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Cardiovascular Research 2003 60(1):58-67; doi:10.1016/S0008-6363(03)00348-1
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Copyright © 2003, European Society of Cardiology

Toll-like receptors in cardiovascular diseases

Dominique de Kleijna,b,* and Gerard Pasterkampa

aExperimental Cardiology Laboratory, University Medical Center (G02 523), Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
bInteruniversity Cardiology Institute of the Netherlands (ICIN), Utrecht, The Netherlands

*Corresponding author. Tel.: +31-30-250-7155; fax: +31-30-252-2693. Email address: d.dekleijn{at}hli.azu.nl

Received 11 December 2002; accepted 14 March 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Innate immunity and...
 3. Toll-like receptors in...
 4. Toll-like receptors and...
 5. A possible role...
 6. TLRs, tolerance and...
 7. TLRs and ligands:...
 Acknowledgments
 References
 
The Toll-like receptor family recognizes mostly exogenous ligands like bacterial lipopolysaccharides or DNA and activates the inflammatory cell. Evidence is accumulating that these Toll-like receptors are important in cardiovascular pathologies. Recently, expression of Toll-like receptors in arterial and myocardial cells has been shown and mouse knockout and human studies on polymorphisms point to a role of Toll-like receptor 4 in neointima formation and atherosclerosis. It is now becoming clear that these receptors not only serve as receptors for pathogen-associated molecular patterns but are also involved in the initiation and progression of cardiovascular pathologies.

KEYWORDS Atherosclerosis; Receptors; Infection/Inflammation; Arteries; Immunology


   1. Introduction
Top
 Abstract
 1. Introduction
2. Innate immunity and...
 3. Toll-like receptors in...
 4. Toll-like receptors and...
 5. A possible role...
 6. TLRs, tolerance and...
 7. TLRs and ligands:...
 Acknowledgments
 References
 
The role of immune mechanisms in cardiovascular disease is supported by studies in genetically modified mice. Experimental research revealed that interference with the immune response to endogenously or exogenously derived triggers or immunization can alter progression of cardiovascular disease in particular atherosclerotic disease [1–4]. The immune system has two approaches to its disposal to recognize and respond to harmful stimuli: the innate and the adaptive immune recognition systems. The innate immune system is limited to the recognition of evolutionary highly conserved pathogen motifs and is considered as a first line of defense. The adaptive immune system involves dynamic adaptation to unique epitopes on pathogens in the environment.

Cell types that are recognized for their role in both immune systems play an important role in the development and progression of cardiovascular disease. Macrophages and T-lymphocytes express receptors that recognize molecular patterns that are foreign to mammalian organism but commonly found on pathogens. Toll-Like receptors (TLRs) recognize exogenous and endogenous ligands and activate the inflammatory cell via the NK-{kappa}B pathway [5–7]. This review will focus on the TLRs in cardiovascular tissue and their expression and role in cardiovascular pathologies with a main focus on atherosclerosis. For more extensive review on the role of adaptive immunity in atherosclerotic disease and its interplay with the innate immune system, we refer the reader to review papers on this issue that have recently been published [8–10].


   2. Innate immunity and TLRs
Top
 Abstract
 1. Introduction
 2. Innate immunity and...
3. Toll-like receptors in...
 4. Toll-like receptors and...
 5. A possible role...
 6. TLRs, tolerance and...
 7. TLRs and ligands:...
 Acknowledgments
 References
 
Unlike adaptive immunity, the innate immune response already appears in primitive organisms in evolution. The induction time of innate immune responses is fast (hours/days) and the principal effector cells are macrophages, natural killer cells and mast cells. The innate immune response is more static than the acquired immune response due to its limitation to recognize highly conserved pathogen motifs entitled pathogen-associated molecular patterns (PAMPs) [11]. The macrophage, one of the principal effector cell of innate immunity, secretes cytokines that guide and regulate function of other cells that are associated with atherogenesis and progression of atherosclerotic disease.

The receptors that are capable of recognition of PAMPs are scavenger receptors and TLRs. In general, the scavenger receptor has the function of capturing physically larger particles as compared to TLRs. The scavenger receptors may remove PAMPs from the blood without triggering inflammatory cellular responses [12]. Macrophage scavenger receptor class A recognizes not only bacterial ligands like lipopolysaccharide (LPS) and lipoteichoic acid (LTA) but also oxidized LDL. Scavenger receptor A knockout mice show inhibition of atherosclerosis formation and enhanced susceptibility to infection, supporting the role of these receptors in atherosclerotic lesion development [13].

2.1. Toll like receptors
The Toll gene was first discovered as encoding for a receptor responsible for the dorsal ventral pattern in Drosophila embryos. Expression of the TLRs is very ubiquitous throughout species like mammals, fruit flies, nematode, chicken and plants [14]. The evolutionary conversation of Toll-Like receptors (TLRs) suggests that this class of receptors was involved in establishing immunity to bacterial infection. The history of research on the role of the TLRs in immunity is short. In 1997, Medzhitov et al. showed that the human homologue of the Drosophila Toll protein is able to initiate an adaptive immune response [15]. Just 1 year later the TLR4 was identified as the gene encoding the LPS receptor which is not functional in LPS insensitive mice [16,17]. The history of research on TLRs in cardiovascular disease is even more recent: just 2 years ago, the first papers were published describing the expression of TLRs in atherosclerotic plaques [18,19]. TLRs are type I transmembrane receptors with extracellular leucine repeats and a carboxy terminal intracellular tail containing a conserved region called Toll/interleukin1 receptor (TIR) homology domain. The extracellular domain is involved in ligand binding but is also necessary for dimerization [20]. A number of ligands have been identified through in vitro systems or knock-out mice (Table 1). Most of these ligands can be classified as PAMPs. However, recent evidence suggests that TLR4 also responds to endogenous factors produced by stress or cell damaging, like heat shock proteins [21], extracellular matrix components of fibronectin [22] and hyaluronan [23].


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  		Table 1 Toll-like receptors and their ligands: modified from Kaisho and Sakira [64]

 
Currently, 10 mammalian TLRs have been discovered. Table 1 summarizes the known ligands that can bind to the different TLRs. TLR1 is ubiquitously expressed but predominantly on monocytes, neutrophils, B-cells and natural killer cells. TLR 2 and 4 will be discussed in more depth. TLR3 is expressed in dendritic cells. TLR 5 expression seems to be restricted to monocytes, neutrophils and dendritic cells [24]. TLR 6 is expressed in thymus, ovary and lung and plays a role in enhancement of TLR2 function by dimerization. TLRs 7, 8 and 9 are expressed in different tissues like lung and spleen. Hemmi et al. described that TLR 9 also identifies bacterial DNA [25]. The observation that multiple TLRs are functional in the recognition and phagocytosis of bacterial epitopes support the idea that these receptors act in concert to elicit an appropriate immune response. Engagement of TLRs transmits transmembrane signals that activate NK-{kappa}B and mitogen-activated protein kinase (MAPK) pathways [5,6,26]. Subsequently, TLR ligation induces expression of a wide variety of genes like genes encoding proteins involved in cytokine production (for TLR4 see Fig. 1), leucocyte recruitment and phagocytosis [26,27].


Figure 1
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  		Fig. 1 Upregulation of cytokine mRNA after stimulation of adventitial fibroblasts derived from Balb/c mice (WT) and Balb/c-C.C3H-Tlr4lps-d (TLR4 def.) mice with 1 and 10 ng/ml LPS. Cytokine mRNA levels and housekeeping mRNAs (L32, GAPDH) were detected using a RNAse protection assay (Pharmingen).

 
2.2. TLR2 and TLR4
Although research on the role of TLRs in immunological response still is in its infancy, TLRs 2 and 4 have been studied most extensively. Also with respect to cardiovascular disease most research has been focussed on these TLRs.

TLR2 plays an important role in the innate recognition of ligands associated with Gram-positive bacteria. Cells that are reported to express TLR2 are macrophages, neutrophils and dendritic cells. Ligands recognized by TLR2 are peptidoglycan, lipoteichoic acid and soluble modulin (Table 1) [28–33]. Also yeast is recognized by TLR2. The broad ligand specificity attributed by TLR2 may be accounted for by the fact that TLR2 must dimerize with other TLRs (like TLR6) to optimally detect these ligands preceding cellular signaling.

TLR4 is well known as the LPS receptor. LPS is the best-characterized ligand for TLR4. However, not only exogenous but also endogenous ligands may bind to TLR4 and activate the innate immune response (Table 1). At first, CD14 was thought to be the primary receptor for LPS. Indeed, CD14 knockout mice show a decreased sensitivity for LPS in vivo and antibodies against CD14 may block LPS signaling [34,35]. However, the function of CD14 is to increase the signaling sensitivity for LPS by the TLR4. Other cofactors that enhance LPS/TLR4 signal transduction are LPS-binding protein and a new LPS-interacting protein, MD-2, which is able to evoke a cellular response upon LPS addition in a TLR4-dependent manner [36,37].

Activation of TLRs 2 and 4 induces NF-{kappa}B-dependent expression of cytokines and chemokines that are known for their roles in leucocyte recruitment and subsequent inflammatory responses and hence, atherogenesis.


   3. Toll-like receptors in the cardiovascular system
Top
 Abstract
 1. Introduction
 2. Innate immunity and...
 3. Toll-like receptors in...
4. Toll-like receptors and...
 5. A possible role...
 6. TLRs, tolerance and...
 7. TLRs and ligands:...
 Acknowledgments
 References
 
The toll-like receptors have been studied for their crucial role in the detection of microbial infection in mammals leading to activation of inflammatory and innate immune responses (for review, see [38]). Recently, accumulating interest emerges from the cardiovascular research field in this toll-like receptor family.

Expression of these receptors has been found in most cardiovascular cells (for human expression see Table 2) like cardiomyocytes [39], endothelial cells [6], adventitial fibroblasts [40], macrophages [41] and dendritic cells [42]. For these cells, studies have been focussed on the role of toll-like receptor 2 and 4.


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  		Table 2 Human TLR expression in cardiovascular cells and atherosclerotic plaques

 
3.1. Myocytes
In neonatal rat cardiomyocytes Tlr2, Tlr3, Tlr4 and Tlr6 are expressed and Tlr2 is involved in NK-{kappa}B activation by oxidative stress suggesting a pro-apoptotic role for Tlr2 [39]. Murine Tlr4 expression increases after myocardial infarction and is located in cardiomyocytes adjacent to the site of ischemic injury and is also found in human idiopathic dilated cardiomyopathy suggesting that Tlr4 is involved in a tissue response to injury [39]. Tlr4 also seems to play a role in sepsis-induced myocardial dysfunction. LPS injection in Tlr4 deficient and control mice resulted in a significant depression of injection phase indexes of contractile function in the wild-type mouse but not in the Tlr4 deficient mouse [43,44].

3.2. Endothelial cell
In relation to sepsis, Tlrs 2 and 4 expression have also been studied in endothelial cells. Compared to cardiomyocytes, endothelial cells have about a 5-fold higher Tlr4 expression [39]. Human endothelial cells express predominantly Tlr4 and very low levels of Tlr2 [6] indicating that Tlr4 might be a potential target for treatment of sepsis and endotoxic shock.

LPS and IFN-{gamma} induce a 2-fold increase in Tlr2 and Tlr4 expression in endothelial cells [45]. This is in contrast with non-vascular gingival fibroblasts which show a small decrease (~20%) in Tlr4 expression after LPS stimulation [46].

3.3. Adventitial fibroblast and macrophage
The adventitial fibroblast has been recently described as an immunologically active cell and is completely equipped to detect Tlr4 ligands and to respond on Tlr4 activation. Tlr4 signaling in these cells leads to NK-{kappa}B activation and production of cytokines [40]. However, Tlr4 expression in these cells did not differ after LPS stimulation (Fig. 2). This identifies the adventitial fibroblast as a potential key-site for cytokine production leading to an active inflammatory process [40].


Figure 2
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  		Fig. 2 Relative TLR4 protein expression in human adventitial fibroblasts after 6, 12 and 24 h. stimulation with 100 ng/ml LPS compared to unstimulated adventitial fibroblasts (0 h). Isolation of human adventitial fibroblasts and TLR4 Western blotting was performed as described earlier [40]. n = 5/timepoint, data are mean±standard deviation.

 
In the healthy and atherosclerotic artery Tlrs1-9 are expressed. Double staining indicated that Tlrs 2 and 4 were primarily expressed in endothelial cells and macrophages in the atherosclerotic lesions [19,18]. Although numbers are small, expression of Tlr4 in human macrophages seems to be stimulated with Ox-LDL in contrast to Tlr2 [19]. After stimulation with LPS, macrophages show a temporary decrease in Tlr4 mRNA expression but return at 24 h LPS treatment to normal Tlr4 mRNA levels as that of non-treated cells [47]. This points to a cell-specific regulation of Tlr4 expression after LPS treatment with an increase in mRNA levels in endothelial cells and a time-dependent decrease or constant Tlr4 mRNA levels in fibroblast and macrophages.

3.4. Dendritic cells and vascular-associated lymphoid tissue (VALT)
In human peripheral blood, monocytes preferentially express TLRs 1, 2, 4, 5 and 8 while immature dendritic cells express high levels of TLRs 1, 2 and 3 and plasmacytoid pre-dendritic cells TLR 7 and 9 [48,49]. Morphological studies show that vascular dendritic cells are rare in the normal artery but accumulate in the atherosclerotic lesions [50] and are most dense in regions subjected to turbulent flow conditions [51,52]. It remains unclear if the vascular dendritic cell is a different subset of dendritic cells with its specific toll-like receptor expression pattern.

Dendritic cells can form together with immunocompetent cells the vascular-associated lymphoid tissue (VALT) [53]. VALT is described in the non-diseased arterial wall in the subendothelial layer and around the vaso vasorum in the adventitia and is probably involved in the detection of harmful antigens [54,55]. Similar to dendritic cells, it remains unclear which TLRs are expressed in VALT. Accumulation of VALT seems to be associated with athero-prone regions and aortic aneurysms [54,55] which might point to a role in atherosclerosis. Adventitial lymphoid infiltrates develop at all sites with extensive atherosclerotic plaque formation and are most prominent in atherosclerotic aneurysms of the abdominal aorta [56]. They consist of B cells and follicular dendritic cells with a potential role in evoking a late humoral immune response [57]; however, a role for TLRs in these adventitial infiltrates is unknown.


   4. Toll-like receptors and atherosclerosis
Top
 Abstract
 1. Introduction
 2. Innate immunity and...
 3. Toll-like receptors in...
 4. Toll-like receptors and...
5. A possible role...
 6. TLRs, tolerance and...
 7. TLRs and ligands:...
 Acknowledgments
 References
 
It is established that atherosclerosis is a complex process with immunological responses at the initiation and progression of this occlusive disease. Atherosclerosis is now considered an inflammatory disease [58,59]. Inflammatory cells, like monocyte derived macrophages and T-lymphocytes, are observed at all stages of atherosclerosis [60,61]. The arterial inflammatory reaction stimulates proliferation and migration of smooth muscle cells that become intermixed with the area of inflammation. Continuous inflammation and the formation of fibrous tissue lead to further progression and enlargement of the lesion. The presence of local plaque rupture is associated with the presence of inflammatory cells that are capable of producing and secreting matrix-degrading enzymes and aggravate the recruitment of additional inflammatory cells and production of chemokines and cytokines [62,63].

Since TLRs can initiate the innate and sequentially the adaptive immune system, both responsible for inflammation, one can assume that TLRs play a role in this process. Especially Tlr4 is interesting, since this receptor has also endogenous ligands like Hsp60 and alternatively spliced fibronectin EDA domain [64].

4.1. The rat and mouse as a model
Although at that time the LPS receptor TLR4 was unknown, it was found that after adventitial LPS application on the rat femoral artery resulted in intimal lesion formation [65] and was the first association between bacterial antigen and neointima formation.

Studies on different strains of inbred mice for their susceptibility for atherosclerosis showed that there are considerable differences between the strains. The natural TLR4-deficient strain C3H/HeJ (C3H) which is insensitive to LPS, is one of the strains that are less susceptible to diet-induced atherosclerosis [66,67]. Based on this, C3H ApoE(–/–) mice and C57Bl ApoE(–/–) were compared in atherosclerotic lesion formation and showed more atherosclerotic lesions in the C57Bl ApoE(–/–) despite the lower plasma lipid concentrations [68]. Atherosclerosis can develop without pathogens [69] supporting a role for the endogenous ligands of TLR4 [64] in atherosclerosis. This study [69] seems to be in contradiction with Grimditch et al. [68], since they show that cholesterol ether levels in the aorta did not differ between ApoE(–/–) and ApoE(–/–) LPS-deficient mice. Unfortunately, atherosclerotic lesion size was not measured in these two groups [69] which is generally accepted as the standard for atherosclerosis.

Bone marrow transplantation of ApoE(–/–) or Apo(+/+) to a C3H host revealed no differences in artherosclerotic lesion formation which points to an important role of the arterial wall cells in artherosclerosis [70]. Perhaps, arterial wall cells of the C3H mouse are unable to recruit monocytes into the arterial wall which would explain the low number of macrophages in C3H artherosclerotic lesions, compared to B6 lesions.

Although the C3H mouse does not respond to exogenous LPS and endogenous EDA [22] due to a TLR4 point mutation, strain differences are based on more genes then the TLR4 gene. Therefore, it is not possible to relate strain differences in atherosclerotic susceptibility with one mutated TLR4 receptor. TLR2, TLR4 and TLR9 knockout mice exist [71] and open the possibility to study the importance of these receptors in different controlled backgrounds including atherosclerotic backgrounds like ApoE(–/–) and ApoE3 Leiden.

The C3H Tlr4 point mutation in a Balb/C background is already commercially (Jackson Lab, www.jax.org) available. Using a femoral cuff model in these mice showed that after adventitial LPS stimulation, neointima formation was reduced by 60% in the TLR4 deficient mouse. This demonstrates that adventitial activation of TLR4 leads to augmentation of neointima formation [40] which is considered the ‘soil’ of atherosclerosis [72].

4.2. Human studies
Several descriptive studies show that TLRs and TLR ligands are present in the human atherosclerotic plaque [18,19] and adventitia [40] pointing to a role of innate immunity in atherosclerosis.

Until now, only Kiechl et al. [73] showed that a Asp299Gly TLR4 polymorphism which attenuates receptor signaling is associated with a lower risk of carotid artherosclerosis. These data provide for the first time epidemiological evidence for the concept that TLR4 is involved in the development of atherosclerosis.

4.3. Matrix turn-over and Toll-like receptors
Several studies show that Toll-like receptor 4 is associated with matrix turn-over. In monocytes, activation of TLR4 by LPS increased the production of matrix metalloprotease 9 (MMP-9) [22], whereas in cervical smooth muscle cells LPS application increased mRNA levels encoding MMP-1, MMP-3 and the elastase cathepsin S [74]. Furthermore, the endogenous Tlr4 ligands, Hsp 60 and EDA, have been observed in arthritic [75] and oncological specimens [76,77] in which matrix turn-over is an important feature while the TLR2 ligand peptidoglycan has been associated with an unstable plaque phenotype [78]. This point to a role of TLR2 and 4 in matrix turn-over. Matrix turn-over and inflammation are two important processes in the progression of atherosclerosis but also in other cardiovascular diseases (Fig. 3).


Figure 3
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  		Fig. 3 Hypothetical scheme for the involvement of Toll-like receptors and its ligands in cardiovascular disease or cardiovascular disease-related processes.

 

   5. A possible role of the adventitial fibroblast in plaque and neointima formation?
Top
 Abstract
 1. Introduction
 2. Innate immunity and...
 3. Toll-like receptors in...
 4. Toll-like receptors and...
 5. A possible role...
6. TLRs, tolerance and...
 7. TLRs and ligands:...
 Acknowledgments
 References
 
Recent studies have led to the concept that fibroblasts are key-sites of cytokine synthesis which can initiate inflammatory processes [79].

Moreover, LPS stimulation of mouse embryonic fibroblasts increased cell migration [80] pointing to a role of TLR4 in fibroblast migration. This is in agreement with the finding that adventitial fibroblasts contribute to neointimal formation [81,82]. Furthermore, adventitial LPS application resulted in neointima formation [40,65] in which TLR4 is involved [40].

In atherosclerotic arteries, adventitial inflammation has been associated with clinical manifest atherosclerotic lesions [83]. Similarly, the presence of C pneumonia staining in the adventitia was associated with the amount of local atherosclerotic plaque [84]. This suggests that inflammation in the adventitia might play a role in luminal plaque/neointima formation.

Fibroblasts are the most prominent adventitial cells in the healthy artery. In healthy pig and rabbit arteries, we could not detect any macrophages (results not shown). Since the adventitial fibroblast contains a functional TLR4 pathway [40] it might be possible that activation of adventitial TLR4 on one hand initiates adventitial inflammation by attracting monocytes via cytokine secretion and on the other hand stimulates migration of adventitial fibroblasts and medial smooth muscle cells to form the neointima (Fig. 4). This does not exclude the luminal contribution via the luminal endothelial cell or the contribution of lipids but adds another possible pathway in the origin of atherosclerosis and neointima formation.


Figure 4
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  		Fig. 4 Hypothetical scheme of an additional adventitial TLR4-dependent pathway in neointima and atherosclerotic plaque formation.

 

   6. TLRs, tolerance and cross-tolerance
Top
 Abstract
 1. Introduction
 2. Innate immunity and...
 3. Toll-like receptors in...
 4. Toll-like receptors and...
 5. A possible role...
 6. TLRs, tolerance and...
7. TLRs and ligands:...
 Acknowledgments
 References
 
The immune system is capable of suppressing the inflammatory response to endogenous and exogenous ligands. Specifically in the case of autoantigens, the inflammatory response is damaging and causally related with the clinical presentation of the auto-immune disease. Thus, downtuning the immune response against atherosclerosis related antigens may theoretically impair the progression of this occlusive disease.

Human TLR4 recognizes LPS. Prolonged exposure of human macrophages or monocytes to bacterial LPS induces a state of tolerance to subsequent LPS challenge [85,86]. In addition, tolerance is also observed for the TLR2 ligand lipotheichoic acid after LPS stimulation (via TLR4) and for the TLR4 ligand LPS after lipotheichoic acid (via Tlr2) showing that LPS-stimulated monocytes are less susceptible to respond to the TLR2 ligand lipoteichoic acid, as well as that lipoteichoic acid-stimulated monocytes respond less to subsequent LPS stimulation [87]. Taken together, these observations indicate the existence of a feedback mechanism that inhibits excessive TLR ligand responses in monocytes. The mechanism of antigen-induced tolerance is not fully understood. In vitro data on TLR expression reveal that (over) expression of TLR2 or TLR4 is not associated with the development of cross-tolerance [86]. Moreover, LPS and lipoteichoic acid-induced tolerance seem to have distinct mechanisms in the sense that unique TLR2 signaling components are disrupted when TLR2 lipoteichoic acid tolerance develops [85]. Tolerance induction is also not caused by paracrine factors [87]. In the search for the mechanisms that are responsible for ligand tolerance it has also been described that surgical interventions induce endotoxin tolerance [88], indicating that endogenous stress-induced ligands may trigger ligand tolerance.

The TLRs also have interacting binding partners in vivo that may help to understand the tolerance development for TLR ligands. For LPS, two well-characterized interacting molecules are lipopolysaccharide binding protein (LBP) and CD14. In vitro, the blood of LBP knockout mice is hyporesponsive to LPS [89] suggesting that this protein is critical to exert its inflammatory potential. However, in vivo LBP knockout mice only reveal slight hyporesponsiveness to LPS [89]. Conflicting with these observations is the finding that high concentrations of LBP in serum of patients with severe sepsis inhibit the LPS response in human monocytes [90]. Literature on the role of CD14 in tolerance development seems to hide contradictions that can be explained by the differential roles of cell bound CD14 and soluble CD14. On one hand, trials with CD14 antibodies revealed a protection against LPS-induced toxicity in rabbits and monkeys [91]. This is confirmed in human studies in which CD14 antibodies are able to inhibit LPS-induced effects like cytokine release and clinical symptoms. On the other hand, soluble CD14 decreases monocyte responses to LPS by transferring cell-bound LPS to plasma proteins with subsequent significant reduction in the ability of monocytes to produce cytokines in response to LPS. The recently described soluble form of TLR4 [92] shares this capability to inhibit excessive LPS responses. In mouse macrophages, expression of the alternatively spliced soluble TLR4 is enhanced after LPS stimulation. In addition, in a mouse macrophage cell line, soluble TLR4 is capable of inhibiting LPS mediated TNF{alpha} and NF-{kappa}B activation. Taken together, these results show that soluble forms of TLR4 and CD14 may function as a feedback mechanism to inhibit the excessive LPS responses in septic conditions.


   7. TLRs and ligands: targets for intervention?
Top
 Abstract
 1. Introduction
 2. Innate immunity and...
 3. Toll-like receptors in...
 4. Toll-like receptors and...
 5. A possible role...
 6. TLRs, tolerance and...
 7. TLRs and ligands:...
Acknowledgments
 References
 
In vitro and in vivo mice knockout studies suggest that TLRs are crucial players in atherosclerotic lesion and intimal hyperplasia formation by modulating the inflammatory response upon exogenous and endogenous triggering. Studies in C3H mice lacking a functional TLR4 strongly indicate that inhibition of TLR4 function impairs the inflammatory response and thereby intima formation. In addition, recently it was discovered that a nucleotide polymorphism in the TLR4 gene, Asp299Gly and Thr399Ile, are associated with reduced extent and progression of carotid atherosclerosis as quantified by B-mode ultrasound [73]. These observations make the TLR a novel potential target to consider for intervention in these processes. However, since functional TLRs are obligatory for a normal response to bacterial infiltration and control of innate, and subsequently adaptive, immunity it might be complicated when TLRs are down regulated systemically. This concept is supported by observations that TLRs and CD14 knock out mice suffer from increased prevalence of bacterial infections. Two options to affect the inflammatory response to TLR-ligand binding could be considered: local treatment with a TLR inhibitor or ligand immunization.

7.1. Local treatment
Except for antibodies that inhibit TLR function, an analogue or inhibitor of TLRs has not been described yet. This makes it difficult to speculate upon the effect of systemically blocking the TLR receptor function in vivo. Assuming that the TLR knock out mice studies can be extrapolated to pharmaceutical interventions in human beings, then it is not likely that TLR-blockers could be developed for long term systemic treatment. Local drug delivery, however, would theoretically be feasible. Drug eluding stents encompassing a TLR4 inhibitor might have beneficial effects on the development of in stent restenosis. This hypothesis is supported by the observations that in stent restenosis seems to be driven by the inflammatory response and that TLR4-deficient mice develop less neointima formation [40]. Atherosclerotic lesions that contain large numbers of inflammatory cells and hide large atheromas could stabilize when local recruitment of macrophages is impaired by blocking the TLR receptor therefore subsequently decreasing cytokine and chemokine release. The recent developments in visualization of these potentially rupture prone lesions might help selecting and targeting these inflammatory lesions [93,94].

7.2. Ligand immunization
Cd4+ T cells are prevalent in atherosclerotic lesions and its presence is associated with enhanced plaque development [10]. CD4+ T cells can be divided in two subsets that seem to counterbalance each other: Th1 cells that induce macrophage activation and promotes inflammation and Th2 cells that have the opposite anti-inflammatory effect. Th2 activation can be induced by antigen administration [95] and suggest a potential role for ligand immunization. Immunizations with disease associated auto-antigens have been successfully performed in experimental models of auto-immune diseases [96]. In atherosclerotic mouse models immunization with two different auto-antigens, ox LDL and HSP65/60 has also been shown to protect against the progression of the disease [97]. HSP60 is a chaperone that protects proteins during maturation and is a ligand for TLR4. Therefore, the search for interventional strategies to block TLR-dependent processes may thus be translated into the search for endogenous ligands. Knowledge on these ligands like HSP60 may help us in the development for antigen-specific immuno-deviating therapies. Most research on antigens that are considered for immunization are auto-antigens. Recently we observed that IgM levels against bacterial peptidoglycan, which is a ligand for TLR2 and present in inflammatory plaques [78], are lower in patients suffering from symptomatic atherosclerosis (data not shown). This observation suggests that there is also a suppression of an immune response against exogenous antigens, in this case recognized by TLR2.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Innate immunity and...
 3. Toll-like receptors in...
 4. Toll-like receptors and...
 5. A possible role...
 6. TLRs, tolerance and...
 7. TLRs and ligands:...
 Acknowledgments
References
 
This work was supported by the Dutch Heart Foundation (99-209 and 2001-162).


   Notes
 
Time for primary review 36 days.


   References
Top
 Abstract
 1. Introduction
 2. Innate immunity and...
 3. Toll-like receptors in...
 4. Toll-like receptors and...
 5. A possible role...
 6. TLRs, tolerance and...
 7. TLRs and ligands:...
 Acknowledgments
 References
 

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