Toll To Be Paid at the Gateway to the Vessel Wall

Editorial
Toll To Be Paid at the Gateway to the Vessel Wall
Göran K. Hansson, Kristina Edfeldt
T
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Hoffmann’s discovery, Toll was, for the first time, associated
with host defense.
Was there a mammalian equivalent of this defense protein in
the fruit fly? Ruslan Medzhitov and Charles Janeway at Yale
University were the first to report the cloning of a mammalian
homologue, a Toll-like receptor (now called TLR4).5 Its ligand
remained unknown. However, by constructing a constitutively
active mutant, Medzhitov et al could determine that TLR
induces NF-␬B activation in a similar way as ligation of the
interleukin (IL)-1 receptor—and similarly to Drosophila Toll.
The Figure displays the signal transduction pathway for mammalian TLR4.
How could mammalian TLR participate in host defense?
Does it bind a “danger molecule” produced by the host on
infection? Or a microbial molecule released from the pathogen?
This important question was answered by Alexander Poltorak
and Bruce Beutler in Dallas, who discovered that TLR4 is the
long-sought receptor for LPS, the active component in endotoxin
from Gram-negative bacteria.6 With this finding, it became
obvious that TLRs constitute a family of pattern recognition
receptors, which ligate “pathogen-associated molecular patterns.” The existence of such receptors had been predicted by
Charles Janeway several years earlier, but their nature had
remained enigmatic.7
Since these fundamental discoveries, the TLR family of
pattern recognition receptors have become a major component in
innate immunity, innate-adaptive crosstalk, infectious diseases,
and inflammatory conditions. Not surprisingly, TLRs are also
involved in cardiovascular diseases. TLR ligation on endothelial
cells is a key step in septic shock.8 Through TLR4, circulating
LPS initiates an NF-␬B signal that leads to nitric oxide production, vasodilation, and hypotensive shock. Ligation of TLR2 by
Gram-positive bacteria may cause a similar reaction, and inflammatory activation can also be induced when bacterial unmethylated CpG DNA binds TLR9.9
Several reports over the last 4 years show that TLRs may also
be involved in atherosclerosis. The entire spectrum of TLRs is
expressed by macrophages and endothelial cells of human
atherosclerotic plaques,10 and TLR4 may be particularly important because it is expressed by macrophages and upregulated by
oxidized LDL.11 Mildly oxidized (minimally modified, mm)
LDL binds to CD14, a glycophosphatidylinositol anchored cell
surface protein; the mmLDL-CD14 complex binds to TLR4 and
triggers a cellular response in the macrophage.12 In addition
to mmLDL, TLR4 also binds heat shock protein-60, another
antigenic protein implicated in atherosclerosis.13
Several lines of evidence suggest that TLR ligation is
proatherogenic. Mice lacking TLR4 develop smaller neointimal
lesions after vascular injury,14 and TLR2 has similar effects.15
Two studies of hypercholesterolemic apoE⫺/⫺ mice show that a
defect in the TLR-associated signal transduction protein,
MyD88, reduces atherosclerosis,16,17 and the absence of TLR4
he innate immune defense is ready to combat invading
microbes whenever they invade our inner territories.
Innate immunity consists of soluble molecules and cellbound receptors, all of which are encoded in the germline DNA.
This contrasts with the adaptive immune system, the molecular
components of which are generated by somatic rearrangement
processes. In atherosclerosis research, much interest has been
focused on a soluble component of innate immunity, the acute
phase protein C-reactive protein (CRP). Other members of the
innate immune family are also involved in the atherosclerotic
process, including the complement cascade, the antimicrobial
peptides, and the pattern recognition receptors (PRR).
See pages 1213 and 1220
The large group of PRRs contains several families of receptors. Their common denominator is a broad specificity with a
capacity to bind many different macromolecules produced by
invading microbes. The first PRRs to be discovered were the
scavenger receptors (ScR), which were identified as transmembrane receptors binding lipopolysaccharide (LPS) of endotoxins,
acetylated LDL, and certain polynucleotides. Michael Brown
and Joseph Goldstein discovered ScR in 1979,1 and the first one
of them, SR-A, was cloned in 1990.2
The next step in PRR discovery came from an entirely
different line of research. Christiane Nüsslein-Volhard of the
Max Planck Institute in Tübingen analyzed mutations in fruit
flies. In 1985, she saw a weird-looking fly larva in which the
ventral portion of the body was underdeveloped. Her spontaneous comment was “Das war ja toll!” meaning “That was weird!”
and she coined the name Toll for the mutated gene. The protein
product of the Toll gene was found to cause ventralization, and
normal functional activity of Toll is necessary for dorsoventral
polarity in the fly. The discovery of Toll was one in a series of
discoveries of genes controlling early embryogenesis, which led
to a Nobel prize for Nüsslein-Volhard in 1995.
A decade after Nüsslein-Volhard’s discovery of Toll, Jules
Hoffmann’s laboratory in Strasbourg reported that Toll not only
controls dorsoventral polarity but also has a role in the immune
defense in Drosophila.3 Without Toll, flies did not survive fungal
infection. Interestingly, Toll activation triggered an NF-␬B
cascade, which mounted the defense against fungi. It was
already known that Spätzle, the protein that induces ventralization by binding to Toll, elicits an NF-␬B cascade.4 With
From the Center for Molecular Medicine and Department of Medicine,
Karolinska Institute, Stockholm, Sweden.
Correspondence to Göran K. Hansson, Center for Molecular Medicine
and Department of Medicine, Karolinska University Hospital L8:03 Karolinska Institute SE-17176 Stockholm, Sweden. E-mail goran.hansson@
cmm.ki.se
(Arterioscler Thromb Vasc Biol. 2005;25:1085-1087.)
© 2005 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000168894.43759.47
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Arterioscler Thromb Vasc Biol.
June 2005
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Schematic view of the TLR4-induced signal transduction pathway.
Ligands bind to the CD14-TLR4 complex, which is linked to a
chain of transducing proteins including MyD88 and IRAK. TRAF6
conveys the signal to a complex that activates the MAP kinase
cascade, which ends on the transcription factor AP-1. In parallel,
the TRAF6 mediated signal activates I␬ kinase (IKK), leading to
phosphorylation, ubiquitination, and degradation of I␬B and the
nuclear translocation of the transcription factor NF-␬B.
itself also reduces disease in such mice.17 Interestingly, the effect
of homozygous TLR4 deficiency was only 24% in the study by
Michelsen et al, while homozygosity for a targeted MyD88
allele was 57%.17 Of note, MyD88 transduces signals not only
from TLRs but also from the IL-1 and IL-18 receptors, both of
which are involved in proatherogenic signaling.18,19 In addition,
both studies were performed using fat-fed apoE⫺/⫺ mice, which
develop excessive hypercholesterolemia, extremely rapid atherosclerosis, and immunologic perturbations,20 all of which
could mask more subtle modifying effects of specific receptors.
It is evident from several studies that TLR ligation induces
inflammatory mediator release, and it may impact on atherosclerosis by promoting inflammatory activity in the plaque. However, recent studies reveal that metabolic effects must also be
considered. In this issue of Arteriosclerosis, Thrombosis, and
Vascular Biology, Kazemi et al show that TLR ligation directly
affects lipid accumulation in macrophages.21 They treated RAW
264.7, a mouse macrophage line, with ligands for TLR2, TLR3,
and TLR4. Both zymosan, a TLR2 ligand, and LPS, which binds
to TLR4, increased expression of adipocyte fatty acid– binding
protein (aP2) and caused accumulation of cholesteryl ester and
triglyceride in the cells. By assisting with the transport of fatty
acids needed for cholesteryl esterification and triglyceride accumulation, aP2 promotes intracellular lipid accumulation in dif-
ferentiated adipocytes and also in lipid-loaded macrophages.
This process is important for atherosclerosis, because aP2
deficiency reduces lesion formation and progression in genetically hypercholesterolemic mice.22–24 The new report by Kazemi
et al identifies a potentially important mechanism for induction
of aP2 in atherogenesis, namely TLR ligation.
aP2 is not the only target gene by which TLR signaling
modulates cellular lipid metabolism. TLR3 and TLR4 inhibit
cholesterol efflux by reducing the expression of ABCA1 and
several other genes.25 This is attributable to an inhibition of the
transcription factor, lipid-X receptor (LXR). Of note, effects on
LXR/ABCA1 were observed with ligands for TLR3 and
TLR4,25 while effects on aP2 were recorded with ligands for
TLR2 and TLR4 but not TLR3.21 This suggests that the crosstalk
between innate immunity and lipid metabolism is complex and
may involve several signal transduction pathways other than the
well-known MyD88-NF-kB pathway. Indeed, the inhibition of
LXR is independent of NF-␬B and mediated through another
transcription factor, IRF-3.25
Another article published in this issue of Arteriosclerosis,
Thrombosis, and Vascular Biology adds additional pieces to the
puzzle. Miller et al26 present an analysis of the signals through
which mmLDL induces proinflammatory gene expression in
macrophages. mmLDL induced robust phosphoinositide-3kinase activation and ERK1/3 phosphorylation, which exceeded
that caused by the TLR4 ligand LPS. In contrast, mmLDL could
not trigger NF-␬B activation. The pattern of induced downstream genes therefore differs between mmLDL and LPS, and it
is concluded that mmLDL induces some of its effects on
macrophages through TLR4 and others independently of TLR4.
The components of mmLDL which trigger these different
pathways remain unclear, although an oxidized phospholipid,
ox-PAPC, was recently reported to target TLR4 and activate
MAP kinases, but not NF-␬B, in endothelial cells.27 Adding to
complexity, ox-PAPC inhibits TLR4 translocation to lipid rafts,
resulting in reduced signaling.28
What to conclude from all this information? The tightly
woven network of molecular interactions between pattern recognition receptors, intracellular and extracellular lipids, and
transcription regulatory pathways has just begun to be unraveled,
and many surprises are undoubtedly waiting ahead of us. But the
fact that such interactions occur, on several different levels,
points to the importance of lipids and lipoproteins in molecular
pattern recognition and to the power of innate immunity as a
regulator of metabolism.
Our understanding of the clinical relevance of pattern recognition in cardiovascular disease is equally limited. Until now,
focus has been on two polymorphic sites in coding regions of
TLR4. The Asp299Gly and Thr399Ile polymorphisms cause
hyporesponsiveness to LPS in macrophages and have been
examined for their effect on atherosclerotic cardiovascular disease in several studies. The first one used ultrasonographic
analysis of intima-media thickness in the common carotid artery
as a surrogate marker for atherosclerosis in 810 individuals of
the Bruneck study.29 55 subjects carrying the 299Gly allele had
lower risk of carotid atherosclerosis and a smaller intima-media
thickness than those carrying the wild-type allele. These data
suggest that the hyporesponsive TLR4 allele protects against
atherosclerosis and hence that TLR4 signaling is proatheroscle-
Hansson and Edfeldt
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rotic. However, a larger study using myocardial infarction as a
hard end point arrived at a different conclusion.30 The 299Gly
and 399Ile were significantly more common among 1213
survivors of a first myocardial infarction than in a matched
control group of 1561 individuals in the Stockholm Heart
Epidemiology Program (SHEEP). The reason for this discrepancy is unknown; one may speculate that TLR4-dependent
activation of the AP-1 and NF-␬B pathways promote smooth
muscle proliferation and the formation of a stable fibrous cap.30
In the Dutch REGRESS study of myocardial infarction, 299Gly
carriers had more severe coronary disease and gained more from
statin treatment than those carrying the wild-type allele.31
At balance, the clinical studies of TLR4 polymorphism
suggest that (1) TLR4 activation promotes lesion growth; (2) it
reduces the risk for myocardial infarction, possibly by promoting
plaque stabilization; and (3) the TLR4 pathway interacts with
statins. These findings demonstrate that innate immunity plays a
role in atherosclerosis and ischemic heart disease, but we do not
yet understand the precise mechanisms. The 2 studies published
in this issue of Arteriosclerosis, Thrombosis, and Vascular
Biology identify molecular mechanisms that may be important in
the immunopathogenesis of atherosclerosis. Therefore, they may
contribute to the phenotypes observed in the epidemiological
studies of cardiovascular disease. These are exciting times at the
heart of immunology.
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Toll To Be Paid at the Gateway to the Vessel Wall
Göran K. Hansson and Kristina Edfeldt
Arterioscler Thromb Vasc Biol. 2005;25:1085-1087
doi: 10.1161/01.ATV.0000168894.43759.47
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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