Down‐regulated IL36RN expression based on peripheral blood mononuclear cells and plasma of periodontitis patients and its clinical significance

Abstract Background The role of IL‐36 receptor antagonist (IL36RN), a mutated gene expression of IL‐36 in periodontitis patients with peripheral blood mononuclear cells (PBMC) and plasma remains to be undetermined. Materials and methods Our study discovered the IL36RN expression through GEO public databases and further validated by PBMC and plasma of periodontitis patients and healthy participants. A total of 194 participants of public datasets, consisting of 97 cases of periodontitis and 97 cases of healthy control were retrospectively evaluated and explored the gene enrichment pathways and clinical significance of IL36RN expression accompanied by three different cytokines. Furthermore, the clinical significance of IL36RN was evaluated in mild‐to‐severe patients of periodontitis by the receiver operating curve (ROC) using the area under the curve (AUC). Results IL36RN expressions were notably down‐regulated in PBMC and plasma of periodontitis patients. Further, a positive correlation of IL36RN expression was significantly observed between PBMC and plasma of periodontitis patients while IL36RN expression was negatively correlated to serum‐based three different cytokines of periodontitis patients. Meanwhile, the ROC‐AUCs achieved a significantly higher range from 0.80 to 0.87 with PBMC of mild‐to‐severe and moderate‐to‐severe periodontitis patients whereas similar patients with plasma obtained a significant AUC range from 0.73 to 0.83. Conclusion IL36RN can distinctively be detectable in periodontitis patients with PBMC and plasma, which can act as a down‐regulated mutated gene that might play an effective role in causing periodontitis. IL36RN may involve by other inflammatory cytokines in the pathogenesis of periodontitis.


| INTRODUC TI ON
Periodontitis, common but largely preventable, is a chronic and multifactorial inflammatory disease that damages the supporting soft tissue and bone of teeth. 1,2 Patients with periodontitis disease may last for a duration of several months or years. The interaction between periodontal pathogens and host inflammatory and immune responses is involved in the pathogenesis of periodontitis. 2 Certain diseases, such as Crohn's disease, 3 asthma, 4 rheumatoid arthritis, 5 and diabetes mellitus, 6 were reported to increase the risk of periodontitis. Complicated immune responses in the body might be playing a critical role in the progression of tissue damagement in periodontitis. 7,8 Interleukin-36 (IL-36), one member of the interleukin-1 (IL-1) superfamily, has subfamily members known as three agonists (IL-36α, IL-36β, and IL-36γ) and two antagonists (interleukin-36 receptor antagonist (IL-36Ra and IL-38)). 9 IL-36Ra is an anti-inflammatory mediator which takes responsibility for the tight regulation of IL-36 signaling. 9 The IL36RN gene encodes IL-36Ra. Various inflammatory diseases, such as inflammatory skin disorders, Crohn's disease, and rheumatoid arthritis have increasingly connected with IL-36 related cytokines. [10][11][12] In periodontitis, IL-1, IL-6, IL-17A, and tumor necrosis factorα (TNFα) known as pro-inflammatory cytokines cause body immune responses to oral bacteria specifically called Porphyromonas gingivalis. 13 Kübra et al. reported that active periodontal disease may cause downregulation of inflammasome regulators and they may increase the activity of IL-1β in periodontal disease including periodontitis. 14 Alexandra et al. found that IL-36γ could be a key inflammatory player in periodontitis and its associated alveolar bone resorption and could be a therapeutic target. 2 Patrick R. et al. checked the serum, saliva, gingival cervical fluid (GCF), and gingival biopsies of patients who suffer from inflammatory periodontal disease and found the presence of elevated levels of IL-35. 15 In this context, several interleukins might be acting as mutated genes in periodontitis disease and may be playing an important role in occurring and progression of periodontitis.
IL-36 has been evaluated in diverse inflammatory diseases. 16 However, the role of IL-36RN, a mutated gene expression of IL-36 in periodontitis patients with peripheral blood mononuclear cells (PBMC) and plasma remains unknown. Our study aims to find IL36RN in PBMC and plasma of the periodontitis patients and its clinical significance.

| Exclusion
Patients suffered from systemic diseases such as diabetes and immune dysfunction. Patients who took anti-inflammatory drugs and anti-tumor drugs three months before treatment; Patients with oral local radiotherapy; Smoking and excessive drinking; Female in pregnancy or lactation.

| Periodontitis severity criteria
Mild periodontitis: ≥2 interproximal sites with clinical attachment loss ≥3 mm, and ≥2 interproximal sites with probing depth ≥4 mm (not on same tooth) or one site with probing depth ≥5 mm.
Moderate periodontitis: ≥2 interproximal sites with clinical attachment loss ≥4 mm (not on same tooth), or ≥2 interproximal sites with probing depth ≥5 mm (not on same tooth).

| RNA isolation
Total RNA was extracted by using the solutions of phenol-chloroform afterward managing the homogenization by guanidine isothiocyanate (Trizol RNA Preparation kit). RNA concentration evaluated by NanoDrop spectrophotometer ND1000 (NanoDrop Technologies Inc.).

| RT-qPCR
The total RNA in the PBMC and plasma samples after transfection was extracted using Trizol reagent according to instructions. Thereafter, the total RNA was reverse-transcribed into cDNA is terminated at 4℃. Three replicates were set for each sample, and 2 −△△Ct was used for relative quantitative analysis of the data.

| Determination of cytokines
The levels of IL-6, TNFα and IL-1β were detected with corresponding Enzyme Linked Immunosorbent Assay (Elisa) kits (ThermoFisher Scientific) according to the instruction of the kit. SPSS 20.0 version (SPSS Inc.) software was utilized for all the statistical analysis of the study and data were expressed as mean ± SD.

| Statistical and datasets analysis
Bioinformatics tools were used to analyze heatmap, volcano map, Gene Ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) enrichment pathways from the GEO database. T-test was performed for comparing the two groups. One-way ANOVA analysis was used to compare multiple groups. Pearson correlation was used for correlation analysis. The clinical significance of IL36RN was evaluated in PBMC and plasma by the receiver operating curve (ROC) using the area under the curve (AUC). p < 0.05 was considered to be representing statistically significant.

| Clinical characteristics
A total of 194 participants were enrolled to evaluate IL36RN expression through PBMC and plasma samples of periodontitis (n = 97) and healthy controls (n = 97). Further, the patients and healthy controls were characterized by clinical parameters such as age, gender, bleeding on probing, oral hygiene index, pocket depth, and clinical achievement level. Among both groups, the parameters of bleeding on probing, oral hygiene index, pocket depth, and clinical achievement level were statistically significant (Table 1).

| Discovery of IL36RN expression in periodontitis
Bioinformatics tools were used to discover IL36RN mutated gene    Figure 4C, (r = 0.409, p < 0.001) Therefore, the expression level of IL36RN in PBMC was directly correlated to plasma of periodontitis patients.

| Three different cytokines expression in PBMC of periodontitis
Serum-based IL-6, TNFα and IL-1β, three different cytokines expression were demonstrated in PBMC of periodontitis and healthy controls. Overall, these three different cytokines were highly expressed in periodontitis patients compared to healthy controls ( Figure 5A-C, p < 0.05). Meanwhile, IL36RN expression in PBMC was negatively correlated to these serum-based three different cytokines of periodontitis patients ( Figure 6A-C, p < 0.05). Thus, IL-6, TNFα, and IL-1β were inversely correlated and up-regulated expressions, which are in contrast to IL36RN.

| Significance of IL36RN in mild, moderate, and severe periodontitis
Besides, IL36RN expression and significance were observed in mild-to-severe periodontitis patients with PBMC and plasma samples. Herein, the mild periodontitis showed significantly higher expression of IL36RN in PBMC and plasma compared to moderate and severe periodontitis ( Figure 7A,B, p < 0.05). Similarly, moderate periodontitis showed higher expression of IL36RN than severe periodontitis whereas lower expression than mild periodontitis patients ( Figure 7A

| DISCUSS ION
Periodontitis is a numerous factorial disease initially due to con- inflammatory process. 22 Thus, cytokines may involve in the pathogenesis of periodontitis.
The present study has discovered and validated the down-

| CON CLUS ION
IL36RN can distinctively be detectable in periodontitis patients with PBMC and plasma, which can act as a down-regulated mutated gene that might play an effective role in causing periodontitis. IL36RN may involve by other inflammatory cytokines in the pathogenesis of periodontitis.

ACK N OWLED G M ENTS
The present study was granted by Science and Technology Research Project of Jilin Provincial Education Department (No.

JJKH20200074KJ) and Jilin Medical and Health Guidance Plan
Project (No. 20200404034).

CO N FLI C T S O F I NTE R E S T S
The authors declared that they have no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.