Pathogen recognition with Toll-like receptors

Taro Kawai1 and Shizuo Akira1,2

The innate immune system is an evolutionarily conserved
system of defense against microbial infections. The family of
Toll-like receptors is a major class of receptors that sense
molecular patterns associated with a broad range of pathogens
including bacteria, viruses, fungi and protozoa. Following
pathogen recognition, Toll-like receptors initiate intracellular
signal transduction that results in the expression of genes
involved in inflammation, antiviral responses and maturation of
dendritic cells. Individual Toll-like receptors activate common
and unique transcription factors through different signaling
pathways to drive specific biological responses against
microorganisms.
Addresses
1
Exploratory Research for Advanced Technology (ERATO), Japan
Science and Technology Agency, 3-1 Yamada-oka, Suita,
Osaka 565-0871, Japan
2
Department of Host Defense, Research Institute for Microbial Diseases,
Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Corresponding author: Akira, Shizuo (sakira@biken.osaka-u.ac.jp)

Current Opinion in Immunology 2005, 17:338344

This review comes from a themed issue on
Hostpathogen interactions
Edited by Bali Pulendran and Robert A Seder
Available online 13th June 2005
0952-7915/$ see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.coi.2005.02.007

Introduction
Innate immune responses against microbial pathogens are
initiated by the recognition of specific structures of invading pathogens, called pathogen-associated molecular patterns (PAMPs), by a limited number of pattern
recognition receptors (PRRs) expressed on macrophages
and dendritic cells (DCs) [1,2]. The recognition of
PAMPs by PRRs triggers an intracellular signaling pathway which culminates in induction of proinflammatory
cytokines, chemokines, type I interferon (IFN-a or -b)
and maturation of DCs that leads to activation of adaptive
immunity. In Drosophila, which lacks an adaptive immune
system, a Toll receptor has a central role in the defensive
response to fungal infection [3]. Identification of a family
of proteins similar to Toll in human and mouse, namely
the Toll-like receptors (TLRs), has revealed that they act
as mammalian PRRs. All TLRs possess amino-terminal
leucine-rich repeats, which are responsible for the recognition of PAMPs, and also possess a carboxy-terminal
Current Opinion in Immunology 2005, 17:338344

Tollinterleukin-1 (IL-1) receptor (TIR) domain, which

is required for initiating intracellular signaling [2].

Recognition of PAMPs by TLRs

Eleven members of the TLR family have been identified
so far (Figure 1). The first TLR member to be discovered
was TLR4, which was demonstrated to be the longsought receptor for bacterial lipopolysaccharide (LPS),
the outer membrane component of Gram-negative bacteria [2]. Subsequent generation of TLR-deficient mice
has revealed that each TLR recognizes different PAMPs,
and even self-molecules. TLR2 recognizes peptidoglycan, in addition to the lipoproteins and lipopeptides of
Gram-positive bacteria and mycoplasma lipopeptide
[2,4]. TLR2 appears to collaborate with its relatives
TLR1 and TLR6 to discriminate between the molecular
structures of diacyl and triacyl lipopeptides, respectively
[2]. Furthermore, TLR2, in concert with non-TLR receptors, even further diversifies its recognition potential.
Innate immune responses against zymosan, a cell-wall
preparation of yeast, are mediated by the collaborative
recognition of TLR2 and dectin-1, a lectin family of
receptors for b-glucans [5]. Signaling through dectin-1
enhanced the TLR2-mediated transcription of target
genes. CD36, a class II scavenger receptor expressed
on the surface of innate immune cells, is involved in
sensing some, but not all, TLR2 ligands [6].
TLR5 recognizes flagellin, a protein component of bacterial flagella [7]. Mouse TLR11, a relative of TLR5,
is expressed abundantly in the kidney and bladder.
TLR11-deficient mice have consistently been shown to
be susceptible to uropathogenic bacterial infection [8
],
indicating that TLR11 senses the products of these
bacteria. In humans, however, TLR11 is thought to be
nonfunctional, owing to the presence of a stop codon.
Synthetic imidazoquinoline-like molecules imiquimod
(R-837) and resiquimod (R-848), and guanosine analogs
such as loxoribine, have potent antiviral activities and are
currently used for treating viral infections. In response to
these molecules, TLR7-deficient mice failed to induce
inflammatory cytokines, type I IFN and DC maturation
[9]. In addition, these molecules activate intracellular
signaling proteins in cells transfected with human and
mouse TLR7. TLR8 is a close relative of TLR7, and a
transfection study has demonstrated that human, but not
mouse, TLR8 confers responsiveness to resiquimod [10].
Taken together, these findings indicate that mouse
TLR7, human TLR7 and human TLR8 mediate the
recognition of these molecules. The structural similarities
between these compounds and ribonucleic acids suggest
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Pathogen recognition with TLRs Kawai and Akira 339

Figure 1

Tri-acyl
lipopeptide

Di-acyl
lipopeptide

LPS

Flagellin

Uropathogenic
bacteria

TLR2 TLR6

TLR4

TLR5

TLR11

dsRNA

Imidazoquinolines
ssRNA

CpG DNA
Hemozoin

TLR1 TLR2
Endosome

Leucine -rich repeats

TIR domain
TLR3

TLR7
TLR8

TLR9

Current Opinion in Immunology

Structures and ligands for Toll-like receptors. TLR2, in collaboration with TLR1 or TLR6, discriminates between the molecular structures of
triacyl and diacyl lipopeptides, respectively. TLR4 recognizes bacterial LPS. TLR5 recognizes bacterial flagellin. TLR11 recognizes uropathigenic
bacteria products. TLR3, TLR7, TLR8 and TLR9 reside in endosomal compartments and recognize nucleic acids; TLR3 recognizes viral dsRNA,
whereas TLR7 and TLR8 recognize viral ssRNA. TLR9 recognizes bacterial and viral CpG DNA motifs. TLR9 also recognizes non-nucleic acids,
such as hemozoin.

the possibility that TLR7 and TLR8 are responsible

for virus detection. Accordingly, activation of DCs by
synthetic single-stranded (ss)RNA rich in guanosine or
uridine derived from RNA viruses was impaired in
TLR7-deficient mice [11
,12
]. Furthermore, ssRNA
activated cells transfected with human TLR8, whereas
mice lacking TLR8 responded normally [12
]. These
findings suggest species differences in the recognition
of RNA viruses between human and mouse TLR8. Moreover, two small-molecule agonists specific for human
TLR7 and TLR8 have been shown to activate different
types of cells and induce different cytokine profiles,
suggesting distinct cellular expressions and functions
between TLR7 and TLR8 in human [13].
TLR9 mediates the recognition of the unmethylated 20 deoxyribo (cytidine-phosphate-guanosine) (CpG) DNA
motifs found in bacteria and DNA viruses [14]. Cells
lacking TLR9 are hyporesponsive to synthetic CpG DNA
and certain DNA viruses [14,15]. TLR9 also recognizes
non-DNA pathogenic components. Malaria parasites
digest host hemoglobin into a hydrophobic heme polymer
known as hemozoin. Hemozoin from Plasmodium falciparum stimulates macrophages and DCs to produce
inflammatory cytokines and chemokines in a TLR9dependent mechanism [16]. However, the precise
mechanism of how TLR9 recognizes both DNA and
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non-DNA crystal is unclear. As noted above, several

self-molecules have been demonstrated to activate cells
via TLRs; notably, TLR recognition of self-molecules
triggers inflammatory responses and in some cases contributes to the pathogenesis of autoimmune diseases. It
has been shown that an immune complex (IC) of mouse
IgG2a and DNA-containing chromatin was bound and
internalized by B cell receptors and that the chromatin
component was sequentially recognized by TLR9 inside
the cells, inducing rheumatoid factor production [17,18].
Similar dual recognition mechanisms have been observed
in DCs, in which the Fcg receptor serves to internalize
DNA-containing ICs within lupus serum and deliver
them to TLR9 [19,20]. Additionally, there is also evidence of TLR-independent activation of DCs by ICs
[19,20].
TLR3 is involved in the recognition of double-stranded
(ds)RNA generated during virus replication [21]. Accordingly, TLR3-deficient mice are hypersusceptible to
mouse cytomegalovirus infection, which indicates that
TLR3 has a protective role against viral infection [22].
TLR3 also mediates the recognition of West Nile virus
(WNV) [23]. WNV infection triggers TLR3-dependent
inflammatory responses that result in a breakdown of the
bloodbrain barrier, followed by enhanced brain infection. TLR3-deficient mice are consistently more resistant
Current Opinion in Immunology 2005, 17:338344

340 Hostpathogen interactions

to lethality after WNV infection, most probably because

of reduced virus entry into the brain. These findings
suggest that TLR-dependent inflammatory responses
might be involved in the pathogenesis of WNV encephalitis, rather than in virus elimination.
TLR3, TLR7 and TLR9, all of which recognize nucleic
acids, are not expressed on the cell surface but are
exclusively expressed in endosomal compartments
[24,25]. Thus, after bacteria and viruses are internalized
to be delivered to the endosome, nucleic acids might be
released, to be recognized by TLRs.

Signal transduction pathway of TLRs

The recognition by TLRs of PAMPs triggers the TIR
domain-dependent association with adapters containing
the TIR domain, including MyD88, TIRAPMAL,
TRIFTICAM1, and TRAMTICAM2 [26]. Individual
TLRs mediate distinctive responses by association with a
different combination of these adapters. MyD88 is utilized by all TLRs, with the exception of TLR3. The
association of TLRs and MyD88 recruits members of the
IL-1 receptor-associated kinase (IRAK) family; two
members, IRAK4 and IRAK1, are sequentially phosphorylated, causing them to dissociate from the receptor
complex, and then associate with TRAF6. TRAF6
forms a complex with ubiquitin-conjugating enzymes
to activate the kinase TAK1, which in turn activates
transcription factors nuclear factor (NF)-kB and activator
protein-1 through the canonical IkB kinase (IKK) complex and the mitogen-activated protein kinase pathway,
respectively. NF-kB activation by this MyD88-dependent pathway results in the expression of inflammatory
cytokine genes, including tumor necrosis factor-a, IL-6
and IL-1b [26,27]. Among these adapters, TIRAPMAL
is required for the activation of the MyD88-dependent
pathway downstream of TLR2 and TLR4 [28,29]
(Figure 2).
In addition to proinflammatory signals, TLRs also trigger
appropriate responses against different PAMPs. Stimulation of macrophages and DCs with LPS and dsRNA
results in the activation of the transcription factor IFN
regulatory factor 3 (IRF3) and subsequent induction of
IFN-b and IFN-inducible genes [30,31]. Notably, these
inductions occur independently of MyD88 [30]. It has
been demonstrated that TRIF is responsible for TLR3and TLR4-mediated IRF3 activation [31,32]. Additionally, TLR4 also requires another adaptor, TRAM, for
IRF3 activation [33] (Figure 2). IRF3 activation occurs
through the actions of the IKK-related kinases TRAFfamily-member-associated NF-kB activator (TANK)binding kinase 1 (TBK1) and IKKi (also called IKKe),
which catalyze IRF3 phosphorylation and stimulate its
nuclear translocation and DNA binding [34,35]. TBK1deficient mice have consistently been shown to be unable
to activate IRF3 and induce IFN-b after exposure to
Current Opinion in Immunology 2005, 17:338344

TLR3- and TLR4 ligands [3638]. Furthermore, TRIF

interacts with two different protein kinases, TBK1 and
receptor-interacting protein-1 (RIP1) [39,40]. In cells
lacking RIP1, TLR3-mediated NF-kB activation and
the subsequent induction of Icam-1 was severely
impaired, whereas IFNb induction was intact. By contrast, responses to LPS were normal in the absence of
RIP1. Thus, TRIF is likely to use TBK1 and RIP1 for
IRF3- and NF-kB activation, respectively, in TLR3
signaling. TRAF6 is also reported to bind TRIF and
cooperatively activate NF-kB [39] (Figure 2). However,
cells doubly deficient in TRAF6 and MyD88 still partially
activated NF-kB in response to LPS, suggesting that
TRIF activates NF-kB through both TRAF6-dependent
and -independent pathways in TLR4 signaling [30].
Among a subset of DCs, plasmacytoid DCs (pDCs) are
known to secrete type I IFN during the infection of DNA
and RNA viruses, suggesting that pDCs express sensors
that detect virus components [41]. TLR7 and TLR9 are
expressed in both pDCs and other conventional DCs,
whereas TLR2, TLR3, TLR4 and TLR5 are expressed
in conventional DCs but not in pDCs. Accordingly, pDCs
respond to CpG DNA, by rapidly secreting large amounts
of IFN-a. Synthesized ssRNA can also induce IFN-a via
TLR7. TLR7 and TLR9 are thus suggested to have
major roles in virus detection in pDCs [41]. It appears
that IFN-a induction by TLR7 and TLR9 depends
entirely on MyD88 [42]. IRF7 is a transcriptional factor,
structurally related to IRF3, that is expressed constitutively in pDCs. Overexpression of IRF7 activates IFN-aand IFN-b-dependent promoters. IRF7 forms a signaling
complex with MyD88 and TRAF6 in the cytoplasm
[43
,44
]. After stimulation, IRF7 translocates into the
nucleus to induce IFN-a [43
,44
]. Similar to IRF3,
phosphorylation is required for the activation of IRF7.
However, in TBK1-deficient cells, TLR9-mediated
IFN-a production was still observed [43
]. In this regard,
IRAK1 has been demonstrated to serve as an IRF7
kinase, and in IRAK1-deficient mice, TLR7- and
TLR9-induced IFN-a production was completely abolished, although inflammatory cytokines were produced
[45
]. Accordingly, IRF7 activation by TLR9 ligand was
impaired in IRAK1-deficient mice, in spite of normal
NF-kB activation. By contrast, pDCs lacking MyD88
and IRAK4 failed to produce inflammatory cytokines
and IFN-a [43
,44
]. IRAK1 is capable of binding and
phosphorylating IRF7. These observations together indicate that IRAK1 specifically mediates IFN-a induction
downstream of MyD88 and IRAK4 [45
] (Figure 2).
More recently, IRF5 has been shown to be involved in a
MyD88TRAF6 complex [46

,47]. Surprisingly, in
macrophages and DCs derived from IRF5-deficient mice,
induction of proinflammatory cytokines in response to
various TLR ligands was severely impaired, whereas
IFN-a induction by the TLR9 ligand was normal
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Pathogen recognition with TLRs Kawai and Akira 341

Figure 2

Transcription
factors

Adapters
TBK1
TLR3

TRIF

IRF3

IRF3

RIP1
NF-B

IFN-
ICAM1

TRAF6
TBK1
TRIF

IRF3

TRAF6

IRF3

IFN-

NF-B

TRAM
TLR4
TIRAP
MyD88

TLR7
TLR9

MyD88

IRAK4

IRAK4

IRAK1

IRAK1

TRAF6
IRF5

IRF5

IRF7

IRF7

TRAF6
IRF5

TLR5

MyD88

IRAK4

IRAK1

TRAF6
IRF5

TLR1
TLR2
TLR6

NF-B

NF-B

Inflammatory
cytokines

IFN-
Inflammatory
cytokines

IRF5
NF-B

Inflammatory
cytokines

IRF5

TIRAP
MyD88

IRAK4

IRAK1

TRAF6
IRF5

Cytoplasm

NF-B

Inflammatory
cytokines

IRF5
Nuclei
Current Opinion in Immunology

Schematic representation of TLR-signaling pathways. All TLRs except for TLR3 share the MyD88-dependent pathway that activates NF-kB and
subsequently induces genes encoding inflammatory cytokines. IRAK1, IRAK4 and TRAF6 are located downstream of MyD88. TIRAP is involved
in the MyD88-dependent pathway downstream of TLR2 and TLR4. TRIF is utilized in the TLR3- and TLR4-mediated activation of IRF3 and the
subsequent induction of expression of IRF3-dependent genes such as that coding for IFN-b. TRAM is specifically involved in IRF3 activation in
TLR4 signaling. TBK1 (together with IKKi) is responsible for the activation of IRF3 downstream of TRIF in TLR3- and TLR4 signaling. TRIF utilizes
RIP1 and TRAF6 to activate NF-kB. IRF7 associates with MyD88, IRAK1 and TRAF6, and mediates TLR7- and TLR9-induced IFN-a production.
IRF5 also associates with MyD88 and TRAF6 to induce genes encoding inflammatory cytokines.

[46

]. These observations indicate that IRF5 is a general

signal transducer that mediates MyD88-dependent gene
induction of proinflammatory cytokines, in contrast to
IRF7, which requires MyD88-dependent IFN-a induction. Upon TLR ligand stimulation, IRF5 was shown to
translocate to the nucleus from the cytoplasm and bind
the potential IFN-stimulated response element (ISRE)
motifs present in the promoter region of proinflammatory
cytokine genes [46

] (Figure 2). Also, IRF8 is known to
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be implicated in TLR9-mediated responses because

pDCs lacking IRF8 failed to produce proinflammatory
cytokines and IFN-a in response to TLR9 ligand. In
these cells, TLR9-stimulated NF-kB activation was
unexpectedly impaired, suggesting that IRF8 mediates
NF-kB activation in TLR9 signaling [48].
Furthermore, differential responses of DCs to distinct
TLR ligands are also reported in terms of adaptive
Current Opinion in Immunology 2005, 17:338344

342 Hostpathogen interactions

immune responses. For example, various TLR2 ligands

induce abundant IL-10 and favor Th2-biased immune
responses both in human and mice, whereas ligands for
TLR4, TLR5 and TLR9 induce Th1-associated cytokines, such as IL-12p70 [49,50]. These observations
suggest that TLR2 signals via unique pathways that
mediate Th2-associated cytokine induction. It was
demonstrated that TLR2 ligands induce a higher phosphorylation of ERK and stabilization of the transcription
factor c-Fos in DCs, which results in induction of IL-10
and suppression of IL-12p70.

TLR systems in the innate immune system will be

required to develop treatments of such disorders.

Conclusions and future perspectives

1.

Janeway CAJ, Medzhitov R: Innate immune recognition. Annu

Rev Immunol 2002, 20:197-216.

2.

Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev

Immunol 2003, 21:335-376.

3.

Hoffmann JA: The immune response of Drosophila. Nature

2003, 426:33-38.

4.

Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T,

Takeda K, Akira S: Differential roles of TLR2 and TLR4 in
recognition of gram-negative and gram-positive bacterial cell
wall components. Immunity 1999, 11:443-451.

5.

Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM:

Collaborative induction of inflammatory responses by
dectin-1 and Toll-like receptor 2. J Exp Med 2003,
197:1107-1117.

6.

Hoebe K, Georgel P, Rutschmann S, Du X, Mudd S, Crozat K,

Sovath S, Shamel L, Hartung T, Zahringer U et al.: CD36 is a
sensor of diacylglycerides. Nature 2005, 433:523-527.

7.

Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR,

Eng JK, Akira S, Underhill DM, Aderem A: The innate immune
response to bacterial flagellin is mediated by Toll-like receptor
5. Nature 2001, 410:1099-1103.

Through the discovery of mammalian TLRs, the molecular mechanisms of pathogen sensing have been elucidated over the past few years. TLRs, alone or in
combination with other TLRs or non-TLRs, are able
to discriminate between the structures of pathogens.
Individual TLRs use different combinations of TIR
domain-containing adapters to activate distinct transcription factors such as NF-kB, IRF3, IRF5 and IRF7, and so
exhibit a variety of biological responses. However, other
pathogen-sensing mechanisms exist that are mediated by
non-TLR receptors. DCs appear to respond to RNA
viruses, by secreting type I IFN via TLR3-dependent
and -independent mechanisms. For example, intracellular introduction of dsRNA stimulates DCs lacking TLR3
to secrete type I IFN. This induction does not require
TRIF but does require TBK1 and IRF3 [31,36]. Retinoic
acid-inducible gene I has recently been identified as an
intracellular sensor for dsRNA, and is composed of the
RNA helicase domain, which binds dsRNA, and the
caspase recruitment domain (CARD), which culminates
in IRF3 activation [51

]. Fas-associated death domain
(FADD) and RIP1 have been demonstrated to be
involved in this pathway [52].
It also appears that recognition of intracellular bacteria is
mediated via TLR-independent mechanisms. The
nucleotide-binding oligomerization domain (NOD) proteins NOD1, NOD2 and CLANIpaf contain one or two
CARDs followed by a central nucleotide-binding domain
(NBSNACHT) and carboxy-terminal leucine-rich
repeats similar to a TLR ectodomain [53]. NOD1 and
NOD2 recognize naturally occurring degradation products and minimal motifs of bacterial peptidoglycan
[53]. Mutations in the human nod2 gene are reportedly
associated with the pathogenesis of inflammatory diseases
such as Crohns disease and Blau syndrome [53]. The
PANNALPPYPAF family shows a similar architecture
to NOD1, except that it contains the pyrin domain
instead of the CARD. This family is also implicated in
intracellular pathogen sensing, and mutations of its members are associated with autoinflammatory disorders [54].
Dysregulation of the innate immune system is known to
cause autoimmunity, autoinflammation, allergy and
tumors. Further understanding of both TLR- and nonCurrent Opinion in Immunology 2005, 17:338344

Acknowledgements
We thank members of the Akira laboratory for helpful discussions, and M.
Hashimoto for excellent secretarial assistance.

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