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The innate immune system recognizes a
broad range of pathogens and initiates
protective responses. Recognition of path-
ogenic microorganisms by the innate im-
mune system relies on pattern recognition
receptors (PRRs) that detect preserved
structures of bacteria, viruses, protozoa,
and fungi, so-called pathogen-associated
molecular patterns (PAMPs) (1).
A major group of PPRs are the Toll-like
receptors (TLRs). After PAMP recogni-
tion, TLRs activate cellular signaling
pathways to induce immune-response
genes, including inflammatory cytokines
(2). Ten different human TLRs have
been identified (3). TLR11, which binds
and recognizes uropathogenic bacteria
and profilin in mice, has been shown to
be nonfunctional in humans owing to a
premature stop codon (4,5). Each TLR
recognizes specific PAMPs, including
recognition of lipoproteins, lipoteichoic
acid, and zymosan by TLR2; dsRNA by
TLR3; lipopolysaccharide (LPS) by
TLR4; flagellin by TLR5; ssRNA by
TLR7/8; and CpG DNA by TLR9 (1,6).
TLR4, considered one of the most impor-
tant PRRs, recognizes LPS of Gram-
negative bacteria; mannans from fungal
pathogens, a soluble component of My-
cobacterium tuberculosis; and endoge-
nous ligands, such as fibronectin and
several heat shock proteins.
ROLE AND SIGNALING MECHANISM
We owe the elucidation of the function
and genetic structure of TLR4 to the dis-
covery of Toll, a transmembrane protein,
initially discovered for its role in the em-
bryonic dorsal-ventral development of
Drosophila (7). Drosophila with a loss-of-
function mutation for Toll exhibited a
high susceptibility to fungal infection,
demonstrating the importance of Toll
in the antifungal response in the insect
(8,9). After this discovery, work by Rock
et al. (10) and Medzhitov et al. (11) inde-
pendently led to the discovery of several
human homolog genes for Toll, the
so-called Toll-like receptors (TLRs).
Structure analysis of TLR4 revealed
that the receptor consists of 3 domains:
an extracellular leucine-rich-repeat (LRR)
domain, a transmembrane domain, and
an intracellular Toll-interleukin-1 receptor
(TIR) domain. The extracellular LRR part
of the receptor is involved in the binding
of LPS. The discovery that LPS is a lig-
and for TLR4 came from studies in mice.
Mice challenged with high doses of LPS
normally develop a shock-like state simi-
lar to Gram-negative septic shock. More
than 30 years ago two mouse strains,
C3H/HeJ and C57Bl/10ScCr, were dis-
covered that were resistant to LPS (12).
The phenotype was thought to be caused
by a mutation in a hypothetical lps gene,
but the location and function of this
gene remained elusive for a long period
(13). The groundbreaking studies of
Poltorak and colleagues (14,15) in 1998
identified TLR4 as the lps gene and
demonstrated that TLR4 is the LPS sen-
sor in both mice and humans.
Overall, recognition of LPS and initia-
tion of signaling by TLR4 is a complex
process, which involves several acces-
sory proteins. LPS is first bound by cir-
protein (LBP), which functions as an op-
sonin for CD14 (16). CD14 then acts as a
Functional Consequences of Toll-like Receptor 4 Polymorphisms
Bart Ferwerda,1,2Matthew BB McCall,1,3Karlijn Verheijen,1,2Bart-Jan Kullberg,1,2André JAM van der Ven,1,2
Jos WM Van der Meer,1,2and Mihai G Netea1,2
Address correspondence and reprint requests to Bart Ferwerda, Department of Medicine
(463), Radboud University Nijmegen Medical Center, P.O. Box 9101, Geert Grooteplein 8,
6500 HB Nijmegen, The Netherlands. Phone: + 31-24-3618819; Fax: + 31-24-3541734; E-mail:
Submitted December 21, 2007; Accepted for publication January 18, 2008; Epub (www.
molmed.org) ahead of print January 25, 2008.
1Department of Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands; 2Nijmegen University
Center for Infectious Diseases and 3Department of Parasitology, Nijmegen, The Netherlands
Toll-like receptor 4 (TLR4) is an important pathogen recognition receptor that recognizes mainly lipopolysaccharide (LPS) of Gram-
negative bacteria, but also structures from fungal and mycobacterial pathogens, as well as endogenous ligands. Two nonsynony-
mous polymorphisms of TLR4, Asp299Gly and Thr399Ile, have been suggested to alter the function of the receptor. Some, but not all,
studies have proposed that these polymorphisms lead to reduced cytokine response and increased susceptibility to Gram-negative
infections. In this review, we compare studies that assessed the effect of the Asp299Gly and Thr399Ile polymorphisms on suscepti-
bility to Gram-negative infections and examine the phenotypic consequences of these polymorphisms. In addition, we review the
geographical distribution of TLR4 polymorphisms and present a model for evolutionary pressures on the TLR4 genetic make-up.
Online address: http://www.molmed.org
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catalyst for the binding of LPS to MD-2
(17-19). After the LPS is transferred to
MD-2, the LPS/MD-2 complex interacts
with TLR4. Recent MD-2/TLR4 crystal-
lography studies have elucidated the
structures of this complex (20). Forma-
tion of the LPS/MD-2/TLR4 complex
eventually results in the dimerization of
the TLR4 TIR domain, initiating down-
stream signaling transduction (21).
Downstream signaling by the TLR4 re-
ceptor involves several intercellular TIR
domain-containing adaptors mediating
proinflammatory gene expression (22,23).
Two pathways that initiate downstream
TLR4 signaling are known, namely the
MyD88- and TRIF-dependent pathways
(see Figure 1). One pathway is mediated
by myeloid differentiation factor 88
(MyD88) and the TIR domain-containing
adaptor protein, also called MyD88
adapter-like protein (TIRAP/MAL). Initi-
ation of MyD88-dependent pathway acti-
vation leads to the activation of nuclear
factor κB (NF-κB) and the transcription
of proinflammatory genes (24). A second
pathway is mediated by the TIR-containing
protein, also called TRIF-related adapter
molecule (TIRP/TRAM), and TIR domain-
containing adapter inducing interferon-β
(TRIF). Initiation of the TRIF-dependent
pathway leads to the activation of inter-
feron regulatory factor 3 (IRF3), and the
expression of interferon (IFN)-β and
IFN-inducible genes (21).
Much research has been performed to
understand the signaling pathways and
innate immune response against Gram-
negative infections mediated by TLR4,
which is therefore one of the best-under-
stood Toll-like receptors. Because of this,
TLR4 also provides an ideal model to
study the consequences of genetic varia-
tion and their relation to the function of
the receptor, and their susceptibility to
diseases. Here we review several genetic
polymorphisms of the TLR4 receptor.
Also, we try to understand if there are
any TLR4 genotypes that result in phe-
GENETIC VARIATION IN HUMAN TLR4
Recognition of pathogens and down-
stream signaling through innate path-
ways involving the TIR domain show
high conservation between organisms
(19). This is in line with the finding that
the innate immune system shows a high
degree of homology between mammals,
insects, and plants, and apparently has
been highly preserved during evolution
(25). Genetic variation between the TLR4s
of different mammals is the highest in
the extracellular LRR region (26). The
stronger variation in this region, which
is involved in the recognition of PAMPs,
is probably the result of the evolutionary
pressure by pathogens on the host.
Sequencing of the human TLR4 re-
vealed that most of the variation in non-
synonymous polymorphisms is located
in the third exon, coding for the LRR do-
main (27). Despite this greater variation
in the TLR4 LRR domain, the frequency
of most nonsynonymous polymorphisms
is low in human populations (<1%). Ex-
ceptions are 2 nonsynonymous polymor-
phisms (SNPs) that have been described
with population frequencies >5%. These
are an A/G transition causing an aspartic
acid/glycine substitution at amino acid
location Asp299Gly (rs4986790), and a
C/T transition causing a threonine/
isoleucine switch at amino acid location
Thr399Ile (rs4986791). Arbour et al. (28)
were the first to report that individuals
with either the Asp299Gly and/or
Thr399Ile polymorphisms had a blunted
response toward inhaled LPS.
The finding that TLR4 polymorphisms
Asp299Gly and Thr399Ile had an effect
Figure 1. Schematic overview of the LPS TLR4 signaling pathway. Recognition of LPS is estab-
lished by the interplay between lipopolysaccharide-binding proteins (LBPs) and CD14 that
transfer the LPS to the MD-2/TLR4 complex. Dimerization of TLR4 then initiates downstream in-
tracellular signaling transduction through Toll-interleukin-1 receptor (TIR) domains and several
adaptor molecules. Eventually, the intracellular signaling results in the activation of specific
pro-inflammatory cytokine profiles by the MYD88-dependent or TRIF-dependent pathway.
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F U N C T I O N A L C O N S E Q U E N C E S O F T L R 4 P O LY M O R P H I S M S
on the responsiveness to bacterial endo-
toxin initiated many genetic association
studies. The results of these studies led
to contradictory conclusions about the
role of the Asp299Gly polymorphism
and its effect on susceptibility to Gram-
negative bacterial infections (29). This
raises the question of the source of these
discrepancies. One possible explanation
may be that these studies looked at the
Asp299Gly and Thr399Ile polymor-
phisms separately. Most studies either
deal with the Asp299Gly or the Thr399Ile
polymorphism, but neglect the fact that
these polymorphisms also exist in a
way (27). This cosegregated state of
TLR4 implies that 4 haplotypes, namely
wt/wt, Asp299Gly/wt, Thr399Ile/wt,
and Asp299Gly/Thr399Ile, are repre-
sented in the population. Our results on
cytokine profiles in LPS-stimulated
whole blood cultures demonstrated that
only the Asp299Gly haplotype differs in
phenotype, showing an increased, rather
than a blunted, pro-inflammatory tumor
necrosis factor (TNF)-α response (30). In-
terestingly, the LPS-induced cytokine re-
sponse in the Asp299Gly/Thr399Ile hap-
lotype does not differ from the wild-type
TLR4 cytokine response (30). Because
the single Thr399Ile haplotype is rare, its
phenotype is still unknown. Therefore,
the TLR4 haplotype that alters the cy-
tokine response to LPS (and thus could
affect the susceptibility to Gram-negative
infection) contains the single TLR4
Asp299Gly mutation (Figure 2).
Population studies also revealed a
specific geographical distribution of the
TLR4 haplotypes (27). All haplotypes
trace their origin to before the out-of-
Africa migration approximately 55,000 to
60,000 years ago. Nowadays, human
populations from Africa have a 10- to 20-
fold higher frequency of the Asp299Gly
haplotype than those from the other con-
tinents. In contrast, white populations
show an almost complete absence of the
Asp299Gly haplotype, but have a higher
frequency of the Asp299Gly/Thr399Ile
haplotype. Asian populations lack all
three of the Asp299Gly, Thr399Ile, and
Asp299Gly/Thr399Ile haplotypes (Fig-
ure 2). This pattern of TLR4 haplotypes
has been the result of differences in envi-
ronmental pathogenic pressure on the
human population during the migration
into Eurasia, Europe, Asia, and the New
World (30). As a consequence of this spe-
cific geographical distribution, most pub-
lished studies performed in populations
with a European genetic background
have analyzed the Asp299Gly/399 hap-
lotype, with no distinct phenotype, in-
stead of the Asp299Gly haplotype. To a
certain extent, the genetic background
of the studied populations could explain
the lack of agreement between published
TLR4 polymorphism studies. We have
therefore reanalyzed published data on
the Asp299Gly/Thr399Ile and Asp299Gly
haplotypes separately, and tested
whether the presented Asp299Gly and
Asp299Gly/Thr399Ile haplotypes corre-
late with the presented phenotypes.
FUNCTIONAL CONSEQUENCES OF THE
Terms used for a PubMed search were
TLR4 polymorphisms, Asp299Gly, and
Toll-like receptor 4 polymorphisms,
which resulted in 164 articles after ex-
clusion of all reviews. These articles
studied the relationship between TLR4
polymorphisms and susceptibility to
sepsis, atherosclerotic disease, asthma,
Candida infections, chronic periodonti-
tis, respiratory syncytial virus (RSV),
transplantation, and Crohn’s disease
(31-51). The pathogenic ligand in most
of these studies is based on the Gram-
negative bacterial LPS, with the excep-
tion of the Candida and RSV studies.
Figure 2. Distribution of TLR4 haplotypes and associated cytokine phenotypes across the 3
continents of the Old World. It shows that Africa, Asia, and Europe each have their own
distinct haplotype, based on the alleles found, shown in white. The phenotypic end point
is shown on the top of the figure, where the expected cytokine production following TLR4
stimulations is presented. When the geographic distribution of Asp299Gly and Thr399Ile
polymorphisms is taken into account, it illustrates that research preformed in Europe has
looked at the cosegregated Asp299Gly/Thr399Ile haplotype, whose cytokine phenotype
does not differ from the wild-type TLR4 cytokine response. This also explains the large
number of studies, performed in Europe, that did not find any association between TLR4
polymorphisms and susceptibility to disease. In contrast, the Asp299Gly/WT haplotype,
that is found almost exclusively in Africa, has a stronger pro-inflammatory cytokine re-
sponse compared with wild-type TLR4.
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The strongest association to date be-
tween TLR4 polymorphisms and dis-
ease susceptibility has been reported in
RVS infections (52), but the functional
relationship between TLR4 polymor-
phisms and RSV virus remains to be
elucidated. Because specific parts of the
LRR involved in the recognition of var-
ious ligands can differ, only the LPS
studies were included. This resulted in
157 research articles about the effect of
the TLR4 Asp299Gly and Asp299Gly/
Thr399Ile SNPs. Of these articles, 62%
reported no association between the
susceptibility to disease and TLR4
polymorphisms (see Figure 3). The ma-
jority of articles included European
populations (90%), and the rest
screened Asian (6%) or African (4%)
populations. The consequence of the
studies involving European popula-
tions is in agreement with the suspi-
cion that these studies did not look at
the Asp299Gly haplotype, as they state,
but reported de facto on the effect of
the Asp299Gly/Thr399Ile haplotype.
Besides the population origin, study
methodology can also influence results.
For the TLR4 studies, three types of
methodology can be distinguished: ge-
netic association studies, functional
stimulation experiments, and cloning
and transfections of the mutated
TLR4. Figure 3 shows that the majority
of published articles are studies of ge-
netic associations. Most of the associa-
tion studies on TLR4 polymorphisms
are small and underpowered, whereas
larger studies tend to be negative and
find no correlation between the TLR4
Asp299Gly/Thr399Ile haplotype and
disease susceptibility (53).
Although genetic association studies
find no correlation between haplotypes
and disease strictly, they do not give in-
sight into the direct functional conse-
quences of TLR4 mutation. Several
studies have investigated the functional
consequence of TLR4 SNPs, and the re-
sults are summarized in Table 1. The
studies of Arbour et al. (28) and Schwartz
(54-56) show that transfected cells with
any of the TLR4 haplotypes have a de-
creased NF-κB activity compared with
normal TLR4. This suggest that the
Asp299Gly/Thr399Ile haplotype should
have an effect on phenotype, leading to
reduced cytokine production by the in-
nate immune response. The question
then is: why are the consequences of
such a functional effect not observed in
genetic association studies?
One reason is that the use of transfec-
tion to investigate the phenotypic effect
of certain mutations meets with several
complications. Transfections represent a
stripped-down model performed in al-
tered cell lines. As a result, transfection
measurements do not give the ideal end
point of phenotypic difference represent-
ing the whole system. Transfections only
represent the homozygous state. Most
population studies of genetic association
and function include TLR4 haplotypes in
heterozygous state, whereas the homo-
zygous state of these polymorphisms is
rare. Disruptions of the receptor trans-
port can influence results by overfeeding
the system (28). Therefore, determina-
tion of the association between the
Asp299Gly/Thr399Ile haplotype and the
phenotype, and its impact on the suscep-
tibility to Gram-negative infection
(based on transfection experiments), is
difficult and prone to artifacts.
Besides transfection experiments, most
functional studies are based on in vitro
stimulation of either whole blood or pe-
ripheral blood mononuclear cells
Figure 3. Circle diagrams with the frequency of the study results and outcomes (below) of
TLR4 polymorphism from the PubMed search. This search revealed that more than half of
the studies do not find any association between susceptibility to infection and TLR4 poly-
morphisms (n = 157). In the top diagrams, the frequencies of the methodologies used are
presented in the studies that showed a positive effect or no association of the investi-
gated TLR4 polymorphisms. This figure shows that the majority of studies are based on ge-
netic association studies, and a minority measured cytokine production following LPS
stimulation of either whole blood or PBMCs. Overall, the contrast between study out-
comes measuring the functional effects of TLR4 polymorphisms and of those looking at
the role these have in susceptibility is revealed.
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F U N C T I O N A L C O N S E Q U E N C E S O F T L R 4 P O LY M O R P H I S M S
(PBMCs) stimulated with LPS. After
stimulation, cytokine release by the cells
is measured. The whole blood cultures
and experiments using isolated PBMCs
are in agreement with the association
studies, as they find little difference be-
tween Asp299Gly/Thr399Ile and the
wild-type haplotype (Table 1). Besides
the in vitro experiments, three in vivo
studies investigated the effects of LPS in
human volunteers. Two of these in vivo
experiments injected 2 ng/kg LPS
(57,58). Neither of them found a differ-
ence in the TNF-α serum concentration
between individuals with different TLR4
haplotypes. However, one study found
decreased IL-6 levels after 6 h in volun-
teers with TLR4 polymorphisms,
whereas the other did not find any dif-
ferences (57,58). The former study also
reported strongly decreased IL-1β pro-
duction after 24-h stimulation, suggest-
ing an effect on the late TRIF-dependent
pathway (58). A third paper measuring
ex vivo and in vivo cytokine production of
the different TLR4 haplotypes also did
not find differences between
Asp299Gly/Thr399Ile and the wild-type
haplotypes (57,59,60). Thus, most studies
of stimulated whole blood and PBMCs
do not find a distinct phenotype of the
FUNCTIONAL CONSEQUENCES OF THE
As mentioned above, studies that in-
clude or define the Asp299Gly haplo-
type are scarce. The best studies of the
Asp299Gly haplotype are those per-
formed in Africa. Unfortunately, no asso-
ciation studies performed in Africa have
focused specifically on the response to
TLR4 ligands. The study by Mocken-
haupt et al. (61) focused on malaria,
whereas that of Newport et al. (62)
looked at susceptibility to Mycobacterium
tuberculosis. Neither study included
stimulated cytokine production, and
therefore it is impossible to say anything
about the LPS responses in the
Asp299Gly haplotypes. Although these
studies indicate a role for the Asp299Gly
haplotype in susceptibility to malaria
and tuberculosis, they do not reveal the
role of the haplotype in the recognition
of Gram-negative bacteria and LPS.
In contrast, Lorenz et al. (63) looked at
sepsis patients versus healthy controls in
France and specifically defined the
Asp299Gly haplotype. The Asp299Gly
Table 1. TLR4 polymorphisms and their impact.
Calvano et al. (57)
Kumpf et al. (65)
Schippers et al. (59,60)
Newport et al. (62)
Heesen et al. (66)
Von Aulock et al. (67)
Chang et al. (68)
Pre- and postoperative cytokines
In vivo and ex vivo response
LPS-induced cytokine release
TLR4 in peripheral neutrophils and
Innate response to candidate
adjuvants RC529 and
monophosphoryl lipid A
Stimulations with different TLR4 ligands
Impact of polymorphisms on
monocytes in patients with Crohn’s
LPS-induced monocytes signaling
Response to LPS
Intima-media thickness and LPS
Insights in stoichiometry, structure, and
Whole blood in vivo
Marsik et al. (58)
Tiberio et al. (69)
Whole blood in vivo
Kroner et al. (70)
Van der Graaf et al. (71)
Peeters et al. (51)
Fagerås et al. (72)
Erridge et al. (73)
Schmitt et al. (74)
Norata et al. (75)
Montes et al. (76)
Kinane et al. (77)
Arbour et al. (28)
Rallabhandi et al. (64)
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haplotype was found only in the group
of patients with septic shock, whereas
the Asp299Gly/Thr399Ile haplotype was
found equally in both patients and con-
trols. This suggests that the Asp299Gly
haplotype has an effect on phenotype, al-
though cytokine measurements are lack-
ing. Until now, only one study reported
cytokine production by individuals with
the Asp299Gly haplotype—it found a
stronger, rather than a blunted, TNF-α
cytokine response (30). Although more
research has to be performed to settle
the functional consequences of the
Asp299Gly haplotype, the evidence so
far indicates that the TLR4 wild-type
haplotype and the Asp299Gly haplotype
have different phenotypes.
EFFECTS OF THE ASP299GLY ON THE
3-DIMENSIONAL STRUCTURE OF TLR4
Crystallography of the TLR4-LPS/MD-2
complex reveals that there are two
highly preserved regions of TLR4 in-
volved in the binding of the LPS/MD-2
complex (20). These preserved domains
are located at the N-terminal and central
domain of the receptor (20). The crystal-
lography shows that the Asp299Gly is
not directly involved in the binding of
MD-2, but the mutation is located close
to the TLR4-MD-2 binding area. Al-
though there is no direct alteration of the
binding location of the LPS/MD-2 com-
plex, it has been suggested using an
ectodomain model of TLR4 that
Asp299Gly polymorphism increases the
rotational freedom of the peptide bond
(64). Interestingly, the same model also
shows that the wild-type TLR4 has a
negatively charged area at position 299,
and this is lost in the Asp299Gly (64).
The modified response of cells of
Asp299Gly polymorphism individuals
could therefore be the result of the in-
creased rotation and change of charge
that may modulate the interaction of
LPS with the TLR4 receptor.
It is tempting to conclude that TLR4
polymorphisms have an effect on the
susceptibility to infections, especially
because of their location in the ligand
recognition area of the receptor.
Despite the tempting theoretical con-
siderations and a number of reports
based on in vitro experiments, most if not
all of the studies using primary cells iso-
lated from individuals bearing the muta-
tion reveal that the Asp299Gly/Thr399Ile
haplotype has little or no effect on re-
sponsiveness to LPS, and the susceptibil-
ity to infections of the bearer is un-
changed. Therefore, we tend to conclude
that the Asp299Gly/Thr399Ile haplotype
has no distinct phenotype. In contrast,
the Asp299Gly haplotype exhibits a
stronger pro-inflammatory TNF-α cy-
tokine response after stimulation with
LPS. In addition, this phenotype seems
to predispose to septic shock. The effect
of the rare Thr399Ile haplotype on func-
tion and susceptibility remains unclear
due to its scarcity in the population.
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