Sensing of Viral Infection and Activation of Innate Immunity by Toll-Like Receptor 3

Abstract

Toll-like receptors (TLRs) form a major group of transmembrane receptors that are involved in the detection of invading pathogens. Double-stranded RNA is a marker for viral infection that is recognized by TLR3. TLR3 triggering activates specific signaling pathways that culminate in the activation of NF-kappaB and IRF3 transcription factors, as well as apoptosis, enabling the host to mount an effective innate immune response through the induction of cytokines, chemokines, and other proinflammatory mediators. In this review, we describe the paradoxical role of TLR3 in innate immunity against different viruses and in viral pathogenesis but also the evidence for TLR3 as a "danger" receptor in nonviral diseases. We also discuss the structure and cellular localization of TLR3, as well as the complex signaling and regulatory events that contribute to TLR3-mediated immune responses.

Full text

CLINICAL MICROBIOLOGY REVIEWS, Jan. 2008, p. 13–25 Vol. 21, No. 1
0893-8512/08/$08.00⫹0 doi:10.1128/CMR.00022-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Sensing of Viral Infection and Activation of Innate Immunity by
Toll-Like Receptor 3
Elisabeth Vercammen, Jens Staal, and Rudi Beyaert*
Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, VIB, Ghent, Belgium,
and Department of Molecular Biology, Ghent University, Ghent, Belgium
INTRODUCTION .........................................................................................................................................................13
PARADOXICAL ROLES OF TLR3 IN VIRAL PATHOLOGY..............................................................................13
A FUNCTION FOR TLR3 BEYOND ITS ROLE IN VIRAL INFECTION?........................................................15
TLR3 STRUCTURE, LIGAND BINDING, AND SPECIFICITY ...........................................................................15
TLR3 LOCALIZATION AND SIGNALING .............................................................................................................17
Localization Holds the Key to TLR3 Activity .......................................................................................................17
TRIF Functions as a TLR3 Adaptor Molecule.....................................................................................................18
Tyrosine Phosphorylation of TLR3 ........................................................................................................................18
Negative Regulation of TLR3 Signaling ................................................................................................................20
CLINICAL RELEVANCE ............................................................................................................................................21
CONCLUSIONS AND FUTURE PERSPECTIVES .................................................................................................22
ACKNOWLEDGMENTS .............................................................................................................................................22
REFERENCES ..............................................................................................................................................................22
INTRODUCTION
Antiviral innate immunity depends on different sensor sys-
tems that detect viral-pathogen-associated molecular patterns
(PAMPs) and initiate specific signaling pathways, including
those leading to the activation of the transcription factors
nuclear factor-␬B (NF-␬B) and interferon regulatory factor 3
(IRF3). NF-␬B mediates the production of several proinflam-
matory cytokines and antiapoptotic proteins (86), whereas
IRF3 regulates the expression of beta interferon (IFN-␤).
IFN-␤ itself activates several other genes, including 2⬘-5⬘-oli-
goadenylate synthetases, protein kinase R, Mx GTPase, and
P56, which contribute to an antiviral effect via the inhibition of
protein synthesis and viral replication. Viral double-stranded
RNA (dsRNA) is a PAMP that is recognized by Toll-like
receptor 3 (TLR3) and several cytosolic sensors, such as pro-
tein kinase R, 2⬘-5⬘-oligoadenylate synthetases, and the re-
cently identified RNA helicases RIG-I (retinoic acid-inducible
gene I) and MDA5 (melanoma differentiation-associated gene
5) (26, 43, 70, 113). TLR3 and the RIG-I/MDA5 RNA heli-
cases differ in their cellular localizations, ligand specificities,
and downstream signaling pathways, which suggests that host
cells have multiple defense mechanisms against viral infection.
During viral replication, dsRNA is produced either as an in-
termediate of the replication cycle or as part of the viral RNA
genome (50). Moreover, based on the observation that mac-
rophages lacking the TLR3 adaptor protein TRIF (Toll/inter-
leukin-1 [IL-1] receptor [TIR] domain-containing adaptor-in-
ducing IFN-␤) are more susceptible to vaccinia virus (41), it
has been suggested that DNA viruses might produce RNA
transcripts that engage TLR3. In addition to dsRNA from viral
origin, endogenous dsRNA that is released from dying cells
activates TLR3 (55). Polyriboinosinic:polyribocytidylic acid
[poly(I:C)] is a stable synthetic dsRNA analogue that is fre-
quently used as a TLR3 ligand to mimic viral infection. In
contrast to the recognition of dsRNA by intracellular mole-
cules, TLR3 preferentially recognizes synthetic poly(I:C)
rather than virus-derived dsRNA, suggesting that TLR3 rec-
ognizes a unique dsRNA structure that largely differs from the
one recognized by other dsRNA-binding proteins (77). The
crucial role of TLR3 in poly(I:C) recognition is reflected in
the observation that TLR3-deficient mice show reduced re-
sponses to poly(I:C), resistance to the lethal effect of poly(I:C)
when sensitized with
D-galactosamine, and reduced production
of inflammatory cytokines (4). Poly(I:C) in a cell-associated
form is even more efficient in triggering TLR3 than soluble
dsRNA (67, 93), suggesting that dsRNA from dying cells is
most likely a more potent and physiologically relevant TLR3
ligand than dsRNA from live cells. Many TLR3 effects rely on
cells of the innate immune system that either express TLR3 or
respond to inflammatory mediators that are produced upon
TLR3 signaling. Immune cells that express TLR3 and contrib-
ute to an innate immune response are dendritic cells, macro-
phages, natural killer cells, and mast cells (37, 64, 78, 104).
Recent work demonstrates that TLR3 is also present in cells
that directly participate in the adaptive immune response (100,
109). In this context, TLR3 ligation was shown to directly
increase IFN-␥ production by antigen-primed CD8
⫹
T cells.
Altogether, this indicates that TLR3 is a “danger” receptor
with a pleiotropic potential in innate and adaptive immunity.
PARADOXICAL ROLES OF TLR3 IN
VIRAL PATHOLOGY
The exact role of TLR3 in viral infection is still controversial
(21, 99). Several reports show that TLR3 contributes to the
elimination of specific viruses, but others demonstrate that
* Corresponding author. Mailing address: Department for Molecu-
lar Biomedical Research, VIB, Ghent University, Technologiepark
927, B-9052 Ghent (Zwijnaarde), Belgium. Phone: 32-9-3313770. Fax:
32-9-3313609. E-mail: rudi.beyaert@dmbr.ugent.be.
13

some viruses can benefit from TLR3 stimulation (Table 1).
The general outcome is probably dependent on several factors,
such as the type of virus, the viral load, its mode of infection
(endoplasmic versus cytoplasmic), the cell type that is infected,
and the stage of infection.
TLR3 has been implicated in viral infections of the respira-
tory tract, which constitutively expresses TLR3. Influenza A
virus infection markedly upregulates the pulmonary expression
of TLR3 and causes acute pneumonia (31, 62). Despite higher
influenza A virus production in the lungs of TLR3-deficient
mice than in those of wild-type mice, TLR3-deficient mice
have an unexpected survival advantage. Due to the absence of
TLR3-mediated inflammatory signaling, influenza A virus-in-
fected mice display significantly lower levels of inflammatory
mediators and a lower number of CD8
⫹
T cells that contribute
to the clearance of infected cells from the lung than uninfected
mice (62). These findings demonstrate that although TLR3
moderates virus production in the lung, it also contributes to
the debilitating effects of a detrimental host inflammatory re-
sponse. Respiratory syncytial virus is another virus that aug-
ments TLR3 expression in lung epithelial cells (30, 83). Unlike
the role of TLR3 in influenza A virus infection, TLR3 is not
required for viral clearance but is necessary to maintain the
proper immune environment in the lung to avoid pathological
development of the infection. In the absence of TLR3, respi-
ratory syncytial virus-infected mice produce a significantly in-
creased number of T helper 2 (TH2) cytokines, which cause
mucus overproduction, a pathological feature of respiratory
syncytial virus infection.
Several studies show that TLR3 is also involved in central
nervous system (CNS) diseases. The CNS broadly comprises
two cell types: glial cells and neuronal cells. Glial cells are
further divided into microglial cells, which are CNS-resident
innate immune cells, and macroglial cells, such as astrocytes
and Schwann cells, which have an ectodermal origin. All these
different neuronal cell types have been shown to express TLR3
and initiate signaling upon being triggered with dsRNA or
viruses, such as rabies virus and herpes simplex virus type 1 (23,
48, 49, 60, 81). This is particularly surprising since it shows that
neurons have the intrinsic machinery to initiate inflammatory
and antiviral responses (57, 81). In contrast to the destructive
role of TLR3 in influenza A virus infection of the respiratory
tract, TLR3 was proposed to have a protective function during
influenza A virus-induced encephalopathy. With the latter vi-
rus, a specific loss-of-function missense mutation (F303S) en-
coded by the TLR3 gene was found in one of three patients
with influenza-associated encephalopathy (40). Upon periph-
eral infection of mice with West Nile virus, TLR3-dependent
inflammatory signaling was shown to facilitate viral entry into
the brain, causing lethal encephalitis (105). Once inside the
brain, the immune response leading to encephalitis is indepen-
dent of TLR3, since wild-type and TLR3-compromised mice
are equally susceptible upon intracerebroventricular adminis-
tration of the virus (105). In vitro infection of astrocytes with
Theiler’s murine encephalomyelitis virus leads to the TLR3-
mediated induction of several IRF- and NF-␬B-dependent
chemokines and cytokines (95), suggesting that TLR3 signaling
is responsible for the initial inflammatory cytokine responses
defining the outcome of Theiler’s murine encephalomyelitis
virus-induced encephalitis. How the TLR3 signaling pathway
influences the outcome of Theiler’s murine encephalomyelitis
virus infection in vivo is not known.
TLR3 positively contributes to the immune response to in-
vading encephalomyocarditis virus. Schulz et al. demonstrated
that TLR3 engagement by dsRNA that is released from dying
encephalomyocarditis virus-infected cells leads to the cross-
priming of myeloid dendritic cells, followed by the cross-pre-
sentation and activation of cytotoxic T cells (93). This function
is proposed to be important in the clearance of viruses that
have no tropism for dendritic cells. Apart from the release of
dsRNA, virus-infected cells can release type I IFNs, which also
promote cross-priming (59). More recently, TLR3-deficient
mice were shown to be more susceptible to encephalomyocar-
ditis virus infection and to have a significantly higher viral load
in the heart than wild-type mice (35). Although encephalomyo-
carditis virus-induced expression of several cytokines and che-
mokines was impaired in TLR3-deficient mice, IFN-␤ produc-
tion was not. The latter finding might reflect a redundant role
of TLR3 and other receptors in the signaling toward IFN-␤
production.
Surprisingly, TLR3 has also been implicated in the immu-
nobiology of skeletal muscle. TLR3 is expressed in muscle cells
both in vitro and in vivo and is upregulated by dsRNA and
IFN-␥. Furthermore, TLR3 levels are elevated in muscle bi-
opsy specimens from patients with inflammatory and human
immunodeficiency virus-associated myopathies (92), suggest-
ing a deleterious role for TLR3 in inflammatory muscle dis-
ease.
A detrimental role for TLR3 was established in the viral
etiology of liver and kidney disease. TLR3-deficient mice dem-
onstrate reduced liver disease and increased resistance to le-
TABLE 1. Role of TLR3 in viral infections
Virus
a
Target system,
organ, tissue, and/or
cells expressing
TLR3
Role
of
TLR3
b
Reference(s)
Influenza A virus Respiratory tract ⫺ 62
CNS ⫹ 40
Respiratory syncytial virus Respiratory tract ⫹ 30
Rabies virus CNS (neuronal cells) ⫺?49
Herpes simplex virus 1 CNS (neuronal cells) ⫺? 49, 81
West Nile virus CNS ⫺ 105
Theiler’s murine
encephalomyelitis virus
CNS (astrocytes) ⫹?95
Encephalomyocarditis virus Heart ⫹ 35
Human immunodeficiency
virus
Skeletal muscle ⫺?92
Punta Toro virus Liver ⫺ 28
Hepatitis C virus Liver ⫹/⫺? 63, 73
Kidney ⫺? 110
Herpes simplex virus 2 Female genital tract ⫹? 8, 25, 39
a
Viruses are listed in the order of their appearance in the text.
b
⫹, protective; ⫺, detrimental; ⫹/⫺, protective or detrimental.
14 VERCAMMEN ET AL. CLIN.MICROBIOL.REV.

thal infection with Punta Toro virus, a hepatotropic phlebovi-
rus. The most dramatic difference upon infection of wild-type
and TLR3-deficient mice with Punta Toro virus was the exag-
gerated release of IL-6 found systemically and in the livers of
infected wild-type animals. Although IL-6 is critical to estab-
lishing antiviral defense, excessive IL-6 release is detrimental
to the liver and thus contributes to viral pathogenesis (28).
Hepatitis C virus is a major cause of liver hepatitis, liver cir-
rhosis, and hepatocellular carcinoma (56). Although several
reports describe a role for TLR3 in hepatitis C virus infection,
the physiological function of TLR3 in hepatitis remains un-
clear. Evidence for the physiological relevance of a hepatitis C
virus-TLR3 interaction has come from biopsy specimens of
patients with hepatitis C virus-positive kidney disease, a fre-
quent complication in hepatitis C virus infections (110). TLR3
mRNA expression was significantly increased in hepatitis C
virus-positive glomerulonephritis and was associated with en-
hanced mRNA levels for the chemokines RANTES and mono-
cyte chemotactic protein 1 (MCP1). TLR3 expression on renal
cells may therefore establish a link between viral infections and
glomerular diseases.
TLR3 has also been implicated in the protection against
herpes simplex virus type 2 infection of the female genital tract
(8, 25, 39). In this context, it was shown that cells of the
reproductive tract express functional TLR3 (6, 74) and that
treatment with dsRNA protects against genital herpes infec-
tion in mice (8). Although an interaction between herpes sim-
plex virus type 2 RNA and TLR3 has not yet been shown, it is
very likely that herpes simplex virus produces dsRNA inter-
mediates that trigger TLR3 (38).
Finally, a role for TLR3 in virus-induced tumor formation
has also been proposed. For example, the Moloney murine and
feline leukemia viruses activate NF-␬B via a specific RNA
transcript from their long terminal repeat region, which is
capable of stimulating TLR3. Since the antiapoptotic and
growth-promoting activities of NF-␬B have been implicated in
leukemogenesis, these data suggest a role for TLR3 in the
promotion of tumor formation under certain conditions (1).
Viruses have evolved multiple ways to modulate TLR3 sig-
naling. Different viruses, including hepatitis C virus, influenza
A virus, respiratory syncytial virus, human immunodeficiency
virus, simian immunodeficiency virus, and measles virus, aug-
ment TLR3 expression, which is in some cases associated with
an increase in its membrane localization (30, 85, 102). Since
upregulation of TLR3 sensitizes the cells to subsequent viral or
dsRNA exposure, we hypothesize that this will contribute to
pathological inflammatory signaling if the host does not cope
appropriately with the increased susceptibility resulting from
increased TLR3 expression. Interestingly, some viruses directly
interfere with intracellular signaling leading to NF-␬B or IRF3
activation as a means of escaping the host immune response
(33, 86). For example, the vaccinia virus A52R protein inhibits
TLR3-induced NF-␬B activation by sequestering key signaling
proteins (tumor necrosis factor [TNF] receptor-associated fac-
tor 6 [TRAF6] and IL-1 receptor-associated kinase 2 [IRAK2])
(36). Another vaccinia virus protein, the A46R protein, inhibits
both NF-␬B and IRF3 activation via its interaction with TRIF
and other TLR adaptor proteins (96). Li et al. showed that the
hepatitis C virus nonstructural protein 3/4A (NS3/4A) protease
can cleave the TLR3 adaptor protein TRIF (also known as
TICAM-1 or Lps2), thereby inhibiting the TLR3 signaling
pathway that leads to NF-␬B and IRF3 activation and subse-
quent IFN-␤ production (see also below) (63). On the other
hand, the hepatitis C virus-encoded RNA-dependent RNA
polymerase NS5B induces IFN-␤ production in a TLR3-de-
pendent manner, probably through the synthesis of dsRNA,
using cellular RNA as a template (73). A possible explanation
for the contrasting effects of NS5B and NS3/4A on IFN-␤
production might be the maintenance of a low, nonlethal level
of hepatitis C virus, which may promote distraction of the host
defense system and enable persistent infection.
A FUNCTION FOR TLR3 BEYOND ITS ROLE
IN VIRAL INFECTION?
In addition to mediating an antiviral host immune response,
TLR3 has been implicated in the protection of immune-privi-
leged sites, including the CNS and the liver. Bsibsi et al.
showed that TLR3 is induced on human astrocytes upon in-
flammation and, when activated, mediates a comprehensive
neuroprotective response rather than a polarized proinflam-
matory reaction (13). TLR3 stimulation also suppresses exper-
imental autoimmune encephalomyelitis by inducing endoge-
nous IFN-␤ (103). TLR3 stimulation was also shown to protect
against lipopolysaccharide-induced liver injury by downregu-
lating TLR4 expression on macrophages (51). Rather than
having this hepatoprotective effect, TLR3 causes the break-
down of hepatic tolerance (58), leading to TLR3-mediated
autoimmune liver disease. In this case, the TLR3 triggering of
myeloid dendritic cells and macrophages leads to the produc-
tion of IFN-␣ and TNF, which induce the secretion of CXC
chemokine ligand 9 by hepatocytes or other cells. CXC che-
mokine ligand 9 serves as an effective chemo-attractant for
autoimmune CD8
⫹
T cells into the liver, where they cause
autoimmune liver damage (16, 58). TLR3 expression is also
associated with lupus nephritis, an autoimmune disease affect-
ing the kidney. Exposure to poly(I:C) can aggravate lupus
nephritis, and this is probably mediated through TLR3, which
is present on both antigen-presenting cells and glomerular
mesangial cells (80). Finally, the observation that poly(I:C)-
induced TLR3 signaling results in pancreatic ␤-cell death and
(unlike other PAMPs, such as single-stranded RNA, lipopoly-
saccharide, or peptidoglycan) the development of diabetes in
mice (19, 108) suggests a role for TLR3 in autoimmune dia-
betes.
All together, these data demonstrate that TLR3 is a crucial
“danger” signaling receptor that, through its presence on both
immune and nonimmune cells, is involved in controlling the
delicate balance between tolerance and inflammation on the
one hand and inflammation and disease on the other hand.
Whether viral RNA is responsible for all TLR3-mediated re-
sponses that have been reported to date or whether there is
also a role for cellular RNA or other molecules that function
as endogenous TLR3 ligands remains to be investigated.
TLR3 STRUCTURE, LIGAND BINDING,
AND SPECIFICITY
Like all other members of the TLR family, TLR3 is a type I
transmembrane receptor protein composed of an extracellular
VOL. 21, 2008 PATHOGEN RECOGNITION BY TLR3 15

domain containing multiple leucine-rich repeats (LRRs), a
transmembrane region, and a cytoplasmic tail containing the
conserved TIR domain (Fig. 1). The transmembrane domain
consists of a single ␣-helix spanning the membrane, while the
TIR domain is made up of a five-stranded ␤-sheet surrounded
by five ␣-helices. These two secondary-structure elements are
connected by loops of which the BB loop (connecting ␤-strand
B with ␣-helix B) is described to interact with the TLR adaptor
molecules. This BB loop contains in all TLRs a conserved
proline residue, except in TLR3, where the proline is replaced
with an alanine (Fig. 1). The importance of this residue is
demonstrated by the failure of the TLR3-Ala795His mutant to
bind the adaptor protein TRIF (79). TLR3 is the sole TLR that
interacts directly with TRIF, whereas other TLRs physically
interact with the adaptor proteins MyD88 adaptor-like,
MyD88, and TRIF-related adaptor molecule, an activity which
is probably related to the divergence in the BB loop. In addi-
tion to the conserved BB loop, three particular boxes that are
highly conserved among TLR family members define the TIR
domain and are involved in TLR3 signaling (20, 64).
The extracellular domain of human TLR3, which supports
ligand binding, consists of 23 tandemly arranged LRRs. Sev-
enteen of the 23 LRRs follow the consensus motif of a 24-
residue repeat, consisting of XL2XXL5XL7XXN10XL12XX
XXF20XXL23X, where L represents an obligate hydrophobic
residue of which leucine is most prevalent, F is a conserved
phenylalanine, and N is a conserved asparagine. The remaining
noncanonical LRRs contain insertions that form large, pro-
truding loops in LRR12 and LRR20 (Fig. 1) (11, 15). Recently,
Bell et al. (11) and Choe et al. (15) described independently
from each other the crystal structure of the TLR3 extracellular
domain. Their studies showed that the LRRs adopt a horse-
shoe-shaped solenoid structure that is heavily glycosylated.
One face of the extracellular domain, residing at the convex
(outer) surface, is glycan free and is predicted to accommodate
nucleotide binding due to the presence of many positively
charged residues (15). Alternatively and in agreement with the
assumed binding mechanism for LRR-containing proteins,
Bell and colleagues proposed that the nucleotide binding site
may reside at the concave surface, which harbors four potential
Asn (N)-linked glycosylation sites and has a predominant neg-
ative charge. It has been suggested that glycosylation of the
concave region could provide a mechanism for controlling
dsRNA binding by TLR3 (11). In addition, the large insertions
in LRR12 and LRR20, which are unique to TLR3 and con-
served in all known mammalian orthologues, might also play
an important role in ligand binding (11, 15). More recently,
mutational analysis of TLR3 located the dsRNA binding site
on the glycan-free surface of the extracellular domain at the
position of LRR20 (Fig. 1) (10). Bell and colleagues proposed
a symmetrical model of receptor chain assembly that takes
advantage of the inherent twofold symmetry of dsRNA. In this
model, two TLR3 molecules dimerize in such a way that the
extracellular domain of the second TLR3 rotates by 180° in
order to bind identically to the opposite strand of the RNA
duplex (79). In consideration of these findings together, TLR3
might provide several areas that accommodate ligand binding,
depending on the glycosylation status of the extracellular do-
main and the structural characteristics of the ligand (e.g., mod-
ifications and duplex length). Studies using different lengths of
the preferential TLR3 ligand poly(I:C) indicated that longer
duplexes are more potent inducers of TLR3 signaling (18, 77).
Differences in potency might be proportional to the ability of
different ligands to mediate TLR3 multimerization. In line
with the symmetrical assembly model, long dsRNA helices are
likely to bind several TLR3 molecules in similar manners (10).
Although TLR3 is monomeric in solution, it can readily form
a stable or transient dimer when embedded in the membrane
and undergo ligand-binding-promoted multimerization (10,
11). Two lines of experimental evidence corroborate this hy-
pothesis. Inactive TLR3 mutant molecules block the activity of
wild-type endogenous TLR3, which is a dominant negative
characteristic of di- or multimerization-induced signaling
events (18). Additional evidence was obtained from a myeloid
U937 cell line stably transfected with a chimeric TLR3-CD32
protein. In this case, the cross-linking of CD32 causes a rapid
rise in intracellular calcium levels, which is mediated by the
cytosolic portion of CD32. Treatment with an anti-TLR3 an-
FIG. 1. Schematic structure of human TLR3. TLR3 is a type I
integral membrane protein of 904 amino acids. The TLR3 extracellular
domain is a horseshoe-shaped solenoid in which LRR forms one turn
of the solenoid. The LRRs are at the N-terminal and C-terminal
regions, flanked by a cysteine-rich Cap domain. The concave surface is
rich in potential N-glycosylation sites and probably heavily glycosy-
lated. Here we represent two N-glycan structures on Asn247 and
Asn413, two residues which are implicated in glycosylation. LRR12
and LRR20 are atypical LRR motifs containing large insertions which
protrude from the solenoid. According to the symmetrical assembly
model, ligand binding occurs at the glycan-free surface involving
LRR20. The transmembrane domain (TM) is made up of one single
␣-helix. The cytoplasmic domain comprises the cytoplasmic linker re-
gion (CYT) (amino acid [Aa] 730 to Aa756) and the TIR domain, from
which the adaptor-binding BB loop protrudes. Ala795 is a conserved
residue residing at the top of the BB loop and is involved in the binding
of TRIF. The three conserved boxes that define the TIR domain are
also indicated.
16 VERCAMMEN ET AL. CLIN.MICROBIOL.REV.

tibody also induced a calcium flux but only when TLR3 was
cross-linked by a secondary antibody, confirming that multi-
merization is required for signaling (18). The overall dimen-
sions of TLR3, based on crystal structure, roughly match the
shape of a CD14 dimer (15). This suggests that TLR3, like
TLR4, might associate with CD14. This was experimentally
confirmed by Lee and coworkers, who showed that TLR3 can
be coimmunoprecipitated with CD14 independently of poly(I:
C). They also observed the intracellular colocalization of TLR3
and CD14 in multiple compartments, including the endoplas-
mic reticulum and the Golgi apparatus. Once poly(I:C) is in-
ternalized, TLR3 and CD14 colocalize in the endosomal and
lysosomal compartments. CD14 is essential for the uptake of
poly(I:C) in these compartments and enhances TLR3 signaling
(61).
The endosomal and lysosomal localization of TLR3 is
thought to be crucial for providing self- versus non-self-dis-
crimination of dsRNA. Under normal conditions, “self-RNA”
is present in the cytoplasm and cannot enter the membrane-
bound vesicles in which the extracellular domain of TLR3 is
present. When present in cellular debris from dying cells, it
can, however, be taken up and delivered to the endosomes and
elicit a potentially hazardous danger response. The immunos-
timulatory potential of RNA is also modulated by nucleoside
modification (54, 77). In this context, suppression of RNA
recognition is proportional to the number of modified nucleo-
sides present in the RNA (54), explaining why mitochondrial
RNA, which is the least modified fraction of mammalian RNA,
is a better TLR3 ligand than mRNA or total RNA. The TLR3-
stimulatory potential of RNA from dying cells is likely a result
of the presence of this mitochondrial RNA (54, 55, 77). The
U1 small nuclear RNA is also capable of TLR3 activation. U1
small nuclear RNA is the endogenous ligand of the 70-kDa
protein subunit of U1 ribonucleoprotein, which is an auto-
antigen frequently associated with rheumatoid arthritis or sys-
temic lupus erythematosus. The potential involvement of
TLR3 may help account for the prominence of antiribonucleo-
protein responses observed in autoimmune diseases (42). Ad-
ditionally, a specific motif of the endogenous tRNA(A-
la)(UGC) that induces TH1 and cytotoxic-T-lymphocyte
immune responses was shown to be effectively recognized by
TLR3 (107). In addition to nucleoside modification and the
endosomal localization of TLR3, glycosylation of the TLR3
extracellular domain might provide a mechanism for discrim-
inating between host and foreign RNA. Since the mutation of
two potential N-glycosylation sites, Asn247 and Asn413 (Fig.
1), or the use of glycosylation inhibitors abrogates TLR3 sig-
naling (18, 98), we hypothesize that the glycosylation of TLR3
is inhibited in the absence of foreign intruders, thus contrib-
uting to the distinction between host and foreign RNA by
keeping TLR3 in an inactive state in the absence of infection.
For viral RNA sensing in the cytoplasm, discrimination be-
tween self-RNA and viral foreign RNA cannot be explained by
a spatial barrier mechanism, as described above for TLR3. The
RNA helicase RIG-I was recently shown to recognize the 5⬘
ends of certain viral RNA genomes, rather than the dsRNA
structure (113). More specifically, RIG-I recognizes and binds
the 5⬘-triphosphate group of cytoplasmic viral RNA that ap-
pears after viral infection or replication. Such 5⬘-triphosphates
are generally removed from, or masked on, host RNA species,
thereby remaining silent to innate immunity and providing a
structural basis for the distinction between self- and non-self-
RNA. Despite structural and functional similarity between
RIG-I and MDA5, RNA sensing by MDA5 does not involve a
5⬘-triphosphate moiety (e.g., in the case of picornaviruses) but
seems to involve the sensing of a dsRNA structure by a still-
unknown mechanism.
TLR3 LOCALIZATION AND SIGNALING
Localization Holds the Key to TLR3 Activity
In contrast to the viral RNA sensors protein kinase R,
RIG-I, and MDA5, whose localization is exclusively cytoplas-
mic (113), TLR3 has been found in endosomal compartments
or at the cell surface. The localization of TLR3 is cell type
dependent, which may reflect the participation of cell-type-
specific pathways in antiviral IFN induction via TLR3. Human
fibroblasts (e.g., the MRC-5 cell line) express TLR3 on the cell
surface, and anti-TLR3 monoclonal antibodies inhibit dsRNA-
induced IFN-␤ secretion by fibroblasts, suggesting that in these
cells TLR3 acts on the cell surface to sense viral infection.
However, in most cell types, including dendritic cells, macro-
phages, and TLR3-transfected HEK293 cells, TLR3 is de-
tected predominantly in intracellular compartments (24, 65,
66, 75). The cytoplasmic linker region (Fig. 1) contains a se-
quence motif that is important for intracellular targeting of
TLR3, since deleting the linker region interferes with normal
intracellular targeting and causes cell surface expression of
TLR3 (24, 75). In the case of human TLR3, Arg740 and
Val741 residues were identified as crucial determinants for
intracellular expression (24), while in murine TLR3, no crucial
residues were found in the linker region (Glu727 to Asp749).
Yet, this sequence was shown to be sufficient for targeting the
plasma membrane protein CD25 to an intracellular location,
indicating that it is responsible for the intracellular targeting of
TLR3 (75). The intracellular targeting sequence of TLR3 leads
this receptor to the same cytoplasmic membranes where TLR7
is localized and adjacent to phagosomes containing apoptotic
cell particles. The fusion of such phagosomes to TLR3-con-
taining membranes might enhance the access of TLR3 to
dsRNA that is derived from apoptotic cells (75). In addition to
having a targeting function, the cytoplasmic linker region con-
tains, at least in human TLR3, several residues that are re-
quired for TLR3-induced NF-␬B and IFN-␤-promoter activa-
tion (24). Phe732, Tyr733, Leu742, and Gly743 are conserved
across human, mouse, and other species, indicating their im-
portance for TLR3 biology.
Although dendritic cells do not express TLR3 on their sur-
faces, exogenously added dsRNA activates the cells to produce
IFN-␣/␤ and IL-12p70, suggesting that after internalization,
dsRNA encounters intracellular TLR3 present in the subcel-
lular compartments and activates TLR3 signals inside the cells.
Supporting this suggestion is the fact that treatment of den-
dritic cells, peripheral blood mononuclear cells, and TLR3-
transfected HEK293 cells with the lysosome maturation and
acidification inhibitors chloroquine and bafilomycin inhibits
the response to poly(I:C) (18, 24, 65). Moreover, the need for
internalization of poly(I:C) also fits with the fact that long-term
incubation with poly(I:C) is required for IFN-␤ induction in
VOL. 21, 2008 PATHOGEN RECOGNITION BY TLR3 17

dendritic cells but not in fibroblasts expressing cell surface
TLR3 (65). Extracellularly delivered dsRNA is internalized by
clathrin-mediated endocytosis, since a dominant negative ver-
sion of Eps15, an essential scaffolding molecule in clathrin-
mediated coat assembly and endocytosis, impairs dsRNA-in-
duced NF-␬B and IFN-␤ activation (52). The localization of
TLR3 in subcellular compartments of the endocytic trafficking
pathway is also in harmony with the observation that the in-
teraction between TLR3 and dsRNA and subsequent TLR3
signaling require an acidic pH ranging from 5.7 to 6.5 (18).
TRIF Functions as a TLR3 Adaptor Molecule
The TIR domain of TLR3 binds the TIR domain-containing
adaptor protein TRIF, which indirectly activates several tran-
scription factors, including NF-␬B, IRF3, and activating pro-
tein 1 (AP-1). TRIF knockout mice show defective responses
to poly(I:C), indicating that TRIF is essential for TLR3-medi-
ated signaling pathways (79, 112). The mechanisms by which
TRIF activates NF-␬B and IRF3 have been reviewed exten-
sively (71). We will therefore focus mainly on the most recently
identified signaling components of the TLR3/TRIF pathway.
To mediate IRF3 activation, the N-terminal domain of TRIF
was originally proposed to engage with two kinases, I␬B kinase ε
(IKKε) (also known as IKKi, where “i” means “inducible”) and
TANK-binding kinase 1 (TBK1) (also known as T2K or NF-␬B-
activating kinase [NAK]), enabling them to phosphorylate IRF3,
which then forms a dimer that translocates to the nucleus to
induce the expression of IFN-␤ (68). However, it is now thought
that TRIF associates with TBK-IKKε through the adaptor pro-
tein NAK-associated protein 1 (NAP1). RNA interference of
NAP1 results in a failure of poly(I:C)-mediated IRF3 activation
and IFN-␤ production, indicating that NAP1 is a TBK1/IKKε
kinase subunit that participates in TRIF-induced IRF3 activation
(Fig. 2) (90). In addition to NAP1, TRAF3 is part of the TBK/
IKKε kinase complex that coprecipitates with TRIF. Moreover,
TLR3 stimulation no longer induces IFN-␤ in TRAF3-deficient
cells, suggesting that TRAF3 is a critical link between TRIF and
the kinases required for IRF3 activation (Fig. 2) (32, 76). Inter-
estingly, both TRAF3 and NAP1 are also critical in the TLR-
independent RIG-I/MDA5 cytoplasmic signaling pathway lead-
ing to IRF3 activation (76, 90).
For NF-␬B activation, two separate pathways mediated by,
respectively, receptor-interacting protein 1 (RIP1) and TRAF6
seem to bifurcate from TRIF. In murine embryonic fibroblasts
deficient in RIP1, poly(I:C)-induced NF-␬B is completely
blocked in deficient mice, indicating that RIP1 is an essential
mediator of the TRIF pathway leading to NF-␬B activation
(69). The interaction of TRIF and RIP1 is mediated through
the RIP homotypic interaction motif (RHIM) present in both
proteins, in the C-terminal part of TRIF and the intermediary
domain of RIP (Fig. 2). TRAF6 is recruited to the N-terminal
domain of TRIF, but the role of TRAF6 is somewhat contro-
versial and probably cell type specific (27, 91). At least in
mouse embryonic fibroblasts, TRAF6 is recruited to TRIF
along with RIP1, followed by polyubiquitination (Ub) of RIP1
(Fig. 2) (17). In a manner similar to what occurs in the TNF
receptor pathway, Ub RIP1 then recruits the ubiquitin re-
ceptor protein transforming growth factor ␤-activating ki-
nase (TAK) binding protein 2 (TAB2) and TAK1. TAK1
phosphorylates IKK␣ and IKK␤, which in turn phosphory-
late the NF-␬B inhibitor I␬B, eventually leading to its deg-
radation and the nuclear translocation of NF-␬B (Fig. 2).
Poly(I:C)-induced NF-␬B activation, but not IRF3 activa-
tion, is decreased in TAK1-deficient mouse embryonic fi-
broblasts, showing that TAK1 is specifically needed for
TLR3-induced NF-␬B activation (94). TAK1 also activates
the mitogen-activated protein kinases c-jun N-terminal ki-
nase, p38, and extracellular signal-regulated kinase, leading
to the phosphorylation and activation of members of the
AP-1 family of transcription factors.
TRIF is the sole TLR adaptor that is able to engage mam-
malian cell death signaling pathways. TRIF-induced cell death
requires caspase activity initiated by the Fas-associated death
domain protein (FADD)/caspase-8 axis and is unaffected by
inhibitors of the intrinsic mitochondrial apoptotic machinery.
The proapoptotic potential of TRIF maps to the C-terminal
RHIM motif that physically interacts with RIP1. Deletion and
mutational analyses revealed that the RHIM in TRIF is essen-
tial not only for TRIF-induced NF-␬B activation but also for
TRIF-induced apoptosis. Yet the activation of NF-␬B can be
blocked by the superrepressor I␬B␣ without blocking apopto-
sis, indicating that the ability of TRIF to induce apoptosis is
NF-␬B independent (34, 53, 82). All together, these data dem-
onstrate that TLR3 is able to induce apoptosis through a
TRIF/RIP1/FADD/caspase-8-dependent pathway (Fig. 2),
which is supposed to represent an important host defense for
limiting the spread of a viral infection. Interestingly, FADD-
deficient as well as caspase-8-deficient B cells were shown to be
defective in proliferative responses induced by dsRNA. There-
fore, in addition to having an apoptotic function, FADD and
caspase-8 also play a role in TLR3-induced proliferative re-
sponses in B cells (9, 46).
Tyrosine Phosphorylation of TLR3
The tyrosine phosphorylation of TLR2, TLR3, and TLR5
has been shown to play a role in the initiation or regulation of
downstream TLR signaling (7, 47, 87, 89). TLR3 harbors five
tyrosine residues in its cytoplasmic tail, two of which (Tyr759
and Tyr858) were shown to be phosphorylated and to contrib-
ute to the full activation of IRF3 and NF-␬B-dependent gene
expression (Fig. 2) (87–89). The phosphorylation of Tyr858 is
presumably involved in TBK1 activation, which induces the
partial phosphorylation and activation of IRF3, accompanied
by IRF3 dimerization and translocation to the nucleus, but
which still needs a second phosphorylation-dependent signal
from the receptor to promote IRF3-dependent reporter gene
induction (88). In this context, phospho-Tyr759 leads to the
recruitment of phosphatidylinositol-3-kinase (PI3K) and acti-
vation of the downstream kinase Akt, which is required to
obtain the full phosphorylation and activation of IRF3 in the
nucleus. In this two-step model of IRF3 activation, both arms
of phosphorylation, one via TRIF and TBK1 and the other via
PI3K, are thus needed to obtain fully active IRF3 (Fig. 2). A
similar two-step model depending on Tyr858 and Tyr759 was
established for TLR3-mediated NF-␬B activation (87). One
signal leads to the phosphorylation of the inhibitory protein
I␬B, which is followed by the release and nuclear translocation
of NF-␬B. The other signal leads to the phosphorylation of the
18 VERCAMMEN ET AL. CLIN.MICROBIOL.REV.

p65 (also known as RelA) subunit of NF-␬B, leading to its
transactivation (Fig. 2). In this model, the role of TLR3 ty-
rosine phosphorylation has been illustrated for the TLR3-in-
duced expression of A20 mRNA, which is known to be NF-␬B
dependent. Mutation of Tyr759 inhibited A20 gene induction,
although NF-␬B was still activated and translocated to the
nucleus. However, NF-␬B failed to bind to the ␬B site of the
target A20 gene promoter. This defect could be attributed to
FIG. 2. TLR3 signaling pathways. Binding of dsRNA to the TLR3-CD14 complex induces the activation of several intracellular signaling
pathways. The activation of NF-␬B and IRF3 is achieved by two different signaling branches emanating from the TLR3 adaptor molecule TRIF,
which binds to the BB loop of the TLR3 TIR domain. Distinct regions of TRIF bind the ubiquitin ligase TRAF6 and the kinase RIP1. Analogously
with the ubiquitin ligase activity of TRAF2 in the TNF receptor pathway, the activity of TRIF-associated TRAF6 might be responsible for the Ub
of RIP1 in the TLR3 pathway. RIP1 ubiquitination is recognized by the ubiquitin receptor proteins TAB2 and TAB3, leading to the activation of
the kinase TAK1, which is part of the same complex. TAK1 phosphorylates and activates IKK␣ and IKK␤, which are part of a bigger IKK complex
with the IKK adaptor protein IKK␥. IKK␤ is known to be the crucial IKK in TLR signaling and phosphorylates I␬B␣, which binds and keeps
NF-␬B (here depicted as a p65/p50 dimer) in an inactive state in the cytoplasm. I␬B␣ phosphorylation leads to its recognition and degradation by
the proteasome, thus allowing NF-␬B to translocate to the nucleus, where it binds and activates specific gene promoters (e.g., A20). TRIF also
binds TRAF3 and NAP1. Whereas the role of TRAF3 is still largely unclear, NAP1 functions as an adaptor for the IKK-related kinases IKKε and
TBK1, which have largely redundant functions. Both kinases phosphorylate IRF3, leading to its dimerization and translocation to the nucleus,
where it binds and activates specific gene promoters (e.g., IFN-␤). Whereas these TRIF-mediated signaling pathways result in the activation of
NF-␬B and IRF3, the phosphorylation of NF-␬B and IRF3 is involved in acquiring the fully activated status of both transcription factors (see the
text for more details). Signaling leading to these events is still largely unclear, but IRF3 phosphorylation is dependent on the kinase Akt, which
is activated by the lipid kinase PI3K, which binds phospho-Tyr759 of TLR3. Interestingly, PI3K also seems to have an inhibitory function on NF-␬B
activation, whereas the phosphorylation of TLR3 on Tyr858 enhances NF-␬B activation by an unknown mechanism. TLR3 also induces apoptosis
via a TRIF- and RIP1-dependent mechanism. The binding of RIP1 to TRIF not only activates NF-␬B but also recruits the DD-containing adaptor
protein FADD via a homotypic DD-DD interaction. FADD in turn interacts with the cysteine protease procaspase-8 through the death effector
domain (DED) present in both proteins. This is believed to result in the proteolytic auto-activation of procaspase-8 and the initiation of cell death.
CYT, cytoplasmic linker.
V
OL. 21, 2008 PATHOGEN RECOGNITION BY TLR3 19

incomplete phosphorylation of the p65 subunit of NF-␬B (87).
Although PI3K has an essential role in TLR3-induced IRF3
activation, it is dispensable for NF-␬B activation, as illustrated
by the insensitivity of A20 mRNA expression to the PI3K
inhibitor LY294002 (87, 88). In contrast, PI3K has been shown
to impair NF-␬B-dependent proinflammatory signaling by in-
teracting with TRIF and interfering with its ability to channel
optimal NF-␬B, but not IRF3, transcriptional activity (Fig. 2
and 3) (3). Altogether, this indicates that PI3K biases the
TLR3 pathway toward IRF3 and the induction of IFN-stimu-
lated genes while impairing NF-␬B-dependent proinflamma-
tory signaling.
The phosphorylated tyrosine residues of TLR3 can also be
expected to bind SH2 domain-containing proteins other than
PI3K. With respect to this, the tyrosine kinase c-Src was re-
cently shown to bind TLR3 and to be necessary for the TLR3-
induced activation of IRF3 through a TRIF-dependent mech-
anism (52).
In conclusion, to initiate intracellular signaling events, TLR3
utilizes, in addition to its conserved boxes and BB loop, at least
two phospho-acceptor tyrosine residues that recruit PI3K,
c-Src, and most likely other unidentified signaling molecules.
Upon TLR3 engagement, these tyrosine residues are phosphor-
ylated by as-yet-unidentified tyrosine kinases. Whichever route
is followed by the phospho-tyrosine signal, it always converges
with the “classical” TRIF-dependent signaling pathway, lead-
ing to the full activation of IRF3- or NF-␬B-dependent gene
expression.
Negative Regulation of TLR3 Signaling
In the section on viral pathologies, we already discussed viral
immune evasion strategies that target the TLR3 pathway.
However, as described above, strong or sustained TLR3 sig-
naling is also potentially harmful or even fatal for the host cell.
It is thus not surprising that mammalian cells have also evolved
several mechanisms for modulating TLR3-mediated responses
(Fig. 3).
We already described above the negative regulatory effect of
the binding of PI3K with TRIF on TLR3-induced NF-␬B ac-
tivation. Similarly, the kinase RIP3 specifically inhibits TLR3-
induced NF-␬B activation by competing with RIP1 for TRIF
binding (69). Other endogenous negative regulators that inter-
act with TRIF include protein inhibitor of activated signal
transducers and activators of transcription (PIASy), TRAF1,
sterile alpha and TIR motif-containing protein (SARM), A20,
and TRAF4 (12, 14, 84, 97, 101, 106, 115). However, these
proteins inhibit NF-␬B as well as IRF3 activation. PIASy is a
member of the SUMO-ligase family that also interacts with
IRF3 and IRF7. Although this protein inhibits TRIF-induced
NF-␬B and IRF3 activation, it has no effect on TRIF-induced
apoptosis (115). TRAF1 is an inducible protein that binds with
the TIR domain of TRIF and is cleaved by a TRIF-activated
caspase. Because caspase inhibition or the expression of a
noncleavable TRAF1 mutant abolishes the inhibitory effect of
TRAF1, it has been suggested that TRIF-induced cleavage of
TRAF1 is essential for the inhibition of TRIF signaling (97).
U
b
U
b
TRAF6
U
b
U
b
T
LR
3
TIR
TIR
T
R
IF
TR
IF
TI
R
RIP1
P
IKKα
P
IKK
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IK
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β
NF- B ACTIVATIONκ
dsR
NA
RHIM
RH
IM
R
HIM
DD
CD14
CD14
TBK1
IKKε
IRF3
IRF3
P
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IRF3 ACTIVATION
P
IA
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FIG. 3. Endogenous and viral (green) inhibitors of TLR3-mediated NF-␬B or IRF3 activation. Most known inhibitors interfere with the
function of TRIF, either by interacting with TRIF (PIASy, TRAF1, SARM, A20, TRAF4, and the vaccinia virus protein A46R) or by degrading
TRIF (hepatitis C virus protease NS3/4A). Other inhibitors interact with TRAF6 (TRAF4, A20, and vaccinia virus protein A52R), RIP1 (RIP3),
TBK1/IKKε (A20, SIKE, and SHP-2), or IRF3 (PIASy). See the text for more details.
20 VERCAMMEN ET AL. C
LIN.MICROBIOL.REV.

The TIR-containing protein SARM also associates with the
TIR domain of TRIF and is a broad inhibitor of TRIF-induced
cytokine and chemokine production (14). A20 is a deubiquitin-
ating enzyme that is induced by several stimuli, including
dsRNA and Sendai virus infection. A20 has been shown to
coprecipitate with TRIF and to inhibit TLR3-mediated NF-␬B
and IRF3 activation. However, its deubiquitinating activity
does not seem to be required for the inhibition of TRIF sig-
naling (106). Additionally, A20 has been shown to deubiquitin-
ate RIP1, TRAF6, and IKK␥ in the TNF and TLR4 signaling
pathway to NF-␬B (111), suggesting that these signaling proteins
might also be targeted in the TLR3 signaling pathway to NF-␬B.
Furthermore, A20 also coprecipitates with TBK1 and IKKε and
inhibits IRF3 phosphorylation and dimerization following the en-
gagement of TLR3 (84). Finally, TRAF4 is another inducible
protein that also physically interacts with TRIF and TRAF6 and
counteracts their function (101).
In contrast to the above-described inhibitors that inhibit
both NF-␬B and IRF3 activation, suppressor of IKKε (SIKE)
interferes uniquely with TLR3-triggered IRF3 activation. Un-
der physiological conditions, SIKE is associated with TBK1
and dissociates upon TLR3 stimulation. The overexpression of
SIKE disrupts the interactions of IKKε or TBK1 with TRIF
and IRF3, without affecting the interactions of TRIF with
TRAF6 and RIP1. Consistently, the overexpression of SIKE
inhibits virus- and TLR3-triggered IRF3 but not NF-␬B acti-
vation (44).
Due to the need for phospho-tyrosine residues in the TIR
domain of TLR3, one might expect that an alternative way of
interfering with TLR3 signaling is dephosphorylation by a ty-
rosine phosphatase. In this context, SH2-containing protein
tyrosine phosphatase 2 (SHP-2) was recently reported to in-
hibit TLR3-activated IFN-␤ production. However, this seems
to occur by a phosphatase activity-independent mechanism, in
which SHP-2 interacts with the kinase domain of TBK1 to
inhibit its activity (5).
Although it is astounding how many different proteins and
mechanisms have evolved to negatively regulate TLR3 signal-
ing, this complexity also underscores the importance of this
process. The diversity of NF-␬B and IRF inhibitory proteins
may have evolved to establish a redundant system in which one
negative-feedback regulator can compensate for the loss or
failure of others. Moreover, specific regulatory proteins might
change the balance between NF-␬B and IRF3 activation. Most
likely, the role of specific negative regulatory proteins also
depends on the cell type or the cell context. For instance, a
restricted expression pattern could confine the effects of the
inhibitory proteins to specific organs or cells. Conditional
gene-targeting studies of negative regulatory proteins will
surely provide the answer to these unresolved questions in the
near future.
CLINICAL RELEVANCE
As our understanding of innate immunity and TLR biology
has developed, so has an interest in applying that understand-
ing to clinical problems. Vaccine adjuvants are perhaps the
most extensively explored applications for TLR agonists. The
rational design of specific TLR agonists with reduced toxicity
but increased potency, compared to those of adjuvant candi-
dates from only a decade ago, offers the opportunity to meet
the stringent safety criteria required for prophylactic vaccines.
At present, two improved adult hepatitis B virus vaccines and
a papillomavirus vaccine that use TLR4 agonists as the adju-
vant have been approved, and there is considerable research
and early development activity exploring the adjuvant activities
of ligands for most other TLRs. Also, the TLR3 ligand
poly(I:C) has already proven to be beneficial as a mucosal
adjuvant for influenza virus vaccine in a murine infection
model (45). In those studies, coadministration of antigen and
poly(I:C) was shown to upregulate the expression of TLR3 and
IFN-␣/␤. Preclinical studies suggest that TLR3 and other TLR
agonists also have the potential to enhance therapeutic vacci-
nation for cancer. In this context, immunization with the mel-
anoma peptide trp2 and adjuvants consisting of cationic lipo-
somes complexed with TLR3 and TLR9 agonists has been
shown to control the growth of established B16 melanoma
tumors in a therapeutic tumor vaccine model (114). Agonists
for TLR3 (as well as for TLR7, TLR8, and TLR9) have shown
promise as a treatment for viral infections. The synthetic non-
toxic poly(I:C) analog poly(I:C
12
U) (Ampligen) is a mis-
matched dsRNA helix in which cytosine is replaced by uridine,
statistically at each 13th residue (2). It has a rapid half-life
compared to that of poly(I:C), which enabled its development
as a clinically useful drug. Unlike poly(I:C), poly(I:C
12
U) is
specifically recognized by TLR3 but not MDA5, which might
account for its reduced toxicity and safe use in clinical trials in
which it has shown anti-human immunodeficiency virus effects.
Poly(I:C
12
U) has been shown to also have various degrees of
antiviral activity against hepatitis B virus, several flaviviruses,
coxsackie B3 virus, and Punta Toro virus (2, 22, 29). Moreover,
a large phase III clinical trial for the treatment of chronic
fatigue syndrome with Ampligen has successfully been com-
pleted (72).
Sustained TLR3 activation is associated with the overpro-
duction of proinflammatory cytokines and can result in sys-
temic inflammatory response syndrome. In addition, excessive
TLR3 expression or triggering is associated with several in-
flammatory diseases, such as inflammation-associated myopa-
thies, lupus nephritis, West Nile virus-driven CNS inflamma-
tion, and viral or autoimmune liver disease (see above for
more-detailed information). TLR3 antagonists might therefore
be quite promising for a number of infectious and inflamma-
tory diseases. Antagonists for TLR3 and several other TLRs
currently under development are structural analogs of agonists
that bind the receptor but fail to signal. Other possibilities
include anti-TLR antibodies and small-molecule antagonists
selected from compound libraries. In addition to direct thera-
peutic targeting of TLR3 by specific TLR3 antagonists, target-
ing the intracellular TLR3 signaling molecules is becoming a
realistic possibility. This might involve targeting the enzymes
that modulate IRF3 or NF-␬B activation (e.g., TBK1). More-
over, the insights gained into the regions of signaling proteins
involved in protein-protein interactions might allow for the
development of specific agents to disrupt these interactions
and thereby limit their signaling capacity. Altogether, we can
conclude that the manipulation of TLR3 responses harbors
therapeutic value for the treatment of a wide range of diseases,
including both infectious and autoimmune disorders in which
TLR3 has been shown to have a role. Moreover, the list of
VOL. 21, 2008 PATHOGEN RECOGNITION BY TLR3 21

disease states for which one or more TLRs represent a rea-
sonable target is growing rapidly. This will surely continue to
be a productive field for drug development in the future.
CONCLUSIONS AND FUTURE PERSPECTIVES
The past couple of years have witnessed tremendous
progress in our understanding of the molecular mechanisms of
TLR signaling. Here we have summarized current knowledge
regarding the function and regulation of TLR3 as a sentinel for
dsRNA-induced responses. Although it is clear that several
viruses stimulate TLR3-dependent signaling, the importance
of TLR3 signaling in antiviral responses or viral pathogenesis
is still far from clear. In many cases, the lack of a clear effect of
TLR3 deficiency on the outcome of a viral infection in mice is
most likely due to redundancy with other dsRNA sensors, such
as RIG-I and MDA5. The use of better and more physiologi-
cally relevant virus infection models, as well as the use of other
readout systems, might clarify the specific role of each receptor
during a viral infection. Moreover, while TLR3 is well known
to activate inflammatory responses to viruses, evidence that
TLR3 fulfils additional roles in the absence of infection is
growing.
Another complication in our current understanding of TLR3
function is that the optimal and physiological ligand for TLR3
is not yet known. Most studies have been done with synthetic
poly(I:C), but the identities of the viral RNA sequences that
trigger TLR3 are still poorly known and depend on the phys-
iological conditions. In this regard, the potential of endoge-
nous RNA (e.g., from dying cells) in mediating TLR3 signaling
and subsequent inflammation or inflammatory disease needs
further attention. In addition, the possibility of the existence of
other still-unidentified TLR3 ligands different from dsRNA
cannot be excluded.
Although our knowledge of TLR3 signaling is already sub-
stantial, there are as yet many outstanding questions that need
to be addressed. It will be important to clarify the place in the
cell from which the TLR3 signals. Although there is ample
evidence that TLR3 signals from within endosomes, the iden-
tities of these vesicular structures are still unclear. Moreover,
in some cell types, TLR3 might also be triggered from the
outside of the cell membrane. It is not unlikely that signaling
pathways initiated from the cell surface or from an intracellular
location are at least partially different. It should also be noted
that a large part of our current knowledge on TLR3 signaling
and the protein-protein complexes that are involved is still
based on overexpression studies. Although knockout mouse
studies confirmed the essential role of most of these signaling
proteins in TLR3 responses, the exact stoichiometry of the
signaling complexes that are formed encourages their charac-
terization at endogenous expression levels.
In conclusion, although the identification of TLR3 and other
mammalian TLRs has truly revolutionized the field of micro-
bial pathogenesis and human immunology, we are just begin-
ning to understand the complexities of this evolutionarily con-
served system and the essential role that it plays in innate and
adaptive immunity. As the basic understanding of microbially
induced TLR signaling reaches a critical level, novel therapies
that can effectively improve the outcomes of infectious and
other inflammatory diseases may arise.
ACKNOWLEDGMENTS
Work in our laboratory is supported in part by grants from the
Interuniversitaire Attractiepolen (IAP6/18), the Fonds voor Weten-
schappelijk Onderzoek-Vlaanderen (FWO; grant 3G010505), and the
Geconcerteerde Onderzoeksacties of the University of Ghent (GOA;
grant 01G06B6). E.V. was supported as a predoctoral research fellow
by the FWO.
REFERENCES
1. Abujamra, A. L., R. A. Spanjaard, I. Akinsheye, X. Zhao, D. V. Faller, and
S. K. Ghosh. 2006. Leukemia virus long terminal repeat activates NF␬B
pathway by a TLR3-dependent mechanism. Virology 345:390–403.
2. Adis, R. D. P. 2004. Mismatched double-stranded RNA: polyI:polyC12U.
Drugs R D 5:297–304.
3. Aksoy, E., W. Vanden Berghe, S. Detienne, Z. Amraoui, K. A. Fitzgerald, G.
Haegeman, M. Goldman, and F. Willems. 2005. Inhibition of phosphoino-
sitide 3-kinase enhances TRIF-dependent NF-␬B activation and IFN-␤
synthesis downstream of Toll-like receptor 3 and 4. Eur. J. Immunol. 35:
2200–2209.
4. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recog-
nition of double-stranded RNA and activation of NF-␬B by Toll-like re-
ceptor 3. Nature 413:732–738.
5. An, H., W. Zhao, J. Hou, Y. Zhang, Y. Xie, Y. Zheng, H. Xu, C. Qian, J.
Zhou, Y. Yu, S. Liu, G. Feng, and X. Cao. 2006. SHP-2 phosphatase
negatively regulates the TRIF adaptor protein-dependent type I interferon
and proinflammatory cytokine production. Immunity 25:919–928.
6. Andersen, J. M., D. Al-Khairy, and R. R. Ingalls. 2006. Innate immunity at
the mucosal surface: role of Toll-like receptor 3 and Toll-like receptor 9 in
cervical epithelial cell responses to microbial pathogens. Biol. Reprod.
74:824–831.
7. Arbibe, L., J. P. Mira, N. Teusch, L. Kline, M. Guha, N. Mackman, P. J.
Godowski, R. J. Ulevitch, and U. G. Knaus. 2000. Toll-like receptor 2-me-
diated NF-␬B activation requires a Rac1-dependent pathway. Nat. Immu-
nol. 1:533–540.
8. Ashkar, A. A., X. D. Yao, N. Gill, D. Sajic, A. J. Patrick, and K. L.
Rosenthal. 2004. Toll-like receptor (TLR)-3, but not TLR4, agonist pro-
tects against genital herpes infection in the absence of inflammation seen
with CpG DNA. J. Infect. Dis. 190:1841–1849.
9. Beisner, D. R., I. L. Ch’en, R. V. Kolla, A. Hoffmann, and S. M. Hedrick.
2005. Cutting edge: innate immunity conferred by B cells is regulated by
caspase-8. J. Immunol. 175:3469–3473.
10. Bell, J. K., J. Askins, P. R. Hall, D. R. Davies, and D. M. Segal. 2006. The
dsRNA binding site of human Toll-like receptor 3. Proc. Natl. Acad. Sci.
USA 103:8792–8797.
11. Bell, J. K., I. Botos, P. R. Hall, J. Askins, J. Shiloach, D. M. Segal, and D. R.
Davies. 2005. The molecular structure of the Toll-like receptor 3 ligand-
binding domain. Proc. Natl. Acad. Sci. USA 102:10976–10980.
12. Boone, D. L., E. E. Turer, E. G. Lee, R. C. Ahmad, M. T. Wheeler, C.
Tsui, P. Hurley, M. Chien, S. Chai, O. Hitotsumatsu, E. McNally, C.
Pickart, and A. Ma. 2004. The ubiquitin-modifying enzyme A20 is re-
quired for termination of Toll-like receptor responses. Nat. Immunol.
5:1052–1060.
13. Bsibsi, M., C. Persoon-Deen, R. W. Verwer, S. Meeuwsen, R. Ravid, and
J. M. Van Noort. 2006. Toll-like receptor 3 on adult human astrocytes
triggers production of neuroprotective mediators. Glia 53:688–695.
14. Carty, M., R. Goodbody, M. Schroder, J. Stack, P. N. Moynagh, and A. G.
Bowie. 2006. The human adaptor SARM negatively regulates adaptor pro-
tein TRIF-dependent Toll-like receptor signaling. Nat. Immunol. 7:1074–
1081.
15. Choe, J., M. S. Kelker, and I. A. Wilson. 2005. Crystal structure of human
Toll-like receptor 3 (TLR3) ectodomain. Science 309:581–585.
16. Colonna, M. 2006. Toll-like receptors and IFN-␣: partners in autoimmu-
nity. J. Clin. Investig. 116:2319–2322.
17. Cusson-Hermance, N., S. Khurana, T. H. Lee, K. A. Fitzgerald, and M. A.
Kelliher. 2005. Rip1 mediates the Trif-dependent Toll-like receptor 3- and
4-induced NF-␬B activation but does not contribute to interferon regula-
tory factor 3 activation. J. Biol. Chem. 280:36560–36566.
18. de Bouteiller, O., E. Merck, U. A. Hasan, S. Hubac, B. Benguigui, G.
Trinchieri, E. E. Bates, and C. Caux. 2005. Recognition of double-stranded
RNA by human Toll-like receptor 3 and downstream receptor signaling
requires multimerization and an acidic pH. J. Biol. Chem. 280:38133–
38145.
19. Devendra, D., J. Jasinski, E. Melanitou, M. Nakayama, M. Li, B. Hensley,
J. Paronen, H. Moriyama, D. Miao, G. S. Eisenbarth, and E. Liu. 2005.
Interferon-␣ as a mediator of polyinosinic:polycytidylic acid-induced type 1
diabetes. Diabetes 54:2549–2556.
20. Dunne, A., and L. A. O’Neill. 2003. The interleukin-1 receptor/Toll-like
receptor superfamily: signal transduction during inflammation and host
defense. Sci. STKE 2003:re3.
21. Edelmann, K. H., S. Richardson-Burns, L. Alexopoulou, K. L. Tyler, R. A.
22 VERCAMMEN ET AL. CLIN.MICROBIOL.REV.

Flavell, and M. B. Oldstone. 2004. Does Toll-like receptor 3 play a biolog-
ical role in virus infections? Virology 322:231–238.
22. Essey, R. J., B. R. McDougall, and W. E. Robinson. 2001. Mismatched
double-stranded RNA (polyI-polyC12U) is synergistic with multiple anti-
HIV drugs and is active against drug-sensitive and drug-resistant HIV-1 in
vitro. Antivir. Res. 51:189–202.
23. Farina, C., M. Krumbholz, T. Giese, G. Hartmann, F. Aloisi, and E. Meinl.
2005. Preferential expression and function of Toll-like receptor 3 in human
astrocytes. J. Neuroimmunol. 159:12–19.
24. Funami, K., M. Matsumoto, H. Oshiumi, T. Akazawa, A. Yamamoto, and T.
Seya. 2004. The cytoplasmic ‘linker region’ in Toll-like receptor 3 controls
receptor localization and signaling. Int. Immunol. 16:1143–1154.
25. Gill, N., P. M. Deacon, B. Lichty, K. L. Mossman, and A. A. Ashkar. 2006.
Induction of innate immunity against herpes simplex virus type 2 infection
via local delivery of Toll-like receptor ligands correlates with beta inter-
feron production. J. Virol. 80:9943–9950.
26. Gitlin, L., W. Barchet, S. Gilfillan, M. Cella, B. Beutler, R. A. Flavell, M. S.
Diamond, and M. Colonna. 2006. Essential role of mda-5 in type I IFN
responses to polyriboinosinic:polyribocytidylic acid and encephalomyocar-
ditis picornavirus. Proc. Natl. Acad. Sci. USA 103:8459–8464.
27. Gohda, J., T. Matsumura, and J. Inoue. 2004. Cutting edge: TNFR-
associated factor (TRAF) 6 is essential for MyD88-dependent pathway
but not Toll/IL-1 receptor domain-containing adaptor-inducing IFN-␤
(TRIF)-dependent pathway in TLR signaling. J. Immunol. 173:2913–
2917.
28. Gowen, B. B., J. D. Hoopes, M. H. Wong, K. H. Jung, K. C. Isakson, L.
Alexopoulou, R. A. Flavell, and R. W. Sidwell. 2006. TLR3 deletion limits
mortality and disease severity due to Phlebovirus infection. J. Immunol.
177:6301–6307.
29. Gowen, B. B., M.-H. Wong, K.-H. Jung, A. B. Sanders, W. M. Mitchell, L.
Alexopoulou, R. A. Flavell, and R. W. Sidwell. 2007. TLR3 is essential for
the induction of protective immunity against Punta Toro Virus infection by
the double-stranded RNA (dsRNA), poly(I:C12U), but not poly(I:C): dif-
ferential recognition of synthetic dsRNA molecules. J. Immunol. 178:5200–
5208.
30. Groskreutz, D. J., M. M. Monick, L. S. Powers, T. O. Yarovinsky, D. C.
Look, and G. W. Hunninghake. 2006. Respiratory syncytial virus induces
TLR3 protein and protein kinase R, leading to increased double-stranded
RNA responsiveness in airway epithelial cells. J. Immunol. 176:1733–1740.
31. Guillot, L., R. Le Goffic, S. Bloch, N. Escriou, S. Akira, M. Chignard, and
M. Si-Tahar. 2005. Involvement of Toll-like receptor 3 in the immune
response of lung epithelial cells to double-stranded RNA and influenza A
virus. J. Biol. Chem. 280:5571–5580.
32. Hacker, H., V. Redecke, B. Blagoev, I. Kratchmarova, L. C. Hsu, G. G.
Wang, M. P. Kamps, E. Raz, H. Wagner, G. Hacker, M. Mann, and M.
Karin. 2006. Specificity in Toll-like receptor signalling through distinct
effector functions of TRAF3 and TRAF6. Nature 439:204–207.
33. Haller, O., G. Kochs, and F. Weber. 2006. The interferon response circuit:
induction and suppression by pathogenic viruses. Virology 344:119–130.
34. Han, K. J., X. Su, L. G. Xu, L. H. Bin, J. Zhang, and H. B. Shu. 2004.
Mechanisms of the TRIF-induced interferon-stimulated response element
and NF-␬B activation and apoptosis pathways. J. Biol. Chem. 279:15652–
15661.
35. Hardarson, H. S., J. S. Baker, Z. Yang, E. Purevjav, C. H. Huang, L.
Alexopoulou, N. Li, R. A. Flavell, N. E. Bowles, and J. G. Vallejo. 2007.
Toll-like receptor 3 is an essential component of the innate stress response
in virus-induced cardiac injury. Am. J. Physiol. Heart Circ. Physiol. 292:
H251–H258.
36. Harte, M. T., I. R. Haga, G. Maloney, P. Gray, P. C. Reading, N. W.
Bartlett, G. L. Smith, A. Bowie, and L. A. O’Neill. 2003. The poxvirus
protein A52R targets Toll-like receptor signaling complexes to suppress
host defense. J. Exp. Med. 197:343–351.
37. Heinz, S., V. Haehnel, M. Karaghiosoff, L. Schwarzfischer, M. Muller,
S. W. Krause, and M. Rehli. 2003. Species-specific regulation of Toll-like
receptor 3 genes in men and mice. J. Biol. Chem. 278:21502–21509.
38. Herbst-Kralovetz, M., and R. Pyles. 2006. Toll-like receptors, innate im-
munity and HSV pathogenesis. Herpes 13:37–41.
39. Herbst-Kralovetz, M. M., and R. B. Pyles. 2006. Quantification of poly(I:
C)-mediated protection against genital herpes simplex virus type 2 infec-
tion. J. Virol. 80:9988–9997.
40. Hidaka, F., S. Matsuo, T. Muta, K. Takeshige, T. Mizukami, and H. Nunoi.
2006. A missense mutation of the Toll-like receptor 3 gene in a patient with
influenza-associated encephalopathy. Clin. Immunol. 119:188–194.
41. Hoebe, K., X. Du, P. Georgel, E. Janssen, K. Tabeta, S. O. Kim, J. Goode,
P. Lin, N. Mann, S. Mudd, K. Crozat, S. Sovath, J. Han, and B. Beutler.
2003. Identification of Lps2 as a key transducer of MyD88-independent
TIR signalling. Nature 424:743–748.
42. Hoffman, R. W., T. Gazitt, M. F. Foecking, R. A. Ortmann, M. Misfeldt, R.
Jorgenson, S. L. Young, and E. L. Greidinger. 2004. U1 RNA induces
innate immunity signaling. Arthritis Rheum. 50:2891–2896.
43. Hornung, V., J. Ellegast, S. Kim, K. Brzozka, A. Jung, H. Kato, H. Poeck,
S. Akira, K. K. Conzelmann, M. Schlee, S. Endres, and G. Hartmann. 2006.
5⬘-Triphosphate RNA is the ligand for RIG-I. Science 314:994–997.
44. Huang, J., T. Liu, L. G. Xu, D. Chen, Z. Zhai, and H. B. Shu. 2005. SIKE
is an IKKε/TBK1-associated suppressor of TLR3- and virus-triggered
IRF-3 activation pathways. EMBO J. 24:4018–4028.
45. Ichinohe, T., I. Watanabe, S. Ito, H. Fujii, M. Moriyama, S.-I. Tamura,
H. Takahashi, H. Sawa, J. Chiba, T. Kurata, T. Sata, and H. Hasegawa.
2005. Synthetic double-stranded RNA poly(I:C) combined with mucosal
vaccine protects against influenza virus infection. J. Virol. 79:2910–2919.
46. Imtiyaz, H. Z., S. Rosenberg, Y. Zhang, Z. S. Rahman, Y. J. Hou, T.
Manser, and J. Zhang. 2006. The Fas-associated death domain protein is
required in apoptosis and TLR-induced proliferative responses in B cells.
J. Immunol. 176:6852–6861.
47. Ivison, S. M., M. A. Khan, N. R. Graham, C. Q. Bernales, A. Kaleem,
C. O. Tirling, A. Cherkasov, and T. S. Steiner. 2007. A phosphorylation
site in the Toll-like receptor 5 TIR domain is required for inflammatory
signalling in response to flagellin. Biochem. Biophys. Res. Commun.
352:936–941.
48. Jack, C. S., N. Arbour, J. Manusow, V. Montgrain, M. Blain, E. McCrea, A.
Shapiro, and J. P. Antel. 2005. TLR signaling tailors innate immune re-
sponses in human microglia and astrocytes. J. Immunol. 175:4320–4330.
49. Jackson, A. C., J. P. Rossiter, and M. Lafon. 2006. Expression of Toll-like
receptor 3 in the human cerebellar cortex in rabies, herpes simplex enceph-
alitis, and other neurological diseases. J. Neurovirol. 12:229–234.
50. Jacobs, B. L., and J. O. Langland. 1996. When two strands are better than
one: the mediators and modulators of the cellular responses to double-
stranded RNA. Virology 219:339–349.
51. Jiang, W., R. Sun, H. Wei, and Z. Tian. 2005. Toll-like receptor 3 ligand
attenuates LPS-induced liver injury by down-regulation of toll-like re-
ceptor 4 expression on macrophages. Proc. Natl. Acad. Sci. USA 102:
17077–17082.
52. Johnsen, I. B., T. T. Nguyen, M. Ringdal, A. M. Tryggestad, O. Bakke, E.
Lien, T. Espevik, and M. W. Anthonsen. 2006. Toll-like receptor 3 associ-
ates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling.
EMBO J. 25:3335–3346.
53. Kaiser, W. J., and M. K. Offermann. 2005. Apoptosis induced by the
Toll-like receptor adaptor TRIF is dependent on its receptor interacting
protein homotypic interaction motif. J. Immunol. 174:4942–4952.
54. Kariko, K., M. Buckstein, H. Ni, and D. Weissman. 2005. Suppression of
RNA recognition by Toll-like receptors: the impact of nucleoside modifi-
cation and the evolutionary origin of RNA. Immunity 23:165–175.
55. Kariko, K., H. Ni, J. Capodici, M. Lamphier, and D. Weissman. 2004.
mRNA is an endogenous ligand for Toll-like receptor 3. J. Biol. Chem.
279:12542–12550.
56. Kato, N. 2001. Molecular virology of hepatitis C virus. Acta Med. Okayama
55:133–159.
57. Lafon, M., F. Megret, M. Lafage, and C. Prehaud. 2006. The innate im-
mune facet of brain: human neurons express TLR-3 and sense viral dsRNA.
J. Mol. Neurosci. 29:185–194.
58. Lang, K. S., P. Georgiev, M. Recher, A. A. Navarini, A. Bergthaler, M.
Heikenwalder, N. L. Harris, T. Junt, B. Odermatt, P. A. Clavien, H.
Pircher, S. Akira, H. Hengartner, and R. M. Zinkernagel. 2006. Immuno-
privileged status of the liver is controlled by Toll-like receptor 3 signaling.
J. Clin. Investig. 116:2456–2463.
59. Le Bon, A., N. Etchart, C. Rossmann, M. Ashton, S. Hou, D. Gewert, P.
Borrow, and D. F. Tough. 2003. Cross-priming of CD8⫹ T cells stimulated
by virus-induced type I interferon. Nat. Immunol. 4:1009–1015.
60. Lee, H., E. K. Jo, S. Y. Choi, S. B. Oh, K. Park, J. S. Kim, and S. J. Lee.
2006. Necrotic neuronal cells induce inflammatory Schwann cell activation
via TLR2 and TLR3: implication in Wallerian degeneration. Biochem.
Biophys. Res. Commun. 350:742–747.
61. Lee, H. K., S. Dunzendorfer, K. Soldau, and P. S. Tobias. 2006. Double-
stranded RNA-mediated TLR3 activation is enhanced by CD14. Immunity
24:153–163.
62. Le Goffic, R., V. Balloy, M. Lagranderie, L. Alexopoulou, N. Escriou, R.
Flavell, M. Chignard, and M. Si-Tahar. 2006. Detrimental contribution of
the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumo-
nia. PLoS Pathog. 2:e53.
63. Li, K., E. Foy, J. C. Ferreon, M. Nakamura, A. C. Ferreon, M. Ikeda, S. C.
Ray, M. Gale, Jr., and S. M. Lemon. 2005. Immune evasion by hepatitis C
virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adap-
tor protein TRIF. Proc. Natl. Acad. Sci. USA 102:2992–2997.
64. Matsumoto, M., K. Funami, H. Oshiumi, and T. Seya. 2004. Toll-like
receptor 3: a link between toll-like receptor, interferon and viruses. Micro-
biol. Immunol. 48:147–154.
65. Matsumoto, M., K. Funami, M. Tanabe, H. Oshiumi, M. Shingai, Y. Seto,
A. Yamamoto, and T. Seya. 2003. Subcellular localization of Toll-like re-
ceptor 3 in human dendritic cells. J. Immunol. 171:3154–3162.
66. Matsumoto, M., S. Kikkawa, M. Kohase, K. Miyake, and T. Seya. 2002.
Establishment of a monoclonal antibody against human Toll-like receptor
3 that blocks double-stranded RNA-mediated signaling. Biochem. Biophys.
Res. Commun. 293:1364–1369.
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67. McBride, S., K. Hoebe, P. Georgel, and E. Janssen. 2006. Cell-associated
double-stranded RNA enhances antitumor activity through the production
of type I IFN. J. Immunol. 177:6122–6128.
68. McWhirter, S. M., K. A. Fitzgerald, J. Rosains, D. C. Rowe, D. T. Golen-
bock, and T. Maniatis. 2004. IFN-regulatory factor 3-dependent gene ex-
pression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc.
Natl. Acad. Sci. USA 101:233–238.
69. Meylan, E., K. Burns, K. Hofmann, V. Blancheteau, F. Martinon, M.
Kelliher, and J. Tschopp. 2004. RIP1 is an essential mediator of Toll-like
receptor 3-induced NF-␬B activation. Nat. Immunol. 5:503–507.
70. Meylan, E., and J. Tschopp. 2006. Toll-like receptors and RNA helicases:
two parallel ways to trigger antiviral responses. Mol. Cell 22:561–569.
71. Meylan, E., J. Tschopp, and M. Karin. 2006. Intracellular pattern recogni-
tion receptors in the host response. Nature 442:39–44.
72. Mitchell, W. 2006. Review of Ampligen clinical trials in chronic fatigue
syndrome. J. Clin. Virol. 37:S113.
73. Naka, K., H. Dansako, N. Kobayashi, M. Ikeda, and N. Kato. 2006. Hep-
atitis C virus NS5B delays cell cycle progression by inducing interferon-beta
via Toll-like receptor 3 signaling pathway without replicating viral genomes.
Virology 346:348–362.
74. Nasu, K., H. Itoh, A. Yuge, M. Nishida, and H. Narahara. 2007. Human
oviductal epithelial cells express Toll-like receptor 3 and respond to double-
stranded RNA: fallopian tube-specific mucosal immunity against viral in-
fection. Hum. Reprod. 22:356–361.
75. Nishiya, T., E. Kajita, S. Miwa, and A. L. Defranco. 2005. TLR3 and TLR7
are targeted to the same intracellular compartments by distinct regulatory
elements. J. Biol. Chem. 280:37107–37117.
76. Oganesyan, G., S. K. Saha, B. Guo, J. Q. He, A. Shahangian, B. Zarnegar,
A. Perry, and G. Cheng. 2006. Critical role of TRAF3 in the Toll-like
receptor-dependent and -independent antiviral response. Nature 439:208–
211.
77. Okahira, S., F. Nishikawa, S. Nishikawa, T. Akazawa, T. Seya, and M.
Matsumoto. 2005. Interferon-beta induction through Toll-like receptor 3
depends on double-stranded RNA structure. DNA Cell Biol. 24:614–623.
78. Orinska, Z., E. Bulanova, V. Budagian, M. Metz, M. Maurer, and S.
Bulfone-Paus. 2005. TLR3-induced activation of mast cells modulates
CD8⫹ T-cell recruitment. Blood 106:978–987.
79. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, and T. Seya. 2003.
TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-me-
diated interferon-beta induction. Nat. Immunol. 4:161–167.
80. Patole, P. S., H. J. Grone, S. Segerer, R. Ciubar, E. Belemezova, A. Henger,
M. Kretzler, D. Schlondorff, and H. J. Anders. 2005. Viral double-stranded
RNA aggravates lupus nephritis through Toll-like receptor 3 on glomerular
mesangial cells and antigen-presenting cells. J. Am. Soc. Nephrol. 16:1326–
1338.
81. Pre´haud, C., F. Me´gret, M. Lafage, and M. Lafon. 2005. Virus infection
switches TLR-3-positive human neurons to become strong producers of
beta interferon. J. Virol. 79:12893–12904.
82. Ruckdeschel, K., G. Pfaffinger, R. Haase, A. Sing, H. Weighardt, G. Hacker,
B. Holzmann, and J. Heesemann. 2004. Signaling of apoptosis through
TLRs critically involves Toll/IL-1 receptor domain-containing adapter in-
ducing IFN-␤, but not MyD88, in bacteria-infected murine macrophages.
J. Immunol. 173:3320–3328.
83. Rudd, B. D., J. J. Smit, R. A. Flavell, L. Alexopoulou, M. A. Schaller, A.
Gruber, A. A. Berlin, and N. W. Lukacs. 2006. Deletion of TLR3 alters the
pulmonary immune environment and mucus production during respiratory
syncytial virus infection. J. Immunol. 176:1937–1942.
84. Saitoh, T., M. Yamamoto, M. Miyagishi, K. Taira, M. Nakanishi, T. Fujita,
S. Akira, N. Yamamoto, and S. Yamaoka. 2005. A20 is a negative regulator
of IFN regulatory factor 3 signaling. J. Immunol. 174:1507–1512.
85. Sanghavi, S. K., and T. A. Reinhart. 2005. Increased expression of TLR3 in
lymph nodes during simian immunodeficiency virus infection: implications
for inflammation and immunodeficiency. J. Immunol. 175:5314–5323.
86. Santoro, M. G., A. Rossi, and C. Amici. 2003. NF-␬B and virus infection:
who controls whom. EMBO J. 22:2552–2560.
87. Sarkar, S. N., C. P. Elco, K. L. Peters, S. Chattopadhyay, and G. C. Sen.
2007. Two tyrosine residues of Toll-like receptor 3 trigger different steps of
NF-␬B activation. J. Biol. Chem. 282:3423–3427.
88. Sarkar, S. N., K. L. Peters, C. P. Elco, S. Sakamoto, S. Pal, and G. C. Sen.
2004. Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in
double-stranded RNA signaling. Nat. Struct. Mol. Biol. 11:1060–1067.
89. Sarkar, S. N., H. L. Smith, T. M. Rowe, and G. C. Sen. 2003. Double-
stranded RNA signaling by Toll-like receptor 3 requires specific tyrosine
residues in its cytoplasmic domain. J. Biol. Chem. 278:4393–4396.
90. Sasai, M., H. Oshiumi, M. Matsumoto, N. Inoue, F. Fujita, M. Nakanishi,
and T. Seya. 2005. Cutting edge: NF-␬B-activating kinase-associated pro-
tein 1 participates in TLR3/Toll-IL-1 homology domain-containing adapter
molecule-1-mediated IFN regulatory factor 3 activation. J. Immunol. 174:
27–30.
91. Sato, S., M. Sugiyama, M. Yamamoto, Y. Watanabe, T. Kawai, K. Takeda,
and S. Akira. 2003. Toll/IL-1 receptor domain-containing adaptor inducing
IFN-␤ (TRIF) associates with TNF receptor-associated factor 6 and
TANK-binding kinase 1, and activates two distinct transcription factors,
NF-␬B and IFN-regulatory factor-3, in the Toll-like receptor signaling.
J. Immunol. 171:4304–4310.
92. Schreiner, B., J. Voss, J. Wischhusen, Y. Dombrowski, A. Steinle, H. Loch-
muller, M. Dalakas, A. Melms, and H. Wiendl. 2006. Expression of toll-like
receptors by human muscle cells in vitro and in vivo: TLR3 is highly
expressed in inflammatory and HIV myopathies, mediates IL-8 release and
up-regulation of NKG2D-ligands. FASEB J. 20:118–120.
93. Schulz, O., S. S. Diebold, M. Chen, T. I. Naslund, M. A. Nolte, L. Alexo-
poulou, Y. T. Azuma, R. A. Flavell, P. Liljestrom, and C. Reis e Sousa. 2005.
Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature
433:887–892.
94. Shim, J. H., C. Xiao, A. E. Paschal, S. T. Bailey, P. Rao, M. S. Hayden,
K. Y. Lee, C. Bussey, M. Steckel, N. Tanaka, G. Yamada, S. Akira, K.
Matsumoto, and S. Ghosh. 2005. TAK1, but not TAB1 or TAB2, plays
an essential role in multiple signaling pathways in vivo. Genes Dev.
19:2668–2681.
95. So, E. Y., M. H. Kang, and B. S. Kim. 2006. Induction of chemokine and
cytokine genes in astrocytes following infection with Theiler’s murine en-
cephalomyelitis virus is mediated by the Toll-like receptor 3. Glia 53:858–
867.
96. Stack, J., I. R. Haga, M. Schroder, N. W. Bartlett, G. Maloney, P. C.
Reading, K. A. Fitzgerald, G. L. Smith, and A. G. Bowie. 2005. Vaccinia
virus protein A46R targets multiple Toll-like-interleukin-1 receptor adap-
tors and contributes to virulence. J. Exp. Med. 201:1007–1018.
97. Su, X., S. Li, M. Meng, W. Qian, W. Xie, D. Chen, Z. Zhai, and H. B. Shu.
2006. TNF receptor-associated factor-1 (TRAF1) negatively regulates Toll/
IL-1 receptor domain-containing adaptor inducing IFN-␤ (TRIF)-medi-
ated signaling. Eur. J. Immunol. 36:199–206.
98. Sun, J., K. E. Duffy, C. T. Ranjith-Kumar, J. Xiong, R. J. Lamb, J. Santos,
H. Masarapu, M. Cunningham, A. Holzenburg, R. T. Sarisky, M. L. Mbow,
and C. Kao. 2006. Structural and functional analyses of the human Toll-like
receptor 3: role of glycosylation. J. Biol. Chem. 281:11144–11151.
99. Tabeta, K., P. Georgel, E. Janssen, X. Du, K. Hoebe, K. Crozat, S. Mudd,
L. Shamel, S. Sovath, J. Goode, L. Alexopoulou, R. A. Flavell, and B.
Beutler. 2004. Toll-like receptors 9 and 3 as essential components of innate
immune defense against mouse cytomegalovirus infection. Proc. Natl.
Acad. Sci. USA 101:3516–3521.
100. Tabiasco, J., E. Devevre, N. Rufer, B. Salaun, J. C. Cerottini, D. Speiser,
and P. Romero. 2006. Human effector CD8⫹ T lymphocytes express TLR3
as a functional coreceptor. J. Immunol. 177:8708–8713.
101. Takeshita, F., K. J. Ishii, K. Kobiyama, Y. Kojima, C. Coban, S. Sasaki, N.
Ishii, D. M. Klinman, K. Okuda, S. Akira, and K. Suzuki. 2005. TRAF4
acts as a silencer in TLR-mediated signaling through the association with
TRAF6 and TRIF. Eur. J. Immunol. 35:2477–2485.
102. Tanabe, M., M. Kurita-Taniguchi, K. Takeuchi, M. Takeda, M. Ayata, H.
Ogura, M. Matsumoto, and T. Seya. 2003. Mechanism of up-regulation of
human Toll-like receptor 3 secondary to infection of measles virus-attenu-
ated strains. Biochem. Biophys. Res. Commun. 311:39–48.
103. Touil, T., D. Fitzgerald, G. X. Zhang, A. Rostami, and B. Gran. 2006.
Cutting edge: TLR3 stimulation suppresses experimental autoimmune en-
cephalomyelitis by inducing endogenous IFN-␤. J. Immunol. 177:7505–
7509.
104. Wang, J., R. Sun, H. Wei, Z. Dong, B. Gao, and Z. Tian. 2006. Poly I:C
prevents T cell-mediated hepatitis via an NK-dependent mechanism.
J. Hepatol. 44:446–454.
105. Wang, T., T. Town, L. Alexopoulou, J. F. Anderson, E. Fikrig, and R. A.
Flavell. 2004. Toll-like receptor 3 mediates West Nile virus entry into the
brain causing lethal encephalitis. Nat. Med. 10:1366–1373.
106. Wang, Y. Y., L. Li, K. J. Han, Z. Zhai, and H. B. Shu. 2004. A20 is a potent
inhibitor of TLR3- and Sendai virus-induced activation of NF-␬B and ISRE
and IFN-␤ promoter. FEBS Lett. 576:86–90.
107. Wang, Z., L. Xiang, J. Shao, and Z. Yuan. 2006. The 3⬘ CCACCA sequence
of tRNAAla(UGC) is the motif that is important in inducing Th1-like
immune response, and this motif can be recognized by Toll-like receptor 3.
Clin. Vaccine Immunol. 13:733–739.
108. Wen, L., J. Peng, Z. Li, and F. S. Wong. 2004. The effect of innate immunity
on autoimmune diabetes and the expression of Toll-like receptors on pan-
creatic islets. J. Immunol. 172:3173–3180.
109. Wesch, D., S. Beetz, H. H. Oberg, M. Marget, K. Krengel, and D. Kabelitz.
2006. Direct costimulatory effect of TLR3 ligand poly(I:C) on human ␥␦ T
lymphocytes. J. Immunol. 176:1348–1354.
110. Wornle, M., H. Schmid, B. Banas, M. Merkle, A. Henger, M. Roeder, S.
Blattner, E. Bock, M. Kretzler, H. J. Grone, and D. Schlondorff. 2006.
Novel role of Toll-like receptor 3 in hepatitis C-associated glomerulone-
phritis. Am. J. Pathol. 168:370–385.
111. Wullaert, A., K. Heyninck, S. Janssens, and R. Beyaert. 2006. Ubiquitin:
tool and target for intracellular NF-␬B inhibitors. Trends Immunol. 27:533–
540.
112. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O.
24 VERCAMMEN ET AL. CLIN.MICROBIOL.REV.

Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, and S. Akira. 2003. Role of
adaptor TRIF in the MyD88-independent Toll-like receptor signaling path-
way. Science 301:640–643.
113. Yoneyama, M., and T. Fujita. 2007. Function of RIG-I-like receptors in
antiviral innate immunity. J. Biol. Chem. 282:15315–15318.
114. Zaks, K., M. Jordan, A. Guth, K. Sellins, R. Kedl, A. Izzo, C. Bosio, and S.
Dow. 2006. Efficient immunization and cross-priming by vaccine adjuvants
containing TLR3 or TLR9 agonists complexed to cationic liposomes. J. Im-
munol. 176:7335–7345.
115. Zhang, J., L. G. Xu, K. J. Han, X. Wei, and H. B. Shu. 2004. PIASy
represses TRIF-induced ISRE and NF-␬B activation but not apoptosis.
FEBS Lett. 570:97–101.
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