REVIEWS

THERAPEUTICS TARGETING
THE INNATE IMMUNE SYSTEM
Richard J. Ulevitch
Proteins that recognize the components and products of microorganisms have an important role
in innate immunity. Here, I focus on recent advances in our understanding of the function of several
such protein families. In particular, I consider how members of the TLR (Toll-like receptor), NOD
(nucleotide-binding oligomerization domain)-protein and MyD88 (myeloid differentiation primary-
response protein 88) families are providing emerging opportunities for the development of new
therapeutics that modify innate immune responses in ways which benefit the host.

Department of
Immunology, The Scripps
Research Institute, La Jolla,
California 92037, USA.
e-mail: ulevitch@scripps.edu
doi:10.1038/nri1396
The modulation of immune responses — achieved by
targeting cell-surface receptors or intracellular pathways
— is one of the main goals in the development of new
therapeutics for human immune or inflammatory dis-
eases. Traditionally, the focus has been on targets that
are considered to be part of the adaptive immune sys-
tem. In this way, major efforts have been made to
develop therapeutics for transplantation, autoimmunity
and cancer. Now, the ever-expanding wealth of informa-
tion about the innate immune system, including the
identification of cognate ligands of innate immune
receptors and the elucidation of their downstream sig-
nalling pathways, provides a new set of targets for drug
development, which might impact on various human
diseases1–9. The innate immune system sits at the inter-
section of the pathways of microbial recognition,
inflammation, microbial clearance and cell death,
thereby offering diverse targets for therapeutics (FIG. 1).
This review limits its scope to gene families that encode
the key receptors of the innate immune system — the
Toll-like receptor (TLR)- and nucleotide-binding
oligomerization domain (NOD)-gene families — their
ligands and their downstream signalling pathways.
Here, the term ligand is used to indicate a molecule
that binds to its cognate receptor, although for TLRs
and NOD proteins proof of the formation of a
ligand–receptor complex is often lacking. TLRs com-
prise a family of cell-surface and endosomally expressed
receptors that recognize conserved products unique to
microorganisms, such as lipopolysaccharide (LPS),
leading to the activation of the innate and adaptive
immune systems. The proteins that are encoded by the
NOD genes (also known as members of the caterpillar
family) are cytosolic proteins that have a role in various
innate and adaptive immune responses. Of particular
interest here are NOD1 and NOD2, which recognize
distinct structures derived from peptidoglycan that are
not ligands for TLRs10–12.
In mammals, the main function of the innate
immune system is to detect the presence of invading
microorganisms. Importantly, another concept of the
function of innate immunity is emerging — that it also
has a role in sterile inflammation. Sterile inflammation
is not driven by the response to infection with micro-
organisms but by ligands derived from damaged cells,
which are not usually present in the extracellular envi-
ronment: for example, heat-shock proteins, β-defensins
and oxidized lipids. It can also be speculated that pro-
teins modified by oxidation or nitration might be TLR
ligands and thereby might promote sterile inflamma-
tion. So, in both infection and sterile inflammation, the
innate immune system uses receptors that include
members of the TLR and NOD-protein families9.
Ligands for these receptor families can be divided into
several groups: naturally occurring molecules that are
released from microbial sources, synthetic structures
based on those of microbial products, small molecules
with no obvious structural relationships to naturally
occurring ligands and endogenous ligands of host ori-
gin. The downstream signalling pathways for the TLRs

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Microbial-clearance Apoptotic and necrotic
and killing pathways cell-death pathways
Innate immune system
Microbial-recognition Sterile-inflammation
pathways pathways
TLR NOD TLR
protein
Exogenous Endogenous
ligands ligands
Figure 1 | Innate immunity at the crossroads. The innate
immune system is at the intersection of several pathways
that influence the balance between health and disease.
These pathways, which are responsible for the recognition
of microorganisms and endogenous host-derived ligands,
trigger the clearance and/or killing of microorganisms, as
well as apoptotic and necrotic cell-death pathways that
depend on pro-inflammatory mediators. NOD, nucleotide-
binding oligomerization domain; TLR, Toll-like receptor.
and NOD proteins show one main difference. TLRs
couple ligand binding to cell activation through mem-
bers of the myeloid differentiation primary-response
protein 88 (MyD88)-adaptor family (BOX 1), whereas
NOD proteins do not seem to use MyD88-dependent
signal-transduction pathways. In the signalling that
occurs in an innate immune response, two central
events are the activation of nuclear factor-κB (NF-κB)
and interferon regulatory factor 3 (IRF3) . The NF-κB
pathway controls the production of pro-inflammatory
molecules, such as interleukin-1 (IL-1) and tumour-
necrosis factor (TNF), whereas the IRF3 pathway leads
to the production of type I interferons (IFN-α and
IFN-β). In this review, I discuss interesting drug targets
that have emerged from recent studies of TLRs and/or
NOD proteins, without considering as targets the
numerous downstream cytokines and other mediators
that are produced following the activation of the innate
immune system.
Signalling from TLRs involves the Toll/IL-1 receptor
(TIR) domain of the receptor interacting with one or
more members of the MyD88-adaptor family, which
also each contain a TIR domain. These interactions
are known as TIR–TIR domain interactions4,13–15. The
engagement of a TLR by its cognate ligand most
probably facilitates the formation of a hetero- or
homodimeric receptor, which then interacts with
cytosolic TIR-domain-containing adaptor proteins.
Numerous studies have investigated the signalling
pathways immediately downstream of the TIR-
domain interactions, showing how the molecules
IRAK1 (IL-1 receptor (IL-1R)-associated kinase 1),
IRAK4 and TRAF6 (TNF-receptor-associated factor 6)
NATURE REVIEWS | IMMUNOLOGY
FOCUS ON TLR SIGNALLING
are involved; however, here, only the steps that include
the MyD88-adaptor family are considered. Ultimately, we
need to define the signalling events and gene-activation
patterns that occur downstream of each of the TLRs so
that we can identify appropriate targets for the selective
activation or inhibition of these pathways.
In contrast to the TLRs, NOD1 and NOD2 seem to
signal through pathways that are independent of
members of the MyD88 family. Instead, after engage-
ment of their cognate ligands, NOD proteins activate
NF-κB through a pathway that requires the kinase
RICK (receptor-interacting serine/threonine kinase; also
known as receptor-interacting protein 2, RIP2) 16,17.
RICK and NOD1 or NOD2 interact through the
caspase-recruitment domains (CARDs) present in each
protein. Further details about other proteins involved in
NOD-protein signalling are unknown at present.
The central question then is how to use the current
information about the signalling pathways of the innate
immune system to design new therapeutics. The desired
feature of any therapeutic that modifies the innate
immune response — either as an agonist or antagonist
— is, of course, specificity. In this review, examples of
agonists and antagonists are provided to illustrate the
potential utility of drugs targeting innate immunity.
Complications that could arise from either blocking or
activating innate immunity are also discussed, as well
as issues that result from targeting innate immune
pathways in acute and chronic settings.
TLR family
Over the past five years, there have been remarkable
advances in the identification and characterization of
the TLRs. This information provides the basis for
considering new ways of modulating the innate
immune response by designing new therapeutic
approaches (FIG. 2).
The Drosophila studies of Hoffmann and col-
leagues 18–21 provided the basis for the discovery
of the TLRs, the mammalian homologues of the
Drosophila protein Toll. The TLR family now consists
of 13 members: although only TLR1 to TLR10 have
been identified in humans. TLR11 does not seem to
be present in humans22, and TLR10 is not present in
mice. TLRs are members of the Toll/IL-1R (TIR)-
domain-containing superfamily, and there is consid-
erable homology between the cytoplasmic domains
of all family members. In particular, a high degree of
similarity exists within the 200 amino-acid region
that comprises the TIR domain; within this domain
are up to three conserved regions that are crucial for
the assembly of downstream signalling complexes
containing one or more members of the MyD88-
adaptor family. Other proteins that transduce signals
or otherwise regulate receptor function probably also
form part of these complexes. Despite the similarity
of TIR-domain-containing superfamily members,
there are substantial differences in the extracellular
regions of these proteins. The extracellular regions of
the IL-1Rs consist of immunoglobulin-like domains.
By contrast, the TLRs have two highly conserved
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REVIEWS
structural features, the most important of which is the
leucine-rich repeat (LRR)-containing ectodomain. This
domain is thought to be involved in ligand recognition
and usually comprises 24–29 amino-acid repeats that
contain the canonical repeat XLXXLXLXX, where X
denotes any amino acid. Both the LRRs and the TIR
domains could be exploited as drug targets.
In addition to the conserved structural features
among the TLRs, there seems to be another level of
organization. The TLRs that are involved in the recogni-
tion of microbial products — TLR1, TLR2, TLR4, TLR5,
TLR6 and TLR11 — are displayed on the cell surface. By
contrast, TLR7, TLR8 and TLR9 are localized intra-
cellularly, and their natural ligands might only be found
within acidic compartments, such as phagolysosomes.
The natural ligand for TLR9 is bacterial DNA, and
recent studies using TLR7-deficient mice, as well as
investigations of human TLR8, have shown that these
TLRs recognize guanosine and uridine (G+U)-rich
single-stranded RNA. Whether TLR3 functions at the
cell surface or intracellularly is still unresolved, although
some reports indicate that signalling from TLR3 does
require endosomal maturation23. These differences
between TLRs indicate that the design of synthetic
agonists will need to take cellular localization into con-
sideration. For example, if high-throughput binding
assays are to be used to identify novel TLR ligands, then
the pH of the binding solutions will need to reflect the
potential differences between the plasma membrane
and intracellular compartments. The precise mecha-
nism by which synthetic ligands enter cells and engage
Box 1 | MyD88-adaptor family
The figure shows the domain
structure of the five known MyD88 DD TIR
members of the MyD88
(myeloid differentiation TIRAP TIR
primary-response protein 88)
-adaptor family: MyD88, TRIF TIR
TIRAP (Toll/interleukin-1
receptor (TIR)-domain- TRAM TIR
containing adaptor protein;
also known as MyD88-
SARM SAM SAM TIR
adaptor-like protein,
MAL), TRIF (TIR-domain-
containing adaptor protein inducing interferon-β), TRAM (TRIF-related adaptor
molecule) and SARM (sterile α- and armadillo-motif-containing protein)15. Evidence
from genetic and biochemical studies has linked signalling from Toll-like receptor 7
(TLR7), TLR8 and TLR9 to a pathway that requires only MyD88. By contrast, signalling
from TLR2, after the formation of a heterodimer with either TLR1 or TLR6, is linked to a
pathway that requires a complex of MyD88 and a second adaptor, TIRAP. The activation
of interferon-regulatory factor 3 (IRF3) and the subsequent induction of interferon-β,
which are elicited by signalling from TLR3 or TLR4, are MyD88-independent and involve
a third adaptor, TRIF. A fourth adaptor, TRAM, is also involved in MyD88-independent
signalling through TLR4 (REF. 74–77), and a fifth adaptor, SARM, has been described, but
its role in TLR signalling is unclear at present. At the most proximal step after ligand
binding, these proteins of the MyD88-adaptor family provide specificity for the outcome
of TLR signalling that is initiated following the engagement of different types of ligands
by different TLRs.
DD, death domain; SAM, sterile α-motif.
514 | JULY 2004 | VOLUME 4
their cognate intracellular TLRs also remains to be
determined. This is of particular interest for the syn-
thetic TLR9 ligand, which is based on non-methylated
CpG sequences that are found in bacterial DNA.
The report by Medzhitov, Janeway and Preston-
Hurlburt98 that described a human homologue of
Drosophila Toll showed that the expression of human
Toll (TLR) in human cell lines leads to the activation of
NF-κB and the expression of cytokines. This data led to
the first model for TLR-dependent cell activation, in
which the expression and multimerization of TLRs is
sufficient to support cell activation. Subsequently, when
the cognate ligands for the TLRs were defined, it became
clear that TLRs associate with other proteins to form a
heteromeric receptor complex. These additional proteins
might be TLRs that form either homo- or heterodimeric
complexes or proteins unrelated to the TLR family.
There are several examples of each type of receptor com-
plex, including the CD14–MD2–TLR4 complex24,25 and
the TLR2–TLR1 (REFS 26–29), TLR2–TLR6 (REFS 26–29)
and TLR4–TLR5 (REF. 30) complexes. Although het-
eromeric complexes have only been identified for TLRs
that are present at the plasma membrane, it seems
probable that further components of these complexes
will be identified for both the cell-surface expressed and
intracellular TLRs.
There is little doubt that the heteromeric nature of the
TLR complexes reflects the downstream physiological
pathways. So, can this knowledge be exploited to identify
new drug targets that do not require the development of
novel agonists or antagonists that directly target the
TLR-ligand-binding sites? To achieve this, agents that
disrupt protein–protein interactions will be required.
NOD-protein family
Recently, a family of mammalian proteins that contain
a nucleotide-binding oligomerization domain (NOD)
and LRRs has been shown to have an important role in
innate immunity, and substantial progress has been
made in identifying the ligands for the two family
members that have known innate immune functions:
NOD1 and NOD2 (REFS 10–12) (FIG. 3). Members of this
family also have structural features that are found in
the large family of R proteins (also known as plant
disease-resistance proteins)31–33. There are more than
20 members of the NOD-protein family, including
proteins of known and unknown functions. One com-
mon feature of these proteins is the presence of specific
structural domains at the amino (N)-terminus,
including the CARD and pyrin sequences. The latter
allow for unique protein–protein interactions and
most probably, the propagation of some downstream
signals. The tissue-distribution patterns of NOD pro-
teins range from ubiquitous to highly restricted.
Mutations in several proteins — including NOD2,
MHC class II transactivator (CIITA), neuronal apop-
tosis inhibitory protein 5 (NAIP5) and cryopyrin —
are associated with increased disease susceptibilities,
chronic inflammatory disease and hypersensitivity to
some bacterial infections. The NOD-protein family
initiates cellular activation through a multi-protein
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 FOCUS ON TLR SIGNALLING

complex known as an inflammasome. The composi- Two of the NOD-protein-family members, NOD1
tion and properties of these multi-protein complexes and NOD2, are intracellular sensors for bacterial
are discussed in detail by Martinon and Tschopp34 and products that include subunit structures found in
are not reviewed here. NOD-protein-family members bacterial peptidoglycans10–12. Similar to the TLRs, the
seem to be involved in a number of autoinflammatory LRR domains of NOD1 and NOD2 are thought to
human diseases, such as Blau syndrome, Muckle-Wells contain the ligand-binding sites. The N-terminal
syndrome and familial cold urticaria, thereby indicating domains might also provide additional sites for drug
their importance in inflammation. This probably also interactions. A recent study by Nunez and col-
depends, in part, on members of the inflammatory- leagues35 highlights the complexity of the domains of
caspase family. The blockade of caspases by selective NOD1 and NOD2 that are required for ligand bind-
inhibitors is a well-developed research area and is not ing. The fact that NOD1 and NOD2 recognize lig-
discussed further here. ands derived from bacterial peptidoglycan, are linked
to NF-κB and contain CARDs indicates that these
proteins are involved in pathways that regulate
Block TLR4 agonists:
TIR domain– inflammation and cell death. The cell-death pathways
MPL, RC-529
TIR domain Block receptor– are almost certainly mediated by members of the cas-
interactions ligand or protein– pase family, including those known as inflammatory
protein interactions
CD14
TLR4 caspases34.
MD2
TLR and NOD-protein ligands
Endosome There are at least four broad types of ligand for the
TLR7/8/9
TLRs, and for NOD1 and NOD2: naturally occurring
TIRAP

TRAM
TRIF
MyD88

molecules that are constituents of microorganisms,

synthetic analogues of naturally occurring substances,
small-molecule fully synthetic compounds, and
endogenous components that are released from host
Agonists: cells during processes such as necrosis, apoptosis and
loxoribine
inflammation. Ligands are generally thought of as agon-
MyD88

(TLR7),
resiquimod IRAK1 ists, but several classes of antagonist have been devel-
(TLR7), RIP1 TRAF6
imiquimod oped using the structure of agonists as a template for
(TLR7, TLR8) TRAF6 synthetic analogues. A survey of TLR ligands is shown
CpG ODNs
(TLR9) in TABLE 1. Most of the ligands are derived from micro-
bial sources, and they include polymeric molecules
such as peptidoglycans (which bind TLR2), bacterial
IKK-γ LPS (TLR4), bacterial and viral DNA (TLR9)36,37 and
double-stranded RNA (TLR3). Nonetheless, there are
IKK-α IKK-β many naturally occurring ligands, these are small mole-
cules and include lipoteichoic acid (TLR2), lipoarabi-
nomannan (TLR2) and taxol (TLR4). Large polymeric
molecules are not ideal drug candidates because of
IκB
NF-κB
many factors, including their pharmacodynamic and
p50 p65
pharmacokinetic properties. By contrast, because
Nuclear membrane
small-molecule ligands have been identified for many
of the TLRs, the design of new small molecules with
drug-like properties seems to be within reach. There are
already good examples of synthetic analogues of natu-
NF-κB- rally occurring ligands, such as molecules based on the
binding motif
structure of lipid A, CpG oligodeoxynucleotides (ODNs)
Figure 2 | TLRs as targets for therapy. Toll-like receptor (TLR) signalling is initiated by plasma modelled on bacterial DNA sequences, peptidoglycan
membrane and intracellular (endosomal) TLRs following ligand recognition. TLR4 is shown as subunits containing muramic acid, and unique peptide
a representative membrane receptor and the endosomal TLRs (TLR7, TLR8 and TLR9) are also sequences38. Compounds of the imidazole quinoline
shown. Stimulation of TLRs triggers interaction with the adaptor molecule MyD88 (myeloid
differentiation primary-response protein 88). Other adaptor molecules, TIRAP (Toll/interleukin-1
series, which function as immunostimulants, provide
receptor (TIR)-domain-containing adaptor protein), TRIF (TIR-domain-containing adaptor proof-of-concept that fully synthetic small molecules are
molecule inducing interferon-β) and TRAM (TRIF-related adaptor molecule) are also involved recognized by TLRs.
in TLR4 signalling. The MyD88-dependent signalling pathway leads to the activation of nuclear The ligands for NOD1 and NOD2 are distinct and
factor-κB (NF-κB), which regulates the expression of target genes that encode pro-inflammatory the minimal structural requirements have been defined.
mediators. Several laboratories have developed agonists of TLR4 and the endosomal TLRs that Both are derived from bacterial peptidoglycan: NOD2
can stimulate TLR signalling and should be useful therapeutics for treating various human
recognizes ligands with muramyl-dipeptidic structures,
diseases. The key steps that could be targeted in the development of molecules to inhibit
TLR signalling are also indicated. IκB, inhibitor of NF-κB; IKK, IκB kinase; IRAK, interleukin-1- whereas NOD1 binds to tripeptide structures in
receptor-associated kinase; MPL, monophosphoryl lipid A; ODN, oligodeoxynucleotide; which the terminal amino acid is diaminopimelic acid.
RIP, receptor-interacting protein; TRAF, tumour-necrosis-factor-receptor-associated factor 6. Interestingly, the binding of tripeptides to NOD1

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REVIEWS
Agonists/
antagonists
Ligand-
recognition
domain
NOD NOD Potential
protein inhibitory
site
CARD
Inhibit
CARD–CARD
interactions
RICK
Kinase domain
NF-κB activation and
caspase activation
Secretion of pro-inflammatory
cytokines (TNF and IL-1β)
Immune responses
Figure 3 | NOD1 and NOD2 as targets for therapy.
Signalling pathways mediated by the nucleotide-binding
oligomerization domain (NOD)-protein-family members
NOD1 and NOD2 are dependent on the receptor-interacting
serine/threonine kinase (RICK) to initiate downstream
signalling. RICK interacts with NOD1 and NOD2 through
homophilic caspase-recruitment domain (CARD)–CARD
interactions. Key steps that could be targeted for the
development of therapeutics are shown. IL, interleukin;
NF-κB, nuclear factor-κB; TNF, tumour-necrosis factor.
does not require the presence of a muramic acid
backbone. Neither NOD1 nor NOD2 seem to recognize
polymeric peptidoglycan structures, regardless of the
bacterial source.
Targeting innate immunity in human disease
Targeting the innate immune system could provide
therapeutic benefit in both acute and chronic human
diseases. Efforts are also being directed towards using
TLR ligands in vaccines. Examples of each of these
applications are discussed here.
Asthma. Considerable effort is going into the design of
new approaches to treat asthma. The prevalence of
allergic diseases, including asthma, has increased
markedly in the past three decades. Unfortunately,
there are few new drugs that offer cures for these prob-
lems, or even provide control or treatment. We still rely
on glucocorticoids and bronchodilators, used in com-
bination and in various forms of administration, as the
standard practice. Although they are effective, these
drugs need to be administered at least twice daily and
are not without long-term effects, especially in the
increasingly young patient population. Furthermore,
corticosteroids that block inflammatory cells do not
516 | JULY 2004 | VOLUME 4
alter the prevailing T helper 2 (TH2)-cell-type response
in sensitized individuals. Therefore, longer-lasting
treatments afforded by manipulating the innate
immune system would provide a great advance.
Immunotherapy can result in decreased symptoms,
reduced medication use and enhanced quality of life. It
can also prevent the transition from allergic disease to
asthma. So, immunotherapy might have therapeutic as
well as preventive effects. However, the present design
of treatments is limited by our lack of understanding of
the essential mechanisms that are involved in triggering
chronic inflammatory disease. For example, in asthma,
we do not have a mechanistic explanation for the selec-
tive activation of TH2 cells39–41. Nonetheless, it is clear
that one aim of treating asthma with immunomodula-
tors would be to try to change the TH-cell balance in asth-
matic individuals: that is, from the dominant TH2-type
inflammation to a TH1-type response. Central to this
approach is the use of synthetic TLR ligands. In partic-
ular, there has been a sustained effort to use oligonu-
cleotides that contain non-methylated CpG motifs to
shift the balance to a TH1-type response40. This strategy
stems from the ‘hygiene hypothesis’, which states that
the marked reduction in infection in children in mod-
ern society produces decreased TH1-type responses and
concomitant TH2-type inflammation. Approaches to
treat patients using TH1-type cytokines have failed and,
in some cases, have caused serious side effects. There
are numerous preclinical studies showing that admin-
istration of the TLR9 ligand CpG ODN induces the
production of TH1-type cytokines, blocks the produc-
tion of TH2 cytokines and prevents the symptoms of
asthma. Treatment with CpG ODNs seems to reduce
disease, without the serious side effects produced fol-
lowing the administration of TH1-type cytokines.
However, at present, there is too little attention paid to
the potential side effects of chronic administration of
immunostimulatory molecules; potential drawbacks
of this type of therapy are discussed later.
Adjuvants/immunostimulation. Another area that
could benefit from targeting the innate immune
response is the development of new adjuvants for vac-
cines. Vaccines are crucial for the management and pre-
vention of infectious disease. Although our endogenous
defences mount effective responses to microorganisms,
it seems that enhanced innate immunity could be bene-
ficial in specific settings. Previously, such enhancement
of immune responses has been achieved using complex
mixtures of microbial products, which most probably
function through the innate immune system to enhance
adaptive immune responses42,43. In addition, we now
know that several types of TLR ligand are efficacious as
vaccine adjuvants44,45. This has been demonstrated for
synthetic TLR7 agonists (imiquimod and resiquimod),
TLR9 agonists (CpG ODNs) and TLR4 agonists (lipid A
analogues), thereby indicating the merits of identifying
synthetic TLR agonists. Here, I discuss a few important
examples that involve TLR4, TLR7 and TLR9 (TABLE 2).
There are now considerable data indicating that the use
of synthetic TLR4 agonists can be beneficial. Some of
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 FOCUS ON TLR SIGNALLING

the most compelling data have been obtained using
monophosphoryl lipid A (MPL)46–48. This compound is
a chemically modified derivative of the lipid A moiety of
LPS. MPL is considerably less toxic than LPS but has a
similar immunostimulatory activity. MPL has been
used extensively in clinical trials, in formulations of
both prophylactic and therapeutic vaccines that have
targeted various diseases, including cancer, infectious
disease and allergies. It seems to have a strong safety
profile and good efficacy. A set of related compounds
that are also lipid A mimetics, aminoalkylglucosaminide
4-phosphates (AGPs), have been found to be TLR4
ligands and some molecules from this new group of
immunostimulants will undoubtedly move into clinical
trials. In particular, one derivative, RC-529, has been
used in a hepatitis B vaccine trial, replacing the standard
alum-based adjuvant. In nearly all of those vaccinated
with an MPL adjuvant, protective antibody levels were
reached after only two doses of the vaccine, in contrast
to the current protocol in which three doses are
required to obtain similar levels of protection.
Recently, single-stranded RNA has been shown to be
a ligand for mouse TLR7 and human TLR8 (REFS 49,50).
However, it is well known that several small-molecule
immunostimulants are also ligands for TLR7 and
TLR8. The prototypical compound, resiquimod,
induces IFN-α and other cytokines. Resiquimod is
one of a class of imidazole quinolines and has been
the subject of several clinical investigations. It has
been shown to be an immune-response modifier that
enhances cutaneous immune responses: it is effective
against external genital warts and other diseases
caused by human papillomavirus51,53. The imidazole
quinolines are the first class of TLR agonists to be
used for several clinical applications in humans.
Another compound, loxoribine (7-allyl-7,8-dihydro-
8-oxo-guanosine), has antitumour activity and func-
tions through a TLR7– MyD88-dependent pathway,
which was demonstrated using TLR7- and MyD88-
deficient mice as a source of cells54–56. The activity of
loxoribine and resiquimod depend on endosomal
maturation and presumably acidification. In this
regard, TLR7 and TLR8 are similar to TLR9 and seem
to form a subgroup within the TLR family, which
functions in endosomal/lysosomal compartments.
TLR7 also recognizes bropirimine, an immuno-
stimulant known to have antitumour effects in super-
ficial transitional-cell carcinoma of the bladder.
Bropirimine might have direct effects on tumour cells
rather than functioning through inducible mediators;
however, more study is required to understand its
mechanism of action.
Infection. Last, one might consider targeting innate
immune pathways in the acute setting, where there is
local or systemic infection. Here, blunting the pro-
inflammatory cascade might be achieved using recep-
tor antagonists based on inhibitory antibodies or
antagonistic ligand mimetics. Another approach could
be to induce protection by activating TLRs and/or
NOD1/2 to elicit nonspecific resistance effects and
enhance host resistance to bacterial or viral infection.
Workers at Corixa Corporation have extended the pre-
vious studies of AGPs (lipid A mimetics) and showed

Table 1 | Toll-like receptor ligands

Receptor Naturally occurring Synthetic analogues Fully synthetic small molecules
Exogenous ligands
TLR1 N.D. Triacyl lipopeptides –
TLR2 Lipoproteins/lipopeptides Di- and triacyl lipopeptides –
Peptidoglycan
Lipoteichoic acid
Lipoarabinomannan
Atypical lipopolysaccharide
TLR3 Double-stranded RNA Poly I:C –
TLR4 Lipopolysaccharide LPS/lipid A mimetics, such as MPL Synthetic lipid A, E5564
Taxol*
HSP60 (Chlamydia pneumoniae)
TLR5 Bacterial flagellin Discontinuous 13-amino-acid peptide –
TLR6 N.D. Diacyl lipopeptides –
TLR7 (G+U)-rich single-stranded RNA* Oligonucleotides Imidazole quinolines (imiquimod, resiquimod)
Guanosine nucleotides (loxoribine)
TLR8 (G+U)-rich single-stranded RNA‡ – Imidazole quinolines (imiquimod)
TLR9 Bacterial DNA CpG oligodeoxynucleotides –
Viral DNA
Other DNA with low content of
non-methylated CpG sequences
Endogenous ligands
TLR2 HSP70 – –
TLR4 HSP60 – –
Oligosaccharides of hyaluronic acid
*Ligand for mouse TLR only. ‡Ligand for human TLR only. HSP, heat-shock protein; LPS, lipopolysaccharide; MPL, monophosphoryl lipid A; N.D., not determined; Poly I:C,
polyinosinic–polycytidylic acid; TLR, Toll-like receptor.

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Table 2 | Toll-like receptors as drug targets in clinical trials
Receptor Ligand Application References
TLR4 MPL adjuvant Adjuvant allergy treatment 78,79
MPL adjuvant Vaccines 46,80,81
Ribi 529 Vaccines 81
E5564 Endotoxaemia, liver disease 82–84
TLR7 Loxoribine Cancer 85
Resiquimod Increased cutaneous immune
responses 86–89
Imiquimod Basal-cell carcinoma 90–92
TLR9 CpG ODNs Vaccine adjuvant 93–96
CpG ODNs Melanoma 97
CpG ODNs Non-Hodgkin lymphoma 97
MPL, monophosphoryl lipid A; ODN, oligodeoxynucleotide; TLR, Toll-like receptor.
that they provide remarkable protection in Listeria or
influenza-virus challenge models, in which there is
no administration of the microorganism itself or
additional antigen57.
Considerable data indicate a role for the innate
immune system in antiviral responses. The work of
Finberg and colleagues58 supports the contention that
viral proteins might directly activate TLRs, such as
TLR4, thereby resulting in an antiviral response.
Subsequently, it was shown that stimulation of TLR3
and TLR4 results in the induction of a subset of genes,
including those that encode type I interferons, which
inhibit viral replication59. Signalling from TLR3 and
TLR4 has been shown to involve both the NF-κB and
IRF3 pathways. More recently, a detailed understanding
of these pathways has emerged from the efforts of
Beutler and others60–65. In particular, it has been shown
that pathways linked to TLR3 and TLR9 contribute to
innate responses to systemic viral infection. Although
neither pathway alone has a dominant role, mutations
affecting both result in markedly decreased antiviral
responses. Therefore, targeting the TLR3, TLR4 and
TLR9 pathways allows for an enhanced response to cer-
tain viral infections. There are numerous small-molecule
agonists that have the potential to enhance resistance to
viral infection. A new strategy involving covalent linkage
of antigen to CpG ODNs promises to provide an
effective way of improving the immunomodulatory
activities of CpG, by enhancing antigen uptake and
stimulation of antigen-presenting cells66,67. This con-
cept has been extended to clinical trials with the use of
allergen–CpG ODN conjugates68.
Targeting intracellular pathways
The important role of NF-κB in TLR signalling is now
well established. The NF-κB-signalling pathways are
one of the main targets for new drug discovery and
development69. Numerous studies have shown that
involvement of the IKK complex (inhibitor of NF-κB
(IκB)-kinase complex), a downstream element in
TLR-signalling pathways, is essential. The two kinases
present in the IKK complex, IKK-α and IKK-β, are
potential targets, and recent studies have been able to
non-selectively block IKK activity using non-steroidal
anti-inflammatory drugs. At present, the most
progress towards developing selective inhibitors is in
the development of selective small-molecule inhibitors
of IKK-β. These might have applications in controlling
events downstream of TLR activation in chronic dis-
ease. In particular, it is possible to envisage the use of
such inhibitors to treat chronic inflammatory diseases,
in which sterile inflammation that results from TLR
activation is important. The use of other selective
kinase inhibitors could also potentially control TLR-
driven inflammation. The kinase targets might include
IRAK4, RICK and the mitogen-activated protein
kinases; this topic is not considered further here.
The current progress in defining the specific roles
of members of the MyD88-adaptor family in TLR sig-
nalling offers new possibilities for the selective block-
ade of pathways downstream of the TLRs. We now
require the development of new therapeutics to target
specific protein–protein interactions. This has proven
to be a formidable barrier, with only limited progress
being made. Screening of conventional chemical
libraries comprising small-molecule drug-like struc-
tures has not provided compounds able to block
protein–protein interactions. In part, this results from
the typical flatness of the interface that characterizes
protein–protein interactions. Large surface areas are
involved, usually 750–1500 Å2, and there are no deep
cavities present that could function as small-molecule-
binding sites. Many researchers, including Wells and
his co-workers70 have defined ‘hot spots’ that represent
the most important regions for high-affinity binding.
As we accumulate knowledge about each type of mole-
cule, the TLRs, NOD-protein-family members and
adaptor proteins should become more tractable tar-
gets. There are several strong examples that support
the contention that progress is being made. One is in
the unrelated area of p53 interactions but the other
provides data about the targeting of TIR domains. In
the former, Vassilev et al.71 have discovered a group of
synthetic compounds, known as nutlins, that specifi-
cally block p53–MDM2 interactions. This finding fol-
lowed a series of elegant studies by others, which were
based on designing peptidic inhibitors and then iden-
tifying the specific binding site of p53 that mediates
MDM2 binding. This work could well provide a model
for future efforts in the design of drugs to target innate
immune pathways.
In fact, Rebek and colleagues72 have provided the
much needed proof-of-concept that TIR domains
could be targets for selective inhibitors. Using the
TIR-domain interactions between MyD88 and the
type 1 IL-1R (IL-1R1) as a model, they synthesized
and characterized a low-molecular-weight MyD88
mimic, hydrocinnamoyl-l-valyl-pyrrolidine. This
compound was developed by modelling a tripeptide
sequence of the BB-loop of the TIR domain —
(F/Y)-(V/L/I)-(P/G). The compound seems to be
selective in both cell-based and in vivo systems.
Development of this low-molecular-weight inhibitor
of the TIR-domain interaction between MyD88 and

518 | JULY 2004 | VOLUME 4 www.nature.com/reviews/immunol

 FOCUS ON TLR SIGNALLING

IL-1R1 indicates that the design of further selective
inhibitors of TIR-domain-containing proteins is
within reach. This promises to yield an entirely new
generation of potential anti-inflammatory compounds
to modulate the innate immune system.
Complications with therapeutics
As therapeutics, CpG ODNs might require administra-
tion over long time periods. The effect of chronic
administration has not yet been addressed in detail.
However, a recent publication reported the conse-
quences of chronic CpG ODN administration73: mice
given daily injections of CpG ODNs developed consid-
erable pathology in their lymphoid organs, with
changes in both structure and function. Specifically, the
report showed destruction of lymphoid-follicle archi-
tecture and suppression of follicular dendritic cells and
germinal-centre B lymphocytes. Furthermore, after
three weeks, multi-focal liver necrosis and haemor-
rhagic ascites developed. These effects were dependent
on both CpG ODNs and TLR9. Additional studies will
be required to determine whether the administration
other TLR ligands induces similar effects and how the
CpG-ODN-induced injury correlates with dosage and
its more-beneficial effects.
The use of TLR antagonists might have applica-
tions in acute settings, in which dampening the pro-
inflammatory response of the innate immune system
is beneficial. In particular, TLR antagonists could be
used to treat septic shock. However, the important
role of the innate immune system in host defence
against microorganisms indicates that only limited
uses of antagonists will be found. Certainly, their use
in any chronic settings will require a high degree of
selectivity among the various TLRs. Determining
how to maintain the balance between host-defence
functions and the potentially harmful effects that
result from TLR activation remains a serious challenge
for those designing new therapeutics.
Conclusions
We now have detailed knowledge of the ligands, receptors
and intracellular-signalling pathways that control innate
immune responses during infection and inflammation,
and this provides new approaches for highly selective
therapeutics. New information about the role of NOD2
in human disease exemplifies how genetic approaches
will increase our understanding of disease pathogenesis
and how best to target individual proteins or pathways.
The potential to modulate the innate immune system is
supported by the first generation small-molecule ligands
of TLR7 and TLR8, the imidazole quinolines. This class of
drug was developed for topical use, targeting papilloma-
virus-induced warts, without understanding its exact
mechanism of action. Furthermore, the ongoing trials
based on targeting TLR9 with CpG ODNs will enhance
our knowledge of the effects of blocking or enhancing
selective pathways of innate immunity. It should now be
possible to use our combined knowledge of the TLR- and
NOD-protein-family members, together with the
recently developed tools for drug discovery, to identify
new therapeutic molecules with selective actions. Our
progress will only be limited by our ability to express the
key proteins of the innate immune system for use in
high-throughput screening assays, and our ability to
develop small-molecule libraries containing chemical
scaffolds that are directed towards the hot spots of pro-
tein–protein interactions. Hopefully, the fruits of
ongoing basic research are within reach of the drug-
development community, and we will soon see how
our newly found knowledge can be used to better treat
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Acknowledgements
I thank J. C. Mathison for comments and editorial support and
P. Rutledge for administrative support. This work was supported
by the National Institutes of Health, The Charles Dana Foundation
and The Novartis Foundation.
Competing interests statement
The author declares competing financial interests: See Web version
for details.
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Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
CD14 | IKK-α | IKK-β | IL-1R1 | IRAK1 | IRAK4 | IRF3 | MD2 |
MyD88 | NOD1 | NOD2 | RICK | SARM | TIRAP | TLR1 | TLR2 |
TLR3 | TLR4 | TLR5 | TLR6 | TLR7 | TLR8 | TLR9 | TLR11 | TRAF6 |
TRAM | TRIF
www.nature.com/reviews/immunol