Review

Correspondence *Universität Würzburg, Medizinische Klinik und Poliklinik I, Herzkreislauf-Zentrum, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany

Email frantz_s@medizin.uni-wuerzburg.de

Introduction

Most microorganisms encountered by healthy individuals are recognized initially by defense mechanisms that are not antigen-specific, a response mediated by the so-called innate immune system. In contrast to adaptive immunity, in which specific antigen receptors are generated by somatic hypermutation and selection, in the innate immune system germline-encoded receptor proteins recognize specific patterns that are shared by groups of pathogens, but not the host. These receptors—'pattern recognition receptors' (PRRs)—detect pathogen-associated molecular patterns (PAMPs) that remain largely unvaried, such as lipopolysaccharide found on the cell surface of Gram-negative bacteria, or double-stranded RNA present in viruses.^{1, 2, 3}

In 1997, vertebrate homologs of the Drosophila spp. transmembrane PRR 'Toll' were identified and termed the 'Toll-like receptors' (TLRs).⁴ To date, 11 human and 13 mouse TLRs have been cloned.³ TLRs have since been shown to be central to the innate immune response. Here, we aim to review the exciting field of TLR activation in the cardiovascular system, and the role individual TLR family members could have in the pathophysiology of cardiovascular diseases.

Toll-like-receptor activation and the 'danger' model

The ligands for TLRs are molecular motifs associated with pathogens and not with the host.³ TLR classes 1, 2, 4 and 6 recognize lipopeptides. The major ligands for TLR2 include the following: specific cell-wall components (such as lipopeptides) of Gram-positive and Gram-negative bacteria; mycobacteria; fungi; parasites; and viruses. TLR4 recognizes specific components of Gram-negative bacteria lipopolysaccharide. In contrast to these plasma-membrane-localized TLRs, the so-called antiviral TLRs—types 3, 7, 8 and 9—are almost exclusively found in intracellular compartments (such as endosomes), and recognize viral PAMPs (e.g. double-stranded RNA in the case of TLR3). Another class of TLR binds protein ligands; flagellin, for example, is detected by TLR5.³

TLRs are commonly accepted to be activated only by microbial patterns; however, in the 'danger' model, Matzinger⁵ proposes that the presence of potentially infectious PAMPs does not necessarily trigger an immune response unless there is evidence of host tissue injury signified by so-called 'alarm' signals (the first potential 'danger' signal was identified as crystalline uric acid in 2003⁶). In support of this hypothesis, Matzinger and colleagues have demonstrated that, in the absence of any foreign pathogens, resting dendritic cells can be activated by virally infected or necrotic cells, but not by healthy cells or cells undergoing programmed cell death (apoptosis).⁷ This model is of particular interest as it could explain the activation of the innate immune system in primarily non-immune-related diseases. TLR4, for example, recognizes the chemotherapeutic agent paclitaxel.⁸ Debate surrounds these studies, however, because of the possibility that lipopolysaccharide or lipopeptide contamination causes subsequent TLR activation. Despite this uncertainty, there is increasing evidence to indicate that TLRs can also detect host-derived molecules under certain circumstances. Heat shock protein 60, a molecular chaperone conserved in both invertebrates and vertebrates, can activate nuclear factor kappa B (NFB) by binding to either TLR2 or TLR4.⁹ Furthermore, upon tissue injury, fragments of hyaluronan and fibronectin are released that can also activate NFB through the same TLRs.^{10, 11} The extracellular matrix can, therefore, stimulate the innate immune response via TLRs when it is altered after tissue destruction, even in the absence of pathogens. Recognition and activation of host-derived molecules by TLRs could be an important link between cardiovascular diseases and the activation of the immune system. Indeed, investigations indicate that TLRs could have a major role in the development and progression of atherosclerosis and heart failure.

Toll-like-receptor signaling

When activated, the TLR signaling network controls the initiation, maintenance, modulation and termination of innate host defenses by several mechanisms.³ First, TLR activation induces the production of proinflammatory cytokines and antimicrobial molecules such as nitric oxide, which activate cellular immune components. This response enables macrophages to eliminate invading microorganisms. Second, TLRs are expressed on dendritic cells and when activated stimulate dendritic cell maturation, which in turn can stimulate T-cell expansion and differentiation, thereby initiating an adaptive immune response. Finally, TLRs induce the expression of costimulatory molecules (e.g. B7-1 [CD80] and B7-2 [CD86]) necessary for sustained activation of adaptive immunity.

TLRs are type 1 membrane-spanning receptors that contain a leucine-rich repeat motif and a signaling motif similar to those of the interleukin (IL-) 1 and IL-18 receptors.³ This common intracellular signaling motif is now termed the 'Toll–IL-1 receptor' (TIR) homology domain, and its presence identifies members of the TIR superfamily.³ As mentioned previously, TLR types 1, 2, 4, 5 and 6 are expressed on the cell surface. Conversely, the antiviral TLRs (types 3, 7 and 9) are localized to intracellular endosomal compartments. TLR8 seems to be found primarily in the intracellular compartment, although a small proportion of the receptors are expressed on the cell surface. This distribution enables recognition of plasmatic as well as intracellular PAMPs.

Each TLR activates a number of signaling pathways, some of which are common to all TLRs and some of which are specific to particular TLR types. This differential induction pattern is dependent largely on which cytoplasmic adaptor molecules are present to associate with the intracytoplasmic domain of the TLRs (Figure 1).¹² These adaptors, which all contain TIR domains, include MyD88 (myeloid differentiation primary response protein), TIRAP (TIR domain-containing adaptor protein), TRIF (TIR-domain-containing adaptor inducing interferon ), and TRAM (TRIF-related adaptor molecule).

Figure 1 The Toll-like receptor signaling pathway

Abbreviations: AP1, activator protein 1; HSP-60, heat shock protein 60; IB, inhibitor of nuclear factor B; IKK, inhibitor of nuclear factor -B kinase ; IKK, inhibitor of nuclear factor -B kinase ; IKK, inhibitor of nuclear factor -B kinase ; IKK, inhibitor of nuclear factor -B kinase ; IRAK1, interleukin 1 receptor-associated kinase 1; IRAK4, interleukin 1 receptor-associated kinase 4; IRF3, interferon regulatory factor 3; IRF5, interferon regulatory factor 5; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; MyD88, myeloid differentiation primary response protein; NFB, nuclear factor B; RIP1, receptor-interacting protein 1; TAB1, TAK1-binding protein 1; TAB2–TAB3, TAK1-binding proteins 2 and 3; TAK1 (M3K7), transforming growth factor--activated kinase 1; TBK1, serine–threonine-protein kinase; TIRAP, TIR domain-containing adaptor protein; TLR4, Toll-like receptor 4; TRAF6, tumor necrosis factor receptor-associated factor 6; TRAM, TRIF-related adaptor molecule; TRIF, TIR-domain-containing adaptor inducing interferon ; Ub, ubiquitin; UB2V1, ubiquitin-conjugating enzyme E2 variant 1; UBE2N, ubiquitin-conjugating enzyme E2N.

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Toll-like receptors and the vascular system

Potential role in atherosclerosis development

Atherosclerosis is characterized by chronic local inflammation of the vascular wall, which results in accumulation of lipids and macrophage-derived foam cells in the subendothelial space.¹⁸ Although the inflammatory nature of the disease process has been widely accepted, the precise components of the atherogenic proinflammatory cascade remain controversial.

Infectious agents

Epidemiologic studies have shown that bacterial infection and atherosclerotic disease are connected,¹⁹ and have suggested there could be a link between atherosclerosis and TLR activation. Common infectious agents such as Chlamydia pneumoniae have been detected within different atherosclerotic lesions and could act as TLR ligands.¹⁸ Seroepidemiologic and pathological observations in animal models have raised the possibility that C. pneumoniae might be an infectious vector that can stimulate atherogenesis and, therefore, that it could be a target for therapy.¹⁹ To date, drug intervention trials of antibiotics have failed to support this hypothesis,²⁰ but these results do not necessarily or conclusively exclude the potential involvement of other infectious agents in the pathogenesis of atherosclerosis.²⁰

Polymorphisms

Several genetic analyses of polymorphisms in the genes encoding TLRs have been carried out to establish a link between TLRs and atherosclerosis (Table 1). Of all the TLR mutations identified, two single-nucleotide polymorphisms of TLR4—Asp299Gly and Thr399Ile—have been studied the most extensively. The biological importance of these polymorphisms is not yet clear. Impaired TLR4 signaling and a blunted response to inhaled lipopolysaccharide were initially described in carriers of these alleles,^{21, 22} but subsequent studies could not reproduce this deficit in TLR signaling.²³ Initial reports found both alleles to be associated with protection from carotid artery atherogenesis; patients with the polymorphisms had a lower risk of carotid atherosclerosis and smaller intima-media thickness as measured by carotid Doppler ultrasonography than did patients without the gene mutations.²² This result, however, could not be reproduced in several larger studies.²⁴

Table 1 A summary of the genetic analyses of polymorphisms in the TLR genes

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Several studies have tried to link the risk of myocardial infarction with TLR polymorphisms, with inconsistent results (Table 1). Although most studies have reported a reduced risk of myocardial infarction in individuals with a TLR polymorphism,^{25, 26} the largest study of nearly 5,000 individuals found no association between the TLR4 Asp299Gly polymorphism and myocardial infarction.²⁷ A concern in all studies is the wide range of allele frequencies in the control groups, which could possibly be caused by population and ethnic differences. This theory is supported by the fact that the Asp299Gly polymorphism could not be detected in a Chinese population.²⁸ In the only meta-analysis of these genetic analyses, which included 7 studies, 5,926 cases and 4,375 control individuals, the pooled odds ratio for myocardial infarction was 0.9 (95% CI 0.68–1.19)—consistent with a reduction in myocardial infarction risk of up to 30% in carriers of the Asp299Gly polymorphism compared with controls.²⁷ Now, larger studies are required to establish whether there is an association between TLR and risk of myocardial infarction.

Clinical evidence

The theory that TLRs are linked with atherosclerosis is further supported by correlations seen in clinical studies. The activation of TLR4 signaling is related to the downstream release of inflammatory cytokines in patients with acute myocardial infarction.^{29, 30} An increase in levels of circulating TLR4-positive monocytes was also observed in patients with unstable angina and acute myocardial infarction.³⁰ Furthermore, TLR4 levels in monocytes were higher in patients with heart failure following acute myocardial infarction than in healthy controls, and were sustained at this higher level.²⁹

Experimental evidence

Experimental data are in line with these clinical findings. Several TLR types (1, 2, 4 and 5) are expressed in atherosclerotic plaques by resident cells and leukocytes that migrate into the arterial wall.^{31, 32} Notably, TLR4 levels can be upregulated by proatherogenic proteins such as oxidized LDL,³³ whereas laminar flow, a well-known protective factor against atherosclerosis progression, downregulates TLR2 expression.³⁴

Studies using loss-of-function approaches have demonstrated convincingly that TLRs have an important role in the development of atherosclerosis. Double homozygous progeny of Tlr4 knockout mice crossed with the atherosclerosis-prone apolipoprotein E (Apoe) knockout mice exhibited reduced atherosclerosis when compared with Apoe knockout controls, even though serum cholesterol levels did not differ.^{35, 36} The knockout of Myd88 also reduced plaque burden when compared with appropriate controls; Myd88–Apoe double-knockout mice had 60% less atherosclerotic lesion than did Apoe knockout mice.³⁶ Inappropriate arterial remodeling, an important cause of vascular pathologies including atherosclerosis and restenosis, could also be related to TLR signaling. In a femoral artery cuff model and a carotid artery ligation model, outward arterial remodeling was blunted in Tlr4-deficient mice.³⁷ In many cell types, TLR4 signaling depends on accessory molecules such as the lipopolysaccharide-binding protein CD14; however, atherosclerosis development is not CD14-dependent. Similar atherosclerosis severity was seen in Cd14–Apoe double-knockout and Apoe knockout mice.³⁵ In conclusion, the evidence that TLR4 has proatherogenic effects is convincing.

TLR2 signaling also seems to be involved in the development of atherosclerosis; Tlr2 knockout mice had a modest reduction in the degree of atherosclerosis when crossed with LDL receptor (Ldlr) knockout mice to produce a double homozygous strain.³⁸ Transplantation of Tlr2 knockout bone marrow tissue into unstimulated Ldlr knockout mice did not reduce atherosclerotic burden; therefore, we can deduce that cells that do not originate from bone marrow are responsible for the proatherogenic effects of TLR2 in the absence of a TLR2 ligand. Conversely, application of a TLR2 agonist dramatically increases disease burden in Ldlr knockout mice (Figure 2). In contrast to atherosclerosis development without TLR2 stimulation, increased plaque burden is dependent on TLR2 expression in bone-marrow-derived cells, as atherosclerotic lesions are decreased in Ldlr knockout mice with Tlr2 knockout bone marrow cells. Evidence also indicates that TLR2 activation contributes to restenosis after stent implantation, as exogenous ligand stimulation of TLR2 induces neointima formation and atherosclerosis development after vascular injury.³⁹ Moreover, TLR2 expression significantly increases after vascular injury, whereas neointima formation is markedly decreased in Tlr2 knockout mice after femoral artery injury.⁴⁰ TLR2, therefore, seems to have an important proatherogenic role.

Figure 2 Aortic atherosclerotic lesions in mice exposed to the synthetic Toll-like receptor 2 agonist Pam3

(A) Aortic atherosclerosis expressed as a fraction of total lesion area after 12 weeks of a high-fat diet and weekly intraperitoneal injections of vehicle, 25 g Pam3 or 50 g Pam3 in female Ldlr knockout and Ldlr–Tlr2 double-knockout mice. Aortas from Ldlr knockout mice treated with (B) vehicle, (C) 25 g Pam3, and (D) 50 g Pam3. A dose–response effect of Pam3 administration on lesion development was observed. Profuse abdominal aortic atherosclerosis was observed in mice exposed to 50 g Pam3. Lesion severity in TLR2-deficient animals was not affected by Pam3. Scale bar 0.5 cm. Permission obtained from American Society for Clinical Investigation © Mullick AE et al. (2005) J Clin Invest 115: 3149–3156. Abbreviations: Ldlr, LDL receptor; TLR2, Toll-like receptor 2; Veh, vehicle.

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Toll-like receptors and myocardial disease

TLRs are not just expressed and functional in immune or vascular cells, but are also readily detectable in cardiac myocytes. Among the classes tested so far, 2, 3, 4 and 6 are expressed in cardiac myocytes, whereas types 1 and 5 are not.^{15, 46} A potential role of TLR2 in the response to oxidative stress has been established in neonatal rat cardiac myocytes in vitro. Blockade of TLR2 function inhibited hydrogen-peroxide-induced NFB activation and diminished the toxic effects of hydrogen peroxide, and consequently apoptosis.⁴⁶ Conversely, TLR4 activation has been shown to reduce apoptosis of cardiac myocytes, an effect mediated by nitric oxide synthase 2,⁴⁷ which suggests that TLR signaling could be important for development of myocardial diseases.

Heart failure

Heart failure is a complex disorder, the development of which involves several pathophysiologic processes including inflammation.^{1, 55} TLRs and their signaling components are activated in heart failure, both in experimental models and in the clinic. TLR4 expression, for example, is increased in the myocardium of patients with advanced heart failure.^{15, 56} In addition, the TLR expression pattern changes in the presence of heart failure. In normal murine and human myocardium, TLR4 expression is diffuse and predominantly confined to cardiac myocytes; in myocardium from patients with advanced heart failure, however, there are focal areas of intense TLR4 staining (Figure 3).^{15, 56} The reason for this change in TLR4 expression in the failing myocardium is not yet known;¹⁵ however, IRAK1 as well as NFB (key components of TLR signaling; Figure 1) are activated by cardiac ischemia, as seen in experimental models and in human heart failure (Figure 4).^{57, 58, 59} Taken together, these observations provide strong evidence that TLRs and their signaling components are activated by cardiac ischemia and heart failure.

Figure 3 Toll-like receptor 4 expression in rat, murine and human myocardium

(A) Primary isolates of adult rat ventricular myocytes 24 h after isolation, stained with a polyclonal antibody targeted to a TLR4-specific epitope adjacent to the cytoplasmic Toll–interleukin 1 receptor domain of the human TLR4 protein. (B) Normal murine cardiac muscle (200 magnification) exhibits diffuse, homogeneous myocyte staining. (C) Cardiac myocytes adjacent to an area of ischemic injury induced by coronary artery ligation exhibit intense sarcolemmal TLR4 staining. (D) Cardiomyocytes from humans with dilated cardiomyopathy displayed intensely stained focal expression of TLR4. Permission obtained from American Society for Clinical Investigation © Frantz S et al. (1999) J Clin Invest 104: 271–280. Abbreviation: TLR4, Toll-like receptor 4.

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Figure 4 Nuclear factor B activation in the heart after ischemic injury

In transgenic mice that express a luciferase reporter the transcription of which is dependent on NFB, light generated at the site of NFB activation within the transgenic mouse is sufficiently intense to be detected externally by a light-sensitive camera upon injection of luciferin. Myocardial infarction induced NFB-dependent in vivo luminescence in the heart of (A) transgenic mice when compared with (B) sham-operated mice. Maximum NFB activity by serial molecular imaging was observed 3 days after myocardial infarction. Reprinted from Biochem Biophys Res Commun, 342, Tillmanns J et al. Caught in the act: In vivo molecular imaging of the transcription factor NF-kappaB after myocardial infarction, pages 773–774, copyright (2006), with permission from Elsevier. Abbreviation: NFB, nuclear factor B.

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The coronary artery ligation model is the best established and most clinically relevant model of heart failure. After coronary artery ligation, mortality and left ventricular dilatation were significantly reduced and left ventricular function was preserved in Tlr2 knockout mice compared with wildtype mice.⁶⁰ These effects might be mediated by TLR-dependent changes in extracellular matrix remodeling.⁶⁰ Further indirect evidence for the link between heart failure and TLR signaling comes from mice with targeted deletion of the NFB subunit p50, which were protected from left ventricular dilatation after myocardial infarction.⁵⁷ TLRs and their downstream signaling components are, therefore, important in left ventricular remodeling after myocardial infarction.

A clinical perspective

TLRs have been recognized as key receptors of the innate immune system. These receptors have been linked to many immune-related diseases, such as rheumatoid arthritis, asthma, inflammatory joint diseases, and lupus erythematosus. Consequently, TLR inhibitors have been a target for drug development for many years.⁶⁶ Indeed, preliminary findings demonstrated that inhibition of TLR signaling was feasible and could represent an effective and well-tolerated treatment for individuals with rheumatoid arthritis.⁶⁷

The effectiveness of immune response inhibition in primarily non-immune-related diseases has not yet been established. The experimental investigations summarized in this Review have shown that TLRs are involved in the development of atherosclerosis, angiogenesis, heart failure, ischemic injury and septic cardiomyopathy, indicating that TLR inhibition could have protective effects in cardiovascular diseases. Although experimentally well proven, so far inhibition of immune responses in cardiovascular nonimmune diseases has failed in clinical practice. For example, the role of the cytokine TNF has been extensively studied experimentally in the context of heart failure, yielding clear evidence that a TNF excess would increase progression of heart failure. Several clinical trials, however, have failed to demonstrate a clinical improvement with TNF inhibition in patients with heart failure.⁶⁸ Inhibition of the complement cascade, although experimentally protective in ischemia–reperfusion injury, had no effect on patients treated with fibrinolysis for acute myocardial infarction.⁶⁹ The failure of immunosuppressive studies in cardiovascular, primary nonimmune diseases is probably related to the fact that activation of the immune system under these circumstances is a double-edged sword—an intact immune system is necessary for many protective pathways, but prolonged immune activation could also activate unfavorable signal cascades, driving disease progression. The major challenge we face is to limit the effect of detrimental innate immune responses, while simultaneously maintaining adequate and appropriate innate immune defense mechanisms.

Conclusions

TLRs are a link between the development of cardiovascular diseases and the immune system. Exciting evidence supporting the theory that TLR activation contributes to the development and progression of atherosclerosis, cardiac dysfunction in sepsis, and congestive heart failure, has come from genetic, clinical and basic science studies. The therapeutic potential of TLRs in cardiovascular disease, however, remains to be defined.

Acknowledgments

Charles P Vega, University of California, Irvine, CA, is the author of and is solely responsible for the content of the learning objectives, questions and answers of the Medscape–accredited continuing medical education activity associated with this article.

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