Association of hypo-responsive toll-like receptor 4 variants with risk of myocardial infarction

Kristina Edfeldta, Anna M Bennetb, Per Erikssonc, Johan Frostegårdd, Björn Wimane, Anders Hamstenc,f, Göran K Hanssona, Ulf de Faireb,f and Zhong-qun Yana,*

a Experimental Cardiovascular Research Unit, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden
b Division of Cardiovascular Epidemiology, Institute for Environmental Medicine, Karolinska Institute, Stockholm, Sweden
c Atherosclerosis Research Unit, King Gustaf V Research Institute, Karolinska Institute, Stockholm, Sweden
d Center of Metabolism and Endocrinology, Center of Infectious Medicine, Karolinska University Hospital, Huddinge, Sweden
e Division of Clinical Chemistry and Blood Coagulation, Department of Surgical Sciences, Karolinska Institute, Stockholm, Sweden
f Department of Cardiology, Karolinska University Hospital, Stockholm, Sweden

Received January 28, 2004; revised April 20, 2004; accepted May 5, 2004 * Corresponding author. Tel.: +46-8-517 73207; fax: +46-8-31 3147
zhong-qun.yan{at}cmm.ki.se

See page 1378 for the editorial comment on this article1


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Aim Toll-like receptor 4 (TLR4) is a receptor for bacterial lipopolysaccharide (LPS) and heat shock protein essential for innate immunity. Recent studies imply that TLR4 polymorphisms might affect atherogenesis. In this study we investigated the impact of LPS-hypo-responsive TLR4 variants on the risk of myocardial infarction (MI).

Methods and results Using TaqMan PCR technology, we determined the prevalence of the Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene, and their association with MI in a study of 1213 survivors of a first MI and 1561 controls from the Stockholm region.

The frequency was 0.096 for carriers of both 299Gly and 399Ile, and 0.006 for carriers of 399Ile alone. Carriers of both 299Gly and 399Ile were more frequent among the male cases than the male controls (10.7% vs 7.9%, ). Compared with wild-type carriers, men with the 299Gly and the 399Ile TLR4 genotype had an increased risk of MI (OR [95% CI]: 1.4 [1.0;1.9]) whereas no association was observed for women. Furthermore a synergistic interaction was found between the TLR4 polymorphism and smoking in men.

Conclusion The association found between TLR4 genotype and risk of MI suggests that TLR4 genetic variants could potentially affect the susceptibility to MI and that TLR4-mediated innate immunity is implicated in the pathogenesis of MI.

Key Words: Toll-like receptor 4 • Myocardial infarction • Immune system • Single nucleotide polymorphism • Genetics


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Myocardial infarction (MI) is a major clinical complication of coronary atherosclerosis, but the mechanisms leading to MI are poorly understood. Inflammation is a key feature of atherosclerosis, and the inflammatory process in the arterial wall is governed by players of the innate as well as the adaptive arms of the immune system.1,2 Thus, both macrophages and T-cells have been identified in lesions, and many mediators such as adhesion molecules and cytokines that recruit and activate immune cells are present in the atherosclerotic vessel wall. Infections have been implicated in the process of atherosclerosis, but the mechanisms involved remain unclear. In addition, microbial heat shock proteins may play a pathogenic role and immunisation with such proteins was recently found to modulate atherosclerosis development in an experimental model of disease.3,4

Toll-like receptors (TLRs) are pathogen-associated molecular pattern receptors responding to invading micro-organisms by initiating an inflammatory reaction.5 Ligation of these receptors leads to activation of nuclear factor-kappa B (NF-{kappa}B) and mitogen-activated protein kinases through the interleukin-1-receptor TLR signalling pathway and transcription of genes important for innate immune responses such as pro-inflammatory cytokines and chemokines as well as co-stimulatory molecules activating adaptive immunity. TLR4 is the receptor responsible for cellular activation, by lipopolysaccharide (LPS), of bacterial endotoxin and also recognises microbial as well as eucaryotic heat shock protein 60.6–8 We and others have shown that TLR4 is expressed in human atherosclerotic lesions and cardiac myocytes.9–11

A total of 29 single nucleotide polymorphisms have been identified in the human TLR4 gene.12 Of them, the congregated Asp299Gly and Thr399Ile polymorphisms have previously been documented to cause hypo-responsiveness to LPS in human alveolar macrophages and airway epithelial cells and increased susceptibility to Gram-negative infections,13 although this effect was not observed in blood-derived monocytes.14,15 While being more susceptible to infections, individuals carrying the 299Gly allele were recently reported to exhibit slower progression of atherosclerosis.16 In addition, a study based on 183 patients suggested an association of reduced risk of acute coronary syndromes with the 299Gly polymorphism.17 Interestingly, the same polymorphism was recently shown not to be associated with coronary artery stenosis.18 To further investigate the importance of TLR4 in MI, in the present study, we screened for the Asp299Gly and the Thr399Ile polymorphisms in a large case-control sample (participants in the Stockholm Heart Epidemiology Program (SHEEP)).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Study population
SHEEP was designed as a case-control study for epidemiological analysis of risk factors for MI.19 The study base comprised all Swedish citizens aged 45–70 and living in the Stockholm county, excluding those with prior MI. The study was conducted by researchers at the Karolinska Institute in collaboration with all the emergency hospitals in the Stockholm County. Cases were men and women admitted for treatment of a first MI using clinical criteria defined by the Swedish Society of Cardiology in 1991. Male cases were identified during 1992–1993 (two years) and female cases during 1992–1994 (three years). During the initial eight months of the study, the upper age limit was set to 65 years, but this was increased to 70 years for the remainder of the study period. One control per case was randomly selected from the Stockholm County population registers after stratification for age, sex and residential area. Five control candidates per case were sampled at the same time so that non-respondent controls could be replaced by another control candidate. Occasionally, both the initial and a substitute control were included due to a late reply of the initial control. With this procedure, more controls than cases were included in the study. The study was conducted in accordance with the Declaration of Helsinki. The ethics committee at the Karolinska Institute approved the study and all participants gave their informed consent.

All participants underwent a physical examination and blood sampling, and answered an extensive questionnaire. The present analyses were restricted to cases who survived 28 days after their first event without further MI before blood sampling, and their controls (1213 cases and 1561 controls in total). SHEEP participants with reinfarctions prior to the blood sampling were excluded.

Blood samples were collected following overnight fasting, approximately 3 months after the MI when acute-phase reactions had subsided, and were subsequently stored at –70 °C until analyses were performed. DNA was isolated using the RapidPrep Macro Genomic DNA isolation kit (Amersham Pharmacia Biotech, Sweden). DNA samples were successfully genotyped for 1172 cases (826 men and 346 women) and 1517 controls (1026 men and 491 women).

Former smokers were defined as persons who smoked regularly but stopped more than two years ago and current smokers were defined as persons who smoked or had stopped smoking within the last two years. Subjects receiving anti-hypertensive drug therapy during the data collection or previously, or with a systolic blood pressure (BP) >=160 mmHg or diastolic BP >=90 mmHg were classified as hypertensive. Serum concentrations of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (ApoA-I) were determined using conventional techniques. Inflammatory markers were measured in serum using commercially available methods as follows: interleukin-6 (IL-6), IL-6 Eli-pair, (Diaclone, Research, Besancon, France); C-reactive protein (CRP) (Dade-Behring, Marburg, Germany); tumor necrosis factor-alpha (TNF-{alpha}) Quantikine HS human TNF-{alpha}, (R & D Systems, MN, USA) and plasma fibrinogen, according to Vermylen et al.20

Genotyping
DNA samples were genotyped using a previously described 5 nuclease assay (TaqMan allelic discrimination test, Perkin Elmer Biosystems).21 Primer and probe sequences for the Asp299Gly and the Thr399Ile polymorphisms are listed in Table 1 . Thr399Ile probes included a minor groove binder to improve mismatch discrimination in the 5 nuclease assay. A total of 12.5 ng DNA was amplified in a total volume of 25 µl containing 1x TaqMan Universal PCR master mix (Perkin-Elmer, Applied Biosystems), 900 nM of each forward and reverse primer and 200 nM of each probe, except FAMTM-labeled allele 1 probe for Asp299Gly which was 50 nM. Amplification was performed by an initial cycle of 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C/92 °C for 15 s and 62 °C/60 °C for 1 min for Asp299Gly and Thr399Ile, respectively. The amplification protocol ended with an indefinite hold at 15 °C in a PTC 225 Peltier Thermal Cycler. A post-PCR endpoint plate read was performed on each plate using the ABI PRISM 7000 Sequence Detection System. The manual calling option in the allelic discrimination application ABI Prism 7000 SDS software version 1 was then used to assign genotypes.


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Table 1 Primer and probe sequences used for genotyping

 
Statistics
Men and women were analysed separately and also together. Baseline demographic variables were expressed as percentages or medians as appropriate. Differences between groups in baseline characteristics were calculated using the Wilcoxon rank-sum test for continuous variables and analysis for categorical variables. Two-sided tests were used. Odds ratios (OR) with 95% confidence intervals (CI), as estimates of the relative risk, were calculated with carriers of TLR4 299Gly and 399Ile variant as the exposed group and using the wild-type population as the unexposed reference group. OR, including adjustments for potential confounders, was calculated using logistic regression. Crude measurements were adjusted for age and residential area. Gender was also adjusted for in analyses that included both men and women. In addition, adjustments were made for diabetes, hypertension, LDL cholesterol, HDL cholesterol, ApoA-I and current and former smoking. Associations between TLR4 genotype and IL-6, CRP, TNF-{alpha} and fibrinogen concentrations were calculated using the Mann–Whitney -test.

Biological interaction between the combined TLR4 299Gly and 399Ile genotype and smoking was evaluated using departure from additivity of effect as criterion for interaction according to Rothman (1986).22 The attributable proportion (AP) due to interaction, with 95% CI, is the proportion of the total effect among those exposed to smoking and the hypo-responsive TLR4 genotype that is attributable to the interaction between the two variables.23 The SAS system for Windows version 8.02 was used for all statistical analyses (SAS Institute Inc.).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Baseline characteristics of all participants are presented in Table 2 . Overall, cases were more exposed to established risk factors than controls, with the exception of hypertension, type II diabetes among women and former smoking among men. The different genotype combinations in the SHEEP material showed the expected frequencies of wild-type TLR4 (0.897) and carriers of the combined 299Gly and 399Ile genotype (0.096). The frequency of carriers of 399Ile alone was 0.006 and only one individual carried 299Gly alone (Table 3 ). In SHEEP, carriers of 299Gly in combination with 399Ile were more frequent among the male cases than the male controls (10.7% vs 7.9%, ) as presented in Table 3. In contrast, the distribution of carriers did not vary between cases and controls among the women (11.0% vs 10.6%, ; Table 3).


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Table 2 Baseline characteristics

 

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Table 3 Genotype frequencies (% [95% CI]) for TLR4 polymorphisms Asp299Gly and Thr399Ile in the study groups

 
The combined TLR4 299Gly and 399Ile genotype was associated with an increased risk of MI in men (crude OR [95% CI]: 1.4 [1.0;1.9]) (Table 4 ). This increased risk was not seen amongst women (crude OR [95% CI]: 1.0 [0.7;1.6]). In order to estimate the overall MI risk, OR for men and women put together was also calculated. This yielded a somewhat lower OR than for men alone but with a narrower CI (Crude OR [95% CI]: 1.3 [1.0;1.6]). Adjustments for potential confounding factors only marginally altered the point estimates and, as expected, yielded somewhat wider confidence intervals throughout. No risk estimates were made for the isolated 299Gly and 399Ile polymorphisms because they were too rare (1 and 17 individuals respectively). Lifestyle-related risk factors like smoking may interact biologically with genetic risk factors to alter the risk for MI. Notably, the ORs were increased 4.6- and 4.0-fold respectively in male and female smokers carrying the combined 299Gly 399Ile polymorphism as compared to non-smoking non-carriers (Table 5 ). A substantial proportion (0.5) of the increased risk in men could be attributed to synergistic interactions between smoking and the genetic variant.


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Table 4 Relative risk of MI estimated by odds ratios (95% CI) in carriers of the 299Gly 399Ile TLR4 variant allele

 

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Table 5 Odds-Ratio (95% CI) for developing MI for subjects exposed to different combinations of cigarette-smoking and TLR4 genotypes compared to wild-type, non-smokers

 
There were no differences in serum IL-6, CRP or plasma fibrinogen concentrations between carriers and non-carriers of the TLR4 polymorphisms as determined by the Mann–Whitney -test (Table 6 and data not shown). However, male carriers of both 299Gly and 399Ile had slightly higher TNF-{alpha} levels than non-carriers (median TNF-{alpha} levels 2.0 vs 1.7 ng/l for carriers and non-carriers respectively, ). This change may reflect the fact that cases were more frequent among carriers than wild-type individuals, and male cases had higher levels of TNF-{alpha} than male controls (unpublished data). Male carriers with 399Ile alone did not differ in their TNF-{alpha} levels compared with non-carriers (median TNF-{alpha} levels 1.6 vs 1.7 ng/l for carriers and non-carriers respectively, ).


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Table 6 Associations between TLR4 polymorphisms and levels of inflammatory markers

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Enhanced expression of TLRs, in particular TLR1, TLR2 and TLR4, has recently been demonstrated in atheroma, and is associated with activation of the transcription factor NF-{kappa}B, which in turn plays an important role in inducing expression of pro-inflammatory genes.9 Given the role of TLRs in recognising pathogen patterns and the potential significance of infection in atherosclerosis as well as its complications, we assessed the impact of TLR4-mediated innate immunity on the risk of myocardial infarction using a molecular genetic approach.

TLR4 is the type 1 transmembrane receptor for LPS and probably also for hsp60 and other ligands.6–8 A single nucleotide substitution in its extracellular leucine-rich domain results in an impaired host immune response towards LPS stimulation in alveolar macrophages and airway epithelial cells.13 By genotyping participants in the SHEEP study, we found an increased prevalence of the TLR4 299Gly and the congregated 399Ile allele in male patients with MI compared with male controls. Although TLR4 gene variations alone cannot account for MI, the identified susceptibility to MI in men carrying both TLR4 299Gly and 399Ile allele implicates that a genetically determined alteration in innate immune responses affects the risk of MI. This notion is further re-inforced by a recent report from a statin trial showing that carriers of the 299Gly allele run a higher risk of cardiovascular events than non-carriers within the placebo group.24

Interestingly, the same TLR4 polymorphism was found to be associated with the progression of atherosclerosis as evaluated by the surrogate measure of intima-media thickness of the common carotid artery. Patients carrying the 299Gly allele of TLR4 exhibited lower levels of circulating inflammatory markers and smaller carotid intima-media thickness.16

These seemingly contrasting findings could be explained by differential pathogenic mechanisms underlying two phases of the disease. A recent study in mice indicated that activation of TLR4 in the vascular wall by LPS markedly promotes neointima development, and this influence of LPS on neointimal formation was diminished in TLR4 deficient mice.25 Similarly, prior blocking of CD14, an accessory protein in the TLR4 complex, abolished LPS-induced neointimal formation after balloon injury of the iliac artery in rabbits.5,26 These neointimal lesions are composed predominantly of proliferating smooth muscle cells, which also prevail in the fibrous cap of atherosclerotic lesions. Taken together, these results clearly indicate that the innate immune response mediated by a functional TLR4 is an important mechanism, not only conferring defense against infectious agents but also promoting the healing process in the artery.

MI is, in most cases, caused by plaque rupture which frequently occurs in atherosclerotic plaques with thin and friable caps because of lack of collagen, but does not usually take place at the most stenotic sites.27–29 It is possible that factors which promote lesion growth also tend to stabilise plaques, while other factors might inhibit plaque growth and at the same time cause instability, plaque rupture and MI.30,31 The findings of an increased risk of MI but decreased lesion size in individuals with a hypo-responsive TLR4 is compatible with this notion, and we speculate that activation of TLR4 may be important for plaque growth and stability.

In a recent study, Sasu and colleagues showed that Chlamydia pneumoniae stimulated proliferation of vascular smooth muscle cells in vitro and that the mitogenic effect was mediated through TLR4.32 They propose that TLR4 recognition of chlamydial heat shock protein 60 might be one of the mechanisms involved. It is therefore plausible that activation of TLR4 by microbial products, or as yet undefined ligands, in the vessel wall stimulate smooth muscle cells to proliferate and form a stabilising cap over the atherosclerotic lesion. Owing to the obvious limitations of the present study, specific mechanisms by which the TLR4 variant confers susceptibility to MI remain to be determined.

Since the results of the first large-scale cholesterol lowering trial using statins,33 the beneficial effects of statin therapy on progression of atherosclerosis and cardiovascular events have been widely appreciated. Recently a report from the REGRESS study showed that, in patients receiving statin therapy, carriers of the 299Gly polymorphism had a lower risk of cardiovascular events than non-carriers, reflecting an interaction between TLR4 genetic variants and efficacy of statin treatment.24 The participants of the present study were recruited during 1992 to 1994 and very few subjects were receiving statin therapy. These few individuals were excluded from our analysis of MI risk. Therefore the current large scale study is the only one assessing an association between TLR4 variants and MI risk, independently of statin treatment. Recently a less-powered study investigating 183 cases with acute coronary syndromes showed a reduced risk of acute coronary syndromes in carriers of the 299Gly polymorphism.17 Another recent study showed no association of the 299Gly polymorphism with coronary artery stenosis.18 However the effects of statin treatment could not be evaluated in the aforementioned studies. Given the altered efficacy of statins in carriers of the 299Gly polymorphism, statin treatment should be taken into consideration when assessing the discrepancy with the results of the current study.

Lifestyle-related risk factors such as smoking have been associated with progression of atherosclerosis. Smoking increases the risk of atherosclerosis and acute cardiovascular events by affecting thrombosis, endothelial dysfunction and inflammation. However, the precise molecular mechanisms involved are not fully resolved. In the current study we found a synergistic interaction between smoking and a hypo-responsive TLR4 genetic variant in men, implying that smoking is of special concern for patients with a hypo-responsive TLR4 genotype.

The patient group in our study was limited to individuals with a first non-fatal MI and no reinfarction. As a result, the present study may be depleted of cases with fatal MI as well as of those with an early recurrent MI; these cases might be affected by the hypo-responsive TLR4 genotype. Therefore the results obtained only relate to individuals with a first nonfatal MI without reinfarction within three months. Taking this limitation into account, the current analysis based on survivors from a first MI may lead to an underestimation of the impact of the screened TLR4 genotypes on MI. It is also possible that fatal and recurrent cases are over-represented amongst non-carriers. However, this could only be investigated in a prospective study. In addition, the differential impact of the TLR4 variant on the risk of MI in men and women should be interpreted with caution due to the potential influence of gender-related factors. A larger study of women would be needed to fully address this question. Another potential limitation could be that blood sampling was undertaken after the MI event. According to our own previous experience it is, however, most likely that MI induced inflammatory responses have subsided, as blood sampling was performed on average 3 months after the MI event. Clearly, a large prospective study on genetically distinct populations will be needed to determine the pathogenetic relevance of these findings.

In summary, the present study suggests that hypo-responsive TLR4 polymorphisms could affect the susceptibility to MI in men and that TLR4-mediated innate immunity may play a role in the pathogenesis of MI.


    Acknowledgments
 
This study was supported by the Swedish Science Foundation (project numbers: 14079, 14071, 6816, 8691, 5193 and 9533), the Heart-Lung Foundation, the AFA Foundation, the King Gustaf V 80th Birthday Foundation, the King Gustaf V and Queen Victoria Foundation, and the Söderberg Foundation.


    Footnotes
 
1 doi:10.1016/j.ehj.2004.06.035. Back

Authors have no conflict of interest.


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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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