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History of Discovery
Microbial Sensing by Toll Receptors
A Historical Perspective
Stanislava Chtarbanova, Jean-Luc Imler
Abstract—The family of Toll-like receptors plays an essential role in the induction of the immune response. These
receptors sense the presence of microbial ligands and activate the nuclear factor-␬B transcription factor. We review the
key studies that led from the formulation of the concept of pattern recognition receptors to the characterization of
Toll-like receptors, insisting on the important role played by the model organism Drosophila melanogaster and on the
increasing evidence connecting these receptors to cardiovascular disease. (Arterioscler Thromb Vasc Biol. 2011;31:
1734-1738.)
Key Words: immune system 䡲 macrophages 䡲 receptors 䡲 viruses
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T
of the antigen receptor with its specific ligand, lymphocytes
require a second signal, coming from the antigen-presenting
cells, to be activated. Because the pattern recognition receptors (PRRs) recognizing these microbial molecules on the
antigen-presenting cells had to be limited in number and
encoded in the germline (as opposed to the rearranging
antigen receptors of the adaptive immune response), he
postulated that these molecules should obey the following 3
rules: (1) they are absent from the host, allowing discrimination between self and nonself; (2) they are conserved among
large numbers of microorganisms, allowing recognition of a
wide array of microorganisms by a limited number of
receptors; and (3) they are essential constituents of the
microorganisms, thus preventing escape from recognition by
the innate immune system through mutation. For example,
the outer membrane of all Gram-negative bacteria is composed of lipopolysaccharides (LPS), whereas most viruses
produce double-stranded RNA molecules in infected cells.
Peptidoglycan, bacterial lipopeptides, and unmethylated CpG
DNA motifs are other examples of microbial molecular
patterns, sometimes called pathogen-associated molecular
patterns, although they are not solely expressed by pathogens.
Most of these molecules were known at the time to induce
signaling cascades activating the transcription factor nuclear
factor-␬B (NF-␬B) and leading to inflammation, but the
nature of their receptors remained unknown.2 The fruitless
search for PRRs led to an alternative model in the mid-1990s,
heralded by P. Matzinger; this model postulated that danger
signals, rather than nonself, was the main driving force for the
immune system.3
Innate immunity is present in both vertebrates and invertebrates, raising the possibility that investigation of host-
oll-like receptors (TLRs) owe their names to a famous
Drosophila gene, Toll. The name comes from the vernacular German Toll, meaning super or fantastic. It was used
in the early 1980s by C. Nüsslein-Volhard to qualify the
phenotype of a new mutant discovered in her pioneering
mutagenesis screen to dissect the genetic pathways controlling embryonic development in the fruit fly Drosophila
melanogaster. The Toll gene is involved in the differentiation
of the dorso-ventral axis of the embryo; some mutant alleles
(loss-of-function) cause dorsalization of the embryo, whereas
others (gain-of-function) cause a ventralization.1 It took
several years to realize that this receptor also has important
immune functions in adult flies and that its orthologues in
mammals play a key role in innate immunity (Figure 1).
Innate immunity is the first-line host defense mechanism
that operates to protect animals (vertebrates and invertebrates) from infectious microorganisms. As such, it plays a
critical role in containing most infections. In addition, molecules induced during the innate immune response, including
cytokines and costimulatory molecules, play a critical role in
the induction of the adaptive immune response in mammals.
C. Janeway was among the first to stress the importance of
this point and to recognize that the immune response to
infectious microbes involves “not only specific antigen determinants, but also certain characteristics or patterns common on infectious agents but absent from the host.”2
Pattern Recognition Receptors: From the
Concept to the Discovery of TLRs
In his concluding lecture of the 1989 Cold Spring Harbor
meeting on immune recognition, Janeway insisted on the fact
that, in addition to the first signal delivered by the interaction
Received on: March 10, 2011; final version accepted on: April 7, 2011.
From the CNRS-UPR9022, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France (S.C., J.-L.I.); Faculté des Sciences de la Vie, Université
de Strasbourg, Strasbourg, France (J.-L.I.).
Correspondence to Jean-Luc Imler, CNRS-UPR9022, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg cedex,
France. E-mail [email protected]
© 2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1734
DOI: 10.1161/ATVBAHA.108.179523
Chtarbanova and Imler
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1981
Discovery of the first antimicrobial peptide, Cecropin in the moth
Hyalophora cecropia
1984
Characterization of the Toll mutation responsible for the dorso-ventral
patterning in Drosophila
1988
Cloning of theToll gene
1989
Concept of Pattern Recognition Receptors (PRR)
1990
Cloning of Cecropin A and Diptericin genes in Drosophila
1991
Identification of NF-κB binding motifs in the promotors of Drosophila
Cecropin and Diptericin genes
1993
Presentation of the "danger" model
1994
Identification of the antifungal peptide Drosomycin in Drosophila
1996
Toll regulates Drosomycin expression and resistance to fungal infections
1997
Identification of a human Toll orthologue regulating NF-κB
1998
Identification of TLR-4 as the receptor of LPS
1999-2004
TLR-4 deficiency is associated with a significant reduction of atherosclerosis in apoE deficient mice
2006
The serine protease Psh senses virulence factors upstream of Toll in
Drosophila
2007
Identification of TLR-3 mutation in humans leading to Herpes simplex
encephalitis
2010
1735
Figure 1. Important milestones in the discovery of
Toll receptors.
Characterization of the family of Toll-like receptors and identification of
ligands for TLR-1,2,3,5,6,7,8,9
2004
2007-2009
History of Discovery: Toll and TLRs
3D structure of TLR-ligand complexes
Atherogenic lipids trigger TLR-2-dependant macrophage apoptosis and
plaque necrosis
defense mechanisms in model organisms prone to genetic
analysis, such as the fruit fly Drosophila, may shed light on
the nature of the elusive PRRs. Infection of insects leads to
the secretion in the hemolymph (insect blood) of a family of
potent cationic antimicrobial peptides. Initially characterized
by H. Boman and his colleagues in 1981 in the moth
Hyalophora cecropia (which gave its name to cecropin, the
first identified antimicrobial peptide),4 these peptides were
subsequently shown to also participate to host-defense in
plants and mammals (eg, defensins, cathelicidins). The analysis of the inducible promoters of the genes encoding these
peptides, performed in parallel on cecropin by Boman and his
collaborators, and diptericin, a novel peptide identified in
dipteran insects (the group to which Drosophila belongs) by
J. Hoffmann et al,5 revealed the importance of DNA motifs
strongly similar to the binding sites for the mammalian
transcription factors of the NF-␬B family. This striking result,
which was the first indication that evolutionary conserved
mechanisms may be involved in the control of innate immunity in mammals and insects and that Drosophila may be a
useful model to define molecularly the PRRs, led to a Human
Frontier Science Program–sponsored collaboration involving
in particular the laboratories of Hoffmann at Strasbourg and
Janeway at Yale.5
At the time, the only known NF-␬B gene in Drosophila
was dorsal, which encodes the transcription factor activated
by Toll on the ventral side of the embryo. However, induction
of diptericin was not affected in dorsal mutant flies or in
other mutants of the Toll pathway. A different picture
emerged when a new marker was included in this analysis,
the gene encoding the antifungal peptide Drosomycin, discovered in 1994.6 Indeed, genetic analysis revealed that the
induction of the drosomycin gene is controlled by a transcription factor closely related to Dorsal, Dorsal-related immunity
factor, whose activity is induced by the Toll pathway. A third
Drosophila NF-␬B family member, Relish, controls expression of diptericin on activation by the immune deficiency
pathway (reviewed by Hoffmann and Reichhart7).
Toll mutant flies, which fail to induce expression of
Drosomycin, are highly susceptible to fungal infections and
1736
Arterioscler Thromb Vasc Biol
August 2011
Drosophila
Mammals
Lys-type ß-Glucans Proteases
PGN
Endosome
lumen
Pathogen Associated Molecular Patterns (PAMPs)
ss
ds
lin
S
A
el
RN
ag
LP
RN
Fl
ifs
ot
m A
G DN
Cp ds
A
Psh
d
te es
yla tid
ac p
Di ope
lip ed
t
la es
cy tid
ia p
Tr ope
lip
Extracellular PGRP-SA GNBP-3
PGRP-SD
space
GNBP-1
Spaetzle
(Cytokine)
Plasma
membrane
Toll
Cytosol
TLR1/2 TLR2/6 TLR4 TLR5
TLR3 TLR7 TLR9
DmMyD88
MyD88/TIRAP/TRIF/TRAM
Tube/Pelle
Cactus
Dif
IRAK-1/IRAK-4
IκB
NFκB
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Nuclear
enveloppe
NFκB
Dif
Antimicrobial peptides
genes
Figure 2. Schematic representation of
Toll/TLR pathways in Drosophila and
mammals. Toll and TLRs activate an evolutionary conserved signaling pathway
involving the Toll/interleukin-1R domain
adapters MyD88 and DmMyD88, the DD
kinases IRAK and Pelle, the inhibitors I␬B
and Cactus, and the Rel family transcription factors NF-␬B and Dif. Mammalian
TLRs are activated on direct binding of
microbial molecular patterns, whereas
Drosophila Toll is activated by the cytokine Spaetzle. Detection of microbial
molecular patterns or virulence factors by
different sets of sensors in Drosophila activates a proteolytic cascade, leading to
Spaetzle activation. See the text for
details. PGN indicates peptidoglycan;
dsRNA, double-stranded RNA; ssRNA,
single-stranded RNA; Dif, Dorsal-related
immunity factor.
Inflammatory cytokines
genes
succumb rapidly to a challenge with the opportunistic fungus
Aspergillus fumigatus.8 When these results were discussed in
the framework of the collaboration between the Strasbourg
and Yale laboratories, they prompted the search for orthologues of Toll in mammals, and the first human Toll was
described in 1997. The scope of that report was limited to the
induction of cytokines and the costimulatory molecule CD80,
which are required to activate naïve T cells, by a constitutively active mutant receptor.9 The crucial data showing that
TLRs carry important immune functions in mammals and
connecting them to the sensing of microbial ligands were
obtained by Bruce Beutler and colleagues, who showed that
mice of the strains C3H/HeJ and C57BL/10ScCr, which do
not sense LPS, carry mutations in the gene encoding TLR4.10
Generation and phenotypic characterization of knockout mutant mice for TLR2 and TLR4 in the group of S. Akira
confirmed a year later that TLRs can mediate induction of
NF-␬B in mammalian cells in response to different sets of
microbial molecules.11 This set the stage for an in-depth
analysis of the role of TLRs in the sensing of infections.
Structure and Function of TLRs
We now know that TLR activation can occur at the cell
surface or in endosomes. At the plasma membrane, TLR2
associates with TLR6 or TLR1 to sense either di- or triacylated lipopeptides, respectively. TLR5 and TLR11 (the latter
present in mice only) sense microbial proteins. TLR5 is a
receptor for flagellin, recognizing a peptide motif essential
for the buildup of the bacterial flagellum, thus explaining why
it is difficult for the bacteria to change this sequence to avoid
recognition. In the endosomal compartment, other TLRs
sense internalized nucleic acids: TLR3 interacts with doublestranded RNA, TLR7 and 8 recognize single-stranded RNAs
enriched in U residues, and TLR9 detects unmethylated CpG
DNA motifs (reviewed by Kawai and Akira12) (Figure 2).
These TLRs play essential roles in the sensing of viral
infections, as illustrated by the development of herpes simplex virus-1 encephalitis in children with TLR3 deficiency.13
Some TLRs need coreceptors to accommodate their ligands,
such as the lipid-binding molecule MD2 and the leucine-rich
repeat protein CD14 for TLR4 and the scavenger receptor
CD36 for TLR2. The TLRs are therefore bona fide PRRs,
interacting with a diverse set of microbial ligands and
signaling to trigger innate immunity.12
How TLRs can accommodate a range of ligands, from
nucleic acids to proteins and lipids, has long been a mystery.
Indeed, TLRs all share the same domain organization, with an
ectodomain composed of leucine-rich repeats and a 150amino-acid intracytoplasmic domain homologous to that of
the interleukin-1 receptor (Toll/interleukin-1 receptor domain). Like all leucine-rich repeat proteins, the ectodomain of
TLRs forms characteristic horseshoe-shaped structures. Interestingly, recent structural analysis of TLRs complexed with
their ligands revealed a remarkable versatility of ligand
interaction between different members of the family. In the
case of TLR1 and TLR2, the acyl chains of lipopeptides bind
to hydrophobic pockets located on the convex side of the
horseshoe in the 2 subunits, thus cross-linking them. In the
case of TLR4, the ligand is not LPS itself but a complex of
LPS and MD2, which associates with the concave surface
provided by the N terminus and the central region of the
TLR4 ectodomain. Finally, in the case of TLR3, the doublestranded RNA helix interacts with 2 distinct sites near the N
terminus and the C terminus of the ectodomain. Although the
emerging picture is that TLRs interact with different ligands
in highly divergent ways, in all cases ligand binding induces
Chtarbanova and Imler
the C termini of the ectodomains to move close to each other,
thus initiating signaling and triggering inflammation (reviewed by Jin and Lee14).
Activation of Innate Immunity: Danger
Versus Nonself Pattern
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Twenty years after the introduction of the PRR concept, the
discovery and characterization of not only TLRs but also the
Nod-like receptors, RIG-I-like receptors, C-type lectin receptors, peptidoglycan recognition proteins, and ␤-Glucan binding proteins have validated the concept and demonstrated that
nonself recognition plays an important role in the activation
of the innate immune response.15,16 However, the alternative
“danger model” is still on the agenda. Indeed, the Toll
pathway in Drosophila is not solely activated by microbial
molecular patterns but can also be activated on sensing
virulence factors. The circulating serine protease Persephone
senses the presence of abnormal proteolytic activities secreted by invading fungi or bacteria and triggers the processing and activation of the Toll ligand Spaetzle independently
of the PRRs.17,18
In mammals, several reports indicate that TLRs can be
activated by endogenous molecules, generated as a result of
cell death or tissue damage. These include oxidized lipoproteins, extracellular matrix components (eg, hyaluronic acid
fragments, versican), or heat-shock proteins (Kawai and
Akira12 and references therein). Of note, the interaction of
TLRs with these endogenous agonists has not yet been
structurally characterized. A complicating issue for these
studies is that because of the exquisite sensitivity of TLRs,
even the slightest contamination with microbial ligands (such
as can occur for LPS and recombinant proteins), can bias the
experiments.19 Self nucleic acids can also be released from dying
cells and activate TLR7 and TLR9, thus triggering type I
interferon production. Interferons then drive the expansion of B
cell clones with specificity for nucleic acids and lead to autoimmune diseases, such as systemic lupus erythematosus.20
The discovery that TLR signaling can be induced by
endogenous molecules opens exciting perspectives for the
treatment of acute and chronic inflammation and the associated diseases, in particular those affecting the cardiovascular
system.
TLRs in Cardiovascular Disease
The association between bacterial infections and atherosclerosis prompted the search for biological functions of TLRs in
blood vessels.21 Several TLRs, including TLR1, -2, and -4,
are expressed by macrophages in atherosclerotic plaques in
mice and humans.22,23 Studies using mutant mice further
established that TLR4 has proatherogenic effects.24,25 Similarly, modulation of TLR2 activity affects atherosclerosis,
with a mild reduction of the disease in low-density lipoprotein receptor (Ldlr) knockout mice in the absence of TLR2
and a strong increase of the atherosclerotic burden in Ldlr
knockout mice treated with the triacylated lipid Pam3CSK4,
which acts as a TLR2 ligand.26 TLR2 also plays a critical role
at later stages during the conversion of atherosclerotic lesions
into necrotic plaques. The macrophage apoptosis accompanying this process is triggered by reticulum endoplasmic
History of Discovery: Toll and TLRs
1737
stress and stimulation with atherogenic lipids, sensed by
TLR2 and its coreceptor CD36.27
Not all TLRs appear to affect atherosclerosis similarly.
Indeed, TLR3, which signals through different adapters than
do TLR2 and TLR4, appears to play a protective, rather than
detrimental, role in the vessel wall of hypercholesterolemic
ApoE⫺/⫺ mice.28 Thus, it is now well established that TLRs
play complex roles in the onset and progression of atherosclerosis. Importantly, these receptors have also been connected to myocardial diseases,21 an area that certainly deserves further investigation in view of its clinical importance.
Concluding Remarks
The discovery of the critical role played by Toll receptors in
innate immunity has been a fantastic adventure, and it
illustrates the importance of invertebrate models to decipher
critical biological pathways. We now know that several
families of PRRs exist and play important roles in the onset
of inflammation and the innate immune response, as once
predicted by Janeway.2 Among these PRRs, the TLRs are the
best studied, genetically and structurally. Interesting potential
clinical and pharmacological developments are in sight.29,30 It
is also becoming apparent that sensing virulence factors or
tissue damage (ie, danger) can activate innate immunity by
mechanisms that remain to be fully investigated, in particular
in mammals. The Drosophila model undoubtedly holds
promise not only for providing insights in this area but also
for other complex topics involving the immune system, such
as gut homeostasis, sterile inflammation, antiviral defenses,
and of course the control of pathogen transmission to humans
by insect vectors.
Disclosures
None.
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Microbial Sensing by Toll Receptors: A Historical Perspective
Stanislava Chtarbanova and Jean-Luc Imler
Arterioscler Thromb Vasc Biol. 2011;31:1734-1738
doi: 10.1161/ATVBAHA.108.179523
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