Abstract
Toll-like receptors (TLRs) contribute to the immune response by recognizing patterns presented by bacteria and other pathogens. These receptors have been implicated in the inflammatory response that contributes to gingivitis and periodontitis. Conflicting reports have suggested that variations in the genes encoding TLRs, particularly TLR2 and TLR4, may influence susceptibility to periodontitis. In this study, the contribution of variations in the genes encoding TLR2 and TLR4 in the context of periodontitis was examined in 254 patients with moderate periodontitis, 418 patients with severe periodontitis, and 260 healthy controls free of gum disease. Genomic DNA was extracted from participants’ whole blood, and genotyping of TLR2/TLR4 as performed using real-time polymerase chain reaction with TaqMan MGB primer. The genotype, allele, and haplotype frequencies were compared among control, moderate periodontitis, and severe periodontitis groups. Statistical analysis was performed using chi-square and logistic regression analyses. Of the 9 polymorphic loci detected in the two genes, one, rs11536889 (G>C) in TLR4, displayed a statistically significant difference in distribution between individuals with moderate periodontitis and severe periodontitis (P<0.05). The distribution of the GG genotype in moderate periodontitis was higher than in the severe periodontitis group (P<0.05). Further, for the haplotype rs7873784, rs1927907, and rs1153688 of TLR4, the distribution of haplotype GCG was statistically different between moderate periodontitis and severe periodontitis (P<0.05, OR=1.501). These findings indicate that variation in TLR4 may affect chronic periodontitis susceptibility in a Han Chinese population.
Keywords: Periodontitis, TLR2, TLR4, polymorphism
Introduction
Periodontitis manifests as chronic inflammation of the tissues supporting the teeth, called the periodontium, in which inflammation of the gums spreads to the deeper tissue layers to reach the periodontal ligaments, alveolar bones, and cementum [1]. The condition arises when one of the most common oral diseases, gingivitis, goes untreated [1]. Indeed, periodontitis has become a serious disease that endangers both the teeth and the overall health. A leading cause of the loss of masticatory organ functions, periodontitis can also lead to increased risk of heart attack and stroke [2,3].
Inflammation that leads to periodontitis begins with plaque and tartar buildup at the gum line. This buildup contains bacteria that are recognized as pathogens by the immune system, which determines subsequent immune responses to the inflammation. Individuals have different inflammatory responses and immune responses to bacteria; indeed, not all bacterial infections lead to periodontitis. Therefore, the host genetic susceptibility, in addition to the pathogens, plays an important role in the pathology of periodontitis.
Toll-like receptors (TLRs) are important immune receptors that bind to ligands and activate cells to trigger the release of inflammatory cytokines, promoting inflammation [4,5]. TLRs can recognize pathogen-associated molecular patterns, i.e., the conserved structure and composition of pathogenic microorganisms; in addition, they can activate adaptive immunity [6]. TLR2 and TLR4 play a crucial role in mediating tissue immune responses to periodontitis [7,8]. These two receptors are expressed in the cells of periodontal tissues such as gingival epithelial cells, and highly-expressed TLR2 and TLR4 are detected in infected gingival tissues [9,10]. When virulence factors produced by periodontal pathogens, such as fimbriae and lipo-polysaccharide (LPS), bind to TLR2 and TLR4, host cells are induced to produce proinflammatory cytokines [11,12]. Thus, TLRs participate in inflammatory responses and innate immune responses following stimulation by bacteria in periodontal tissues.
However, some debate exists about the extent to which these receptors contribute to individual differences in susceptibility to periodontitis. Some studies have indicated that variations in the genes encoding TLR2 and TLR4 may affect susceptibility to and/or severity of periodontitis, but the results have been conflicting [13,14]. To help understand the potential contribution of genetic variation in TLR2 and TLR4 to periodontitis, the current study used a case-control approach to explore the correlation between polymorphisms in TLR2 and TLR4 and the degree of susceptibility to and severity of chronic periodontitis.
Participants and methods
Participants
Patients visiting the School of Stomatology, Jiamusi University between Dec. 2012 and Sept. 2014 were recruited as study participants. Patients were included if having moderate or severe chronic periodontitis according to the classification criteria related to periodontal health in the Third National Health and Nutrition Examination Survey in the United States [15,16]. Additional inclusion criteria were as follows: Han nationality; no prior periodontal treatment; absence of chronic systemic diseases including cardiovascular diseases, diabetes, and endocrine disorders; no prior use of antibiotics or immunosuppressant agents within the preceding six months; absence of fever, cough, and inflammatory lesions of other regions in the preceding month; not pregnant or lactating; and having at least 15 remaining teeth. A total of 672 patients with periodontitis were included (254 cases had moderate periodontitis, 418 cases had severe periodontitis). An additional 260 individuals receiving physical exams in the same period were selected as a healthy control group; they were free of periodontal disease. The participants included a total of 932 individuals; 641 were males (68.8%) and 291 were females (31.2%). The mean participant age was 62.2±9.7 years. This study was approved by the Hospital Ethics Committee, and informed consent was obtained from all participants.
Periodontal health examination
Periodontal health was evaluated for all participants by periodontal specialists. Two sites of each remaining tooth were examined. Clinical indexes comprised the sulcus bleeding index (SBI), probing depth (PD), periodontal attachment loss (AL), and plaque index (PLI), as well as the status of bone resorption and loosening of teeth. The median value of distal lingual sides and the median value of proximal buccal sides were recorded.
Genomic DNA extraction
To identify genetic variation in TLR2 and TLR4, DNA was obtained from blood samples of each participant. Four mL of venous blood was drawn from the forearm of each participant, treated with EDTA anticoagulant, and stored at -20°C. Blood samples were thawed, and red blood cells were lysed using an erythrocyte lysate (Boster Biotech, Wuhan, China). Samples were centrifuged, then precipitated white blood cells were collected and evenly mixed with 10% sodium lauryl sulphate (Sangon Biotech, Shanghai), Te buffer [10 mM Tris-HCl (pH 7.8); 1 mM EDTA (pH 8.0)], and proteases. The mixture was stored overnight at 37°C. After salt fractionation and centrifugation, the supernatant was collected, treated with chloroform, and centrifuged for extraction. DNA was precipitated with ethanol, washed with 70% Ethanol, and transferred to an Ependorf tube (Generay Biotech, Shanghai). After the DNA was dried in the open air, 200 μL of TE buffer were added to resuspend it, and then samples were stored at -80°C.
Genotyping
Single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 were genotyped with a high-throughput real-time quantitative PCR method using the 7900HT instrument (Applied Biosystems, Foster City, CA, USA) and the TaqMan MGB probe (Biocoen Biotechnology, Beijing). This method is suitable for large-scale screening for SNPs. The reaction was performed in a total volume of 25 μL, comprising 0.5 μL DNA template, 2.5 μL TaqMan universal PCR master mix 2× (Applied Biosystems, Foster City, CA, USA), 0.125 μL 40× TaqMan genotyping assay mix, and 1.875 μL ultrapure water. The reaction was performed under the following conditions: predenaturation at 95°C for 10 min; and denaturation at 95°C for 15 s, and annealing at 60°C for 1 min, for a total of 50 cycles. SDS2.3 software was then used for genotyping. Multiple experiments were conducted on certain samples, where a blank control was designed to improve the accuracy of genotyping, and the results of repeated genotyping were consistent with that of original genotyping.
Statistical analysis
Double data entry was performed using EpiData version 3.1 to create a data bank, and logic checks were performed. SAS 9.2 (SAS Institute, Cary, NC, USA) was used to analyze data. Chi-squared, Hardy-Weinberg equilibrium, and unconditional logistic regression analyses were used to assess distributions and variables. Odds ratio (OR) and 95% confidence intervals (95% CI) were used to assess the risk of periodontitis. Testing Haplotype Effects In Association Studies (THESIAS) software (http://gene canvas.ecgene.net/downloads) was used to analyze haplotype differences between affected participants and healthy controls. P<0.05 was considered to indicate a difference was statistically significant.
Results
Distribution of genotypes at 9 loci of TLR2 and TLR4
Nine loci (3 in TLR2; 6 in TLR4) were identified to have genetic variation among individuals in the study. Hardy-Weinberg equilibrium was tested for the genotype frequencies of these polymorphic loci (Table 1). The distributions of genotypes and alleles at each locus did not significantly differ (each P value >0.05) between the control group and the periodontitis group. However, a significant difference in genotype distributions was detected between patients with moderate periodontitis and patients with severe periodontitis for the SNP rs11536889 (TLR4) (P<0.05). Specifically, the frequency of the GG genotype compared to the combined GC + CC genotypes was significantly higher in moderate periodontitis than in severe periodontitis (P<0.05) (Table 2).
Table 1.
Genotype and allele frequencies of TLR2 and TLR4 variants in individuals with periodontitis and healthy controls [n (%)]
| SNP locus | Genotype | Control | Moderate periodontitis | Severe periodontitis | x2 | P |
|---|---|---|---|---|---|---|
| TLR2 | ||||||
| rs1898830 | AA | 62 (24.31) | 69 (27.94) | 121 (28.95) | 3.638 | 0.457 |
| AG | 131 (51.38) | 132 (53.44) | 207 (49.52) | |||
| GG | 62 (24.31) | 46 (18.62) | 90 (21.53) | |||
| Allele A | 255 (50.00) | 270 (54.66) | 449 (53.71) | 2.551 | 0.279 | |
| Allele G | 255 (50.00) | 224 (45.34) | 387 (46.29) | |||
| rs13150331 | AA | 64 (24.90) | 68 (27.20) | 109 (26.08) | 1.790 | 0.774 |
| AG | 129 (50.20) | 128 (51.20) | 222 (53.11) | |||
| GG | 64 (24.90) | 54 (21.60) | 87 (20.81) | |||
| Allele A | 257 (50.00) | 264 (52.80) | 440 (52.63) | 1.083 | 0.582 | |
| Allele G | 257 (50.00) | 236 (47.20) | 396 (47.37) | |||
| rs3804100 | CC | 63 (25.20) | 66 (28.21) | 110 (26.96) | 3.230 | 0.520 |
| CT | 108 (43.20) | 94 (40.17) | 151 (37.01) | |||
| TT | 79 (31.60) | 74 (31.62) | 147 (36.03) | |||
| Allele C | 234 (46.80) | 226 (48.29) | 371 (45.47) | 0.967 | 0.617 | |
| Allele T | 266 (53.20) | 242 (51.71) | 445 (54.53) | |||
| TLR4 | ||||||
| rs10759930 | CC | 33 (12.84) | 40 (16.00) | 50 (12.11) | 2.151 | 0.708 |
| CT | 123 (47.86) | 114 (45.60) | 197 (47.70) | |||
| TT | 101 (39.30) | 96 (38.40) | 166 (40.19) | |||
| Allele C | 189 (36.77) | 194 (38.80) | 297 (35.96) | 1.092 | 0.579 | |
| Allele T | 325 (63.23) | 306 (61.20) | 529 (64.04) | |||
| rs11536879 | AA | 205(79.77) | 198(79.84) | 328 (80.00) | 2.324 | 0.676 |
| AG | 44 (17.12) | 47 (18.95) | 72 (17.56) | |||
| GG | 8 (3.11) | 3 (1.21) | 10 (2.44) | |||
| Allele A | 454 (88.33) | 443 (89.31) | 728 (88.78) | 0.248 | 0.883 | |
| Allele G | 60 (11.67) | 53 (10.69) | 92 (11.22) | |||
| rs10983755 | AA | 24 (9.37) | 25 (10.00) | 35 (8.54) | 0.768 | 0.943 |
| AG | 96 (37.50) | 95 (38.00) | 164 (40.00) | |||
| GG | 136 (53.13) | 130 (52.00) | 211 (51.46) | |||
| Allele A | 144 (28.13) | 145 (29.00) | 234 (28.54) | 0.095 | 0.954 | |
| Allele G | 368 (71.87) | 355 (71.00) | 586 (71.46) | |||
| rs1927907 | AA | 29 (11.60) | 30 (12.15) | 40 (10.31) | 0.959 | 0.961 |
| AG | 103 (41.20) | 95 (38.46) | 155 (39.95) | |||
| GG | 118 (47.20) | 122 (49.39) | 193 (49.74) | |||
| Allele A | 161 (32.20) | 155 (31.38) | 235 (30.28) | 0.540 | 0.763 | |
| Allele G | 339 (67.80) | 339 (68.62) | 541 (69.72) | |||
| rs7873784 | CC | 0 (0.00) | 2 (0.81) | 2 (0.49) | 0.084* | |
| CG | 49 (19.60) | 32 (12.96) | 54 (13.24) | |||
| GG | 201 (80.40) | 213 (86.23) | 352 (86.27) | |||
| Allele C | 49 (9.80) | 36 (7.29) | 58 (7.11) | 3.439 | 0.179 | |
| Allele G | 451 (90.20) | 458 (92.71) | 758 (92.89) | |||
| rs11536889 | CC | 11 (4.28) | 11 (4.40) | 23 (5.61) | 8.800 | 0.066 |
| CG | 97 (37.74) | 69 (27.60) | 151 (36.83) | |||
| GG | 149 (57.98) | 170 (68.00) | 236 (57.56) | |||
| Allele C | 119 (23.15) | 91 (18.20) | 197 (24.02) | 5.841 | 0.051 | |
| Allele G | 395 (76.85) | 409 (81.80) | 623 (75.98) |
Fisher’s exact test.
Table 2.
Genotype and allele frequencies of TLR4 rs11536889 [n (%)] in individuals with periodontitis
| rs11536889 | Moderate periodontitis | Severe periodontitis | P | P1=GG vs GC + CC | P2=CC vs GC + GG |
|---|---|---|---|---|---|
| CC | 11 (4.40) | 23 (5.61) | 0.028 | 0.362 | |
| CG | 69 (27.60) | 151 (36.83) | |||
| GG | 170 (68.00) | 236 (57.56) | 0.011 | ||
| Allele C | 91 (18.20) | 197 (24.02) | 0.013 | ||
| Allele G | 409 (81.80) | 623 (75.98) |
Haplotype analysis and population characteristics
There were 6 haplotypes at 3 adjacent polymorphic loci (rs7873784, rs1927907 and rs1153688) of TLR4, wherein the GCG haplotype (at rs7873784, rs1927907, and rs1153688) showed statistical significance (P<0.05, OR=1.501) in terms of its frequency distributions in patients with moderate periodontitis and patients with severe periodontitis (Table 3). All participants were compared regarding their status of smoking and toothbrushing. Smoking status was significantly different in patients with periodontitis (P<0.05) (Table 4). A stratified analysis on the smokers found no difference in the number of packs of cigarettes smoked per day between groups (P>0.05) (Table 5). Additionally, no difference was detected in the frequency of toothbrushing between groups (P>0.05) (Table 6).
Table 3.
Haplotype distributions for TLR4 (rs1927907-rs11536889-rs7873784) in individuals with periodontitis
| Haplotype | Moderate periodontitis | Severe periodontitis | OR (95% CI) | P |
|---|---|---|---|---|
| AGC | 0.024 | 0.014 | ||
| AGG | 0.259 | 0.267 | ||
| ACG | 0.006 | 0.012 | ||
| GGC | 0.041 | 0.049 | ||
| GGG | 0.478 | 0.420 | ||
| GCG | 0.169 | 0.230 | 1.501 (1.067-1.986) | 0.021 |
OR-odds ratio; 95% CI, 95% confidence interval.
Table 4.
Relationship between smoking and periodontitis [n (%)]
| Smoking status | Control | Moderate periodontitis | Severe periodontitis | x2 | P |
|---|---|---|---|---|---|
| Current smoker (n=143) | 22 (8.46) | 27 (10.63) | 94 (22.49) | 32.280 | <0.001 |
| Ever smoker (n=362) | 102 (39.23) | 102 (40.16) | 158 (37.80) | ||
| Non-smoker (n=427) | 136 (52.31) | 125 (49.21) | 166 (39.71) |
Table 5.
Relationship between the degree of smoking and periodontitis [n (%)]
| Number of cigarettes | Control | Moderate periodontitis | Severe periodontitis | x2 | P | |
|---|---|---|---|---|---|---|
| <1 pack/day | Current smoker | 25 (26.04) | 22 (22.45) | 64 (35.36) | 5.870 | 0.053 |
| Ever smoker | 71 (73.96) | 76 (77.55) | 117 (64.64) | |||
| ≥1 and ≤2 pack/day | Current smoker | 2 (9.52) | 6 (26.09) | 15 (31.25) | 3.697 | 0.158 |
| Ever smoker | 19 (90.48) | 17 (73.91) | 33 (68.75) | |||
| >2 pack/day | Current smoker | 1 (14.29) | 1 (12.50) | 7 (30.43) | 0.653* | |
| Ever smoker | 6 (85.71) | 7 (87.50) | 16 (69.57) | |||
Fisher’s exact test.
Table 6.
Relationship between the frequency of brushing and periodontitis [n (%)]
| Frequency of brushing | Control | Moderate periodontitis | Severe periodontitis | P |
|---|---|---|---|---|
| 1 time/day | 85 (32.69) | 97 (38.19) | 167 (39.95) | 0.372* |
| 2 times/day | 139 (53.46) | 133 (52.36) | 209 (50.00) | |
| 3 times/day | 25 (9.62) | 21 (8.27) | 33 (7.89) | |
| 1 time/several days | 2 (0.77) | 1 (0.39) | 2 (0.48) | |
| Never brushing | 9 (3.46) | 2 (0.79) | 7 (1.67) |
Fisher’s exact test.
Discussion
The occurrence of periodontitis is mediated, at least in part, by the host response to pathogenic bacteria. Bacteria, particularly bacterial LPS, can be recognized by TLRs, which are pattern recognition receptors. TLR2 and TLR4 thereby mediate the innate immune responses of related host cytokines. TLR gene polymorphisms may change the structural domains of receptors and alter their responses to pathogenic bacteria. TLR4, upon recognizing Gram-negative bacteria, binds to LPS and subsequently induces cells to produce inflammatory cytokines including tumor necrosis factors and interleukins [9,10]. This stimulation leads to more serious inflammatory responses; in the case of gum tissue inflammation, a chronic and heightened response produces more serious periodontal damage [17-19]. Interestingly, no responses to stimulation by LPS are observed in TLR4-deficient mice [20]. Further, the expression levels of TLR2 and TLR4 on the surface and interior of gingival epithelial cells are higher in periodontitis patients than in healthy individuals. After TLR ligands stimulate the gingival epithelial cells, they produce inflammatory cytokines [9,10,21]. A study in European populations found that TLR4 polymorphisms were risk factors for periodontitis [22].
This study found that one SNP locus, rs11536889, of TLR4 displayed different genotype distributions between patients with moderate periodontitis and patients with severe periodontitis. Patients carrying the GG genotype at rs11536889 are more prone to developing severe periodontitis. There were 6 haplotypes at 3 adjacent polymorphic loci (rs7873784, rs1927907, and rs1153688) of TLR4, wherein the GCG haplotype differed in distribution between patients with moderate periodontitis and patients with severe periodontitis. This finding suggests that TLR4 polymorphisms are closely correlated with moderate and severe chronic periodontitis. Indeed, TLR4 SNPs appear to be a genetic susceptibility factor for periodontal diseases, influencing the progression of periodontal diseases and leading to more serious inflammatory responses. The production of additional inflammatory cytokines and bacterial products leads to more serious damage to periodontal tissues.
In addition, this study detected a difference in the smoking status between controls and those with periodontitis, in that the proportion of smokers in the periodontitis group was higher. However, a stratified analysis did not detect a difference in the number of packs of cigarettes smoked per day. Interestingly, although toothbrushing is a good approach to mechanical plaque control, no difference was found here in the frequency of toothbrushing between individuals with periodontitis and healthy controls. We will further explore this finding in following-up studies.
In summary, this study found that TLR4 polymorphisms were correlated with periodontitis susceptibility in a Han Chinese population, and the GCG haplotype (at rs7873784, rs1927907, and rs1153688) may be a risk factor for periodontitis development. Genetic susceptibility to periodontitis might be correlated with other polymorphic loci of TLRs, but those regions were not explored in this study. Further, chronic periodontitis may involve multiple gene interactions and synergy effects of factors including surrounding environments and individual health conditions. Currently, a more in-depth study is required to investigate the degree of the impact of TLR polymorphisms on susceptibility to periodontal diseases, and we will further investigate the impact of TLR polymorphisms on protein functions in follow-up studies.
Disclosure of conflict of interest
None.
References
- 1.Darveau RP. Periodontitis: a polymicrobial disruption of host homeostasis. Nat Rev Microbiol. 2010;8:481–490. doi: 10.1038/nrmicro2337. [DOI] [PubMed] [Google Scholar]
- 2.Kassebaum NJ, Bernabé E, Dahiya M, Bhandari B, Murray CJ, Marcenes W. Global burden of severe periodontitis in 1990-2010: a systematic review and meta-regression. J Dent Res. 2014;93:1045–1053. doi: 10.1177/0022034514552491. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Richards D. Review finds that severe periodontitis affects 11% of the world population. Evid Based Dent. 2014;15:70–71. doi: 10.1038/sj.ebd.6401037. [DOI] [PubMed] [Google Scholar]
- 4.Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol. 2014;5:461. doi: 10.3389/fimmu.2014.00461. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Clark K. Protein kinase networks that limit TLR signalling. Biochem Soc Trans. 2014;42:11–24. doi: 10.1042/BST20130124. [DOI] [PubMed] [Google Scholar]
- 6.Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 2011;34:637–650. doi: 10.1016/j.immuni.2011.05.006. [DOI] [PubMed] [Google Scholar]
- 7.Swaminathan V, Prakasam S, Puri V, Srinivasan M. Role of salivary epithelial toll-like receptors 2 and 4 in modulating innate immune responses in chronic periodontitis. J Periodontal Res. 2013;48:757–765. doi: 10.1111/jre.12066. [DOI] [PubMed] [Google Scholar]
- 8.Lappin DF, Sherrabeh S, Erridge C. Stimulants of Toll-like receptors 2 and 4 are elevated in saliva of periodontitis patients compared with healthy subjects. J Clin Periodontol. 2011;38:318–325. doi: 10.1111/j.1600-051X.2011.01702.x. [DOI] [PubMed] [Google Scholar]
- 9.Lappin DF, Sherrabeh S, Erridge C. Stimulants of Toll-like receptors 2 and 4 are elevated in saliva of periodontitis patients compared with healthy subjects. J Clin Periodontol. 2011;38:318–325. doi: 10.1111/j.1600-051X.2011.01702.x. [DOI] [PubMed] [Google Scholar]
- 10.Buduneli N, ÖZcaka Ö, Nalbantsoy A. Salivary and plasma levels of Toll-like receptor 2 and Toll-like receptor 4 in chronic periodontitis. J Periodontol. 2011;82:878–884. doi: 10.1902/jop.2010.100467. [DOI] [PubMed] [Google Scholar]
- 11.Yun CS, Choi YG, Jeong MY, Lee JH, Lim S. Moutan Cortex Radicis inhibits inflammatory changes of gene expression in lipopolysaccharide-stimulated gingival fibroblasts. J Nat Med. 2013;67:576–589. doi: 10.1007/s11418-012-0714-3. [DOI] [PubMed] [Google Scholar]
- 12.Wang PL, Azuma Y, Shinohara M, Ohura K. Toll-like Receptor 4-mediated signal pathway induced by Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts. Biochem Biophys Res Commun. 2000;273:1161–1167. doi: 10.1006/bbrc.2000.3060. [DOI] [PubMed] [Google Scholar]
- 13.Zheng J, Gao L, Hou T, Liu Z, Wu C, Wu S, Wang P, Wen Y, Yao X, Guo X. Association between TLR4 polymorphism and periodontitis susceptibility: a meta-analysis. Crit Rev Eukaryot Gene Expr. 2013;23:257–64. doi: 10.1615/critreveukaryotgeneexpr.2013007496. [DOI] [PubMed] [Google Scholar]
- 14.Song GG, Kim JH, Lee YH. Toll-like receptor (TLR) and matrix metalloproteinase (MMP) polymorphisms and periodontitis susceptibility: a meta-analysis. Mol Biol Rep. 2013;40:5129–41. doi: 10.1007/s11033-013-2616-1. [DOI] [PubMed] [Google Scholar]
- 15.Dye BA, Li X, Lewis BG, Iafolla T, Beltran-Aguilar ED, Eke PI. Overview and quality assurance for the oral health component of the National Health and Nutrition Examination Survey (NHANES). 2009-2010. J Public Health Dent. 2014;74:248–256. doi: 10.1111/jphd.12056. [DOI] [PubMed] [Google Scholar]
- 16.Arora N, Papapanous PN, Rosenbaum M, Jacobs DR Jr, Desvarieus M, Demmer RT. Periodontal infection, impaired fasting glucose and impaired glucose tolerance: results from the continuous National Health and Nutrition Examination Survey 2009-2010. J Clin Periodontol. 2014;41:643–652. doi: 10.1111/jcpe.12258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Kayagaki N, Wong MT, Stowe IB, Ramani SR, Gonzalez LC, Akashi-Takamura S, Miyake K, Zhang J, Lee WP, Muszynski A, Forsberg LS, Carlson RW, Dixit VM. Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science. 2013;341:1246–1249. doi: 10.1126/science.1240248. [DOI] [PubMed] [Google Scholar]
- 18.Dunzendorfer S, Lee HK, Soldau K, Tobias PS. Toll-like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS responses. FASEB J. 2004;18:1117–1119. doi: 10.1096/fj.03-1263fje. [DOI] [PubMed] [Google Scholar]
- 19.Sun Y, Guo QM, Liu DL, Zhang MZ, Shu R. In vivo expression of Toll-like receptor 2, Toll-like receptor 4, CSF2 and LY64 in Chinese chronic periodontitis patients. Oral Dis. 2010;16:343–350. doi: 10.1111/j.1601-0825.2009.01630.x. [DOI] [PubMed] [Google Scholar]
- 20.Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol. 1999;162:3749–3752. [PubMed] [Google Scholar]
- 21.Ren L, Leung WK, Darveau RP, Jin L. The expression profile of lipopolysaccharide-binding protein, membrane-bound CD14, and toll-like receptors 2 and 4 in chronic periodontitis. J Periodontol. 2005;76:1950–1959. doi: 10.1902/jop.2005.76.11.1950. [DOI] [PubMed] [Google Scholar]
- 22.Schröder NW, Meister D, Wolff V, Christan C, Kaner D, Haban V, Purucker P, Hermann C, Moter A, Göbel UB, Schumann RR. Chronic periodontal disease is associated with single-nucleotide polymorphisms of the human TLR-4 gene. Genes Immun. 2005;6:448–451. doi: 10.1038/sj.gene.6364221. [DOI] [PubMed] [Google Scholar]