Correlation between Salivary Lipopolysaccharide ofPorphyromonas gingivaliswith Circulatory Immunoglobulin-E and Immunoglobulin-G4in Periodontally Healthy Children with House Dust Mite Allergy

The presence of periodontal pathogens in periodontally healthy children is often overlooked or ignored. Since house dust mite allergy often appears among children with chronic gum disease, it is important to understand the role of lipopolysaccharide—a major immunodominant antigen of Porphyromonas gingivalis—in stimulating atopic inflammatory markers of allergies. The aim of the present study is to investigate whether any correlation exists between salivary lipopolysaccharide of Porphyromonas gingivalis with circulatory Immunoglobulin-E and Immunoglobulin-G4 in periodontally healthy children with house dust mite allergy. We did an analytic observational study between January and December 2017. We recruited 20 periodontally healthy children (aged 6–16 years) from pediatric allergy-immunology clinic at Dr. Soetomo General Hospital (Indonesia). Lipopolysaccharide of Porphyromonas gingivalis was obtained from salivary secretion, while Immunoglobulin-E and Immunoglobulin-G4 were obtained from venous puncture simultaneously. Immunoglobulin analyses were performed by direct-sandwich ELISA, and lipopolysaccharide analyses were performed by limulus amebocyte lysate (LAL) assay. The average of salivary lipopolysaccharide was 7.21 ± 3.06 μg/ml, Immunoglobulin-E was 114.44 ± 26.19 pg/ml, and Immunoglobulin-G4 was 31.02 ± 9.09 ng/ml. There was a strong correlation between salivary lipopolysaccharide and Immunoglobulin-E (r = 0.695, p < 0.001), and a very strong correlation between salivary lipopolysaccharide and Immunoglobulin-G4 (r = 0.796, p < 0.001). Conclusion: data revealed significant correlations between salivary lipopolysaccharide of Porphyromonas gingivalis with circulatory Gingival Disease A Professional Approach for Treatment and Prevention 2 Immunoglobulin-E and Immunoglobulin-G4 in periodontally healthy children with house dust mite allergy.


Introduction
Given the complexity of community-based health care systems in the era of national insurance and high rate of poverty in Indonesia, it is not surprising that limited health literacy is common especially in rural areas [1]. Numerous studies show a link between limited parental health literacy with poor oral health among their child [2][3][4]. Parents' health literacy is often positively correlated with the frequency of dental visits and their knowledge/understanding of preventive measures in terms of oral or gingival diseases [5]. Thus, problems in the mouth range from bacterial biofilm, dental caries, chronic gingivitis, and locally aggressive gingivitis often reported in children whose parents have limited health literacy [6].
The presence of bacterial biofilm has an association with the presence of periodontal pathogen, which may lead to a periodontal disease when left untreated or undertreated [6]. Bacterial biofilm is often found among children with limited health literacy [7]. When left untreated or undertreated, these periodontal pathogens can spread below the gum line and may develop into gingivitis and periodontitis in the future [8]. Gingivitis is a reversible dental plaque-induced inflammation limited to the gingiva [9], while periodontitis is usually accompanied by gingivitis but involves irreversible destruction of the supporting tissues [10].
While many people believe that periodontal pathogen is an adult issue, nowadays this kind of pathogen is also a pediatric issue, since its presence is often reported in children aged 6-16 years [11,12]. Even though the origin and transmission of these pathogens are not fully understood, few researches have linked them with salivary transmission. Saliva is the most probable vehicle for person-to-person transmission of periodontal pathogens, such as Porphyromonas gingivalis [13]. Porphyromonas gingivalis, a black-pigmented gram-negative anaerobic rod, is one of the most crucial periodontal pathogen, not only found in adults but also common in children [14]. It can be cultured occasionally from periodontally healthy mouths [14]. Their lipopolysaccharide and whole cell were detected in the gum and oral cavities of approximately 37% of people and at similar frequencies across ages and genders [15]. This fact highlights that Porphyromonas gingivalis may be acquired in the first days of life [16]. These results are intriguing, while some researchers argued that children may acquire these periodontal pathogens from their parents, especially if the parent has periodontitis [17].
Evidence suggests that the composition of the oral microbiome differs between children with and without allergy [18], and disruption of the bacterial biofilm in children leads to allergic responses following allergen challenge in subjects not previously sensitized to the allergen [19]. It is likely that the greatest concentration of Porphyromonas gingivalis lipopolysaccharides can be found in saliva, since it is a prerequisite for their transmission [20]. Hygiene hypothesis explained a protective role of microbiome (including oral microbiome) in the development of asthma and allergy [21]; on the other hand, in periodontally healthy and diseased mouths, Porphyromonas gingivalis in subgingival plaque elicit both local and systemic immune responses [22]. To our knowledge, however, studies investigating the direct role of periodontal pathogens in terms of atopic inflammatory marker have not been reported.
The aim of the present study is to investigate whether any correlation exists between salivary lipopolysaccharide of Porphyromonas gingivalis with circulatory Immunoglobulin-E (Ig-E) and Ig-G 4 . To accomplish this, we simultaneously measured salivary lipopolysaccharide, circulatory Ig-E, and Ig-G 4 in periodontally healthy children with house dust mite allergy.

Ethical considerations
This is the grant number for ethics approval (20/Panke.KKE/I/2017). A written informed consent was obtained from every parent to be included in the study. This study was approved by Institutional Review Boards of Dr. Soetomo General Hospital Ethics Committee for Health Research at Surabaya, January 20, 2017. Results will be disseminated through peer-reviewed publications within 1 year after experiment has been finished.

Research design and setting
This is an analytic observational study with cross-sectional study design to assess the correlation between salivary lipopolysaccharide of Porphyromonas gingivalis with the circulatory Ig-E and Ig-G 4 . The study has been conducted by the support from Faculty of Dental Medicine Universitas Airlangga (Indonesia). Overall study started on January 1, 2017, and finished on December 31, 2017, at pediatric allergy-immunology clinic at Dr. Soetomo General Hospital (Indonesia) for data collection and Airlangga Oral and Dental Laboratory for IgE and IgG 4 measurement.

Sample size estimation
The sample size is based on a substantial meaningful change in IgE level observed in the group [23]. Korn et al. [23] stated that ELISA is able to assay for free IgE in a concentration range of 1-2000 pg/ml from peripheral blood samples with a substantial meaningful change of 0.5 pg/ml and the expected standard deviation of IgE concentration is assumed to be 0.01 pg/ml based on findings by Korn [23]. For this randomized controlled trial design, the formula is: All parameters were assumed as follows: mean change of IgE in intervention group = 10 pg/ml; mean change of IgE in control group = 0 pg/ml; α = 0.05; β = 0.20; δ = 10 pg/ml; s = 7; and s 2 = 49.
We calculated and found the value of N = 17.71. This estimate requires 18 patients, to obtain 80% statistical power with 5% significance level for an independent samples and paired t-test. Due to the nature of this study being a pilot study, we aimed to recruit a minimal of 22 subjects in order to compensate for an estimate of 20% drop outs.

Participants
Study participants are recruited by direct invitation and pamphlets from pediatric allergy-immunology clinic at Dr. Soetomo General Hospital (Indonesia). Data collectors reviewed the clinic appointment schedules to identify subjects with HDM allergy and met with subjects and their parents or families after their physician visit for a screening to assess their eligibility. Patients were enrolled after considering various inclusion and exclusion criteria such as mentioned in (Table 1). By total sampling, 32 subjects were obtained from this hospital. They were prescreened from January 31 to February 28, 2017 at pediatric allergyimmunology clinic at Dr. Soetomo General Hospital (Indonesia). Based on the inclusion and exclusion criteria mentioned in (Table 1), 22 subjects were eligible to participate in the study. Informed consent is given as a voluntary agreement to participate in this research, out of which, 20 parents agreed and 2 parents declined to participate in the study.

Data collection
Data were collected by taking venous puncture for measuring total serum IgE and IgG 4 from subjects' blood samples and taking saliva for measuring lipopolysaccharide of Porphyromonas gingivalis concentrations [23]. Total serum IgE and IgG 4 were assessed by direct-sandwich ELISA (R&D System Europe Ltd., Abingdon, UK) according to the manufacturer's protocol. Briefly, total serum IgE was detected by diluting plasma (1:200), transferring it to pre-coated plates, and adding the supplied conjugate. However, total serum IgG 4 were detected using monoclonal antibody against IgG 4 , followed by additions of blocking solution, diluted plasma sample (1:100,000) or standards, and conjugate, with washing between the steps. Total serum IgE and IgG 4 ELISAs were developed with the supplied TMB substrate and stop solutions. Total serum IgE and IgG 4 concentrations were determined using assay-specific 7-point calibration curves generated with the

Inclusion Criteria Exclusion Criteria
Children aged 6-16 years at any gender and ethnic manufacturer-supplied standard. A value of 0.01 pg/ml was assigned for concentrations below the limit of detection [23].
We used a simple sample-processing method for PCR to detect Porphyromonas gingivalis from subjects' saliva [23]. We obtained 500 μL of bacterial culture from salivary secretion, added to 49.5 ml of sterile saline [23]. From this suspension, two serial 1:100 dilutions were made, and 0.1 ml was plated onto plate count agar from the last dilution [23]. Following that, lipopolysaccharides of Porphyromonas gingivalis were extracted by commercially optimized assay coupled with chromogenic substrate from pellet. We measured the concentration of extracted lipopolysaccharide by Pierce LAL Chromogenic Endotoxin Quantitation Kit. All measurements were done in duplicate and values averaged for analysis [23].

Statistical analysis
The obtained data were tabulated and analyzed using Statistical Package of Social Science (SPSS version 17, IBM, New York, USA). First, univariable linear regression procedures were conducted to examine associations between circulatory Ig-E and all determinants. In advance, dummy variables were created for all categorical determinants. Second, a multiple linear regression analysis with a stepwise exclusion method was conducted with all continuous and dummy variables. Determinants that seemed relevant for prediction of activation were kept in the model. Statistical significance was set at p < 0.05 [23].

Results
A total of 20 periodontally healthy children accepted to participate in this study; 8 were males and 12 were females. The average age was 10.52 ± 2.78 years (range, 6-16 years). The patients' general characteristics are as shown in Table 2.

Correlation between salivary LPS-Pg and Ig-E levels
Analysis of correlation between salivary LPS-Pg and Ig-E levels showed a strong correlation (r 2 = 0.695; p < 0.001, n = 20), and there was a positive correlation as shown in Figure 1.

Correlation between salivary LPS-Pg and Ig-G 4 levels
Analysis of correlation between salivary LPS-Pg and Ig-G 4 levels showed a very strong correlation (r 2 = 0.796; p < 0.001, n = 20), and there was a positive correlation as shown in Figure 2.

Results of univariate logistic regression analysis
As shown in Table 3, univariate logistic regression analysis uses gender, age, height, weight, BMI, family size, insurance status, number of colony-forming unit (CFU) of Pg, level of salivary LPS-Pg, level of circulatory Ig-G 4 as the independent variables, and circulatory Ig-E as the dependent variable. Data revealed that number of colony-forming units (CFU) of Pg, level of salivary LPS-Pg, and level of circulatory Ig-G 4 were associated with circulatory Ig-E (p < 0.05).

Results of multivariable regression analysis
Multivariable regression analysis was performed using indexes which were demonstrated to be related to circulatory Ig-E in univariate analysis. The results showed that only level of salivary LPS-Pg and level of circulatory Ig-G 4 remained a risk factor for circulatory Ig-E ( Table 4).

Discussion
Despite existing literature pointing to a potential role of lipopolysaccharide in modulating allergic reactions, it remains a relatively underresearched subject matter. Notably, the majority of existing research has only investigated the interactions between periodontitis and clinical symptoms of allergies [24]. In this research, we had observed a direct correlation between lipopolysaccharide of Porphyromonas gingivalis with atopic inflammatory markers.
Lipopolysaccharide is a gram-negative endotoxin, ubiquitous in the environment and can therefore exacerbate allergic responses [25]. Existing literature has demonstrated the pathogenic role of Porphyromonas gingivalis dental biofilm to stimulate lipopolysaccharide-driven inflammatory responses [26], and therefore, lack of dental plaque and calculus in supra-gingival surface, sub-gingival surface, as well as human epithelial cell rests of Malassez account for the lack of response to lipopolysaccharide to induce host inflammatory responses [27].
As a unique endotoxin, lipopolysaccharide-driven inflammatory responses can induce a more pronounced pro-inflammatory cytokine response [28]. At very low concentration, lipopolysaccharide may induce atopic inflammatory responses by Th-1 shifting into Th-2, which is more potent to stimulate antibodies production [29]. Lipopolysaccharide binds to Toll-like receptor (TLR) 4 and greatly enhances the response of TLR4-transfected cells [30]. Thus, damage from lipopolysaccharide extends beyond the exhaustion of host innate immunity [31].
Lipopolysaccharide activates macrophage via the TLR4/NF-κB pathway [32]. In turn, TLR4 activation increases SOCS3 mRNA expression [33]. Since SOCS3 is an inducible endogenous negative regulator of JAK/STAT pathway [34], therefore administration of lipopolysaccharide in a model of experimental periodontal disease will be correlated with dynamics of the atopic inflammatory reaction [35]. Meanwhile, given the unique lipopolysaccharide-induced atopic inflammatory responses and lipopolysaccharide-triggered mast cell derived, we can predict that lipopolysaccharide of this periodontal pathogen may stimulate the level of circulatory Ig-E and Ig-G 4 , even in the healthy populations [36,37].
In line with the hypothesis, our present study confirms the existence of a significant correlation between salivary lipopolysaccharide of Porphyromonas gingivalis with the circulatory Ig-E and Ig-G 4 in periodontally healthy children with house dust mite allergy. Importantly, this association remained even after adjusting for baseline and clinical parameters, suggesting an independent association between salivary lipopolysaccharide and allergic biomarkers.
This study had several limitations. Firstly, this study had small sample size, limiting the generalizability of the results. Perhaps more observations with longer periods would have been statistically significant with a larger sample size. Secondly, the cross-sectional approach of this current study cannot draw any  conclusions concerning direct relationships. Given the correlational nature of the analysis, we cannot clarify whether salivary lipopolysaccharide of Porphyromonas gingivalis is the direct cause of high circulatory Ig-E and Ig-G 4 in periodontally healthy children with house dust mite allergy. Lastly, this study did not assess the levels of house dust mite-specific serum Ig-E and the effect of increasing Ig-E level with the occurrence of allergic manifestation. Indeed, studies have recognized that evaluating an allergic manifestation and quantifying the levels of house dust mite-specific serum Ig-E are critical requisites when trying to establish an association with salivary lipopolysaccharide of Porphyromonas gingivalis. Nevertheless, this was beyond the scope of the present study, which aimed to investigate the correlation between salivary lipopolysaccharide and atopic inflammatory markers.

Conclusion
In conclusion, salivary lipopolysaccharide of Porphyromonas gingivalis might serve as a predictor for circulatory Ig-E and Ig-G 4 in periodontally healthy children with house dust mite allergy. These data might guide future studies on the actual role of these periodontal pathogens with the progression and sensitization of allergic diseases and help to establish a more effective treatment for child allergies. With increasingly more studies indicating the association of Porphyromonas gingivalis colonization and its lipopolysaccharide in an allergic child in the future, clinicians should be more aware about these periodontal pathogens in children's saliva and gum tissue. Despite a rare progression into a localized aggressive periodontitis, chronic gingivitis should be treated well in children, since its lipopolysaccharide may be linked with allergic diseases in the future.

Funding source
This study was supported partly by Faculty of Dental Medicine Universitas Airlangga Health Research Projects. The funding agencies had no role in data collection, analysis, interpretation, or the decision to submit the results.