TLR2 2029C/T and TLR3 1377C/T and −7C/A Polymorphisms Are Associated with the Occurrence of Abdominal Aortic Aneurysm

An abdominal aortic aneurysm (AAA) is a relatively common and potentially life-threatening vascular disease among the elderly population. AAAs are usually asymptomatic and are characterized by progressive aortic dilation, which often leads to a spontaneous rupture in the advanced stages and, as a consequence, to the patient’s death (1–3). It is known that the pathogenesis of AAA is complex and multifactorial; however, the exact molecular mechanism leading to the formation and progression of AAA is still not fully elucidated. Numerous studies have consistently demonstrated that the initiation of AAA involves an inflammatory response, which is modulated by the attraction of exogenous immune cells and enforced by the upregulation of adhesion molecules (4–7). Degradation of the extracellular matrix, especially changes in elastin and collagen properties, which is caused by increased activity of matrix metalloproteinases (MMPs) and serine proteases, as well as smooth muscle cell (SMC) apoptosis are the predominant features associated with the progression of AAA (2, 8–13). Recently, an important link between the family of TLRs and the pathogenesis of AAA has been observed (14–22). TLRs are type I transmembrane proteins expressed on various immune cells, which recognize molecular patterns unique to pathogens or endogenous molecules released from dying or injured cells (23). It was found that TLR2 induced an inflammatory response and promoted AAA progression in TLR2-deficient mice with Porphyromonas gingivalis infection, whereas TLR4 knockout mice displayed a less-progressive effect (19). The TLR2-deficient mice also showed attenuation of vascular fibrosis and aneurysmal remodeling, which led to a regression in already established AAA (18). Furthermore, TLR2 plays a crucial role in the regulation of vascular inflammation and reactive oxygen species production in injured vessels (22). Marked upregulation of vessel wall inflammation in giant cell arteritis was observed after stimulation with the TLR4 ligand LPS (21). Triggering TLR4-induced dendritic cell differentiation in the vessel wall, with subsequent initiation of adaptive immune responses (stimulation of T cells by MHC molecules, cluster of differentiation [CD]83 and CD86) and enhanced progression of vasculopathy (21). Lai et al. (15) showed that human AAA exhibited an elevated level of TLR4 expression that was localized to vascular SMCs (VSMCs). The authors suggested that TLR4 signaling contributes to the proinflammatory environment, specifically IL-6 and MCP-1 of VSMCs, and induces proteinase release from VSMCs, which promotes AAA formation and progression (15). It is noteworthy that TLR4 deficiency stimulated angiotensin II (AngII)–induced AAA formation and/or atherosclerosis development in other studies (16, 20). Immunohistochemical analysis of human aortas revealed a high level of TLR4 and TLR3 expression in SMCs and endothelial cells, whereas no differences in TLR3 transcript expression within the aneurysmal wall were observed compared with that in nonaneurysmal aortas (16). Our previous study (14) demonstrated a higher level of TLR3 mRNA in the aortic wall than in the blood of individuals with AAA, which confirmed a potential role of TLR3 in the pathogenesis of AAA. However, studies focusing on the effect of TLR single-nucleotide polymorphisms (SNPs) on AAA formation are insufficient. Recently, a significant epistatic effect between the TLR4 rs1927914 and MMP9 rs17576 SNPs and the risk of overall aortic aneurysm, including AAA and thoracic aortic aneurysm, was described (24). The authors revealed that males with the TLR4 rs1927914 heterozygous (TC) genotype had an elevated risk of AAA formation, whereas the rs11536889 SNP could interact with dyslipidemia to increase the risk of thoracic aortic aneurysm initiation (25).

In this preliminary study, we elucidated the TLR2 (2029C/T and 2258G/A), TLR3 (1377C/T, 1234C/T, and −7C/A), TLR4 (896A/G, 1196C/T, and 3266G/A), and TLR9 (−1237T/C, −1486T/C, 1174G/A, and 2848C/T) genotype distribution and investigated the correlation between the specific TLR SNPs and the occurrence of AAA.

The study populations consisted of 104 patients with AAAs (89.4% male, mean age 70.5 ± 7.0 y) recruited from the Department of Surgery, Medical University of Vienna (Vienna, Austria) and 112 healthy, unrelated volunteers (66.1% male, mean age 69.7 ± 9.6 y). Subjects were consecutively enrolled between May 2015 and September 2017. The participants had a baseline aneurysm diameter of 53.6 ± 11.3 mm, and 84.6% (88/104) of them received statin treatment. The demographic and clinical characteristics of the AAA patients are summarized in Table I. Healthy volunteers had a body mass index of 25.3 kg/m² (range: 20.5–29.3) for women and 27.6 kg/m² (range: 20.7–40.1) for men. Potential participants were excluded if they did not consent to provide blood, were diagnosed with chronic inflammatory diseases or malignant disease, or had a history of alcohol abuse or recreational drug intake. Individuals not diagnosed with coronary artery disease (CAD), diabetes, and hypertension; without a family history of AAAs; and with no history of alcohol consumption and cigarette smoking were classified as healthy controls. All study participants gave written informed consent to participate in the study. The study was approved by the Ethics Committee of the Medical University of Vienna and was conducted in accordance with the Declaration of Helsinki and the good clinical practice guideline.

Genotyping was performed according to methods modified in our laboratory and described in detail elsewhere (26–28). Briefly, genomic DNA was extracted from fresh peripheral blood using a QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Polymorphisms of the genes TLR2 (2029C/T and rs121917864; 2258G/A and rs5743708), TLR3 (1377C/T, F459F, and rs3775290; 1234C/T, L412F, and rs3775291; −7C/A and rs3775296), TLR4 (896A/G and rs4986790; 1196C/T and rs4986791; 3266G/A and rs1927914), and TLR9 (−1237T/C and rs5743836; −1486T/C and rs187084; 1174G/A and rs352139; 2848C/T and rs352140) were detected by PCR-RFLP analysis and were analyzed using the QIAxcel system (QIAGEN). The TLR SNPs were discriminated based on the different fragment lengths (Fig. 1A–C). All identified genetic variants were confirmed via Sanger sequencing by using a 96-capillary 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA).

Total RNA from fresh peripheral blood was extracted using a QIAamp RNA Blood Mini Kit (QIAGEN), and the mRNA expression of the TLR genes was quantified by real-time PCR technology. First-strand cDNA for quantitative RT-PCR was synthesized from 1 μg RNA by using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative RT-PCR was performed in a 25-μl reaction mixture containing 50 ng cDNA, gene-specific primers (29, 30), Power SYBR Green PCR Master Mix (Applied Biosystems), and nuclease-free water with a 7900HT Fast Real-Time PCR System (Applied Biosystems). Initial denaturation was performed at 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 60 s. The relative expression level of the TLR genes was calculated by the comparative C_t method. Relative TLR mRNA expression in blood samples was normalized to the mRNA level of the housekeeping gene GAPDH. Each sample and nontemplate controls were run in triplicate.

Serum IFN-α, IFN-β (PBL Assay Science, Piscataway, NJ), IL-4, TNF (TNF-α; Thermo Fisher Scientific, Frederick, MD), IL-6 (R&D Systems, Minneapolis, MN), and IL-10 (Thermo Fisher Scientific, Rockford, IL) were determined using commercially available ELISAs according to the manufacturer’s instructions. The absorbance values of the samples and standards at a wavelength of 450 nm were read using a Victor³ plate reader (PerkinElmer, Waltham, MA), and the cytokine concentrations were calculated using a standard curve.

The variables are reported primarily as the mean ± SEM. The χ² test and the Mann–Whitney U test, as well as one-way ANOVA with Dunnett post hoc test, were used for statistical analyses of TLR genotype frequencies and the mRNA levels of the TLR genes. All calculations were performed, and graphs were constructed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA). Hardy–Weinberg equilibrium, linkage disequilibrium, and haplotype calculations were carried out using SNPStats software (https://www.snpstats.net/start.htm). The odds ratio (OR) with a 95% confidence interval (95% CI) for assessing the effect of potential AAA confounders (e.g., age, sex, and nicotine consumption) on the risk of AAA incidence was determined by a logistic regression model. Moreover, possible associations between the selected TLR polymorphisms and the risk of cardiovascular disease (e.g., heart insufficiency, hypertension, and peripheral artery disease) in unadjusted and adjusted multivariate models were studied. These data were analyzed using the SPSS software package for Windows 25.0 (SPSS, Chicago, IL). Any p values ≤0.05 were considered significant, whereas the p value after Bonferroni correction (p^c) for multiple testing was 0.006 (raw p value/8).

To examine the frequencies of TLR2 (2029C/T and 2258G/A), TLR3 (1377C/T, 1234C/T, and −7C/A), TLR4 (896A/G, 1196C/T, and 3266G/A), and TLR9 (−1237T/C, −1486T/C, 1174G/A, and 2848C/T) polymorphisms, molecular typing was performed in 104 patients with AAA (Table I) and 112 healthy, unrelated volunteers. The expected allele and genotype frequencies for the TLR4 896A/G, 1196C/T, and 3266G/A SNPs and the TLR9 −1486T/C SNP did not display Hardy–Weinberg equilibrium in healthy individuals (p < 0.05) and were excluded from further analysis. The heterozygous genotype of the TLR2 2029C/T SNP was four times more common in patients with AAA than in healthy subjects (50.0% versus 11.6%; p < 0.0001). The wild-type CC, heterozygous CT, and homozygous recessive TT genotypes were detected in 43.3% (45/104), 50.0% (52/104), and 6.7% (7/104) of patients with AAA, respectively (Figs. 1, 2). Among healthy volunteers, almost all examined individuals, 88.4% (99/112), had the wild-type genotype for this SNP, whereas the heterozygous genotype was detected in 11.6% (13/112) of individuals. For the TLR3 1377C/T SNP, heterozygous and homozygous recessive genotypes were detected more frequently in healthy individuals than in patients with AAA (71.4% versus 40.4%; p < 0.0001). The wild-type CC, heterozygous CT, and homozygous recessive TT genotypes were detected in 28.6% (32/112), 58.0% (65/112), and 13.4% (15/112) of healthy subjects, respectively (Fig. 2). In the case of the TLR3 −7C/A SNP, the wild-type CC genotype was detected in 69.2% (72/104) of patients with AAA, whereas the heterozygous CA and homozygous recessive AA genotypes occurred in 18.3% (19/104) and 12.5% (13/104) of subjects, respectively. Among the healthy volunteers, 46.4% (52/112) had a mutation present in at least one allele of the TLR3 −7C/A SNP, whereas the wild-type genotype for this SNP was detected in 53.6% (60/112) of volunteers. In the case of the TLR9 2848C/T SNP, the heterozygous and homozygous recessive genotypes were more common in patients with AAA than in healthy individuals (79.8% versus 66.0%; p = 0.024). No significant differences in the distribution of the TLR2 2258G/A, TLR3 1234C/T and TLR9 −1237T/C and 1174C/A SNPs were found (Fig. 2).

In summary, considering the allelic distribution of the TLR2, TLR3, and TLR9 SNPs, no significant differences in the frequency of the TLR2 2258G/A, TLR3 1234C/T, and TLR9 −1237T/C and 1174C/A alleles were observed (Table II). In the case of the TLR2 2029C/T SNP, the T allele was more common in patients with AAA than in healthy subjects (31.7% versus 5.8%; p < 0.0001; Table II). For the TLR3 1377C/T SNP, the C allele was detected more frequently in patients with AAA than in healthy individuals (68.3% versus 57.6%; p = 0.022).

Next, we analyzed the association between TLR polymorphisms and the risk of AAA formation and development, applying models for codominant, dominant, recessive, and overdominant allele settings. The heterozygous genotype of the TLR2 2029C/T SNP was associated with at least an 8-fold increased risk of AAA incidence (OR: 8.80; 95% CI: 4.36–17.77; p < 0.001, in the codominant model; Table III). This SNP showed an enhanced risk of AAA occurrence even after Bonferroni correction for multiple testing (p^c = 0.006). In contrast, a decreased risk of AAA incidence in patients with heterozygous genotypes of the TLR3 1377C/T and −7C/A SNPs was observed (OR: 0.14; 95% CI: 0.07–0.28; p < 0.001; and OR: 0.35; 95% CI: 0.19–0.66; p = 0.0012, respectively, in the codominant model; Table III). Furthermore, the heterozygous genotype of the TLR9 2848C/T SNP was associated with a 2-fold increased risk of AAA incidence (OR: 2.03; 95% CI: 1.09–3.77; p = 0.023, in the dominant model; Table III). However, this association did not reach statistical significance after Bonferroni correction for multiple testing. No association between the TLR2 2258G/A SNP, TLR3 1234C/T SNP, and TLR9 −1237T/C and 1174G/A SNPs and the risk of AAA occurrence was observed.

To investigate the potential link between TLR2 and TLR3 polymorphisms and the incidence rates of cardiovascular disease symptoms, logistic regression analysis was performed. The wild-type genotype of the TLR3 −7C/A SNP was associated with at least a 3-fold increased risk of hypertension in both unadjusted and adjusted models (OR 3.466; 95% CI: 1.157–10.386; p = 0.026; and OR 3.954; 95% CI: 1.081–14.461; p = 0.038, respectively) (Table IV). For the TLR3 1377C/T SNP, the risk of heart insufficiency was almost four times greater for patients with AAA carrying the homozygous recessive genotype than for subjects with wild-type or heterozygous genotypes of this polymorphism (OR 3.833; 95% CI 1.104–13.311; p = 0.034, unadjusted model; OR 4.180; 95% CI 0.994–17.572; p = 0.051, adjusted model; Table IV). In addition, the homozygous recessive genotype of the TLR3 1234C/T SNP was associated with at least 3-fold increased risk of right peripheral artery disease (OR 3.724; 95% CI: 1.033–13.425; p = 0.044, unadjusted model; OR 4.504; 95% CI: 0.748–27.113; p = 0.100, adjusted model; Table IV). No association between TLR2 and TLR9 SNPs and incidence rates of cardiovascular disease symptoms was observed (p > 0.05).

To address the possibility that some TLR SNPs may alter the level of mRNA expression, we determined the relative expression levels of the TLR genes compared with that of the reference GAPDH gene. Analysis of blood cell gene expression revealed that the carriers of the homozygous recessive TLR3 −7C/A genotype exhibited significantly different and 7-fold lower mRNA levels than heterozygous and wild-type SNP genotypes carriers, respectively (p = 0.046, one-way ANOVA with Dunnett post hoc test; Fig. 3A). A trend toward a nearly 3-fold higher and significantly different gene expression level in the carriers of the heterozygous or homozygous recessive genotypes of the TLR2 2029C/T than that in the carriers of the wild-type genotype of this SNP was also observed (p = 0.063, one-way ANOVA with Dunnett post hoc test; Fig. 3B). No statistically significant associations between the TLR2 2258G/A, TLR3 1377C/T and 1234C/T, and TLR9 −1237T/C, 1174C/A, and 2848C/T SNPs and their relative gene expression levels were found (p > 0.05).

To investigate whether the polymorphism in the TLR genes may be related to altered cytokine release, the concentrations of IFN-α, IFN-β, IL-4, IL-6, IL-10, and TNF-α were measured in the serum of all individuals. The serum level of IL-6 was significantly higher in patients with heterozygous or homozygous recessive genotypes of the TLR2 2029C/T SNP than in subjects with wild-type genotypes of this polymorphism (median 15.02 pg/ml, interquartile range [IQR] 19.42 pg/ml versus median 8.42 pg/ml, IQR 4.02 pg/ml; p = 0.008; Fig. 4A). Moreover, the serum of patients with mutations detected in at least one allele of this SNP also had a significantly higher level of TNF-α compared with that of subjects with the wild-type genotype of this polymorphism (median 107.35 pg/ml, IQR 82.72 pg/ml versus median 48.72 pg/ml, IQR 79.49 pg/ml; p = 0.038; Fig. 4B). The IL-6 level was also higher in patients with the wild-type genotype of the TLR3 −7C/A SNP than in heterozygous and homozygous recessive subjects (median 12.29 pg/ml, IQR 17.71 pg/ml versus median 8.27 pg/ml, IQR 4.95 pg/ml; p = 0.005). IFN-α and IFN-β were not detected in the serum samples from patients with AAA and healthy volunteers.

Haplotype analysis of the TLR2 2029C/T and 2258G/A; TLR3 1377C/T, 1234C/T, and −7C/A; and TLR9 −1237T/C, 1174G/A, and 2848C/T SNPs showed that the most frequent haplotype was CGCCCTCA (10.5 and 10.6% for patients with AAA and healthy volunteers, respectively). The CACCCTCA haplotype was detected at a minor frequency in 0.2% of patients with AAA and in 0.8% of healthy volunteers. No association with an increased risk of AAA incidence was found for all examined SNPs (p > 0.05). No evidence of linkage disequilibrium in the investigated TLR SNPs was found (r² < 0.2).

Despite considerable advancements in AAA diagnosis and surgical treatment over the past few decades, surgical interventions are associated with high rates of morbidity and mortality. To date, the exact molecular mechanism of AAA formation and development has not been fully elucidated, and better understanding can improve the clinical outcomes of patients with AAA, enhance their quality of life, and provide more cost-effective healthcare. In the present preliminary study, we report an association between polymorphisms of the TLR2 and TLR3 genes and the risk of AAA incidence in humans. The heterozygous genotype of the TLR2 2029C/T SNP was more common in patients with AAA than in healthy subjects and was associated with an increased risk of AAA occurrence. In contrast, the heterozygous genotype of the TLR3 1377C/T and −7C/A SNPs was less common in patients with AAA than in healthy individuals and was associated with a decreased risk of AAA incidence. An association between serum IL-6 and TNF-α levels and a mutation detected in at least one allele of the TLR2 2029C/T SNP was also observed.

So far, several studies have been performed to assess the association of TLR SNPs and predisposition to chronic inflammatory diseases, such as atherosclerosis, CAD, and myocardial infarction (31–37). However, no studies have determined whether TLR2 and TLR3 variants are risk factors for AAA formation. In this preliminary study, we demonstrated that the heterozygous genotype of the TLR2 2029C/T SNP was associated with an increased risk of AAA incidence and a higher level of TLR2 mRNA expression, whereas the TLR3 1377C/T and −7C/A polymorphisms seem to exert an independent protective effect. Furthermore, the TLR9 2848C/T SNP was associated with an increased risk of AAA, whereas no relationship between TLR4 SNPs and AAA risk was found. It was previously described that the TLR2 2258G/A SNP was associated with CAD in the Turkish population, whereas no association for the TLR2 2029C/T SNP was noticed (33). No significant correlation between the TLR2 2029C/T and 2258G/A polymorphisms and type 2 diabetes and CAD or myocardial infarction was observed in other studies (35, 37). In Chinese elderly individuals in Singapore, no association between the TLR3 1234C/T variant and choroidal neovascularization and polypoidal choroidal vasculopathy was observed (38). This SNP was also not associated with macrovascular complications, such as cardiovascular heart disease, in patients with type 2 diabetes mellitus (39). Interestingly, HEK293 cells expressing 1234C/T and 1660C/T mutations of the TLR3 gene infected in vitro with coxsackievirus B3 suffered an abrogation of type I IFN activation, with subsequently increased virus replication (36). The authors suggested that individuals carrying these polymorphic variants may have a blunted TLR3-mediated innate immune response to enteroviral infection and an increased risk of myocarditis and dilated cardiomyopathy (36). However, the functional effect of the TLR3 1234C/T SNP on human immunity in chronic inflammatory disease, including enteroviral myocarditis and dilated cardiomyopathy, is still not fully elucidated.

We then studied the impact of TLR SNPs on gene expression to provide a potential link to the susceptibility for AAA formation. We found that carriers of the homozygous recessive genotype of the TLR3 −7C/A SNP exhibited lower mRNA expression than carriers of the heterozygous or wild-type genotypes of this SNP. In contrast, a trend toward higher gene expression of the carriers of the heterozygous or homozygous recessive genotypes of the TLR2 2029C/T SNP was noticed. To our knowledge, there are no studies regarding the relationship between TLR2 and TLR3 gene polymorphisms and/or the protein function of these SNPs and AAA in the literature. However, Vorkapic et al. (17) revealed a higher expression of TLR3 in human aortas with AAA in association with macrophages and T lymphocytes than in healthy aortas. Furthermore, they postulated that cytoplasmic adaptor protein TIR-domain-containing, adaptor-inducing IFN-β (TRIF) is important for the signal transduction of TLR3 in AAA formation (17). Consistent with this hypothesis, TLR3 deficiency in hematopoietic immune cells in low-density lipoprotein receptor (LDLR)–deficient mice leads to reduced atherosclerotic plaques with decreased levels of macrophage and T lymphocyte infiltration, which suggests that TRIF mediates its proatherogenic role downstream of TLR3 (40). It was previously reported that the TLR3 1377C/T SNP influences the receptor–cognate ligand interaction by modifying the ectodomain of TLR3 and thus functionally attenuating the receptor (41). Interestingly, the TLR2 2029C/T polymorphism was previously suspected to be an artifact because of the presence of variations in the duplicated noncoding pseudogene homologous in the exon 3 sequence of the TLR2 gene (42). Furthermore, the TLR2 2029C/T variation is located in close proximity to the TLR2 Pro681His polymorphic site that abolished the binding of the general adaptor molecule myeloid differentiation factor 88 (MyD88) to TLR2 (43). Taken together, these data lead us to speculate that TLR2 and TLR3 polymorphisms might be an integral part of the mechanism of AAA formation by affecting the receptor–ligand interaction.

Although our study had many strengths, some drawbacks warrant consideration. The first limitation of this study is the relatively small number of subjects, which may not be representative of the entire population. Second, we had a difference in sex-matched similarity in patients with AAA and healthy volunteers. However, the adjusted analysis revealed that the sex of the individuals did not influence the TLR-AAA associations.

In conclusion, our study demonstrated a potential role of the TLR2 2029C/T and the TLR3 1377C/T and −7C/A SNPs in modulating the risk of AAA incidence. We found that the TLR2 2029 CT genotype seems to be a predictive factor for AAA occurrence, whereas the TLR3 1377C/T and −7C/A SNPs appear to be associated with a decreased risk of AAA incidence. The preliminary study offers novel perspectives regarding the pathogenesis of AAA. Further studies with a larger number of individuals are required to confirm these intriguing results.

We thank all volunteers for valuable contribution to the study.

The authors have no financial conflicts of interest.