E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

Role of MyD88‐dependent and MyD88‐independent signaling in Porphyromonas gingivalis‐elicited macrophage foam cell formation

Clinical studies and experimental modeling identify a potential link between periodontal disease and periodontal pathogens such as Porphyromonas gingivalis and atherosclerosis and formation of macrophage foam cells. Toll‐like receptors and molecules governing their intracellular signaling pathways such as MyD88 play roles in atherosclerosis, as well as host response to P. gingivalis. The aim of this study was to define roles of MyD88 and TRIF during macrophage foam cell formation in response to P. gingivalis. In the presence of human low‐density lipoprotein (LDL) mouse bone‐marrow‐derived macrophages (BMφ) cultured with P. gingivalis responded with significant reduction in tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). The BMφ stained strongly with oil red O, regardless of whether bacterial challenge occurred concurrent with or before LDL treatment. Heat‐killed P. gingivalis stimulated foam cell formation in a similar way to live bacteria. The BMφ from MyD88‐knockout and Lps2 mice revealed a significant role for MyD88, and a minor role for TRIF in P. gingivalis‐elicited foam cell formation. Porphyromonas gingivalis‐elicited TNF‐α and IL‐6 were affected by MyD88 ablation and to a lesser extent by TRIF status. These data indicate that LDL affects the TNF‐α and IL‐6 response of macrophages to P. gingivalis challenge and that MyD88 and TRIF play important roles in P. gingivalis‐elicited foam cell formation.     

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.     

Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway,but not through the p38 MAPK pathway

Zhang D, Zheng H, Zhao J, Lin L, Li C, Liu J, Pan Y. Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway, but not through the p38 MAPK pathway. J Periodont Res 2011; 46: 31–38. © 2010 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The aim of this study was to investigate whether endothelial intracellular adhesion molecule‐1 (ICAM‐1), an inflammation biomarker for periodontitis, could be modified by infection with either of two strains of P. gingivalis with different virulence capacities: avirulent ATCC 33277 and virulent W83. Material and Methods: We examined the expression of ICAM‐1, IκBα, phospho‐p38 MAPK and nuclear factor‐kappaB (NF‐κB) p65 in an umbilical vein endothelial cell line (ECV‐304) treated with ATCC 33277 and W83, with or without the NF‐κB antagonist MG132 and/or a specific p38 inhibitor (SB203580), by real‐time PCR, western blotting and immunofluorescence. Results: Both strains could induce ICAM‐1 expression; additionally W83 was able to increase ICAM‐1 expression more significantly than ATCC 33277. In P. gingivalis‐infected endothelial cells, both p38 MAPK and NF‐κB signaling pathways were triggered by a rapid increase of p38 MAPK phosphorylation and a more delayed degradation of IκBα, followed by the nuclear translocation of NF‐κB. It was found that ICAM‐1 production in endothelial cells was abrogated by inhibition of the NF‐κB pathway, but not by inhibition of the p38 MAPK pathway, using the inhibitors of the latter two molecules. Conclusion: The induction of ICAM‐1 by infection of umbilical vein endothelial cells with P. gingivalis might be mediated through the NF‐κB pathway, but not by the p38 MAPK pathway.     

Porphyromonas gingivalis antigen preferentially stimulates T cells to express IL‐17 but not receptor activator of NF‐κB ligand in vitro

Although T cells have been implicated in the pathogenesis and are considered to be central to both their progression and control of chronic inflammatory periodontal diseases, the precise contribution of T cells to tissue destruction has not been fully clarified. Recently, interleukin (IL)‐17 and receptor activator of Nuclear factor κB NF‐κB ligand (RANKL) have received much attention as a result of their proinflammatory and bone metabolic roles, respectively. We therefore investigated the effect of outer membrane protein (OMP) from Porphyromonas gingivalis (P. gingivalis) on the expression of IL‐17 and RANKL in peripheral blood mononuclear cells (PBMCs) and compared these between gingivitis and periodontitis, which are representative of stable and progressive lesions, respectively. The in situ expression of these molecules was also examined. P. gingivalis OMP stimulated PBMCs to express IL‐17 at both the mRNA and protein level. Although the mean expression of mRNA was not different between the two groups, the mean level of IL‐17 in the culture supernatants was higher in gingivitis patients than in periodontitis patients. However, the frequency of IL‐17‐positive samples was higher in the periodontitis patients. This stimulatory effect was not evident for RANKL expression in either periodontitis or gingivitis patients. In gingival tissue samples, IL‐17 mRNA was detected in gingivitis more frequently than in periodontitis. The expression of RANKL mRNA was much lower than that of IL‐17 in terms of both level and frequency. These results suggest that IL‐17 but not RANKL may be involved in the pathogenesis of periodontal diseases. However, there may be negative regulatory mechanisms for IL‐17 in gingivitis.     

Azithromycin may inhibit interleukin-8 through suppression of Rac1 and a nuclear factor-kappa B pathway in KB cells stimulated with lipopolysaccharide

Background: Recent studies have shown that the 15‐member macrolide antibiotic azithromycin (AZM) not only has antibacterial activity, but also results in the role of immunomodulator. Interleukin (IL)‐8 is an important inflammatory mediator in periodontal disease. However, there have been no reports on the effects of AZM on IL‐8 production from human oral epithelium. Therefore, we investigated the effects of AZM on IL‐8 production in an oral epithelial cell line. Methods: KB cells were stimulated by Escherichia coli or Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS) with or without AZM. IL‐8 mRNA and protein expression and production in response to LPS were analyzed by quantitative polymerase chain reaction, flow cytometry, and enzyme‐linked immunosorbent assay. The activation of nuclear factor‐kappa B (NF‐κB) and Rac1, which is important for IL‐8 expression, was analyzed by enzyme‐linked immunosorbent assay and Western blotting, respectively. Results: IL‐8 mRNA expression, IL‐8 production, and NF‐κB activation in LPS‐stimulated KB cells were inhibited by the addition of AZM. LPS‐induced Rac1 activation was also suppressed by AZM. Conclusions: This study suggests that AZM inhibits LPS‐induced IL‐8 production in an oral epithelial cell line, in part caused by the suppression of Rac1 and NF‐κB activation. The use of AZM might provide possible benefits in periodontal therapy, with respect to both its antibacterial action and apparent anti‐inflammatory effect.     

Effect of cytokine and antigen stimulation on peripheral blood lymphocyte syndecan‐1 expression

Introduction: Cytokines are not only produced by activated lymphocytes but also interact with a number of cell‐surface molecules on the same cells. Syndecan‐1 is one such cell‐surface molecule, which has the capacity to bind a variety of growth factors as well as cytokines. The aim of this study was to examine the effects of transforming growth factor β (TGF‐β), interleukin‐1 (IL‐1), IL‐2, IL‐4, lipopolysaccharide (LPS) from Porphyromonas gingivalis and tetanus toxoid on syndecan‐1 expression by B and T lymphocytes. Methods: B and T lymphocytes were obtained from the peripheral blood of healthy donors. Following exposure to the above growth factors, cytokines and antigens, syndecan‐1 expression was determined by flow cytometry. Results: Subjects could be categorized as high or low expressors of syndecan‐1. In the high‐responder group TGF‐β1 alone resulted in a significant increase in syndecan‐1 expression by both B and T cells. None of the other cytokines and antigens produced a significant response. When analysed in combination, TGF‐β1 in combination with IL‐2, IL‐4, P. gingivalis LPS and tetanus toxoid all produced significant increases in syndecan‐1 expression by B cells. For T cells, combinations of TGF‐β1 with IL‐2 and tetanus toxoid resulted in increased syndecan‐1 expression. Conclusions: Both B and T lymphocytes synthesize the cell‐surface proteoglycan syndecan‐1 and its expression can be modulated by TGF‐β1, either alone or in combination with IL‐2, IL‐4 and LPS from P. gingivalis and tetanus toxoid. While these may reflect general responses under inflammatory conditions their biological significance requires further investigation.     

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.     

An acute injection of Porphyromonas gingivalis lipopolysaccharide modulates the OPG/RANKL system and interleukin‐6 in an ovariectomized mouse model

Background/aims: In the present study, we attempted to develop a simulated model to explore the causal effects of periodontal pathogens on skeletal homeostasis in postmenopausal osteoporosis. Methods: Fifty‐three female adult ICR mice were randomly assigned to an experimental group (ovariectomized) or a control group. A single injection of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis‐LPS, ATCC 33277) or Escherichia coli lipopolysaccharide (E. coli‐LPS) was administered intraperitoneally 4 weeks after an ovariectomy. Concentrations of interleukin‐6 (IL‐6), osteoprotegerin (OPG), and the receptor activator of nuclear factor‐κB ligand (RANKL) in serum were subsequently analyzed using an enzyme‐linked immunosorbent assay (ELISA). Results: Under stimulation with P. gingivalis‐LPS or E. coli‐LPS, the concentration of OPG rose in both groups. The serum level of RANKL showed a decreasing trend 24 h after the injection in both groups. After injection of P. gingivalis‐LPS in both the experimental and control animals, the OPG : RANKL ratio increased 24 h after the booster (22.26–620.99, P < 0.05). The serum level of IL‐6 in the experimental group significantly increased 1–6 h after administration of E. coli‐LPS and 1–3 h after administration of P. gingivalis‐LPS (P < 0.05). Conclusions: A single booster injection of P. gingivalis‐LPS induced short‐term changes in OPG, RANKL, and IL‐6 serum levels in this ovariectomized mouse model.     

Resveratrol attenuates the pathogenic and inflammatory properties of Porphyromonas gingivalis

Porphyromonas gingivalis has been strongly associated with chronic periodontitis, which affects tooth‐supporting tissues. This Gram‐negative anaerobic bacterium produces a repertoire of virulence factors that modulate tissue destruction directly or indirectly by the induction of inflammatory processes. The aim of this study was to investigate the effects of resveratrol, a major polyphenol found in grapes and wine, on the growth and virulence properties of P. gingivalis as well as on gingival keratinocyte tight junction integrity and the host inflammatory response. Resveratrol exhibited antibacterial activity that may result from damage to the bacterial cell membrane. Resveratrol also killed a pre‐formed P. gingivalis biofilm and reduced bacterial adherence to matrix proteins. In addition, resveratrol had a protective effect on the integrity of the keratinocyte tight junctions by inhibiting its breakdown by P. gingivalis. This may be related to the ability of resveratrol to inhibit the protease activities of P. gingivalis. Lastly, resveratrol reduced P. gingivalis‐mediated activation of the NF‐κB signaling pathway and attenuated TREM‐1 gene expression as well as soluble TREM‐1 secretion in monocytes. The effect on NF‐κB activation likely results from the ability of resveratrol to act as a PPAR‐γ agonist. In summary, the antibacterial, anti‐adherence, and antiprotease properties of resveratrol, as well as its ability to protect the gingival keratinocyte barrier and attenuate the inflammatory response in monocytes suggest that it may be a promising novel therapeutic agent for treating periodontal disease.     

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.     

Porphyromonas gingivalis inhibits M2 activation of macrophages by suppressing α‐ketoglutarate production in mice

Reprograming of metabolic pathways is critical in governing the polarization of macrophages into classical proinflammatory M1 or alternative anti‐inflammatory M2 phenotypes in metabolic diseases, such as diabetes. Porphyromonas gingivalis, a keystone pathogen of periodontitis, causes an imbalance in M1/M2 activation, resulting in a hyperinflammatory environment that promotes the pathogenesis of periodontitis. However, whether P. gingivalis infection modulates metabolic pathways to alter macrophage polarization remains unclear. Bone‐marrow‐derived macrophages (BMDMs) were collected from 6‐week‐old female C57BL/6 mice and stimulated with P. gingivalis, P. gingivalis‐derived LPS or IL‐4. Relative gene expression and protein production were measured by quantitative real‐time PCR, RNA sequencing and western blotting. Colorimetric assays were also performed to assess the amounts of α‐ketoglutarate (α‐KG) and succinate. P. gingivalis or P. gingivalis‐derived LPS‐induced inflammatory responses enhanced M1 macrophages and suppressed M2 macrophages, even in the presence of IL‐4. P. gingivalis inhibited Idh1/2 and Gpt1/2 mRNA expression, and increased Akgdh mRNA expression, thus decreasing the ratio of α‐KG/succinate. Supplementation of cell‐permeable dimethyl‐α‐KG dramatically restored M2 activation during P. gingivalis infection. Our study suggests that P. gingivalis maintains a hyperinflammatory state by suppressing the production of α‐KG by M2 macrophages.