Modulation of Matrix Metalloproteinase and Cytokine Production by Licorice Isolates Licoricidin and Licorisoflavan A: Potential Therapeutic Approach for Periodontitis

Background: Inflammatory cytokines and matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in response to periodontopathogens play a major role in the tissue destruction observed in periodontitis, which is a disease that affects tooth‐supporting structures. In the present study, we investigate the effects of licorice‐derived licoricidin (LC) and licorisoflavan A (LIA) on the secretion of various cytokines and MMPs by human monocyte‐derived macrophages stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS). Methods: Macrophages were treated with non‐toxic concentrations of LC or LIA before being stimulated with A. actinomycetemcomitans LPS. The secretion of cytokines and MMPs and the activation of nuclear factor‐kappa B (NF‐κB) p65 and activator protein (AP)‐1 were assessed by enzyme‐linked immunosorbent assays. Results: LC and LIA inhibited the secretion of interleukin (IL)‐6 and chemokine (C‐C motif) ligand 5 in a concentration‐dependent manner but did not affect the secretion of IL‐8 by LPS‐stimulated macrophages. LC and LIA also inhibited the secretion of MMP‐7, ‐8, and ‐9 by macrophages. The suppression of cytokine and MMP secretion by LC and LIA was associated with the reduced activation of NF‐κB p65 but not that of AP‐1. Conclusion: The present study suggests that LC and LIA have potential for the development of novel host‐modulating strategies for the treatment of cytokine and/or MMP‐mediated disorders such as periodontitis.     

DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines

Toll‐like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP‐1). THP‐1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA‐initiated cytokine production was determined either by blocking TLR9 signaling in THP‐1 cells with chloroquine or by measuring IL‐8 production and nuclear factor‐κB (NF‐κB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL‐1β, IL‐6, and TNF‐α) was increased significantly in bDNA‐stimulated cells compared with controls. Chloroquine treatment of THP‐1 cells decreased cytokine production, suggesting that TLR9‐mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9‐transfected cells demonstrated significantly increased IL‐8 production (P < 0.001) and NF‐κB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF‐κB genes in response to periodontal bDNA in THP‐1 cells, suggesting that cytokine induction is through NF‐κB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.     

MAPKs, activator protein-1 and nuclear factor-κB mediate production of interleukin-1β-stimulated cytokines, prostaglandin E₂ and MMP-1 in human periodontal ligament cells

Murayama R, Kobayashi M, Takeshita A, Yasui T, Yamamoto M. MAPKs, activator protein‐1 and nuclear factor‐κB mediate production of interleukin‐1β‐stimulated cytokines, prostaglandin E ₂ and MMP‐1 in human periodontal ligament cells. J Periodont Res 2011; 46: 568–575. © 2011 John Wiley & Sons A/S Background and Objective: Determination of the interleukin‐1 (IL‐1) signaling cascades that lead to the production of various inflammatory mediators and catabolic factors may clarify attractive targets for therapeutic intervention for periodontitis. We comprehensively assessed the involvement of MAPKs, activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB) in IL‐1β‐induced production of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin E₂ (PGE₂) and MMP‐1 in human periodontal ligament cells. Material and Methods: Human periodontal ligament cells were pretreated with an inhibitor for each of the MAPKs or NF‐κB and subsequently treated with IL‐1β. Following treatment, phosphorylation of three types of MAPK (ERK, p38 MAPK and c‐Jun N‐terminal kinase), IκB kinase (IKK) α/β/γ and IκB‐α, as well as the DNA binding activity of AP‐1 and NF‐κB and the production of IL‐6, IL‐8, PGE₂ and MMP‐1, were determined by western blotting, a gel mobility shift assay and ELISA, respectively. Results: The three MAPKs, simultaneously activated by IL‐1β, mediated the subsequent DNA binding of AP‐1 at various magnitudes, while IKKα/β/γ, IκB‐α and NF‐κB were also involved in the IL‐1 signaling cascade. Furthermore, IL‐1β stimulated the production of IL‐6, IL‐8, PGE₂ and MMP‐1 via activation of the three MAPKs and NF‐κB, because inhibitors of these significantly suppressed the IL‐1β‐stimulated production of these factors. Conclusion: Our results strongly suggest that MAPK, AP‐1 and NF‐κB mediate the IL‐1β‐stimulated synthesis of IL‐6, IL‐8, PGE₂ and MMP‐1 in human periodontal ligament cells. Therefore, inhibition of activation of MAPK, AP‐1 and/or NF‐κB may lead to therapeutic effects on progression of periodontitis.     

Rosuvastatin Inhibits Interleukin (IL)‐8 and IL‐6 Production in Human Coronary Artery Endothelial Cells Stimulated With Aggregatibacter actinomycetemcomitans Serotype b

Background: Rosuvastatin exhibits anti‐inflammatory effects and reduces periodontal diseases and atherosclerosis; however, its role in regulating periodontopathogen‐induced endothelial proinflammatory responses remains unclear. The purpose of this study is to determine whether rosuvastatin can reduce the proinflammatory response induced by Aggregatibacter actinomycetemcomitans (Aa) in human coronary artery endothelial cells (HCAECs). Methods: HCAECs were stimulated with purified Aa serotype b lipopolysaccharide (LPS) (Aa‐LPS), heat‐killed (HK) bacteria (Aa‐HK), or live bacteria. Expression of Toll‐like receptors and cellular adhesion molecules were evaluated by fluorometric enzyme‐linked immunosorbent assay. Endothelial cell activation was evaluated by quantifying nuclear factor (NF)‐kappa B‐p65 and cytokine expression levels by quantitative polymerase chain reaction and flow cytometry. Effect of rosuvastatin in expression of the atheroprotective factor Krüppel‐like factor 2 (KLF2) and cytokines were also studied using similar approaches. Results: HCAECs showed increased interleukin (IL)‐6, IL‐8, intercellular adhesion molecule 1, and platelet endothelial cell adhesion molecule 1 expression when stimulated with Aa‐LPS or Aa‐HK. NF‐κB‐p65 activation was induced by all antigens. Aa‐induced IL‐6 and IL‐8 production was inhibited by rosuvastatin, particularly at higher doses. Interestingly, reduced IL‐6 and IL‐8 levels were observed in HCAECs stimulated with Aa in the presence of higher concentrations of rosuvastatin. This anti‐inflammatory effect correlated with a significant increase of rosuvastatin‐induced KLF2. Conclusions: These results suggest Aa‐induced proinflammatory endothelial responses are regulated by rosuvastatin in a mechanism that appears to involve KLF2 activation. Use of rosuvastatin to prevent cardiovascular disease may reduce risk of endothelial activation by bacterial antigens.     

Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo

Nakamura H, Ukai T, Yoshimura A, Kozuka Y, Yoshioka H, Yoshinaga Y, Abe Y, Hara Y. Green tea catechin inhibits lipopolysaccharide‐induced bone resorption in vivo. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2008.01198.x. © 2009 John Wiley & Sons A/S Background and Objective: Bone resorption is positively regulated by receptor activator of nuclear factor‐κB ligand (RANKL). Pro‐inflammatory cytokines, such as interleukin (IL)‐1β, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co‐culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)‐induced bone resorption. Material and Methods: Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner. Results: The alveolar bone resorption and IL‐1β expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14‐expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS‐induced nuclear factor‐κB (NF‐κB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF‐κB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony‐stimulating factor with or without GTC. The number of osteoclasts was decreased in dose‐dependent manner when GTC was added to the culture medium. Conclusion: These results suggest that GTC suppresses LPS‐induced bone resorption by inhibiting IL‐1β production or by directly inhibiting osteoclastogenesis.     

Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts

Tamai R, Kiyoura Y, Sugiyama A. Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts. J Periodont Res 2011; 46: 13–20. © 2010 John Wiley & Sons A/S Background and Objective: Nitrogen‐containing bisphosphonates (NBPs) are widely used as anti‐bone‐resorptive drugs. However, use of NBPs results in inflammatory side‐effects, including jaw osteomyelitis. In the present study, we examined the effects of alendronate, a typical NBP, on cytokine production by human peripheral blood mononuclear cells (PBMCs) and gingival fibroblasts incubated with lipid A. Methods: The PBMCs and gingival fibroblasts were pretreated with or without alendronate for 24 h. Cells were then incubated in the presence or absence of lipid A for a further 24 h. Levels of secreted human interleukin (IL)‐1β, IL‐6, IL‐8 and monocyte chemoattractant protein‐1 (MCP‐1) in culture supernatants were measured by ELISA. We also examined nuclear factor‐κB (NF‐κB) activation in both types of cells by ELISA. Activation of Smad3 in the cells was assessed by flow cytometry. In addition, we performed an inhibition assay using SIS3, a specific inhibitor for Smad3. Results: Pretreatment of PBMCs with alendronate promoted lipid A‐induced production of IL‐1β and IL‐6, but decreased lipid A‐induced IL‐8 and MCP‐1 production. In human gingival fibroblasts, alendronate pretreatment increased lipid A‐induced production of IL‐6 and IL‐8, and increased NF‐κB activation in gingival fibroblasts but not PBMCs stimulated with lipid A. In contrast, alendronate activated Smad3 in both types of cells. Finally, SIS3 inhibited alendronate‐augmented IL‐6 and IL‐8 production by human gingival fibroblasts but up‐regulated alendronate‐decreased IL‐8 production by PBMCs. Conclusion: These results suggest that alendronate‐mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF‐κB and Smad3 activity.     

Specificity of Antimicrobial Peptide LL‐37 to Neutralize Periodontopathogenic Lipopolysaccharide Activity in Human Oral Fibroblasts

Background: The antimicrobial peptide LL‐37 is known to have a potent lipopolysaccharide (LPS)‐neutralizing activity in various cell types. Because of observed heterogeneity within periodontopathogenic LPS, the authors hypothesized that LL‐37 had specificity to neutralize such LPS activity. The present study, therefore, aims to investigate the LPS‐neutralizing activity of LL‐37 to various periodontopathogenic LPS in interleukin‐8 (IL‐8) production after challenging them in human oral fibroblasts. Methods: Human periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) were cultured from biopsies of periodontal ligament and gingival tissues. After cell confluence in 24‐well plates, LPS (10 μg/mL) from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were added with or without LL‐37 (10 μg/mL). After 18 hours, the supernatant was collected and analyzed in IL‐8 production by enzyme‐linked immunosorbent assay. Results: All periodontopathogenic LPS statistically significantly induced IL‐8 production in both PDLF and GF (P <0.01). After neutralization with LL‐37, both PDLF and GF showed a statistically significant reduction in IL‐8 production compared with LPS‐treated groups without LL‐37 (P <0.01), and the percentage of reduction in IL‐8 production in PDLF appeared to be higher than in GF. In addition, the percentage of reduction in IL‐8 production varied considerably according to each periodontopathogenic LPS. Conclusions: The antimicrobial peptide LL‐37 had an ability to suppress periodontopathogenic LPS‐induced IL‐8 production in both PDLF and GF. Its LPS‐neutralizing activity revealed specificity to periodontopathogenic LPS and seemed to be dependent on the heterogeneity within LPS between different genera.     

The Proteasome Inhibitor Bortezomib Inhibits Inflammatory Response of Periodontal Ligament Cells and Ameliorates Experimental Periodontitis in Rats

Background: Periodontitis is a chronic inflammatory disease initiated by bacteria and their virulence factors. Bortezomib (BTZ) is the first proteasome inhibitor for clinical treatment of malignancies. Its anticancer activity is accompanied by an anti‐inflammatory effect. However, there are few reports about its anti‐inflammatory effect and underlying mechanism in periodontal disease, especially on human periodontal ligament cells (hPDLCs), which are considered a promising cell‐based therapy for treating periodontitis. Methods: hPDLCs were treated with lipopolysaccharide (LPS) and pretreated with BTZ. mRNA and protein levels of tumor necrosis factor (TNF)‐alpha, interleukin (IL)‐1β, IL‐6, and IL‐8 were determined. The anti‐inflammatory mechanism of BTZ was studied. Further, experimental rat periodontitis was induced with ligature and LPS injection, and simultaneously and locally treated with BTZ (three injections/week). Four weeks after treatment, microcomputed tomography, immunohistochemical, and histopathologic analyses were performed. Results: Bortezomib administration at safe concentrations (≤1 nM) inhibited production of proinflammatory cytokines in LPS‐stimulated hPDLCs via nuclear factor (NF)‐kappa B, p38/extracellular signal‐regulated kinase, and mitogen‐activated protein kinase/activator protein‐1 pathways. Moreover, in the LPS and ligature‐induced periodontitis rat model, BTZ suppressed expression of TNF‐α, IL‐1β, IL‐6, and IL‐8, reduced the ratio of receptor activator of NF‐κB ligand/osteoprotegerin, and prevented alveolar bone absorption. Conclusion: These findings demonstrate the anti‐inflammatory activity of BTZ against periodontal inflammatory response and present BTZ as a promising therapy for periodontal disease.