High Glucose-Mediated Cytokine Regulation in Gingival Fibroblasts and THP-1 Macrophage: a Possible Mechanism of Severe Periodontitis with Diabetes.

BACKGROUND/AIMS: Diabetic patients are susceptible to severe periodontitis, but the precise mechanism is not fully understood. Aim of this study was to explore the biological pathogenesis of severe periodontitis in diabetic patients focusing on the crosstalk of human gingival fibroblasts (HGFs) and macrophages. METHODS: A total of 70 periodontitis patients with or without diabetes mellitus (DM) were enrolled, and the statistical relationships of diabetic conditions to the periodontal inflammatory parameters were examined by cross-sectional study. In in vitro study, HGFs cell line CRL-2014® (ATCC) and differentiated THP-1 macrophages were cultured with normal glucose (NG: 5.5 mM) or high glucose (HG: 25 mM) condition, and treated with indicated inflammatory factors such as calprotectin (CPT), interleukin (IL)-1ß and IL-6. To examine the effects of HG on soluble IL-6 receptor (sIL-6R) production in THP-1 macrophages, the supernatants were collected and the sIL-6R levels were measured by ELISA. To examine the effects of HG on IL-1ß or IL-6-induced matrix metalloproteinase (MMPs) production in HGFs, the supernatants were collected. Levels of MMP-1 and tissue inhibitor of MMP-1 (TIMP-1) were measured by ELISA. Finally, after conditioned medium (CM) from THP-1 macrophages cultured with NG or HG conditions was collected, HGFs were treated with the CM. The supernatants were collected 24 hours later and the levels of MMP-1 and TIMP-1 were measured. To examine the specific effects of IL-1ß contained in CM on MMP-1 and TIMP-1 production in HGFs, IL-1 receptor antagonist (IL-1ra) was used. RESULTS: There were statistical correlation between IL-1ß and sIL-6R levels in gingival crevicular fluid (GCF) and HbA1c in periodontitis patients with DM (IL-1ß: P=0.035, sIL-6R: P=0.040). HG and CPT significantly induced sIL-6R production in THP-1 macrophages. HG significantly enhanced IL-1ß or IL-6/sIL-6R-induced MMP-1 production in HGFs. The increase of MMP-1 by both IL-1ß and IL-6/sIL-6R was significantly inhibited by specific ERK or IκB inhibitors. Corresponding to the regulation of MMP-1 production, HG condition increased the phosphorylation of p44/42 MAPK and IκBα in HGFs treated with IL-1ß or IL-6/sIL-6R. Finally, MMP-1 production in HGFs cultured with HG increased significantly by CM from THP-1 macrophages cultured with HG. The induction of MMP-1 by the CM from THP-1 macrophages cultured with HG was significantly inhibited by dose dependent of IL-1ra in HGFs cultured with HG. CONCLUSION: Diabetic conditions such as HG induce IL-1ß and sIL-6R production from macrophages in inflammatory periodontal tissues and may exacerbate the periodontitis synergistically via MMP-1 production from HGFs.

ß-carotene suppresses Porphyromonas gingivalis lipopolysaccharide-mediated cytokine production in THP-1 monocytes cultured with high glucose condition.

Periodontitis is associated with development of diabetes mellitus. Although lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a major pathogen of periodontitis, may lead the progression of diabetes complications, the precise mechanisms are unclear. We, therefore, investigated the effects of ß-carotene on production of Pg LPS-induced inflammatory cytokines in human monocytes cultured high glucose (HG) condition. THP-1 cells were cultured under 5.5 mM or 25 mM glucose conditions, and cells were stimulated with Pg LPS. To investigate the productivity of TNF-α, IL-6, and MCP-1, cell supernatants were collected for ELISA. To examine the effects of NF-kB signals on cytokine production, Bay11-7082 was used. HG enhanced Pg LPS-induced production of TNF-α, IL-6, and MCP-1 via NF-kB signals in THP-1. ß-carotene suppressed the enhancement of the Pg LPS-induced cytokine production in THP-1 via NF-κB inactivation. Our results suggest that ß-carotene might be a potential anti-inflammatory nutrient for circulating Pg LPS-mediated cytokine production in diabetic patients with periodontitis.

Calprotectin Induces IL-6 and MCP-1 Production via Toll-Like Receptor 4 Signaling in Human Gingival Fibroblasts.

Calprotectin, a heterodimer of S100A8 and S100A9 molecules, is associated with inflammatory diseases such as inflammatory bowel disease. We have reported that calprotectin levels in gingival crevicular fluids of periodontitis patients are significantly higher than in healthy subjects. However, the functions of calprotectin in pathophysiology of periodontitis are still unknown. The aim of this study is to investigate the effects of calprotectin on the productivity of inflammatory cytokines in human gingival fibroblasts (HGFs). The HGFs cell line CRL-2014® (ATCC) were cultured, and total RNAs were collected to examine the expression of TLR2/4 and RAGE mRNA using RT-PCR. After the cells were treated with S100A8, S100A9, and calprotectin, supernatants were collected and the levels of IL-6 and MCP-1 were measured using ELISA methods. To examine the intracellular signals involved in calprotectin-induced cytokine production, several chemical inhibitors were used. Furthermore, after the siRNA-mediated TLR4 down-regulated cells were treated with S100A8, S100A9, and calprotectin, the levels of IL-6 and MCP-1 were also measured. HGFs showed greater expression of TLR4 mRNA, but not TLR2 and RAGE mRNA compared with human oral epithelial cells. Calprotectin increased significantly the production of MCP-1 and IL-6 in HGFs, and the cytokine productions were significantly suppressed in the cells treated with MAPKs, NF-κB, and TLR4 inhibitors. Furthermore, calprotectin-mediated MCP-1 and IL-6 production were significantly suppressed in TLR4 down-regulated cells. Taken together, calprotectin induces IL-6 and MCP-1 production in HGFs via TLR4 signaling that involves MAPK and NF-κB, resulting in the progression of periodontitis. J. Cell. Physiol. 232: 1862-1871, 2017. © 2016 Wiley Periodicals, Inc.

Clinical significance of GCF sIL-6R and calprotectin to evaluate the periodontal inflammation.

Background Periodontitis is an inflammatory disease. The aim of this study was to investigate whether the soluble form of interleukin-6 receptor (sIL-6R) and calprotectin concentrations in gingival crevicular fluid are useful biomarkers in the evaluation of periodontitis. Methods First, a cross-sectional study was performed. A total of 34 periodontitis patients were enrolled and the gingival crevicular fluid samples were collected from the healthy and inflamed sites of periodontal pockets in each patient. The relationship between periodontal condition and gingival crevicular fluid sIL-6R and calprotectin concentrations was analysed statistically. The cut-off values of gingival crevicular fluid sIL-6R and calprotectin concentrations for the evaluation of periodontitis were determined using a receiver operating characteristic curve. Next, by using enzyme-linked immunosorbent assay, it was examined whether calprotectin induces sIL-6R production in THP-1 macrophages. Results Both gingival crevicular fluid sIL-6R and calprotectin concentrations were significantly higher in the inflamed sites than in the healthy sites ( P < 0.0001). The cut-off values of gingival crevicular fluid sIL-6R and calprotectin concentrations for the evaluation of periodontal inflammation were as follows: sIL-6R: 43.5 pg/site; calprotectin: 134.3 ng/site. In the in vitro study, calprotectin significantly induced sIL-6R production in THP-1 macrophages ( P < 0.01). Conclusions Both gingival crevicular fluid sIL-6R and calprotectin concentrations are significant biomarkers in the evaluation of periodontal inflammation.

Glycated albumin and calprotectin levels in gingival crevicular fluid from patients with periodontitis and type 2 diabetes.

BACKGROUND: Patients with diabetes mellitus (DM) have a high prevalence of periodontitis. Periodontitis in these patients is characterized by severe inflammation and tissue breakdown, and its diagnosis is important for cures of periodontitis and DM. The purpose of this study is to investigate the levels of glycated albumin (GA), a DM marker, and calprotectin, an inflammatory marker, in gingival crevicular fluid (GCF) from patients with periodontitis and DM (DM-P). METHODS: The 78 participants in this study were patients with DM, chronic periodontitis (CP), DM-P, and healthy individuals (H). GCF and blood were collected, and GA and calprotectin in GCF were analyzed using Western blotting and enzyme-linked immunosorbent assay. Levels were compared among H, DM, CP, and DM-P groups. Blood GA and glycated hemoglobin (HbA1c) were measured, and the correlation among GCF GA and blood HbA1c or GA levels was investigated. Receiver operating characteristic (ROC) analysis for GCF GA to predict DM was performed. RESULTS: GA was identified in GCF, and its amount/concentration in GCF samples from DM and DM-P were significantly higher than those of non-DM groups (H and CP). Calprotectin amounts in GCF from CP and DM-P were significantly higher than in H and DM groups. GCF GA level was positively correlated with blood HbA1c and GA level. ROC analysis of GCF GA showed an optimal cutoff value to predict DM. CONCLUSIONS: GA showed a high level in GCF from patients with DM. Examination of GA and calprotectin in GCF may be useful for predicting DM-P.