Evaluation of Salivary Il-38 Levels in Periodontitis: A Cross-Sectional Study

Abstract

The goal of the current study was to assess levels of salivary interleukin (IL)-38, IL-1β, and IL-10 in various periodontal clinical conditions. In total, 60 (20 healthy, 20 gingivitis, and 20 stage II–III, grade A–B periodontitis) subjects were included in the study. Demographic and clinical periodontal parameters were recorded. Samples were examined for IL-38, IL-1β, and IL-10 levels by means of enzyme-linked immunosorbent assay. Results demonstrated that the periodontitis group had significantly lower salivary IL-38 levels (P < 0.05) than the healthy group. Salivary IL-10 levels did not differ significantly between the groups (P > 0.05). The salivary IL-1β levels of gingivitis (P < 0.001) and periodontitis groups (P < 0.01) were significantly higher than those of the healthy group. The present study indicated that IL-38 level is decreased in periodontal disease. The results suggested a possible role of IL-38 in the periodontal inflammation process. Clarifying the mechanisms of IL-38 in the inflammatory process may contribute to the development of novel treatment strategies in periodontal diseases.

Introduction

Periodontitis is a common health problem, and the main cause of tooth loss in adults. It is a chronic inflammatory disease with multifactorial etiology and pathogenesis in which dental-supporting tissues are affected. As a result of the interaction between microorganisms the primary etiological factors, and the host response, impaired homeostasis in the periodontium may lead to periodontal tissue loss (Darveau, 2010). Local over-production of cytokines and other mediators is critical in the pathogenesis of periodontal disease (Graves, 2008). After the recognition of microorganisms and their presentation to appropriate cells, interleukin (IL) 1β, IL-6, and tumor necrosis factor-alpha (TNF-α) are the first pro-inflammatory cytokines to appear in the periodontal disease process (Garlet, 2010). Regulatory immune cells produce anti-inflammatory cytokines to control the inflammatory process and tissue destruction. IL-10 and transforming growth factor-β are anti-inflammatory cytokines that have a prominent role in the periodontal inflammation process (Cardoso et al., 2008).

The IL-1 family (IL-1F) has a major function in immune responses and inflammatory processes to physical injury and pathogenic microorganisms (Preshaw and Taylor, 2011). IL-1F comprises 11 members with both proinflammatory (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ) and anti-inflammatory properties such as the IL-1 receptor antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38 (Dinarello, 2018). IL-1β, a potent stimulator of periodontal tissue destruction, participates in immune regulation, inflammation, and bone resorption in periodontal disease (Garlet, 2010). Periodontal pathogens and their products stimulate IL-1β production from macrophages, leukocytes, gingival fibroblasts, periodontal ligament cells, and osteoblasts (Liu et al., 2010). Periodontitis was reported to cause systemically (serum) and locally (gingival crevicular fluid and saliva) high production of IL-1β, and the biological effects of IL-1β were attributed to increased tissue concentration (Engebretson et al., 2002; Kim et al., 2021; Preshaw et al., 2020).

IL-38, identified in 2001 (previously also called IL-1F10, IL1HY2, FIL-1θ or IL-1θ) is the 10th member of IL-1F (Lin et al., 2001; Palomo et al., 2015). Expression of IL-38 was detected in the fetal liver, spleen, thymus, tonsils, salivary glands, and skin (Ciccia et al., 2015; Lin et al., 2001). It has been suggested that IL-38 is an IL-36 antagonist and also inhibits the proinflammatory activities of IL-36 by acting as a receptor antagonist similar to IL-36Ra and IL-1Ra (van de Veerdonk et al., 2012). IL-38 was shown to have an anti-inflammatory impact in vivo and in vitro in models (Boutet et al., 2017; Mora et al., 2016; van de Veerdonk et al., 2012). In recent studies, IL-38 expression was reported to be abnormal in chronic inflammatory diseases, such as systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, and psoriasis (Xu and Huang, 2018). IL-38 overexpression was shown to downregulate TNF-α and IL-10 levels in experimental arthritis (Boutet et al., 2017). In mouse arthritis models, IL-1β and IL-6 expression in ankle joints was higher in IL-38-deficient mice than in control mice (Takenaka et al., 2015). IL-38 expression was shown to reduce on the palatal mucosa of periodontally healthy smokers (Wang et al., 2020). In addition to tissues, some studies have shown the presence of IL-38 in body fluids (such as synovial fluid, serum/plasma, and saliva in primary Sjögren syndrome) (Boutet et al., 2017; Liu et al., 2020; Luo et al., 2020). To the best of our knowledge, there is only one study in the literature evaluating the human salivary IL-38 levels in periodontal inflammation (Korkmaz et al., 2023). Although the mechanisms by which IL-38 exerts its anti-inflammatory effects are not fully understood, it has been suggested that it has a regulatory role in chronic inflammatory diseases (Jiang et al., 2021; van de Veerdonk et al., 2018). These results suggest that IL-38 may have a role in the pathogenesis of periodontitis, an inflammatory disease.

Biomarkers are important determinants in observing normal biological or pathogenic processes and are quite practical and easy to evaluate in body fluids. Proteins leaking from tissues as a result of tissue damage can be detected in body fluids. Saliva is one of the body fluids frequently used in the evaluation of biochemical markers related to periodontal disease (Gursoy and Kantarci, 2022). In the current study, we investigated the salivary IL-38 levels in various periodontal clinical conditions and their correlation with the IL-1β and IL-10 levels.

Materials and Methods

Study groups

The current study was conducted in accordance with the ethical principles of the Helsinki Declaration. It was approved by the Health Sciences University, Hamidiye Clinical Research Ethics Committee (21/46). NCT05568290 is the registration number for this study at Clinicaltrials.gov.

Sixty volunteers who signed the consent form were recruited from the School of Dentistry (Periodontology, İzmir Katip Çelebi University), between April and December 2021, in this cross-sectional study. All subjects were examined in our clinic for the first time for treatment and did not receive supportive periodontal therapy. By considering “IL-1β” as the primary outcome variable, 20 subjects per group were determined according to the previous study (Tobón-Arroyave et al., 2008) for 88% power, α = 0.05, and effect size 0.46, because there were no human studies evaluating salivary IL-38 for periodontal diseases.

Periodontal examination

A single calibrated examiner (L.S.) examined each subject. Calibration of the examiner was performed by repeated measurements (after 7 days) of 6 patients with periodontitis who were not included in the study. The intra-examiner calibration was measured for clinical attachment loss (CAL; k = 0.85). The periodontal parameters were recorded, including plaque index (PI) (Silness and Loe, 1964), gingival index (GI) (Loe, 1967), bleeding on probing (BOP) (Ainamo and Bay, 1976), probing depth (PD), and CAL at six surfaces of all teeth (except for third molars). Periodontal examination of all subjects was performed by a Williams periodontal probe and evaluated radiographic results.

Determination of periodontal status

Subjects were classified into periodontal health (healthy: 9 females and 11 males), gingivitis (12 females and 8 males), and periodontitis (10 females and 10 males). Healthy subjects had no systemic disease, took no drugs, and exhibited periodontal health (absence of signs of inflammation and bone and attachment loss) (Chapple et al., 2018). Gingivitis was defined as the absence of CAL and the presence of an average GI of ≥1 for the whole mouth and BOP in ≥10% sites, with PD of ≤3 mm in teeth (Chapple et al., 2018). Periodontitis was defined as the presence of interdental CAL at ≥2 non-adjacent teeth or buccal/oral CAL of ≥3 mm with PD >3 mm in ≥2 teeth (Tonetti et al., 2018). In this study, the periodontitis group included stage II grade B (n = 3), stage III grade A (n = 9), and stage III grade B (n = 8). Exclusion criteria included: (1) pregnancy and lactation; (2) on antibiotics and/or anti-inflammatory therapy within the past 3 months; (3) subjects with systemic diseases, (4) history of periodontal treatment within the past 6 months; (5) smoking (determined by patient’s declaration); and (6) edentulous patients.

Collection of samples

The unstimulated whole saliva sampling was performed by the examining physician (L.S.) in the morning. The collection was performed before eating, drinking, and oral hygiene practices. Salivary samples were centrifuged for 10 min at 800g, and then frozen at −80°C until analysis.

Biochemical analysis

The IL-38, IL-1β, and IL-10 levels in saliva samples were measured using enzyme-linked immunosorbent assay (ELISA) kits (MyBioSource Human ELISA Kit, San Diego, California, USA) following the manufacturer’s instructions. The limit of detection (LOD) value was 4.69 pg/mL for IL-1β, 4.69 pg/mL for IL-10, and 1.0 pg/mL for IL-38. Optical density was monitored by a microplate reader set to 450 nm. The unit of data was defined as picograms per milliliter (pg/mL).

Statistical analysis

Data were analyzed using SPSS for Windows 20.0. Mean (standard deviation) and median were given for descriptive statistics. P ≤ 0.05 was considered a statistical significance level. GI, PI, and BOP (ordinal data) and IL-10 were analyzed using the Kruskal–Wallis and post hoc tests because these did not exhibit a normal distribution. Other data were normally distributed, and differences between groups were assessed by analysis of variance (ANOVA) and post hoc Tukey’s tests. An analysis of covariance (ANCOVA), controlling for age was used to confirm the significant differences for biochemical data. Spearman rho (for non-normally distributed variables) correlation coefficients were used to explore associations among data. A univariate linear regression analysis was applied to evaluate the association of the IL-1β concentrations with the clinical periodontal parameters. Age was additionally assessed as a covariate for IL-1β and interaction between clinical periodontal parameters in a model with an analysis of multicollinearity.

Results

Demographic and clinical characteristics

Demographic and clinical values of subjects are displayed in Table 1. Mean ages (range, 20–33 years) were statistically higher in the periodontitis group than in the gingivitis and healthy groups (P < 0.001). Mean values of GI, PI, PD, and BOP in the gingivitis groups were significantly higher than in the healthy group (P < 0.001). BOP, PD, and CAL values of patients with periodontitis were significantly higher than in those with gingivitis (P < 0.01 for BOP, P < 0.001 for PD and CAL).

Table 1.

Between-Group Comparisons of Demographic, Clinical, and Biochemical Data Obtained from the Study Participants

Parameters	Clinical groups			P^X	P^Y
Parameters	Healthy (n = 20)	Gingivitis (n = 20)	Periodontitis (n = 20)	P^X	P^Y
Age, years	26.7 ± 4.4Median = 27	33.7 ± 9.6^d Median = 34.5	42.1 ± 10.4^f,a Median = 41.5	<0.001
GI	0.4 ± 0.2Median = 0.3	1.4 ± 0.3^f Median = 1.4	1.5 ± 0.4^f Median = 1.4	<0.001
PI	0.7 ± 0.4Median = 0.7	1.3 ± 0.3^f Median = 1.4	1.5 ± 0.4^f Median = 1.5	<0.001
BOP (%)	6.8 ± 4.8Median = 6.1	42.9 ± 13.9^f Median = 38.6	58.1 ± 20.9^f,b Median = 54.9	<0.001
PD (mm)	1.3 ± 0.2Median = 1.4	2.0 ± 0.3^f Median = 2.0	3.2 ± 0.6^f,c Median = 3.2	<0.001
CAL (mm)	0	0	3.1 ± 0.5^f,c Median = 3.1	<0.001
Salivary IL-38 levels (pg/mL)	7.2 ± 5.8Median = 3.8	5.0 ± 1.4Median = 4.5	4.3 ± 0.6^d Median = 4.1	0.027	0.24
Salivary IL-1β levels (pg/mL)	7.0 ± 1.1Median = 7.0	43.7 ± 18.7^f Median = 46.3	40.8 ± 40.3^e Median = 29.7	<0.001	0.02
Salivary IL-10 levels (pg/mL)	13.4 ± 2.2Median = 12.6	16.1 ± 8.0Median = 13.1	18.0 ± 10.5Median = 14.5	0.35

Statistically significant difference (P < 0.05, Tukey honest significant difference [HSD] multiple-comparison test) compared with G group.

Statistically significant difference (P < 0.01, Tukey HSD multiple-comparison test) compared with G group.

Statistically significant difference (P < 0.001, Tukey HSD multiple-comparison test) compared with G group.

Statistically significant difference (P < 0.05, Tukey HSD multiple-comparison test) compared with H group.

Statistically significant difference (P < 0.01, Tukey HSD multiple-comparison test) compared with H group.

Statistically significant difference (P < 0.001, Tukey HSD multiple-comparison test) compared with H group.

Analysis of variance (ANOVA) test.

Analysis of covariance (ANCOVA), adjusted for age. Presented data were reported as mean ± standard deviation.

G group, Gingivitis; H group, Healthy.

Biochemical findings

Values of IL-1β, IL-38, and IL-10 are shown in Table 1. Salivary IL-38 levels were statistically lower the periodontitis group than in the healthy group (P < 0.05). Salivary IL-1β levels of gingivitis and periodontitis groups were statistically higher than those in the healthy group (P < 0.001, P < 0.01, respectively).

Correlations

Table 2 reveals correlation analysis results. No significant correlations were seen between IL-38 levels and other parameters. In this study, IL-1β levels exhibited a positive correlation with clinical parameters, except for CAL. IL-10 levels were significantly positively correlated with IL-1β levels (r = 0.430, P = 0.001), PD (r = 0.278, P = 0.035) and CAL (r = 0.268, P = 0.042) parameters. The univariate regression analysis evidenced that there was a significant direct impact of GI, PI, BOP, and, PD on salivary L-1β (both P < 0.05; Table 3). Additionally, the association of GI, PI, and BOP values with IL-β levels remained significant when age was included as a covariate in the multiple regression analysis (P < 0.001, P = 0.011, P = 0.001, respectively; Table 4).

Table 2.

Spearman Correlation Analysis Among Clinical Parameters and Biochemical Salivary Levels

	IL-38	IL-10	IL-1β
GI	−0.190	0.098	0.632 (<0.001)^a
PI	−0.70	0.254	0.419 (0.001)^a
BOP	−0.233	0.301	0.526 (<0.001)^a
PD	−0.254	0.235 (0.035)^b	0.485 (<0.001)^a
CAL	−0.231	0.268 (0.042)^a	0.237
IL-1β	−0.162	0.394 (0.002) ^b
IL-10	−0.122

Presented data was reported as Spearman rho (P value) and n = 20 patients for per group.

Correlation is significant at the 0.01 level (2-tailed).

Correlation is significant at the 0.05 level (2-tailed).

Table 3.

Univariate Linear Regression Analysis of Clinical Periodontal Measurements with Salivary IL-1β Levels in All Enrolled Subjects

Regression coefficients			95% CI for B		Collinearity statistics
Variable	ß	B	Lower	Upper	t	P value
GI	0.498	25.626	13.689	37.563	4.3	<0.001 ^*
PI	0.388	22.826	8.319	37.332	3.152	0.003 ^*
BOP	0.484	0.570	0.294	0.846	4.142	<0.001 ^*
PD	0.314	10.384	1.993	18.774	2.479	0.016 ^*
CAL	0.232	4.707	−0.574	9.988	1.785	0.08

ß, standardized regression coefficient; B, coefficient; CI, confidence interval; t, test statistics.

P value < 0.05, statistically significant, n = 20 patients for per group.

Table 4.

Multiple Linear Regression Analysis of Clinical Parameters with Salivary IL-1β Levels When Age Was Included as Covariant

Regression coefficients			95% CI for B		Collinearity statistics
Variable	ß	B	Lower	Upper	P value	VIF
GI	0.481	24.761	11.386	38.136	<0.001 ^*	1.234
AGE	0.039	0.111	−0.638	0.860	0.767	1.234
PI	0.344	20.208	4.839	35.577	0.011 ^*	1.123
AGE	0.135	0.388	−0.365	1.140	0.307	1.123
BOP	0.467	0.549	0.238	0.860	0.001 ^*	1.251
AGE	0.039	0.113	−0.648	0.874	0.766	1.251
PD	0.254	8.385	−1.102	17.873	0.082	1.274
AGE	0.131	0.376	−0.452	1.204	0.366	1.274
CAL	0.141	2.860	−3.312	9.031	0.357	1.373
AGE	0.175	0.503	−0.373	1.380	0.255	1.373

ß, standardized regression coefficient; B, coefficient; CI, Confidence Interval; t, test statistics; VIF, variance inflation factor.

P value < 0.05, statistically significant, n = 20 patients for per group.

Discussion

In this study, salivary IL-38, IL-1β, and IL-10 levels were evaluated in patients with gingivitis and periodontitis and compared with those of periodontal healthy individuals. Our results showed that salivary IL-38 levels were significantly decreased in patients with periodontitis compared with healthy subjects. To the best of our knowledge, this is the first study to evaluate saliva IL-38 levels together with IL-1β and IL-10 in different periodontal conditions.

Although the mechanism of IL-38, which is one of the new members of IL-1F, is not clear, it has been suggested that it exerts antagonistic effects on inflammatory cytokine induction (Mora et al., 2016). Studies have shown that IL-38 degradation or overexpression is strongly associated with various chronic inflammatory diseases (Boutet et al., 2017; Ciccia et al., 2015; Rudloff et al., 2015). It was reported that the mRNA and protein of IL-38 levels are reduced in inflammatory skin diseases such as psoriasis (Mercurio et al., 2018). In inflammatory bowel disease, IL-38 expression levels were shown to increase in the colon (Xie et al., 2020). Wang et al. (2020) showed that IL-38 levels were significantly downregulated in the palatal mucosa of periodontally healthy smokers compared with periodontally healthy non-smokers. Ciccia et al. (2015) showed that mRNA expression of IL-38 was increased in the salivary glands of patients with primary Sjögren syndrome. Jiang et al. (2021) determined that patients with osteoarthritis had significantly higher IL-38 levels in the synovial fluid and serum than healthy controls. Luo et al. (2020) found that the mRNA and protein levels of IL-38 levels in serum and salivary were significantly lower in primary Sjögren syndrome group than in nonprimary Sjögren syndrome and healthy control group. In this current study, the periodontitis group exhibited significantly lower salivary IL-38 levels than the healthy group. Our result supports studies reporting decreased IL-38 levels in some chronic inflammatory diseases (Jiang et al., 2021; Luo et al., 2020; Mercurio et al., 2018; Wang et al., 2020; Xie et al., 2020). In contrast to our study results, Korkmaz et al. (2023) observed, in their study evaluating patients with stage II–III grade B and C periodontitis, that levels of salivary IL-38 were significantly higher in the periodontitis group than in the healthy and gingivitis group. This contrast may be explained by the variable systemic and periodontal status of the study groups. In addition, this situation may be because of distinct reasons that may have an important impact on salivary biochemical markers, for example, dietary habits or differences in the stages and grades of individuals with periodontitis. Owing to this heterogeneity in results, further studies are needed to elucidate the relationship between IL-38 and periodontal disease. The ANCOVA of IL-38 showed that when age is adjusted for, the significant difference between groups is lost. We could not compare with others because of the lack of similar studies where age has been considered as a factor for IL-38 levels.

Inflammatory and anti-inflammatory factors regulate the inflammatory environment in periodontal diseases. IL-1β is a very substantial cytokine in the periodontal inflammation process (Garlet, 2010; Graves, 2008). Studies stated that patients with more severe BOP and deeper PD had elevated salivary IL-1β levels (Ebersole et al., 2015; Reddahi et al., 2022). In a clinical study (Teles et al., 2009), it was demonstrated that salivary IL-1β levels were higher in patients with periodontitis than in healthy subjects. The results of another study (Tan et al., 2020) showed that salivary IL-1β levels were elevated in subjects with gingivitis and in stage I and III periodontitis compared with healthy subjects. Reddahi et al. (2022) reported that patients with stage III grade B and C periodontitis had higher salivary IL-1β levels than healthy subjects, but the difference between grade B and grade C was not statistically significant. In our study, gingivitis and periodontitis groups had significantly higher saliva IL-1β levels than the healthy group. Our ANCOVA results, which were adjusted for age, showed that IL-1β was a biomarker related to periodontal disease and were consistent with the results of Kim et al. (2021). A statistically significant correlation between clinical measurements and salivary IL-1β levels was shown (Kim et al., 2021; Tobón-Arroyave et al., 2008). In addition, studies have reported a strong association between IL-1β and clinical indices in linear regression models and that IL-1β indeed reflected periodontal status (Tobón-Arroyave et al., 2008; Zhang et al., 2021). In our study, salivary IL-1β levels showed a significant correlation with clinical parameters. The significant relationship between GI, PI, and BOP and IL-β levels continued when age was implemented as a covariant. These results support results of previous studies (Ebersole et al., 2015; Miller et al., 2006; Reddahi et al., 2022; Tan et al., 2020; Teles et al., 2009; Tobón-Arroyave et al., 2008; Zhang et al., 2021). A study reported that IL-1β levels of individuals with chronic periodontitis did not show a significant difference compared with patients with gingivitis (Ramseier et al., 2009). In our study, there were no significant differences between gingivitis and periodontitis in salivary IL-β levels, and no correlation was found between salivary IL-1β and IL-38 levels.

IL-10 by T helper 2 (Th2) is a crucial anti-inflammatory cytokine in the inflammatory process (Iyer and Cheng, 2012). Information on IL-10 levels in periodontal disease in the literature is still conflicting. Some studies (Reddahi et al., 2022; Teles et al., 2009) have shown that there is no significant difference in salivary IL-10 levels in the periodontitis group compared with the healthy group. Tan et al. (2020), in their study, determined that salivary IL-10 levels of stage I periodontitis were not significantly different from those of the gingivitis group. On the other hand, subjects with stage I periodontitis and gingivitis had increased salivary IL-10 levels compared with healthy subjects. In the current study, we observed an increase in the salivary IL-10 levels in the gingivitis and periodontitis groups compared with the healthy group, but these differences were not significant between groups. A study reported that overexpression of IL-38 reduced the secretion of IL-10 induced by lipopolysaccharide (LPS) in human leukemic cell line (THP-1) (Boutet et al., 2017). Overexpression of IL-38 was reported to downregulate IL-10 levels (Boutet et al., 2017; Chu et al., 2016; Jiang et al., 2021). We detected that there was a negative correlation between salivary IL-10 and IL-38 levels, but it was not statistically significant in this study.

The limitations of this study should be examined in further research. First, owing to the cross-sectional nature of this study, it was difficult to assess all aspects of the relationship between salivary IL-38 levels and the progression of periodontal disease. Second, the sample size may have been inadequate to reveal the relationship between IL-38, IL-1β, and IL-10, if any. Third, because the population of this study was small, we were not able to determine changes in IL-38 levels at different stages of periodontitis. Therefore, large-scale studies should be performed to get more precise statistical results. Moreover, the relationship between IL-38 levels and other proinflammatory and anti-inflammatory cytokines should be investigated in further studies.

Conclusion

In the current study, we found lower salivary IL-38 levels in periodontal inflammation. The results of this study indicate a possible function of IL-38 in the pathogenesis of the periodontal disease. Nevertheless, further longitudinal clinical and laboratory studies should be performed to better reveal the relation between salivary IL-38 levels and periodontal disease severity, and the possible effect mechanisms in the periodontal inflammation process of IL-38.

Footnotes

Acknowledgments

The authors are thankful to Professor Dr. Mehmet SAĞLAM from the Department of Periodontology, Faculty of Dentistry, İzmir Katip Çelebi University, İzmir, Türkiye, for many valuable opinions during the study.

Authors’ Contributions

A.T.: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Data Curation, Writing—Original Draft, Writing—Review & Editing, Visualization, Final approval of the version to be submitted. E.S.: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Writing—Original Draft, Visualization, Final approval of the version to be submitted. L.S.: Methodology, Formal analysis, Investigation, Writing—Original Draft, Visualization, Final approval of the version to be submitted. S.K.: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Writing—Original Draft, Visualization, Final approval of the version to be submitted.

Ethics Declarations

The current study was conducted in accordance with the ethical principles of the Helsinki Declaration. It was approved by the Health Sciences University, Hamidiye Clinical Research Ethics Committee (21/46). Sixty volunteers who signed the consent form were recruited from the School of Dentistry (Periodontology, İzmir Katip Çelebi University).

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Availability of Data and Materials

All data generated or analyzed during this study are included in this published article.

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