The indicative role of inflammatory index in the progression of periodontal attachment loss

Objective To explore the forewarning immunological indicators during periodontal attachment loss progression in American adults. Methods A total of 5744 participants with periodontal attachment loss were enrolled from the National Health and Nutrition Examination Surveys (NHANES) 2009–2014. In which, dependent variable was the counts of teeth with severe attachment loss (depth of periodontal probing was above 5 mm). Independent variables were circulatory immunological indexes, including counts of white blood cells (WBC), Lymphocytes, Monocytes, Neutrophils, Eosinophils, and Basophils. The association among variables was examined using multivariable linear regression models, fitting with smoothing curves, and generalizing additive models. Results Based on the indicators of 5744 subjects, we found that severe attachment loss tended to occur in the elderly or males and was accompanied by higher WBC, Monocytes, and Neutrophils, as well as lower poverty-income ratio and educational qualification. WBC (above the inflection point: 6200 cells/µL) and Neutrophils (above the inflection point: 3300 cells/µL) counts were positively associated with attachment loss progression in each multivariable linear regression model. On subgroup analyses, stratified by sex and race, the positive correlation of WBC or Neutrophils with severe attachment loss was stable in both men and women, as well as in all races except blacks (WBC β = − 0.0576, 95% CI − 0.1945 to 0.0793, Neutrophils β = − 0.0527, 95% CI − 0.2285 to 0.1231). Conclusion Increasing WBC (above 6200 cells/µL) and Neutrophils (above 3300 cells/µL) counts were risk indicators of severe periodontal attachment loss among all races, except in blacks.


Introduction
With a prevalence of 47% in the American population over 30 years old [1,2], periodontitis impaired the aesthetic and oral function [3,4], mainly manifested as attachment loss and alveolar bone resorption [5][6][7].Current studies focused on the correlated inflammation or immunity factors of severe periodontitis [8], but the indepth monitoring of immunological indicators during attachment loss progression remains unknown.
On accounts of the abundant systemic circulation of the maxillofacial region, periodontitis causes or exacerbates comorbidities and resulting changes in circulating immune indicators (including white blood cells, Neutrophils, etc.) [7,9,10].Previous studies showed that severe periodontitis was accompanied by changes in the levels of inflammatory indicators [11,12], for example, patients with periodontitis are often accompanied by an increase in mean platelet volume and a decrease in eosinophilic counts.Meanwhile, circulatory immune indexes were widely utilized as forewarning or diagnostic criteria of infectious diseases.For instance, white blood cell (WBC) counts were generally expected to be closely associated with a respiratory infection, which diagnoses pneumonia, determines its etiology, and predict disease progression [13,14].To forewarn the occurrence and progression of severe periodontitis, it is necessary to explore the potential relationship between circulatory immunological indexes and periodontal attachment loss progression.
In this study, we aimed to explore the association between severe attachment loss progression and circulatory immunological indicators by multivariable linear regression based on the NHANES database.Severe periodontal attachment loss tended to occur in males and was accompanied by higher WBC, Monocytes, and Neutrophils.Furthermore, WBC, Monocytes, and Neutrophils counts were closely associated with periodontal attachment loss.This study sought to shed light on the correlation between the progression of severe periodontitis and immune-related indicators.

Study population
Data of the current study were abstracted from the National Health and Nutrition Examination Survey (NHANES).In total, 14,071 participants were enrolled from NHANES between 2009 to 2014.After excluding participants (n = 2336) with incomplete records of periodontal examination, 11,735 participants were available.Further, excluding participants who had the attachment loss < 5 mm or > 2 mm (n = 5771), and with missing data of the immune cell examination (n = 222), 5744 participants were finally enrolled in the study.The study was approved by the review board of the National Center for Health Statistics.

Dependent variable
Periodontal attachment loss was identified by dental examiners, who were dentists (D.D.S./D.M.D.) licensed in at least one U.S. state.All oral health assessments occurred in a designated room at the mobile examination center (MEC).In this experiment, attachment loss ≥ 5 mm was defined as severe attachment loss [15].In addition, dependent variable was counts of teeth with severe attachment loss.

Statistical analysis
All statistical analyses were performed by using R (http:// www.R-proje ct.org), with statistical significance set at P < 0.05.All estimates were calculated by using sample weights following the analytical guideline edited by NCHS.Three multivariable linear regression models were constructed by hierarchical multiple regression to test the significance of variables: model 1, no covariates were adjusted; model 2, age, gender, and race were adjusted; model 3, all covariates presented in Table 1 were adjusted.Furthermore, smooth curve fittings and generalized additive models were used to address the nonlinearity.The inflection points were calculated using a recursive algorithm, with a two-piecewise linear regression model conducted on both sides of the inflection point when nonlinearity was detected.

Results
The demographic and laboratory data of the 5744 participants (3217 men and 2527 women), with the weighted characteristics of the participants subclassified based on quartiles of attachment loss teeth counts (Q1: 0; Q2: 1; Q3: 2-5; and Q4: 6-27), as presented in Table 1.There were significant differences in baseline characteristics between the attachment loss teeth quartiles, except for the counts of Eosinophils, Basophils, and Lymphocytes.Participants with generalized periodontal attachment loss tend to occur in the elderly or males and are accompanied with higher BMI, WBC, Monocytes, Neutrophils, and Eosinophils, as well as lower PIR and educational qualification.

Discussion
Accompanied with attachment loss, severe periodontitis caused serious aesthetic problems and reduced chewing efficiency [16,17].Previous studies suggested that the progression of attachment loss was accompanied with increasing circulatory immunological indexes in severe periodontitis [13,14], but immune cell counts serve as forewarning or diagnostic criteria for severe attachment loss or periodontitis progression remains unclear.Our multivariate logistic regression analyses indicated that elevated WBC or Neutrophils was correlated with attachment loss progression.However, on subgroup analysis, we identified the negative relationship between WBC or Neutrophils and attachment loss among blacks.
Phagocytosis by WBC, Neutrophils, and Monocytes constitutes the main defense mechanism against bacterial challenges in periodontitis [18].As the enriched immune cell killing bacteria and destroying inflamed tissues [19], immunity homeostasis was essential for periodontal attachment maintenance [20].In consist with our results, a previous study showed that severe periodontitis accompanied with higher WBC or Neutrophils levels than the healthy population [6].On the other hand, infectious viruses, including Human simplex virus-1, Epstein-Barr virus, and Human cytomegalo virus, were commonly involved in the occurrence and development of periodontitis or attachment loss [21].Simple viral infections accompanied with the decrease of WBC and Neutrophils [22], while combination of bacterial infections promotes WBC elevation [23].Recent identified inflection point of fluctuating confounder was utilized as forewarning of periodontitis risk, and periodontal status [24].Our multivariable linear regression analyses suggested that participants with WBC above 6200 cells/µl or Neutrophils above 3300 cells/µl obtained higher risk of periodontal attachment loss, which can serve for severe periodontitis prevention and treatment.
Previous studies identified the racial differences of WBC or Neutrophils counts between blacks and other races [25,26].In which, WBC or Neutrophils counts were significantly lower in blacks during dental plaque accumulation, but hyperactivity of circulating neutrophils appeared in blacks [26].Importantly, the reactivity of oxidative burst was significantly lower in neutrophils from blacks, especially from African American males [27].Periodontal attachment loss-related racial differences can provide accurate guidance for subsequent community prevention.
This study demonstrated the association between circulatory immunological indicators and periodontal attachment loss.However, several limitations are noticeable.First, all self-reported information might cause recall bias or misclassification bias.Second, further studies with larger sample sizes using more relevant variables (such as gingival sulcus fluid data and bone destruction) may discover more accurate factors in periodontal attachment loss.Third, circulating immune cells counts were affected by the whole-body state, periodontal pocket related immunological indexes may have better reproducibility.

Conclusion
Our study suggested that increasing WBC (above 6200 cells/µL) and Neutrophils (above 3300 cells/µL) counts were potential risk indicators of periodontal attachment loss progression, which provides an early warning for severe periodontitis.

(a) (b)
Fig. 3 The association between WBC, Neutrophils and periodontal attachment loss, stratified by race

Fig. 1 Fig. 2
Fig. 1 The association between WBC periodontal loss.a Each black point represents a sample.b Solid rad line represents the smooth curve fit between variables.Blue bands represent the 95% of confidence interval from the fit

Table 1
Characteristics of the study population

Table 2
Association between WBC and periodontal attachment loss progression

Table 3
Association between Neutrophils and periodontal attachment loss

Table 4
Threshold effect analysis of WBC on the progression of attachment loss using the two piecewise linear regression model

Table 5
Threshold effect analysis of Neutrophils on the progression of attachment loss using the two piecewise linear regression model