The innate immune defense is ready to combat invading microbes whenever they invade our inner territories. Innate immunity consists of soluble molecules and cell-bound 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-κB 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-κB cascade.4 With 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-κB 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. 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 Iκ kinase (IKK), leading to phosphorylation, ubiquitination, and degradation of IκB and the nuclear translocation of the transcription factor NF-κB.

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-κB 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 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 differentiated 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-κB 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-3-kinase activation and ERK1/3 phosphorylation, which exceeded that caused by the TLR4 ligand LPS. In contrast, mmLDL could not trigger NF-κB 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-κB, 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 proatherosclerotic. 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-κB 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.

Footnotes