Download Toll-like receptors and human pathology

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Revisión
Inmunología
Vol. 23 / Núm 4/ Octubre-Diciembre 2004: 328-338
Toll-like receptors and human pathology
E. Sánchez*, G. Orozco*, and J. Martín
Instituto de Parasitología y Biomedicina «López-Neyra», CSIC, Granada.
* The authors have contributed equally to this work.
RECEPTORES TIPO TOLL Y PATOLOGÍA HUMANA
Recibido: 8 Noviembre 2004
Aceptado: 1 Diciembre 2004
RESUMEN
El sistema inmunitario innato desarrolló uno de los mecanismos de reconocimiento frente a las infecciones microbianas y
el inicio de defensa del hospedador. Éste mecanismo está basado
en el reconocimiento de patrones moleculares presentes en los
distintos componentes microbianos así como en determinados
ligandos endógenos. Estos patrones son reconocidos, entre otros
receptores, a través de los llamados receptores Toll-like (TLRs),
los cuales son responsables de la activación de reacciones inflamatorias y tienen un papel crítico en el inicio de la respuesta inmunitaria innata. La función de los TLRs en varias enfermedades
humanas ha sido investigada a través del estudio de polimorfismos de genes que participan en la señalización a través de TLR.
Estos estudios sugieren que varias enfermedades, incluidas enfermedades infecciosas, autoinmunitarias, ateroesclerosis e inflamatorias de las vías respiratorias están afectadas por la función
de estos TLR. Los futuros estudios en este campo continuarán
aumentando el conocimiento de la patogénesis y etiología de estas
enfermedades así como revelarán información sobre las distintas
opciones terapéuticas.
PALABRAS CLAVE: Receptores tipo toll / Polimorfismo genético / Enfermedades infecciosas / Autoinmunidad / Aterosclerosis / Enfermedades inflamatorias de las vías respiratorias.
328
ABSTRACT
The innate immune system has evolved one of the mechanisms of recognition of microbial infection and initiation of host
defence. This is based on the recognition of molecular patterns
that are present in microbial components or endogenous ligands.
These patterns are recognized, among other, through the Toll-like
receptors (TLRs), which activate inflammatory reactions and are
critical in the initiation of adaptive immune responses. The function of TLRs in various human diseases has been investigated by
the study of several polymorphisms in genes that participate in
TLR signalling. These studies have suggested that several diseases, including infectious, autoimmune, atherosclerosis and inflammatory airway diseases are affected by the TLR function. The future studies in this field will continue to improve the understanding
of the pathogenesis and aetiology of these diseases and they will
as well reveal information about therapeutic options.
KEY WORDS: Toll-like receptors / Genetic polymorphism / Infectious diseases / Autoimmunity / Atherosclerosis / Inflammatory airway diseases.
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INTRODUCTION
Innate immunity is the first-line of host defence of
multicellular organisms that rapidly operates to limit infection
upon exposure to infectious agents. Accumulating evidence
indicates that activation of the innate immune system is a
prerequisite for the induction of acquired immunity,
particularly for the induction of a T helper 1 (Th1)-cell
response(1, 2). Toll-like receptors (TLRs) play a crucial role
in innate immunity, through their ability to bind pathogenassociated molecular patterns (PAMPs)(3). The receptors of
the innate immune system that recognise PAMPs are called
pattern-recognition receptors (PRR)(4). PRRs are expressed
on the cell surface, activate signalling pathways that induce
antimicrobial effector responses and inflammation upon
recognition of PAMPs. Therefore TLRs control multiple
functions and activate signals that are critically involved
in the initiation of adaptative immune responses(5, 6). Different
TLRs appear to recognise specific microbial products
(PAMPs), including lipopolysaccharide, bacterial lipoproteins,
peptidoglycan and bacterial DNA(7). Numerous studies,
conducted mainly in vitro, have led to the identification of
a large number of different ligands for each member of the
TLR family(7, 8). Recognition of a single ligand has been
demonstrated for TLR3 in responses to poly(I:C) (doublestranded viral RNA)(9), TLR5 for responses to flagellin and
flagellated bacteria(10), and TLR9 for mediating responses
to CpG bacterial DNA(11). On the contrary, TLR2 and TLR4
have a broad specificity for the recognition of microbial
patterns. TLR2 is responsible for the recognition of Grampositive bacteria (peptidoglycan, lipoteichoic acid) (12),
mycobacterial species(13, 14), protozoan parasites(15, 16), as well
as microbial lipoproteins, glycoproteins, glycolipids, and
nonenteric LPS(17-21). TLR4 recognizes LPS(22, 23), the LPS
mimetic drug Taxol(24), the fusion protein of respiratory
syncytical virus (RSV)(25), as well as fungal ligands(26, 27).
TLRs can also dimerise upon interaction with the proper
ligand. Dimers consisting of TLR4-TLR4, TLR2-TLR6 and
TLR1-TLR2 have been described(28). The Drosophila protein
Toll was originally described as a type I transmembrane
receptor that controls the dorsal-ventral patterning of the
fly embryo(29). Toll signalling has been identified as an
essential element of an effective anti-fungal immune response
in the fly(30). Several studies have identified mammalian
homologs of Toll, proteins now referred to as Toll-like
receptors (TLR) (31-33). TLRs are grouped into the same
gene family based on their sequence similarity. Eleven
mammalian members have been described, namely TLR1
to TLR11. All of them are type I transmembrane proteins
that show peculiar structural features. Several leucine-rich
repeats (LRRs) are present in the extracellular domain of
E. SÁNCHEZ, G. OROZCO, J. MARTÍN
IL-1RI
TLR4
TLR3
MyD88
IRAK 4
MyD88independent
pathway
TRIF
IRAK 1
TRAF6
MKK
JnK
IKKγ
IKK
complex
IKKβ
IKKα
IκB
Degradation
IκB
NF-κB
AP-1
NF-κB Nucleus
Figure 1. The TLR signalling pathway.
the molecule(31). TLRs cross the cytoplasmatic membrane
once, and their intracellular portion is extremely similar to
the cytoplasmatic domain of the IL-1R and related molecules,
which is designated Toll-IL-1R or TIR domain(34). By contrast,
the extracellular region of the TLRs and IL-1R differs
markedly: the extracellular region of TLRs contains LRRs
motifs, whereas the extracellular region of IL-1R contains
three immunoglobulin-like domains.
Upon ligand binding, TLRs (homo- hetero-) dimerise
and undergo a conformational change required for the
recruitment of downstream signalling molecules. After TLR
ligation, the adaptor molecule myeloid-differentiation
primary-response protein 88 (MyD88) is recruited to the
cytoplasmatic TIR domain, where it facilitates the association
of IL-1R-associated kinase 4 (IRAK4) with the receptor
complex (Fig. 1). The binding of MyD88 to IRAK4 facilitates
IRAK4-mediated phosphorylation of crucial residue/s in
the kinase-activation loop of IRAK1, which induces the
kinase activity of IRAK1. Activated IRAK1 then
autophosphorylates, and this enables tumour-necrosis factor
(TNF)-receptor-associated factor 6 (TRAF6) to bind to the
complex. The IRAK1-TRAF6 complex then disengages from
the receptor and interacts with another preformed complex
consisting of transforming growth factor-β (TGF-β)-activated
kinase (TAK1), TAK1-binding protein 1 (TAB1) and TAB2
or TAB3. Next, IkB kinases (IKKs) are activated, and
phosphorylate the inhibitor of NF-kB (IkB). This
phosphorylation leads to the degradation of IkB and
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VOL. 23 NUM. 4/ 2004
TABLE I. Polymorphisms in TLRs genes and their relationship with human pathologies
TLR
SNP
TLR2
Arg753Gln
-16934 A/T
-196 to –174 del,
-191 G/A, 597 T/C,
1350 T/C
TLR3
-7 A/C, +71 C/A,
Leu412Phe, 1377 C/T
TLR4
Asp299Gly
Thr399Ile
TLR5
392 Stop
TLR6
Ser249Pro
TLR9
-1237 C/T
TLR10 +1031 G/A, +2322 G/A
Disease
Association
Staphylococcal, Mycobacterium leprae and Mycobacterium Tuberculosis infection
Asthma and allergies
Asthma
Yes (53-56)
Yes (131)
No (135)
Asthma
Response to inhaled endotoxin, Gram negative sepsis, severe RSV bronchiolitis, Yes (42-46, 60, 80, 92,
rheumatoid artritis, diabetic neuropathy, Crohn´s disease, ulcerative colitis,
95, 110, 111)
atherosclerosis
Meningococcal disease, rheumatoid artritis, systemic lupus erythematosus,
No (8, 47, 81, 82, 101,
multiple sclerosis, atherosclerosis, asthma
112, 113, 130)
Response to inhaled endotoxin, severe RSV bronchiolitis, diabetic neuropathy,
Yes (42, 60, 92, 96)
ulcerative colitis
Rheumatoid artritis, systemic lupus erythematosus, multiple sclerosis, asthma
No (81, 101, 130)
Legionella pneumophila infection
Yes (58)
Asthma
Yes (132)
Crohn´s disease, asthma
Asthma
consequently the release of NF-kB to the nucleus(35). MyD88
is essential for responses against a broad range of microbial
components. However, a closer study of MyD88-deficient
cells has revealed the existence of MyD88-dependent and
–independent pathways, both of which mediate signalling
in response to LPS(36).
Due to the fact that TLRs are clearly at the top of the
immune-system pyramid, great efforts have been made to
elucidate their possible role on human diseases. Here, we
review some of the findings that have arisen from these
investigations, relating to infectious diseases, autoimmunity,
atherosclerosis and inflammatory airway diseases (Table I).
TLRs AND INFECTIOUS DISEASES
Recognition of microbial non-self plays a crucial role in
host defence. This recognition strategy is based on the detection
of PAMPs that are essential products of microbial physiology,
unique to microbes and recognised by PRRs. The TLRs are
a part of this innate immune defence. The role of the TLR
family in host defence against microbes bearing one or more
of these PAMPs has been confirmed by experimental assays
with mice bearing spontaneous or genetically engineered
defects of various TLR signalling components(8). Regarding
Gram-negative infection genetic differences in susceptibility
to LPS are well established; C3H/HeJ and C57BL/10ScCR
mice strains are hyporesponsive to LPS. Since the C3H/HeJ
330
No (135)
Yes (97, 133)
Yes (134)
mouse strain is exquisitely sensitive to progressive overwhelming
infection by Salmonella typhimurium(37), extensive studies led
to the identification of tlr4 as the gene encoded by Lps, with
C3H/HeJ mice harbouring a spontaneous point mutation
(Pro712His) in the cytoplasmic signalling domain (38, 39).
Two other mouse strains bear mutations of tlr4; C57BL10/ScN
and its subline C57BL10/ScNCr carry a genomic deletion
and do not express TLR4, while motor neuron degeneration
(mnd) mutant mice have a large insertion within exon 2 that
results in an aberrantly spliced transcript predicting a truncated
protein(40). Relative to mice bearing a wild-type TLR4, C3H/HeJ
and C57BL10/Sc mutant mice have also been shown to
develop prolonged bacteremia after experimental infection
with Neisseria meningitidis, another Gram-negative bacteria
recognized for its exuberant production of LPS(41). The role
of TLR4 seems clear in response to Gram-negative LPS in
mice, and several groups have studied the effect of the two
cosegregating missense mutations (Asp299Gly and Thr399Ile)
on humans at risk for sepsis. These polymorphisms have
been associated with a blunted physiological response to
inhaled endotoxin(42), for this observation the Asp299Gly
allele has been associated with a predisposition with Gramnegative sepsis(43-46). The biological significance of the Asp299Gly
and Thr399Ile has been demonstrated by transfection of THP1 cells with either the wild-type or the mutant allele of the
TLR4 gene(46). Although these data support the relevance of
TLR4 to human LPS responsiveness, a retrospective analysis
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of the Asp299Gly mutation failed to demonstrate an association
of this polymorphism with the frequency or severity of
documented meningococcal disease(8, 47).
On the other hand a critical role for TLRs in host defence
against Gram-positive bacteria was strongly suggested by
several experiments in which TLR2-deficient mice were
unable to respond to peptidoglycan and lipoproteins derived
from these organisms (Staphylococcus aureus)(48, 49). Extensive
in vitro data strongly suggest that TLR2 participates in
mycobacterial infection(50-52). Several studies have suggested
that a mutation in the TLR2 gene (Arg753Gln) may predispose
individuals to staphylococcal, Mycobacterium leprae and
Mycobacterium tuberculosis infections(53-56). Underhill et al.(52)
reported that TLR2 is the principal mediator of macrophage
activation in response to mycobacteria and might cause
defective mycobacterial phagocytosis by macrophages,
resulting in an impaired Th1 response characteristic of
patients with lepromatous leprosy. These findings suggest
that this TLR2 polymorphism plays an important role in
defence against Gram-positive infections.
Recently a study has demonstrated that TLR5 recognizes
the flagellin protein, a potent inflammatory stimulus present
in the flagellar structure of many bacteria(10, 57). The author
also suggested that a common TLR5 stop codon polymorphism
is associated with susceptibility to infection with Legionella
pneumophila, a flagellated bacterium(58).
Regarding fungal infection, there is relatively little
evidence that implicate mammalian TLRs in antifungal host
defence. In vitro assays have shown that TLR2 recognizes
zymosan, a yeast cell wall particle, and that TLR4 plays a
role in the induction of cytokines and intracellular signalling
in response to stimulation with Aspergillus fumigatus and
Cryptococcal polysaccharide capsule, respectively(8).
There is evidence for the participation of TLR in host
defence against viral infection. Some studies using a model
of RSV infection of tlr4-deficient C57BL/10ScNcr mice
implicated TLR4 and CD14 in the recognition of RSV fusion
(F) protein(25, 59). TLR4 mutations (Asp299Gly, Thr399Ile) are
associated with an increased risk of severe RSV bronchiolitis(60).
TLR3 is another candidate for viral recognition, arising from
studies using poly (I:C), a synthetic double-stranded RNA
(dsRNA) analogue that is a molecular pattern associated
with viral infection(9). Recent reports also suggest that mouse
TLR7 and human TLR7 and TLR8 may participate in antiviral
host defence(61, 62).
TLRs AND AUTOIMMUNITY
Autoimmune disease is an attack on self-tissues by the
adaptative immune system(63). Systemic autoimmune diseases
E. SÁNCHEZ, G. OROZCO, J. MARTÍN
such as rheumatoid arthritis (RA) and systemic lupus
erythematosus (SLE) are characterized by the production
of autoantibodies, including rheumatoid factor (RF), antinuclear
antibodies and antibodies to DNA(64). The production of
circulating antibodies in response to many pathogens depends
on recognition of those pathogens by both T and B cells.
This constraint greatly reduces the risk of autoantibody
production, as T and B cells would have to break their
tolerance to the same self-antigen simultaneously. Some
antigens from pathogens are T-cell-independent, being
lipopolysaccharide (LPS) the best understood of them. LPS
is recognized by TLR4, present on all mouse B cells(38) but
not human B cells(65). High concentrations of LPS stimulate
the proliferation of mouse B cells. But at lower concentrations,
the simultaneous recognition of LPS by antigen receptors
and by TLR4 causes a synergistic signal, which provokes
proliferation and production of circulating antibodies
selectively by bacteria-specific B cells(66, 67).
The mechanisms that lead to autoantibody production
remain relatively obscure, but a report by Leadbetter et al
points to a similar mechanism in which B cells seem to
produce RF independently of T cells(68). In a mouse model
of systemic autoimmune disease, the simultaneous activation
of cell-surface antigen receptors and another toll-like receptor,
TLR9, causes a particular subclass of self-immunoglobulin
(IgG2a) to be recognized by B cells as if it were a pathogen.
This triggers T-cell-independent proliferation of B cells. In
the bloodstream of the autoimmune mice, self-IgG2a
accumulates in complexes with self-DNA. This DNA fails
to be cleared from the bloodstream for unknown reasons.
TLR9 detects bacterial DNA(11). Bacterial and vertebrate
DNAs differ by the absence of CpG methylation in bacteria.
The immune system uses TLR9 to detect the presence of
unmethylated CpG dinucleotides as a signal of infection.
In fact, vertebrate DNA is not completely methylated: only
70-80% of the CpG dinucleotides in vertebrate genomes are
actually methylated. It seems possible that those unmethylated
CpGs might be causing the observed effects. Interestingly,
DNA methylation is decreased in cells from autoimmune
mice and humans(69), which supports the hypothesis that
unmethylated self-DNA may actually be a pathogenic factor
in autoimmunity(70). In addition, drugs that can induce SLE
in humans inhibit CpG methylation(71), strengthening the
link between hypomethylated self-DNA and autoimmunity.
Although we do not know how rheumatoid factors are
involved in diseases, a high titre at diagnosis correlates with
the severity of both later RA and disease in tissues other
than joints, such as blood vessels (72). The finding that
mammalian DNA can stimulate TLR9 when present in
immune complexes was corroborated by Viglianti et al(73).
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Although TLRs are usually thought to be crucial for the
recognition of pathogens, they can also bind to self-antigens,
such as heat shock proteins, fibrinogen and others(74), released
by cells undergoing stress, damage or necrotic death. So,
B cells may simultaneously recognize these self-antigens
through cell-surface antigen receptors and TLRs, provoking
other autoantibodies to nuclear antigens such as selfDNA itself, in autoimmune diseases that are characterized
by prominent tissue damage.
Recently, another point of view about the role of TLRs
on autoimmunity has been reported(75). In this work, it is
proposed that in healthy individuals, tolerance to self antigens
is maintained by the balanced crosstalk between C-type
lectin receptors (CLRs), which sample specific carbohydrated
structures on self-antigens and induce tolerance(76), and
TLRs, which sense danger signals by PAMPS and induce
immune activation. The normally balanced CLR-TLR crosstalk
can be disturbed by overstimulation of TLRs and/or
understimulation of CLRs, leading to dendritic cell (DC)
maturation, and ultimately autoimmunity. They hypothesize
that disturbed glycosilation of self-antigens might set the
stage for autoimmunity by weakening the capacity of CLRs
to buffer «danger signals» through TLRs.
Next, we will review some of the most relevant findings
concerning the role of TLRs in some common autoimmune
diseases, such as rheumatoid arthritis (RA), systemic lupus
erythematosus (SLE), type 1 diabetes (T1D), inflammatory
bowel disease (IBD) and multiple sclerosis (MS).
Activation of the innate immune system triggers the
release of proinflammatory cytokines and chemokines
(including TNF-α) that can contribute to the initiation and/or
perpetuation of inflammatory responses, such as those seen
in RA. Active infection of the joint stimulates a strong innate
immune response, and such infection commonly culminates
in a destructive arthritis. Although the etiology of RA is
largely unknown, we know that antigen-presenting cells
(APCs) such as macrophages and DCs play an important
role in disease pathogenesis(77-79). APCs are well equipped
with several receptors involved in antigen uptake and
processing, including TLRs. TLR4 is the most investigated
TLR in RA. This receptor interacts with endogenous ligands
released by cells undergoing stress, damage or necrotic
death, which are present in inflamed synovial joints of
patients with RA, activating immune cells to produce factors
that are involved in the breakdown of the cartilage. The
most studied SNPs within the TLR4 gene are Asp299Gly
and Th399Ile, but their role on RA susceptibility is unclear.
Although Radstake et al find an association between Asp299Gly
polymorphism and RA(80), other reports show no association(81,
82). It is worth noting that the frequency of TLR4 Asp299Gly
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heterozygous is of 17% in Radstake´s manuscript, which is
considerable higher that the reported in most studies.
It has been reported that synovial cells express low levels
of both TLR2 and TLR9(83). The former is up-regulated by
exposure to peptidoglycans (PG), which is consistent with
this PAMP contributing to the development of RA. Synovial
fibroblasts from RA patients cultured with PGs increased
their expression of intercellular adhesion molecule 1 (ICAM1) and various metalloproteinases, and produced the
proinflammatory cytokines IL-6 and IL-8. This is consistent
with the evidence that PG injected intraarticularly induces
inflammation(84). Recently, it has been reported that the
nominal antigen-independent, polyclonal activation of
preactivated T cells via TLR2 has a pivotal role in the initiation
and perpetuation of pathogen-induced chronic inflammatory
joint disease(85).
Deng et al. demonstrated that intraarticular injection of
bacterial DNA induces an inflammatory arthritis(86, 87). They
showed that CpG DNA, recognised by TLR9, triggers TNFα
production, facilitating the development of CpG mediated
arthritis. This was followed by a study by Zeuner et al
documenting that suppressive oligodeoxynucleotides
specifically inhibited the immune activation induced by
CpG DNA, blocked production of TNFα, and thereby
prevented development of RA(88).
A typical characteristic of SLE is the presence of
autoantibodies against self-antigens, including nucleic acids.
Signalling through TLR9 might be responsible for that(68).
This hypothesis has been supported by Anders et al, who
found that CpG-oligodeoxynucleotides (CpG-ODN) or E.
coli DNA aggravate renal disease in MRLlpr/lpr mice, a murine
model for SLE(89). CpG-DNA increased serum levels of DNA
autoantibodies of the IgG2a isotype in the MRLlpr/lpr mice,
caused massive proteinuria, and worsened renal pathology.
Injected exogenous DNA or ODN bound to TLR9-positive
macrophages and DC in glomeruli and renal interstitium
of MRLlpr/lpr mice caused enhanced renal chemokine and
chemokine receptor expression. Thus activation of TLR9 by
unmethylated synthetic or E. coli DNA triggers exacerbation
of autoimmune lupus nephritis.
TLR4 is believed to recognize, in addition to microbial
constituents, host-derived molecules(74). A recent publication
followed this idea by showing that cell surface-expressed
GP (a paralogue of the human HSP90, which has been linked
to active SLE) as a ligand of TLR2 and TLR4 leads to a
MyD88-dependent development of lupus-like autoimmune
disease in mice, independent from lymphocyte activity(90).
Polymorphisms of TLR4 and TLR2 have been studied
concerning SLE predisposition and severity, but no association
has been found(81).
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Recently, the important role of TLR3 in the development
of T1D has been shown(91). Wen et al found that treatment
with poly (I:C), a synthetic dsRNA that can stimulate immune
responses similar to those produced by viral infections, plus
immunization with whole insulin-protein induced a high
incidence of diabetes in B6/RIP-B7.1 mice. This effect is
performed through TLR3 recognition of poly (I:C). When
they investigated TLR expression in pancreatic islets in
humans, TLR3 showed the highest expression level in all
the individuals studied.
Of note, Asp299Gly and Thr399Ile polymorphisms of
the TLR4 gene have been shown association with reduced
prevalence of diabetic neuropathy in patients with T2D(92).
Primary human intestinal epithelial cells (IECs) of normal
mucosa constitutively express TLR2, TLR3, TLR4 and TLR5
and their expression is selectively altered in patients with
IBD(93). TLR4 is strongly up-regulated in both Crohn´s disease
and ulcerative colitis, while the expression of TLR2 and
TLR5 remains unchanged. Furthermore, the expression of
TLR3 in the intestinal epithelium is down-regulated in active
Crohn´s disease, but not in ulcerative colitis. These results
suggest that IBD may be associated with distinctive changes
in selective TLR expression in the intestinal epithelium.
Interestingly, a recent study by Kobayashi et al., using
multiple genetic manipulations of the myeloid cell-specific
deletion of Stat3 mouse model, showed that enterocolitis is
significantly improved in TLR4/Stat3 double-deficient
mice(94). Furthermore, polymorphisms in TLR genes have
shown association with IBD. TLR4 Asp299Gly polymorphism
is associated with both Crohn´s disease and ulcerative
colitis(95); Thr399Ile mutation in the TLR4 gene is associated
with ulcerative colitis(96) and a promoter SNP in the TLR9
gene is associated with Crohn´s disease(97).
Activation of APCs through TLR9 is also important in
the development of MS(98). TLR4 is necessary for LPS-induced
oligodendrocyte injury in the central nervous system(99).
In mice, a transcriptional activation of TLR2 has been
associated with the clinical course of experimental autoimmune
encephalomyelitis(100). Nevertheless, Asp299Gly and Thr399Ile
polymorphisms in TLR4 do not predispose to the development
of MS(101).
TLRs AND ATHEROSCLEROSIS
Atherosclerosis is a consequence of a complicated
inflammatory process with immune reactions at the initiation
and progression of this disease. Evidence is accumulating
that shows expression of TLRs on a variety of cells, including
cells of the arterial wall(102). Human endothelial cells in normal
arteries express TLR4 and low levels of TLR2(103, 104) and in
E. SÁNCHEZ, G. OROZCO, J. MARTÍN
the atherosclerotic plaque, expression of TLR2 and TLR4
has been described in macrophages and endothelial cells(105).
It is now clear that endogenous ligands are also able to
activate immune responses through TLRs. The extra domain
A of cellular fibronectin (in vivo experiments have demonstrated
that this domain is expressed in injured arteries), chlamydial
LPS or human heat shock protein 60 (hsp60) are endogenous
ligands of TLR4 that have been involved in the induction of
atherosclerosis(106-108). The expression of innate immune
receptors in healthy arteries and atherosclerotic plaques,
and the presence of these ligands in atherosclerotic and
injured arteries, suggest that the TLR are involved in the
development and progression of atherosclerotic disease(109).
Recently, the Asp299Gly TLR4 polymorphism has been
associated with atherosclerosis progression. In a case-control
study it was found that subjects with the Asp299Gly allele
had lower levels of some of the inflammatory cytokines,
acute-phase reactants, soluble adhesion molecules, and other
mediators of inflammation; also this allele was associated
with a decreased risk of atherosclerosis(110). Another study
in patients with acute coronary syndromes confirm previous
results, since the Asp299Gly allele was associated with a
decreased risk of acute coronary events independently of
standard coronary risk factors (OR 0.41; 95% CI 0.18-0.95;
P= 0.037)(111). Both results suggest that this TLR4 polymorphism
protects against the progression of atherosclerosis disease.
But conflicting results have also been reported. A study
found no difference in the frequency of TLR4 polymorphisms
in patients requiring carotid endarterectomy, compared to
controls(112) and another study suggested that predisposition
to and progression of coronary artery stenosis are not related
to this TLR4 polymorphism(113), although Gly299 allele carriers
had a greater reduction in cardiovascular events when treated
with pravastatin(114).
The role of TLRs in the cardiac function has also been
demonstrated in tlr2-knockout mice. This study suggests
that TLR2 is involved in cardiac remodelling after myocardial
infarction(115).
The conflicting reports do not exclude, however,
involvement of TLR4 in atherosclerosis; the lack of association
in some studies provide additional evidence against the
importance of TLR4 polymorphism in either cardiac or
carotid artery stenosis(112, 114). It is possible that polymorphisms
within genes in these pathways, other than TLR4, are important
in the pathogenesis of coronary artery disease; other candidate
genes have been associated with coronary atherosclerosis
or acute coronary syndromes, such as the genes encoding
CD14(116, 117), IL-6(118) or MyD88(119).
In conclusion, these data support the concept that an
efficient innate immune defence against bacteria and associated
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long-term intravascular inflammatory stress are involved
in the development of atherosclerosis.
TLRs IN INFLAMMATORY AIRWAY DISEASES
The role of TLR proteins in inflammatory airway diseases,
such as asthma and allergy is being intensively studied.
These studies are based on two contradictory lines of reasoning.
First, exposure to LPS increases the severity of asthma. A
study on people sensitive to house dust mite allergen showed
that the severity of their asthma correlated more closely
with levels of LPS than with those of the allergen itself(120).
People with allergic asthma are also more sensitive to the
bronchoconstrictive effects of inhaled endotoxin(121) than are
non-asthmatics. Thus, LPS can exacerbate asthma, probably
by increasing the extent of airway inflammation, through
its recognition by TLRs. In contrast to its exacerbating effect
on asthma, exposure to LPS and other TLR ligands in early
childhood may, paradoxically, decrease the incidence of
asthma later in life (122, 123). The essence of the «hygiene
hypothesis»(124) is the idea that exposure to specific infections,
or perhaps endotoxins, drives the maturing immune system
in infancy/childhood towards a Th1 phenotype, and away
from the Th2 phenotype associated with atopy. Since TLRs
are implicitly involved in responses to many pathogenrelated molecules, they may be critical receptor pathways
involved in maturation of the normal adult immune system.
There is some evidence for this hypothesis, albeit indirect,
since polymorphisms in CD14, the correceptor involved in
LPS signalling via TLR4, correlate with IgE levels(125). It is
clear that LPS can either exacerbate or diminish the severity
of asthma, depending on the timing of LPS exposure and
whether the disease or its exacerbations result primarily
from LPS or allergens. In addition, the type of TLR stimulation
during the initial phase of immune activation determines
the polarization of the adaptative immune response and
may play a role in the initiation of Th2-mediated immune
disorders, such as asthma(126). These results may also be
explained by the finding that high doses of LPS activate the
CD4+ CD25+ T regulatory (Treg) cells that may prevent
the activation of pathogenic T cell clones(127). On the contrary,
low doses of LPS activate DCs that in conjunction with IL6 production, release T cells from the inhibitory effect of the
Treg cells(128), thereby leading to the activation of the pathogenic
T cell clones.
The importance of TLRs in allergic diseases is less well
understood. Studies of polymorphisms in TLR genes have
the potential to advance our understanding of the response
to endotoxin and the pathogenesis of allergic disease. The
best-studied polymorphism is the Asp299Gly amino acid
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substitution of TLR4. This SNP is associated with differences
in LPS responsiveness in humans, demonstrating that genesequence changes can alter the ability of the host to respond
to environmental stress(42). In addition, Asp299Gly and
Thr399Ile polymorphisms in TLR4 gene have been shown
to be associated with a modified response to endotoxin in
asthmatic patients(129). On the other hand, other studies
analysing TLR4 polymorphisms-asthma associations have
yielded heterogeneous results. Raby et al.(130) found no
evidence that genetic variation in TLR4 contributes to asthma
susceptibility. Yang et al.(113) could confirm the previously
observed lack of association of TLR4 polymorphisms with
asthma. Nevertheless, their findings suggest that genetically
determined hyporesponsiveness to endotoxin may increase
atopy severity. These findings are consistent with the known
ability of LPS to exacerbate existing asthma and to decrease
atopy and suggest that the Asp299Gly polymorphism is
predictive of airway and atopic responses in a specific subset
of the population.
Polymorphisms mapping to other TLRs genes have been
studied in relation to asthma and allergy susceptibility, but
not as widely as the TLR4 genetic variants. Regarding to the
TLR2 gene, -16934A/T genetic variation is a major determinant
of the susceptibility to asthma and allergies in children of
farmers (131). Furthermore, SNPs in TLR6, TLR9 and TLR10
genes have shown association with asthma(132-134). Recently,
Noguchi et al.(135) screened the 5´ flanking and coding regions
of TLR2, TLR3, TLR4 and TLR9 for polymorphisms. They
found 16 variants, even though none of the alleles or haplotypes
were associated with asthma or total IgE levels in a Japanese
population.
CONCLUDING REMARKS
TLRs are a family of transmembrane receptors, some of
which have been clearly demonstrated to play a key role in
innate immunity. The first focus of research was on the TLR
signalling pathway. The identified LPS signalling mediators
may be important pharmacological targets, and the future
identification of all the component of the LPS signalling
cascade will be an invaluable platform for designing
therapeutical interventions. Also, the effect of various TLR
polymorphisms on disease progression becomes better
understood, a patient’s genotypic profile will comprise an
increasingly important factor in the consideration of various
therapeutic options. Future studies will need to address the
biological importance of the known differences in TLR
function, including expression patterns, ligand specificities
and the relationships among TLR signalling, genetic
polymorphism and human disease.
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CORRESPONDENCE TO:
Javier Martín MD, PhD
Instituto de Parasitología y Biomedicina «López Neyra», CSIC
Parque Tecnológico de Ciencias de la Salud.
Avenida del Conocimiento s/n
18100 Armilla (Granada), Spain.
Phone: 34-958-181669. Fax: 34-958- 181633
E-mail: [email protected]
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