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Toll‐like receptor‐1, ‐2, and ‐6 genotypes in relation to salivary human beta‐defensin‐1, ‐2, ‐3 and human neutrophilic peptide‐1

Abstract Aim To examine whether functional gene polymorphisms of toll‐like receptor (TLR)1, TLR2, and TLR6 are related to the salivary concentrations of human beta‐defensins (hBDs)‐1, ‐2, ‐3, and human neutrophilic peptide (HNP)‐1. Materials and Methods Polymorphisms of TLR1 (rs5743618), TLR2 (rs5743708), and TLR6 (rs5743810) were genotyped by PCR‐based pyrosequencing from the salivary samples of 230 adults. Salivary hBD‐1, ‐2, ‐3, and HNP‐1 concentrations were measured using enzyme‐linked immunosorbent assay. General and periodontal health examinations, including panoramic radiography, were available for all participants. Results The genotype frequencies for wild types and variant types were as follows: 66.5% and 33.5% for TLR1, 95.5% and 4.5% for TLR2, and 25.1% and 74.9% for TLR6, respectively. The TLR2 heterozygote variant group exhibited higher salivary hBD‐2 concentrations than the TLR2 wild‐type group (p = .038). On the contrary, elevated hBD‐2 concentrations were detected in the TLR6 wild‐type group compared with the TLR6 heterozygote and homozygote variant group (p = .028). The associations between TLR6 genotypes and salivary hBD‐2 concentrations remained significant after adjusting them for periodontal status, age, and smoking. Conclusion hBD‐2 concentrations in saliva are related to TLR2 and TLR6 polymorphisms, but only the TLR6 genotype seems to exhibit an independent association with the salivary hBD‐2 concentrations.

Principal findings: Here, we demonstrated that salivary hBD-2 concentrations associate with the TLR6 genotype.
Practical implications: Understanding the genetic background behind the activation mechanisms of salivary immune-regulatory peptides may help us define individuals who could have an increased risk of developing periodontal disease.
Human beta-defensins (hBDs) are cysteine-rich cationic antimicrobial peptides that are expressed and secreted by epithelial cells.
We hypothesized that polymorphisms in the TLR2 gene and in the genes of TLR2 heterodimer pairs, TLR1 and TLR6, have an impact on defensin concentrations in saliva. Therefore, we aimed to determine the relation between TLR1, TLR2, and TLR6 gene polymorphisms and salivary concentrations of hBD-1, -2, -3, and HNP-1.

| Study design and salivary samples
The salivary samples of the present study originated from the national Finnish health survey "Health 2000" sample collection (BRIF8901). A detailed information on the survey design and protocols can be found elsewhere (Gursoy, Könönen, Huumonen, et al., 2013;Liukkonen et al., 2017). Briefly, the study protocols of the survey had been Digital panoramic radiographs, which were available for all subjects, were used in the detection of alveolar bone loss on the mesial and distal surfaces of each tooth as well as on the furcation areas of multi-rooted teeth. The radiographical examination protocols had been approved by the Advisory Board for Radiation Safety, and the Radiation and Nuclear Safety Authority and the Ethics Committees for Human Studies of the National Public Health Institute and the Institute of Epidemiology and National Health, Finland granted the safety licences. The participation in the radiographical examination was voluntary, and pregnancy was the only exclusion criterium. Radiographs were taken with a dental panoramic X-ray device (PM 2002 CC proline apparatus;Planmeca, Helsinki, Finland), and the measurements were performed using an inbuilt measuring tool of the Dimaxis™ software (Planmeca). A detailed description of the measurements regarding alveolar bone loss has been previously described (Gursoy, Könönen, Huumonen, et al., 2013).
Paraffin-stimulated whole saliva samples were collected and placed in carbonic acid ice at the field, and then stored at À70 C until their further use. A subset of 230 salivary samples, collected from a population of 40-60 (mean of 48.7) years of age, having a minimum of 20 teeth, was included in the present study for genotyping and the determination of antimicrobial peptide concentrations. The selection of saliva samples was based on the number of teeth with PPD ≥4 mm to form three periodontally distinct groups as follows: 81 participants had PPD ≥4 mm at 14 or more teeth; 65 participants had PPD ≥4 mm at two or seven teeth; and 84 participants had no teeth with PPD ≥4 mm.
The saliva samples were centrifuged at 10,000 rpm for 5 min, and the pellets were used for the assays of genotyping and the supernatants for hBD 1-3 and HNP-1 measurements. The samples were analysed blindly in the laboratory.
2.2 | Exposure variable TLR1, TLR2, and TLR6 genotypes were defined as exposure variables. Genotyping of the selected TLRs was performed by an automated pyrosequencer (PSQ™96MA Pyrosequencer, Biotage, Uppsala, Sweden). The isolated genomic DNA was used for PCR.
The PCR and sequencing primers were designed as described elsewhere (Vuononvirta et al., 2011;Nuolivirta et al., 2013) and ordered from Sigma-Aldrich, Finland. To guarantee the specificity of pyrosequencing, three negative controls were included in each run. PCR products with potential SNPs were recognized as the template in the pyrosequencing reactions, using a commercial reagent kit (PSQ™96 Pyro Gold Q96 reagent kit) according to the manufacturer's protocol. The following polymorphisms of TLRs (SNP id) were examined: TLR1 (rs5743618), TLR2 (rs5743708), and TLR6 (rs5743810).

| Outcome variables
Salivary hBD-1, hBD-2, hBD-3, and HNP-1 concentrations (pg/ml) were defined as outcome variables. hBD-3 and HNP-1 concentrations were measured by in-house sandwich-enzyme-linked immunosorbent T A B L E 1 The distribution of age, gender, the percentage of smokers, number of teeth, mean, number of teeth with probing pocket depth (PPD) of ≥4 mm, number of bleeding sextants, and total alveolar bone loss among genotypes of TLR1, TLR2, and TLR6

| Statistical analyses
The  .957 Note: Data are presented as median (min-max). Significant differences (p < 0.05) are presented in bold.
1-3 and HNP-1 concentrations differed significantly between the periodontally healthy and two diseased groups (Table 2). HNP-1 concentrations were elevated along with increased number of teeth with PPD ≥4 mm, whereas higher hBD-3 concentrations were measured only in the localized periodontitis group (PPD ≥4 mm at two or seven teeth) in comparison to the controls (no teeth with PPD ≥4 mm). Table 3 presents the defensin concentrations in saliva according to the TLR genotypes. Salivary hBD-2 concentrations were significantly lower among TLR2 wild types in comparison to the TLR2 heterozygote and homozygote group (p = .038). In the TLR6 wild-type group, in contrast, salivary hBD-2 concentrations were higher than those in the TLR6 heterozygote and homozygote group (p = .028) (Table 3). A simultaneous presence of TLR1/TLR2 or TLR2/TLR6 genotypic variants did not affect salivary hBD 1-3 or HNP-1 concentrations (Table 4).
According to linear regression analysis, the implementation of periodontal status, age, and smoking in the regression model diminished the association between TLR2 variants and salivary hBD-2 concentrations. The association between TLR6 variants and salivary hBD-2 concentrations remained significant after adjustments (Table 5).

| DISCUSSION
It was previously demonstrated that the simultaneous presence of microbial pattern recognition receptor polymorphisms and the colonization of periodontitis-associated bacteria is associated with periodontitis (Gursoy et al., 2016;Liukkonen et al., 2017), but the possible mechanism behind this outcome was not explained. To our knowledge, this study is the first to demonstrate a relationship between TLR polymorphisms and salivary hBD concentrations. Based on the current findings, TLR2 (rs5743708) and TLR6 (rs5743810) polymorphisms influence salivary concentration of hBD-2; however, only TLR6 has an independent association with hBD-2 concentrations.
According to Nuolivirta et al. (2013), low (<3%) TLR2 (rs5743708) and common ($50%) TLR6 (rs5743810) frequencies are general among European populations. The outline of the present study was hBD 1-3 and HNP-1 concentrations in saliva. Further studies are needed to demonstrate disturbances of the hBD 1-3 and HNP-1 activation at mRNA and protein levels in individuals with different TLR polymorphisms.
Expressions of gingival hBDs are regulated by infectious or inflammatory stimuli (Özdemir et al., 2020). The activation of various TLRs, such as TLR2, TLR3, and TLR4, has been linked to the hBD2 secretion in uterine, vaginal, or in bronchial epithelial cells (Fruitwala et al., 2019). It is notable that the functioning receptors and pathways related to hBD response of epithelial cells vary in different tissues and demonstrate a cell-type dependency (Fruitwala et al., 2019). In periodontal tissues, F. nucleatum stimulates gene and protein expressions of hBD-2 via its protein FAD-I through TLR1/ TLR2 and TLR2/TLR6 heterodimerizations (Bhattacharyya et al., 2016).
These heterodimers are thought to be pre-formed on the cell surface (Oliveira-Nascimento et al., 2012). In the present study, we hypothesized that single or simultaneous polymorphisms in TLR1, TLR2, or TLR6 genes lead to alterations in pattern recognition and hBD activation. There is some evidence that TLR2 polymorphisms are linked to the extent and severity of urinary tract infections or rheumatoid arthritis; however, these relations are prone to confounding factors, such as ethnicity, environmental exposures, or level of infection (Corr & O'Neill, 2009). These assumptions are in line with our study, where the association observed between the TLR2 polymorphism and T A B L E 4 Salivary human beta-defensin (hBD)-1, hBD-2, hBD-3, and human neutrophil peptide (HNP)-1 concentrations in groups carrying either of the TLR1/TLR2 or TLR2/TLR6 variants hBD-2 concentration in saliva was disrupted when the confounding factors (periodontal status, age, and smoking) were included in the analyses. Although this observation does not rule out the effect of the TLR2 genotype on the hBD-2 protein profile, it provides evidence that the hBD-2 expression profile is multifactorial. A few studies exist in the literature as regards to the association between TLR activation in neutrophils and their HNP expression. Over-expressions of genes TLR2 and TLR4 were related to suppressed gene expression of hBD-1, hBD-2, and HNP-1 in children with adenoid hypertrophy (Gankovskaya et al., 2018). Surprisingly, it has been shown that Vitamin D increased pneumococcal killing of neutrophils by stimulating the HNP protein expression via TLR2 activation (Subramanian et al., 2017). Although these findings indicate that the TLR-mediated HNP expression is dependent on the level of TLR activation, it is still unclear how much various TLR polymorphisms may modify the HNP production and secretions by neutrophils.
A novel finding in our study was the association between TLR6 polymorphism and salivary hBD-2 concentration. hBD-2 expression in human colorectal adenocarcinoma cells is regulated by TLR6 (Vora et al., 2004). In primary human oral epithelial cells, hBD-2 expression stimulated by FAD-I was mediated by TLR2/TLR6 heterodimerization, but the role of TLR6 alone in hBD expression was not defined (Bhattacharyya et al., 2016). There are controversial results in terms of hBD levels in saliva samples collected from periodontitis patients from whom elevated, steady, or suppressed hBD levels were reported (Yilmaz et al., 2020). One explanation could be the effect of TLR6 polymorphisms on hBD-2 concentrations, as this polymorphism is widely (around 50%) present in populations. Another explanation may be due to the different bacterial profile in individuals with TLR6 polymorphisms, indirectly affecting salivary hBD-2 concentrations. Unfortunately, the present study design does not allow us to test these potential explanations or to clarify the connection between hBD-2 expression and TLR6 pattern recognition.
In conclusion, while TLR2 (rs5743708) and TLR6 (rs5743810) polymorphisms relate to salivary hBD-2 concentration, only TLR6 (rs5743810) polymorphism has an independent association with it. Further studies are required to examine this association in terms of the bacterial colonization profiles, dysbiosis, and initiation of periodontal disease.