. TLR2 is also reported to be involved in the
recognition of atypical LPS from nonenterobacteria, whose
structures are different from typical LPS of gram-negative
bacteria [3, 10]. However, a recent report has indicated that
lipoproteins contaminated in a LPS preparation from Por-
phyromonas gingivalis stimulated TLR2 and that LPS from P.
gingivalis itself had poor activity for TLR4 stimulation [11].
There are also controversial reports regarding peptidoglycan
recognition by TLR2. More careful analyses will be needed to
exclude any possibility of contaminates remaining.
TLR1 and TLR6 are structurally related to TLR2 [12].
TLR2 and TLR1 or TLR6 form a heterodimer, which is
involved in the discrimination between the molecular
structure of diacyl and triacyl lipopeptides [13–15]. CD36,
a member of the class II scavenger family of proteins was
shown to serve as a facilitator or coreceptor for diacyl
lipopeptide recognition through the TLR2/6 complex [16].
TLR5 is responsible for the recognition of flagellin, the
major portion of bacterial flagella [17]. Gewirtz et al. [18]
reported that TLR5 is expressed on the basolateral surface,
but not the apical side of intestinal epithelial cells,
suggesting that TLR5 serves as the sensor for the
pathogenic bacteria that invade across the epithelium. A
common stop codon polymorphism in the ligand-binding
domain of TLR5 (TLR5 392STOP SNP) is unable to
mediate flagellin signaling and is associated with suscep-
tibility to pneumonia caused by Legionella pneumophila
[19]. However, researchers in Vietnam have reported that
the TLR5 392STOP SNP is not associated with suscepti-
bility to typhoid fever [20]. A recent work has reported that
some commensal bacteria, such as a and e Proteobacteria,
change the TLR5 recognition site of flagellin without losing
flagellar motility [21]. This modification may contribute to
the persistence of these bacteria on mucosal surfaces.
Bacterial DNA, which contains unmethylated CpG
motifs, is a potent stimulator of the host immune response.
In vertebrates, the frequency of CpG motifs is severely
reduced and the cysteine residues of CpG motifs are highly
methylated, which leads to abrogation of the immunostim-
ulatory activity. Analysis of TLR9-deficient mice showed
that TLR9 is a receptor for CpG DNA [22].
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Fig. 1 TLR signaling pathways. TLR signaling pathways originate
from the cytoplasmic TIR domain. A TIR domain-containing adaptor,
MyD88, associates with the cytoplasmic TIR domain of TLRs and
recruits IRAKs to the receptor upon ligand binding. IRAKs then
activates TRAF6, leading to the activation of TAK1. TAK1 activates
the IKK complex consisting of IKKα, IKKβ, and NEMO/IKKγ. The
IKK complex phosphorylates IκB, resulting in nuclear translocation of
NF-kB, which induces expression of inflammatory cytokines. Among
the TLR family members, TLR4 on cell surfaces and TLR3, 7, 8, and
9 on endosomal membrane can induce type I IFNs. Stimulation of
macrophages and DCs with LPS and dsRNA results in the activation
of IRF3 and subsequent induction of IFN-β and IFN-inducible genes.
A TIR domain-containing adapter, TRIF is responsible for TLR3- and
Mouse TLR11, a relative of TLR5, is expressed abun-
dantly in the kidney and bladder. TLR11-deficient mice are
susceptible to uropathogenic bacterial infections, indicating
that TLR11 senses the component of uropathogenic bacteria.
However, TLR11 is thought to be nonfunctional in human
because of the presence of stop codon [23].
Fungi
TLRs are involved in the recognition of fungal pathogens
such as Candida albicans, A. fumigatus, Cryptococcus
neoformans, and Pneumocystis carinii [3, 24] (Table 1).
J Mol Med (2006) 84:712–725
TLR4-mediated IRF3 activation. In addition, TLR4 also requires
another adaptor, TRAM, for IRF3 activation. TRIF interacts with
TBK1, IKKi, RIP1, and TRAF6. IRF3 is activated by TBK1 and
IKKi, which catalyze IRF3 phosphorylation and stimulate its nuclear
translocation and DNA binding. pDCs express TLR7 (also TLR8 in
human) and TLR9 and secrete large amounts of IFN-αs and IFN-β in
response to ssRNA and CpG DNA. This induction by TLR7 and
TLR9 depends entirely on MyD88. After stimulation, IRF7 trans-
locates into the nucleus to induce type I IFNs. IRF7 is activated
through forming a signaling complex with MyD88, IRAKs, and
TRAF6 in the cytoplasm. IRAK-1 but not IRAK-4 is an essential
enzyme for phosphorylation and activation of IRF7 in TLR7- and
TLR9-mediated type I IFNs induction
Several components located in the cell wall or cell surface
of fungi were identified as potential ligands for TLRs. Yeast
zymosan activates TLR2/TLR6 heterodimers, whereas
mannan, derived from Saccharomyces cerevisiae and C.
albicans is detected by TLR4. Phospholipomannan,
present on the cell surface of C. albicans, is recognized
by TLR2, while TLR4 mainly interacts with glucuronox-
ylomannan, the major capsular polysaccharide of C.
neoformans [24].
Dectin-1 is a lectin family receptor for the fungal cell
wall component, b-glucan [25]. Dectin-1 functionally
collaborates with TLR2 and induces a strong immune
J Mol Med (2006) 84:712–725 715

Table 1 Pathogen recognition of TLRs TLR2 is also involved in the recognition of viral
components such as measles virus, human cytomegalovirus,
TLRs Ligands
and herpes simplex virus type 1 (HSV-1) [35–37].
TLR1 Triacyl lipopeptides (bacteria) During viral replication, dsRNA is generated, and TLR3
TLR2 Peptidoglycan, lipoprotein, lipopeptides, atypical is involved in the recognition of a synthetic analog of
LPS (bacteria) dsRNA, polyinosine–deoxycytidylic acid (poly I:C), a
Zymosan, phospholipomannan (fungi) potent inducer of type I IFNs [38, 39]. TLR3-deficient
GPI anchor (protozoa)
mice are susceptible to mouse cytomegalovirus [40]. On the
Envelope protein (virus)
TLR3 Poly (I:C), dsRNA (virus) other hand, TLR3-deficient mice show more resistance to
TLR4 LPS (bacteria) West Nile virus (WNV) infection. This single-stranded
Mannan, glucuronoxylomannan (fungi) RNA (ssRNA) flavivirus triggers inflammatory responses
Glycoinositolphospholipids (protozoa) via TLR3, which leads a disruption of the blood brain
RSV fusion protein (virus) barrier, followed by enhanced brain infection [41]. These
TLR5 Flagellin (bacteria) findings suggested that WNV utilizes TLR3 to efficiently
TLR6 Diacyl lipopeptides (bacteria)
enter the brain.
TLR7/TLR8 Synthetic imidazoquinoline-like molecules,
Mouse splenic DCs are divided into CD11c high B220−
ssRNA (virus)
TLR9 CpG DNA (bacteria, protozoa, virus) and CD11c dull B220+ cells. The latter contain plasmacytoid
Hemozoin (protozoa) DCs (pDCs), which induce large amounts of IFN-α during
TLR11 Component of uropathogenic bacteria (bacteria) viral infection. CpG DNA motifs are also found in genomes
Profilin like molecule (protozoa) of DNA viruses, such as HSV-1, HSV-2, and murine
cytomegalovirus (MCMV). Mouse pDCs recognize CpG
TLRs recognize molecular patterns associated with a broad range of
pathogens including bacteria, fungi, protozoa, and viruses DNA of HSV-2 via TLR9 and produce IFN-α [42]. TLR9-
deficient mice are susceptible to MCMV infection, suggest-
ing that TLR9 induces antiviral responses by sensing CpG
response to yeast via recruitment of the tyrosine kinase, Syk DNA of DNA virus [40, 43, 44].
[26–28]. TLR2 recognizes a variety of microbial products TLR7 and TLR8 are structurally highly conserved
through functional cooperation with several proteins, some proteins [12]. The synthetic imidazoquinoline-like mole-
of which are structurally related to TLR2 whereas others cules imiquimod (R-837) and resiquimod (R848) have
are not. potent antiviral activities and are used for clinical treatment
of viral infections. Analysis of TLR7-deficient mice
Protozoa showed that TLR7 recognizes these synthetic compounds
[45]. Human TLR7 and TLR8, but not murine TLR8,
TLRs also sense the components of protozoa including recognize imidazoquinoline compounds [46]. Furthermore,
Trypanosoma cruzi, Trypanosoma brucei, Toxoplasma murine TLR7 recognizes guanosine analogs such as
gondii, Leishmania major, and Plasmodium falciparum loxoribine, which has antiviral and antitumor activities
(Table 1). GPI anchors, glycoinositolphospholipids, and [47]. All these compounds are structurally similar to
genomic DNA-derived from T. cruzi are recognized by ribonucleic acids. After a short while, TLR7 and human
TLR2, TLR4, and TLR9, respectively [29]. Murine TLR11 TLR8 also were shown to recognize guanosine- or uridine-
senses the profilin-like molecule of T. gondii [30]. Malaria rich ssRNA from viruses such as human immunodeficiency
parasites digest host hemoglobin into a hydrophobic heme virus, vesicular stomatitis virus (VSV), and influenza virus
polymer known as hemozoin. TLR9 recognizes hemozoin [48, 49].
from P. falciparum [31]. However, the precise mechanism
of how TLR9 recognizes both DNA and non-DNA crystals Signaling pathways
remains unclear.
The activation of TLR signaling pathways originates
Virus from the cytoplasmic TIR domains. Four TIR domain-
containing adaptors (MyD88, TIRAP/MAL, TRIF, and
TLR4 recognizes not only bacterial components but also TRAM) were shown to play an important role in
viral envelope proteins (Table 1). The fusion protein from TLR signaling pathway. These adaptors are associated
respiratory syncytial virus (RSV) is recognized by TLR4 with TLRs through homophilic interaction of TIR
[32]. TLR4-mutated C3H/HeJ mice are sensitive to RSV domains. Each TLR mediates distinctive responses in
infection [33]. The envelope protein of mouse mammary association with a different combination of these
tumor virus directly activates B cells via TLR4 [34]. adapters (Fig. 1).
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MyD88-dependent pathway
TLR family members use many of the same signaling
components as IL-1R because both have a conserved
cytoplasmic domain, the TIR domain. The TIR domain-
containing adapter MyD88 is utilized by all TLRs with the
exception of TLR3. MyD88 possesses a TIR domain in its
C terminus and a death domain in its N terminus. Upon
stimulation, MyD88 recruits IL-1 receptor kinase (IRAK) to
TLRs through the interaction of both molecules’ death
domains. Two members of the IRAK family, IL-1 receptor-
associated kinase (IRAK-1) and IRAK-4 are activated by
phosphorylation, and associate with TRAF6. TRAF6 forms
a complex with ubiquitin-conjugating enzymes to activate
TAK1, leading to the activation of nuclear factor (NF)-κB
(IκB) and activator protein-1 through the canonical IκB
kinase (IKK) complex and the mitogen-activated protein
kinase pathway, respectively. The IKK complex phosphor-
ylates the inhibitor of IκB, which sequesters the transcrip-
tion factor NF-κB to induce target genes. This signaling
pathway is called the “MyD88-dependent pathway” and is
essential for the expression of inflammatory cytokine
genes, including TNF-α, IL-6, IL-12, and IL-1β, and
costimulatory molecules [3]. In the case of the TLR2 and
TLR4 signaling pathways, TIRAP/MAL is also required for
the activation of the MyD88-dependent pathway [50, 51].
MyD88-independent pathway
In addition to proinflammatory signals, some members of
the TLR family trigger the induction of type I IFNs. In
particular, TLR3 and TLR4 have the ability to induce IFN-
β and IFN-inducible genes in a MyD88-independent
manner [52]. Thus, this signaling pathway is called the
MyD88-independent pathway. A third adapter, TRIF [53,
54], is essential for the TLR3- and TLR4-mediated
MyD88-independent pathway [39, 55]. TRIF is also
required for the induction of inflammatory cytokines in
the TLR4 signaling pathway.
A fourth adapter, TRAM is essential for the TLR4-
mediated MyD88-independent pathway [56–58]. The N
terminus of TRAM has a myristoylation site, the mutation
of which alters its normal membrane localization and
abolishes the TLR4 signaling. TRAM acts as a bridging
adapter between TLR4 and TRIF [58].
TLR3- and TLR4-mediated signaling activates a member
of the IRF family of transcription factors, IRF3 to induce
IFN-β and IFN-inducible genes. Two noncanonical IKKs,
inducible IKK (IKKi)/IKKɛ and TRAF family member-
associated NF-κB activator-binding kinase 1 (TBK1)/NF-
κB-activating kinase (NAK)/TRAF2-associated kinase
(T2K), were shown to be responsible for TLR3- and
TLR4-mediated IRF-3 activation [59–63]. Upon stimula-
J Mol Med (2006) 84:712–725
tion, TRIF interacts with TBK1 and IKKi, which phos-
phorylates IRF3, allowing IRF3 to translocate into the
nucleus and activate the IFN-β promoter.
TRAF6 is reported to bind TRIF and cooperatively
activate NF-κB [64]. However, cells doubly deficient in
TRAF6 and MyD88 still partially activated NF-κB in
response to LPS, suggesting that TRIF activates NF-κB
through both TRAF6-dependent and TRAF6-independent
pathways in TLR4 signaling [65, 66].
Receptor-interacting protein-1 (RIP1) was implicated in
the TLR3-mediated NF-κB response to dsRNA. RIP1 binds
the C terminus of TRIF via a RIP homotypic interaction
motif (RHIM). In cells lacking RIP1, TLR3-mediated NF-
κB activation and the subsequent induction of intercellular
adhesion molecule 1 is severely impaired, whereas IFN-β
induction is intact. By contrast, responses to LPS were
normal in the absence of RIP1. However, LPS fails to
stimulate NF-κB activation in rip−/−MyD88−/− cells,
revealing that RIP1 is also required for the TRIF-dependent
TLR4-induced NF-κB pathway. In the TNF-α pathway,
RIP1 interacts with the E3 ubiquitin ligase TRAF2 and is
modified by a polyubiquitin chain. Upon TLR3 activation,
RIP1 is also modified by polyubiquitin chains and is
recruited to TLR3 along with TRAF6 and TAK1, suggest-
ing that RIP1 uses a similar ubiquitin-dependent mecha-
nism to activate IKK-β in response to TNF-α and TLR3
ligands [67].
Specific pathway in pDCs
pDCs are specialized for producing large amounts of type I
IFNs during viral infection [68]. pDCs highly express
TLR7 and TLR9 and produce high levels of IFN-α in
response to TLR7 and TLR9 ligands [47]. It appears that
IFN-α induction by TLR7 and TLR9 depends entirely on
MyD88 [69]. IRF7 is a transcriptional factor structurally
related to IRF3, which is expressed constitutively in pDCs.
Overexpression of IRF7 activates IFN-α- and IFN-β-
dependent promoters. IRF7 forms a signaling complex with
MyD88 and TRAF6 in the cytoplasm [70, 71]. After ligand
stimulation, IRF7 translocates into the nucleus to induce
IFN-αs [70, 71]. Similar to IRF3, IRF7 is activated by its
phosphorylation. However, in TBK1-deficient cells, TLR9-
mediated IFN-α production is still observed [70]. Mouse
pDCs lacking IRAK-4 fails to produce both inflammatory
cytokines and IFN-αs [71]. Human TLR7-, TLR8-, and
TLR9-mediated induction of IFN-α/β and -λ was also
IRAK-4 dependent [72]. Recently, IRAK-1 was shown to
serve as an IRF7 kinase. In IRAK-1-deficient mice, TLR7-
and TLR9-induced IFN-α production was completely
abolished, although inflammatory cytokines are normally
produced. Furthermore, IRF7 activation by TLR9 ligand is
impaired in IRAK-1-deficient mice, in spite of normal
J Mol Med (2006) 84:712–725
NF-κB activation. IRAK-1 but not IRAK-4 can directly
bind and phosphorylate IRF7; thus, IRAK-1 specifically
mediates IFN-α induction downstream of MyD88 and
IRAK-4 [73].
IRF8 is involved in TLR9-mediated responses because
pDCs lacking IRF8 fails to produce proinflammatory
cytokines and IFN-α in response to TLR9 ligand; in
IRF8-deficient cells, TLR9-stimulated NF-κB activation is
unexpectedly impaired, suggesting that IRF8 mediates
NF-κB activation in TLR9 signaling [74].
NLR
Structure
Mammalian NLR family members are also thought to
recognize PAMPs. NLR proteins contain three characteristic
domains. The first is a LRR domain, which is involved in
ligand recognition. The second is a centrally located NOD
(also known as a NACHT domain), which acts as an ATP-
dependent dimerization domain. The last N-teminal domain is
composed of protein–protein interaction cassettes. NLRs are
divided into subgroups according to the N-terminal domain:
caspase-recruitment domain (CARD), pyrin domain (PYD),
baculoviral inhibitory repeat (BIR)-like, and unclassified.
NODs, NALPs, and IPAFs contain CARDs, PYDs, and BIR,
respectively. All these domains are involved in the regulation
of proapoptotic and proinflammatory signaling [75].
Pathogen recognition and signaling pathway
Recent reports showed that some members of the NLR
family are involved in intracellular recognition of microbial
products. Although initial studies identified LPS as a
NOD2 ligand [76], it is well established now that NOD1
and NOD2 recognize peptidoglycan-derived peptides, γ-D-
glutamyl-meso-diaminopimelic acid (iE-DAP) [77] and
muramyl dipeptide (MDP) [78, 79], respectively. Recogni-
tion of ligands through the LRR domains activates NOD
proteins, which then recruit the serine–threonine kinase
RICK (also known as RIP2 or CARDIACK) through a
CARD–CARD interaction [80]. In the NOD2 signaling
pathway, activation of RICK leads to K63 (Lys63)-linked
polyubiquitylation of NEMO/IKKγ, which is followed by
phosphorylation of IKKβ and the phosphorylation of IκB,
leading to the translocation of NF-κB to the nucleus [81,
82]. CARD12 (also known as IPAF or CLAN) binds both
NOD1 and NOD2 through NOD–NOD interactions [83]
and inhibits NOD1- and NOD2-mediated activation of
NF-κB in vitro [84]. However, recent reports show that
invasive bacteria lead to the activation of CARD12 and the
induction of expression of IL-1β [85]. CARD6 was shown
717
to physically interact with NOD1 and RICK (but not with
NOD2) and suppress NOD1/RICK-mediated NF-κB activa-
tion [86]. In addition to NF-κB activation and NOD1 and
NOD2 signaling give rise to the activation of the mitogen-
activated protein kinase (MAPK) pathway although the
precise mechanism remains unknown [4] (Fig. 2).
Inflammasome is a multiprotein complex and is respon-
sible for the activation of caspase 1 and 5, leading to the
processing and secretion of the proinflammatory cytokines
IL-1β and IL-18 [87]. Two types of the inflammasome
were reported to date: NALP1 inflammasome and NALP2/
3 inflammasome (Fig. 3). Whereas NALP1 inflammasome
is composed of NALP1, ASC, caspase 1, and caspase 5,
NALP2/3 inflammasome contains NALP2/3, ASC, caspase
1, and Cardinal. NALPs are the chief platforms for
inflammasome [88]. ASC is an essential adapter molecule
that connects the NALPs to caspase 1. Because NALP2/3
do not have FIND domain and CARD domains in the C
terminus, the NALP2/3 inflammasome contains Cardinal,
which has the same structural organizations as the C-
terminal region of NALP1 [88]. All of the inflammasome
components reside in the cytoplasm. Activation of inflam-
masome is thought to occur through the recognition of
PAMPS by NALPs. However, the precise activation
mechanism remains unknown. After activation of inflam-
masome, activated caspases cleave the proinflammatory
cytokines, IL-1β and IL-18. Processing leads to the
secretion of mature IL-1β and IL-18 and induces inflam-
matory responses. IL-1β production is tightly controlled to
avoid excessive inflammation. Caspase-1 activation is
inhibited by CARD containing ICEBERG, COP, and
DASC (POP1) and possibly by human caspase-12. Acti-
vated caspase-1 is blocked by PI-9 and IL-1β activity is
inhibited by IL1Ra [89].
The NALP3/cryopyrin/CIAS1 inflammasome is activated
by MDP. Recent reports showed that NALP3/cryopyrin/
CIAS1 is involved in the recognition of bacterial RNA, ATP,
and uric acid crystals, and is essential for caspase-1
activation and IL-1β and IL-18 production in response to
these ligands [90–92] (Fig. 3).
Another CARD-containing NOD-LRR protein, Ipaf is
responsible for Salmonella typhimurium-induced, but not
TLR-induced, caspase-1 activation. ASC but not RICK/
RIP2 is also essential for S. typhimurium-induced caspase-1
activation [85] (Fig. 3).
The Lgn1 locus was found to control the intracellular
replication of L. pneumophila in murine macrophages.
NAIP5/Birc1e, a NLR protein with BIR domains, was
identified to be responsible for the Lgn1 effect [93].
NAIP5/Birc1e is assumed to function as a cytoplasmic
sensor of L.pneumophila and is responsible for L.pneumo-
phila-mediated caspase-1 activation. The pathway used for
caspase-1-dependent processing and secretion of IL-1 was
718 J Mol Med (2006) 84:712–725

Fig. 2 Signaling pathways of

NOD1 and NOD2. NOD1 and
NOD2 recognize iE-DAP and
MDP, respectively, and activate
NF-κB via RIP2/RICK. NOD1
and NOD2 signaling gives rise
to the activation of MAPKs by
unknown mechanism. CARD12
negatively regulates RICK/
RIP2-mediated NF-κB
activation by both NOD1 and
NOD2. CARD6 also negatively
regulates RICK/RIP2-mediated
NF-κB activation by NOD1

distinct from the pathway used for caspase-1-dependent Diseases

inhibition of L. pneumophila growth. It is interesting to note
that the adapter molecule ASC is essential for the secretion
of IL-1β after L. pneumophila infection but not for the
control of L. pneumophila replication. These data suggest
that Ipaf and Asc have separate and distinct functions in
controlling innate immune responses to L. pneumophila [94]
(Fig. 3).
The importance of NLRs was highlighted by findings that
mutations affecting the function of these proteins are
associated with the occurrence of autoimmune diseases.
NOD2 is associated with two autoinflammatory disorders.
A missense point mutation in the human NOD2 gene is
correlated with susceptibility to Crohn’s disease [4]. Blau
syndrome, an inheritable disorder characterized by granu-

Fig. 3 Bacterial recognition of

NLRs and their signaling. NLRs
recognize bacterial proteins in
the cytoplasm and trigger signal-
ing pathways. MDP, RNA, ATP,
and uric acid crystal are recog-
nized by NALP3, which forms
an inflammasome comprised of
ASC, CARDINAL, and caspase-
1. NALP1 also forms an inflam-
masome composed of ASC,
caspase-1, and caspase-5. Little
is known about the natural
stimuli that lead to the activation
of NALP1 inflammasome.
Activated caspase-1 cleaves
pro-IL-1β for the maturation of
IL-1β. Another NOD-LRR
protein, IPAF, is activated by S.
typhimurium and induces
maturation of IL-1β associating
with ASC. Naip5/Birc1e is acti-
vated by L. pneumophila and
induces caspase-1 activation in
an IPAF/ASC-dependent manner
J Mol Med (2006) 84:712–725
lomatous polyarthritis, panuveitis, and exanthema, was
shown to share a mutated form of CARD15 (which encodes
NOD2) [95].
NALP3/cryopyrin/CIAS1 is associated with three auto-
inflammatory syndromes: familial-cold autoinflammatory
syndrome, Muckle–Wells syndrome, and neonatal-onset
multisystem inflammatory disease [96, 97]. These diseases
are now referred as cryopyrin-associated periodic syndrome
(CAPS). CAPS patients have systemic inflammation with
increased levels of C-reactive protein, serum amyloid A,
and inflammatory cytokines. However, it is not well
understood how the mutated NALP3/cryopyrin/CIAS1
contributes to the induction of systemic infection [75].
CIITA is a master transcription regulator of MHC class
II genes [98]. Mutations in CIITA lead to MHC class II
deficiency or a rare form of autosomal recessive hereditary
immunodeficiency, group A bare lymphocyte syndrome
[99, 100].
Familial Mediterranean fever (FMF) is an autosomal
recessive inherited disease that is characterized by recurrent
episodes of fever accompanied with topical signs of
inflammation. The gene, Mediterranean fever, which
encodes the pyrin (also known as marenostrin) protein is
mutated in patients with FMF [75].
Antiviral RNA helicases
Whereas TLR2 and TLR4 recognize viral components at
the cell surface, TLR3, TLR7, TLR8, and TLR9 are
exclusively expressed in endosomal compartments. After
phagocytes internalize viruses or virus-infected apoptotic
cells, viral nucleic acids are released in phagolysosomes
and are recognized by these TLRs [3]. Recent reports
showed that hosts also have a mechanism to detect
replicating viruses in the cytoplasm in a TLR-independent
manner [101].
RIG-I
RIG-I was identified as a cytoplasmic receptor for viral
dsRNA. RIG-I is a DExD/H box RNA helicase, which
possesses C-terminal helicase domain and two tandem
caspase-recruiting domains at the N terminus (Fig. 4).
RIG-I binds to RNA with its C-terminal helicase domain
and unwinds dsRNA in an ATPase dependent manner. The
binding of the helicase domain and RNA is likely to induce
conformational changes that exposes the N-terminal
CARDs to recruit downstream signaling proteins, leading
to the activation of IRF3 and NF-κB [5].
The functional significance of RIG-I was shown in vitro
and in vivo [5, 6]. Overexpression of RIG-I blocks
replication of VSV and encephalomyocarditis virus
719
(EMCV), and RNAi of RIG-I type I inhibited induction of
type I IFNs in response to Newcastle disease virus (NDV),
Sendai virus, and EMCV [5]. Most RIG-I-deficient mice
are embryonic lethal because of severe liver degeneration.
Conventional DCs and fibroblasts derived from RIG-I-
deficient mice are almost completely unable to produce
type I IFN and inflammatory cytokines after infection with
NDV, Sendai virus, or VSV. However, RIG-I-deficient
pDCs produced type I IFNs normally in response to NDV.
In pDCs, TLR systems are responsible for RNA virus-
mediated type I IFN induction. Thus, RIG-I and the TLR
system exert antiviral responses in a cell-type-specific
manner [6].
Other molecules
Melanoma differentiation-associated gene 5 (MDA5, also
called Helicard) was identified as the DExD/H box RNA
helicase and contains two CARD-like domains and a single
helicase domain [102–105]. Like RIG-I, overexpression of
MDA5 enhanced NDV-induced type I IFN production and
antiviral responses in response to VSV or EMCV. RNAi of
MDA5 blocks NDV-induced activation of type I IFN
promoters [105]. MDA5 was identified as a binding target
for V proteins of paramyxoviruses [104]. Although the
precise mechanism is unclear, these V proteins inhibit the
dsRNA-induced activation of the IFN-β gene through
MDA5. Virus-encoded proteins may specifically target
these RNA helicases to escape from antiviral detection by
the host cells.
An RNA helicase Lgp2 is also related to RIG-I and
MDA5, but lacks the CARD-like domains. Overexpression
of Lgp2 inhibits Sendai virus-induced activation of IFN-β
promoter, suggesting that Lgp2 is a negative regulator of
RIG-I and MDA5 [105, 106] (Fig. 4).
Signaling pathway of antiviral RNA helicases
The signaling pathway of RIG-I and MDA5 ultimately
engages the IKK complex (IKKα/β/γ) and TBK1/IKKi,
which leads to phosphorylation and activation of NF-κB
and IRF3 and IRF7, respectively. IFN-β promoter stimula-
tor 1 (IPS-1) was identified as a molecule that acts
downstream of RIG-I and MDA5. IPS-1 consists of two
domains characterized as an N-terminal CARD domain and
a C-terminal effector domain. IPS-1 associates with RIG-I
and MDA5 with a CARD domain. Overexpression of IPS-1
activates IFN-β, IFN-α4, IFN-α6, and NF-κB promoters
and inhibits VSV replication. RNAi of IPS-1 blocks type I
IFN production in response to intracellular administration
of dsRNA or infection with VSV or NDV. TBK1 and IKKi
are required for IPS-1-mediated activation of the type I IFN
promoters, although it does not directly bind to these
720 J Mol Med (2006) 84:712–725

Fig. 4 Signaling pathways of

antiviral RNA helicases. Viruses
produce dsRNA during
replication in cytoplasm. RIG-I
and Mda5 recognize dsRNA
to initiate antiviral signaling.
IPS-1 interacts with RIG-I and
Mda5 via the CARD-like
domain, followed by the
activation of IRF3 and IRF7 via
TBK1- and IKKi-dependent
phosphorylation. IPS-1 also
activates NF-κB via FADD/
RIP1-dependent pathways.
Synthetic dsDNA also activates
type I IFN promoters, although
a receptor responsible for
DNA recognition was not
identified

protein kinases. IPS-1 interacts with FADD and RIP1 via its
effector domain to facilitate NF-κB activation. Thus, IPS-1
is an adaptor protein that mediates RIG-I- and MDA5-
dependent antiviral responses [107].
IPS-1 was independently identified by three different
groups: mitochondrial antiviral signaling protein (MAVS)
[108], virus-induced signaling adaptor [109], and CARD-
adaptor-inducing IFN-β (Cardif) [110]. The MAVS study
shows that MAVS localizes to the outer mitochondrial
membrane. The mitochondrial-targeting transmembrane
domain is essential for MAVS signaling, implying the
importance of mitochondria in innate immunity [108]
(Fig. 4).
Viral modification of the host immune system
Viruses have various mechanisms to escape from host
immunity. Vaccinia virus (VV) protein A52R was shown to
block the activation of NF-κB by multiple TLRs, in
particular, TLR3 [111, 112]. A52R associates with both
IRAK2 and TRAF6, and disrupts signaling complexes
containing these proteins. Furthermore, deletion of the
A52R gene from VV reduced virus virulence [112]. The
other VV protein, A46R, has a TIR domain, and as such is
the only viral member of the IL-1R/TLR family identified
to date [111]. Recent studies revealed that A46R inhibited
IL-1R/TLR, but not TNF-induced NF-κB activation, by
associating with TIR-domain containing adaptor molecules
[111, 113]. A46R interacts with multiple TIR-containing
adaptors and thereby inhibits the activation of IRF3 and
NF-κB [113]. NS3-4A, a multifunctional protein of
hepatitis C virus that has serine protease activity essential
for the production of mature viral proteins, was recently
shown to block the activation of IRF3. NS3-4A counteracts
not only TLR3-dependent pathways by targeting Trif for
proteolytic cleavage, but also counteracts TLR3-indepen-
dent pathways by targeting an undefined protein of the
RIG-I-dependent pathway [114, 115]. Meylan et al. [110]
showed that Cardif is cleaved by wild-type but not by a
catalytically inactive form of NS3-4A, which cleaves the
C-terminal region of Cardif, causing disruption of IRF3 and
NF-κB activation, probably by causing mislocalization of
cleaved Cardif from mitochondria. Also, the V proteins of
paramyxoviruses, such as simian virus 5, human para-
influenza virus 2, mumps virus, Sendai virus, and Hendra
virus, associate with MDA5 and block the downstream
signaling of MDA5 [104, 105]. Thus, for their survival
many viruses antagonize important components of the host
antiviral defense.
DNA recognition in cytoplasm
DNA, including host DNA, can be recognized indepen-
dently of TLR9. DNase II, a chief DNase present in the
phagosome of macrophages, clears DNA from phagocy-
tosed apoptotic cells or debris [116]. In DNase II-deficient
mice, macrophages begin to produce IFN-β, which lead to
subsequent TLR9-independent inflammatory events [117].
Furthermore, dsDNA but not ssDNA derived from either
pathogens or the host activates both immune and nonim-
mune cells when introduced into the cytoplasm by
transfection [118, 119]. Double-stranded breaks form of
J Mol Med (2006) 84:712–725
DNA (B-DNA) but not Z-form DNA stimulated mouse and
human stromal cells and DCs, resulting in the production of
type I IFN and chemokines. B-DNA activated IRF3 and the
IFN-β promoter via both TBK1 and IKKi, whereas it
activated transcription factor NF-κB independently of both
TBK1 and IKKi. Both of these pathways required the
adaptor molecule IPS-1 in vitro but not TLRs or RIG-I and
MDA5. In addition, B-DNA activation of fibroblasts
conferred resistance to viral infection, suggesting that
B-DNA-induced IFN genes might be important in antiviral
innate immune responses. Furthermore, B-DNA activation
of the signaling pathways may be involved in the pathogen-
esis of nucleic acid-dependent autoimmune diseases [120]
(Fig. 4). It is thus necessary to identify a cytoplasmic
detector of DNA.
Conclusion
Since the discovery of mammalian TLRs, there was a rapid
clarification of the molecular mechanisms of pathogen
recognition. Research on TLRs has also revealed that
recognition of intracellular pathogens is also mediated via
a TLR-independent mechanism. Newly described NLR
family members, which recognize pathogens in the cyto-
plasm, are genetically linked to several human immuno-
logical disorders. Innate immune responses via RIG-I are
essential for the control of viral replication in the
cytoplasm. Furthermore, TLR system and RIG-I system
were tactfully utilized against viral infection in a cell-type-
specific manner. Thus, hosts have developed the skillful
and complicated innate immune system to counter the
various pathogens with various modes of infection. Future
studies on PRRs should provide a more comprehensive
understanding of innate immune responses.
Acknowledgements We thank M. Hashimoto for secretarial assis-
tance. This work was supported by grants from Special Coordination
Funds, the Ministry of Education, Culture, Sports, Science and
Technology, and the Japan Research Foundation for Clinical
Pharmacology.
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