Actinomycetemcomitans Lipopolysaccharide Research Articles

Aggregatibacter actinomycetemcomitans induces a proatherosclerotic response in human endothelial cells in a three‐dimensional collagen scaffold model

AbstractThe role of periodontopathogens in inflammatory endothelial dysfunction is not known. This study characterizes a three‐dimensional model with human coronary artery endothelial cells on three‐dimensional (HCAEC‐3D) type I collagen scaffolds to evaluate whether infection with Aggregatibacter actinomycetemcomitans induces a proinflammatory response associated with atherosclerosis. The HCAEC‐3D culture was physicochemically characterized with regard to biocompatibility and barrier function. Then, the culture was infected with A. actinomycetemcomitans strain ATCC 29522 at multiplicities of infection (MOIs) of 1:1, 1:10, and 1:100. Cultures without infection and stimulated with A. actinomycetemcomitans lipopolysaccharide were used as controls. The secretion of soluble factors (IL‐6, IL‐1β, MCP‐1, RANTES, MIP‐1, IL‐8, IL‐1α, and TNF‐α) was evaluated via flow cytometry; TGF‐β1 was evaluated via enzyme‐linked immunosorbent assay (ELISA). The adhesion and migration of fluorescent human THP‐1 monocytes was evaluated. IL‐8, MCP‐1, and IL‐6 secretion increased in a dose‐dependent manner with A. actinomycetemcomitans infection and was significantly greater than that under control treatment. The concentration of TGF‐β1 was significantly higher at MOI 1:100 than in controls. Treatment of the 3D cultures with A. actinomycetemcomitans at different MOIs induced significant differences in the adhesion of monocytes to the endothelium compared to the control without infection. Lastly, conditioned media from 3D cultures treated with A. actinomycetemcomitans induced monocyte migration. The effects of IL‐8, MCP‐1, IL‐6, and TGF‐β1 on the endothelium indicate the ability of A. actinomycetemcomitans to induce an inflammatory response through a mechanism of monocyte adhesion and migration and endothelial dysfunction.

Inhibition of the histone demethylase KDM4B leads to activation of KDM1A, attenuates bacterial-induced pro-inflammatory cytokine release, and reduces osteoclastogenesis

ABSTRACTPeriodontal disease (PD) afflicts 46% of Americans with no effective adjunctive therapies available. While most pharmacotherapy for PD targets bacteria, the host immune response is responsible for driving tissue damage and bone loss in severe disease. Herein, we establish that the histone demethylase KDM4B is a potential drug target for the treatment of PD. Immunohistochemical staining of diseased periodontal epithelium revealed an increased abundance of KDM4B that correlates with inflammation. In murine calvarial sections exposed to Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa-LPS), immunohistochemical staining revealed a significant increase in KDM4B protein expression. The 8-hydroxyquinoline ML324 is known to inhibit the related demethylase KDM4E in vitro, but has not been evaluated against any other targets. Our studies indicate that ML324 also inhibits KDM4B (IC50: 4.9 μM), and decreases the pro-inflammatory cytokine response to an Aa-LPS challenge in vitro. Our results suggest that KDM4B inhibition-induced immunosuppression works indirectly, requiring new protein synthesis. In addition, fluorescence-stained macrophages exhibited a significant decrease in global monomethyl histone 3 lysine 4 (H3K4me) levels following an Aa-LPS challenge that was prevented by KDM4B inhibition, suggesting this effect is produced through KDM1A-mediated demethylation of H3K4. Finally, ML324 inhibition of KDM4B in osteoclast progenitors produced a significant reduction in Aa-LPS-induced osteoclastogenesis. These data link histone methylation with host immune response to bacterial pathogens in PD, and suggest a previously unreported, alternative mechanism for epigenetic control of the host inflammatory environment. As such, KDM4B represents a new therapeutic target for treating hyper-inflammatory diseases that result in bone destruction.

Regulation of matrix metalloproteinase secretion by green tea catechins in a three-dimensional co-culture model of macrophages and gingival fibroblasts

ObjectivesElevated levels of matrix metalloproteinases (MMPs) have been associated with the active phases of tissue and bone destruction in periodontitis, an inflammatory disease characterized by a significant breakdown of tooth support. In the present study, we used a three-dimensional (3D) co-culture model of macrophages and gingival fibroblasts to investigate the ability of a green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) to regulate the secretion of MMP-3, -8, and -9. MethodsThe 3D co-culture model was composed of gingival fibroblasts embedded in a type I collagen matrix overlaid with macrophages. Two arbitrary ratios were tested. The ratio composed of 1 macrophage to 10 fibroblasts was used to mimic a slightly inflamed periodontal site while the ratio composed of 10 macrophages to 1 fibroblast was used to mimic a severely inflamed periodontal site. The 3D co-culture model was pre-treated for 2h with either the green tea extract or EGCG. It was then stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). The model was also first stimulated with LPS for 2h and then incubated with the green tea extract or EGCG. The concentrations of secreted MMP-3, -8, and -9 were quantified by enzyme-linked immunoassays. ResultsWhen the 3D co-culture model was stimulated with A. actinomycetemcomitans LPS, the 10:1 ratio of macrophages to gingival fibroblasts was associated with a highest secretion of MMP-3 and -9 and, to a lesser extent, MMP-8, than the 1:10 ratio. Non-cytotoxic concentrations of the green tea extract or EGCG reduced the basal secretion levels of all three MMPs. A 2-h treatment with the green tea extract or EGCG prior to the stimulation with LPS resulted in a dose-dependent decrease in MMP secretion, with MMP-9 showing the most significant decrease. A decrease in MMP secretion was also observed when the green tea extract or EGCG was added following a 2-h stimulation with LPS. ConclusionsOur results suggested that green tea catechins, and more specifically EGCG, offer promising prospects for the development of a novel adjunctive treatment for periodontitis because of their ability to decrease the secretion of MMPs, which are important tissue-destructive enzymes produced by mucosal and immune cells.

Green tea polyphenol epigallocatechin-3-gallate and cranberry proanthocyanidins act in synergy with cathelicidin (LL-37) to reduce the LPS-induced inflammatory response in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts

ObjectivesThe human antimicrobial peptide cathelicidin (LL-37) possesses anti-inflammatory properties that may contribute to attenuating the inflammatory process associated with chronic periodontitis. Plant polyphenols, including those from cranberry and green tea, have been reported to reduce inflammatory cytokine secretion by host cells. In the present study, we hypothesized that A-type cranberry proanthocyanidins (AC-PACs) and green tea epigallocatechin-3-gallate (EGCG) act in synergy with LL-37 to reduce the secretion of inflammatory mediators by oral mucosal cells. MethodsA three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts treated with non-cytotoxic concentrations of AC-PACs (25 and 50μg/ml), EGCG (1 and 5μg/ml), and LL-37 (0.1 and 0.2μM) individually and in combination (AC-PACs+LL-37 and EGCG+LL-37) were stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). Multiplex ELISA assays were used to quantify the secretion of 54 host factors, including chemokines, cytokines, growth factors, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). ResultsLL-37, AC-PACs, and EGCG, individually or in combination, had no effect on the regulation of MMP and TIMP secretion but inhibited the secretion of several cytokines. AC-PACs and LL-37 acted in synergy to reduce the secretion of CXC-chemokine ligand 1 (GRO-α), granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6), and had an additive effect on reducing the secretion of interleukin-8 (IL-8), interferon-γ inducible protein 10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1) in response to LPS stimulation. EGCG and LL-37 acted in synergy to reduce the secretion of GRO-α, G-CSF, IL-6, IL-8, and IP-10, and had an additive effect on MCP-1 secretion. ConclusionThe combination of LL-37 and natural polyphenols from cranberry and green tea acted in synergy to reduce the secretion of several cytokines by an LPS-stimulated 3D co-culture model of oral mucosal cells. Such combinations show promising results as potential adjunctive therapies for treating inflammatory periodontitis.

Cleavage of host cytokeratin-6 by lysine-specific gingipain induces gingival inflammation in periodontitis patients.

Background/PurposeLysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion.MethodsK6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.ResultsWe identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.ConclusionKgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.

Metabolic Syndrome Exacerbates Inflammation and Bone Loss in Periodontitis

Clinical studies have shown that metabolic syndrome (MetS) is associated with increased risk of developing periodontitis. However, the underlying mechanisms remain largely unknown. Since it is known that lipopolysaccharide (LPS)–activated toll-like receptor 4 signaling pathways play a crucial role in periodontitis, we hypothesized that MetS enhances LPS-induced periodontal inflammation and alveolar bone loss. In this study, we induced MetS in C57BL/6 mice by feeding them high-fat diet (HFD), and we induced periodontitis by periodontal injection of Aggregatibacter actinomycetemcomitans LPS. We found that mice fed a HFD had significantly increased body weight, plasma lipids, insulin, and insulin resistance when compared with mice fed regular chow, indicating that the mice developed MetS. We also found that a HFD markedly increased LPS-induced alveolar bone loss, osteoclastogenesis, and inflammatory infiltration. Analysis of gene expression in periodontal tissue revealed that HFD and LPS injection cooperatively stimulated expression of cytokines that are known to be involved in periodontal tissue inflammation and osteoclastogenesis—such as interleukin 6, monocyte-chemotactic protein 1, receptor activator of nuclear factor kappa-B ligand, and macrophage colony-stimulating factor. To further understand the potential mechanisms involved in MetS-boosted tissue inflammation, our in vitro studies showed that palmitic acid—the most abundant saturated fatty acid (SFA) and the major SFA in the HFD used in our animal study—potently enhanced LPS-induced proinflammatory gene expression in macrophages. In sum, this study demonstrated that MetS was associated with increased periodontal inflammation and alveolar bone loss in an LPS-induced periodontitis animal model. This study also suggests that SFA palmitic acid may play an important role in MetS-associated periodontitis by enhancing LPS-induced expression of inflammatory cytokines in macrophages.

Synergistic anti-inflammatory activity of the antimicrobial peptides human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts.

Given the spread of antibiotic resistance in bacterial pathogens, antimicrobial peptides that can also modulate the immune response may be a novel approach for effectively controlling periodontal infections. In the present study, we used a three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) to investigate the anti-inflammatory properties of human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) and to determine whether these antimicrobial peptides can act in synergy. The 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to LPS stimulation compared to fibroblasts and epithelial cells alone. The 3D co-culture model was stimulated with non-cytotoxic concentrations of hBD-3 (10 and 20 µM) and LL-37 (0.1 and 0.2 µM) individually and in combination in the presence of A. actinomycetemcomitans LPS. A multiplex ELISA assay was used to quantify the secretion of 41 different cytokines. hBD-3 and LL-37 acted in synergy to reduce the secretion of GRO-alpha, G-CSF, IP-10, IL-6, and MCP-1, but only had an additive effect on reducing the secretion of IL-8 in response to A. actinomycetemcomitans LPS stimulation. The present study showed that hBD-3 acted in synergy with LL-37 to reduce the secretion of cytokines by an LPS-stimulated 3D model of gingival mucosa. This combination of antimicrobial peptides thus shows promising potential as an adjunctive therapy for treating inflammatory periodontitis.

Lipopolysaccharide derived from Aggregatibacter actinomycetemcomitans inhibits differentiation of osteoblasts

Background and objectiveAlveolar bone defects in aggressive periodontitis are generally caused by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) through osteoclast differentiation facilitated by the interaction between the host's immune system, stimulated by the bacterial outer components of A. actinomycetemcomitans, and osteoblasts. Recently, some reports on the direct effects of A. actinomycetemcomitans on osteoblast differentiation have been published. However, the mechanisms in detail have still remained unknown. We herein demonstrated that A. actinomycetemcomitans might inhibit differentiation of osteoblasts and identified a causative substance. Materials and methodsThe following culture supernatants of A. actinomycetemcomitans were added to mouse osteoblasts, MC3T3-E1: supernatants fractionated by the molecular weight, supernatant supplemented with proteinase K and then heated, and supernatant supplemented with polymyxin B. Subsequently, RNA was extracted to determine the expressions of bone differentiation markers, alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP). Additionally, lipopolysaccharide (LPS) was added to MC3T3-E1 culture to measure the markers. ResultsA. actinomycetemcomitans culture supernatant, added to the MC3T3-E1, inhibited the expressions of bone differentiation markers at all concentrations. Only the fraction with a molecular weight of >100 kDa inhibited the bone differentiation. Neither proteinase K nor heating had an effect. However, polymyxin B completely abrogated the differentiation inhibitory activity. A. actinomycetemcomitans LPS concentration dependently inhibited the expressions of the bone differentiation markers. ConclusionsA. actinomycetemcomitans LPS suppressed osteoblast differentiation. This suggests that suppressed bone differentiation is involved in the alveolar bone destruction.

The Influence of Cortical Perforation on Guided Bone Regeneration Using Synthetic Bone Substitutes: A Study of Rabbit Cranial Defects

Titanium (Ti) is frequently used in dental implants because of its excellent corrosion resistance, mechanical properties, and biocompatibility. However, Ti ions may be slowly released as a result of corrosion, contributing to peri-implantitis, a major cause of dental implant failure. This study examined the influence of Ti ions on cytokine levels in murine splenocytes, an immunocompetent cell type, stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) and the influence of Ti ions on splenocyte viability. Splenocytes were prepared from 5- to 10-week-old male C57BL/6 mice and BALB/c mice. Cytokine levels in culture supernatants from murine splenocytes stimulated with A actinomycetemcomitans LPS were determined with enzyme-linked immunosorbent assay. A tetrazolium salt assay was carried out to evaluate the cytotoxicity of Ti ions against murine splenocytes. The present study demonstrated that Ti ions influence cytokine levels in LPS-stimulated splenocytes. Significantly higher values (P < .05) were observed for interleukins 1β, 6, and 10; interferon-γ; tumor necrosis factor alpha; and granulocyte macrophage colony-stimulating factor in the culture supernatants of LPS-stimulated splenocytes of both mouse strains in the presence of Ti ions, as compared to the absence of Ti ions. Tetrazolium salt assay confirmed that the Ti ions used in this study did not affect the viability of murine splenocytes. This study suggests that Ti ions enhanced cytokine production induced by periodontopathic bacterial LPS.

Simvastatin Inhibits LPS-induced Alveolar Bone Loss during Metabolic Syndrome

Studies in recent years have shown a positive relationship between metabolic syndrome (MS) and periodontal disease (PD). Given that patients with MS take statins to reduce cholesterol, and statins also have anti-inflammatory effects, it is important to determine if statin intake hinders the progression of MS-associated PD. In this study, PD was induced in Zucker fat rats (ZFRs), an animal model for MS, and in control lean rats by periodontal injection of Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS), while simvastatin was given to some of the rats via gavage. After 4 wk of treatment, alveolar bone loss was determined by micro-computed tomography. To explore the underlying mechanisms, we determined the effect of simvastatin on tissue inflammation and the expression of molecules involved in osteoclastogenesis. Results showed that while bone loss was increased by LPS in both ZFRs and the control lean rats, it was significantly more in the former than the latter. Simvastatin effectively alleviated bone loss in both ZFRs and the control rats. Results also showed that LPS stimulated leukocyte tissue infiltration and expression of molecules for osteoclastogenesis, but simvastatin significantly modulated the stimulation. This study demonstrated that simvastatin inhibited LPS-induced alveolar bone loss and periodontal tissue inflammation in rats with MS.

Simvastatin inhibits lipopolysaccharide‐induced osteoclastogenesis and reduces alveolar bone loss in experimental periodontal disease

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase and have anti-inflammatory effects independent of cholesterol lowering. Recent clinical studies have indicated that statin intake has a beneficial effect on periodontal disease. However, the underlying mechanisms have not been well understood. In the current study, we employed a rat model with lipopolysaccharide (LPS)-induced periodontal disease and determined the effect of simvastatin, a commonly prescribed statin, on osteoclastogenesis, gingival inflammation and alveolar bone loss. Sprague-Dawley rats were injected with Aggregatibacter actinomycetemcomitans LPS in periodontal tissue three times per week for 8 wk and part of the rats with LPS injection were also given simvastatin via gavage. After the treatments, the rat maxillae were scanned by microcomputed tomography and the images were analyzed to determine alveolar bone loss. To explore the underlying mechanisms, the effect of simvastatin on osteoclastogenesis and gingival expression of proinflammatory cytokines were also determined by tartrate-resistant acid phosphatase staining and real-time polymerase chain reaction assays, respectively. Results showed that LPS treatment markedly increased bone loss, but administration of simvastatin significantly alleviated the bone loss. Results also showed that LPS treatment stimulated osteoclastogenesis and the expression of inflammatory cytokines, but simvastatin significantly modulates the stimulatory effect of LPS on osteoclastogenesis and cytokine expression. This study demonstrated that simvastatin treatment inhibits LPS-induced osteoclastogenesis and gingival inflammation and reduces alveolar bone loss, indicating that the intake of simvastatin may hinder the progression of periodontal disease.

Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulated epithelial cells produce interleukin-15 that regulates T cell activation

ObjectiveOral epithelial cells act not only as mechanical barriers but also as immunological barriers by producing various mediators such as cytokines. Since, in periodontal disease, limited information is available regarding the role of oral epithelial cell-derived cytokines on T cell activation, we investigated the responses of human T cells (Jurkat cell) to cytokines in KB cells (an oral epithelial cell line) that had been stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). DesignTo evaluate T cell activation in response to the culture supernatant of KB cells, we examined cell proliferation and interferon gamma (IFN-γ) production, which is closely related to periodontal disease, in Jurkat cells. Culture supernatant of LPS-stimulated KB cells enhanced cell proliferation and IFN-γ production in Jurkat cells. To determine the active component within the culture supernatant, the production of epithelial cell-derived cytokines, interleukin-12 (IL-12), IL-15 and IL-18, in LPS-stimulated KB cells was analysed. ResultsIL-15, but not IL-18, was significantly increased in the culture supernatant of LPS-stimulated KB cells. Moreover, additional anti-IL-15 neutralizing antibody abolished culture supernatant-induced IFN-γ expression in Jurkat cells. ConclusionThese results suggest that periodontal pathogens induce the production of IL-15 from epithelial cells, and leading the activation of T cells in periodontal lesions.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up‐regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin‐1 receptor antagonist‐deficient mice

The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10μg/mL of Aggregatibacter actinomycetemcomitans LPS for 24h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. The levels of IL-1α, IL-1β, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A.actinomycetemcomitans LPS were significantly increased by approximately 4- (p<0.05), 5- (p<0.05), 1.3- (p<0.05) and 6- (p<0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p<0.05), 1.6- (p<0.05), 2.5- (p<0.05), 1.6- (p<0.05) and 1.9- (p<0.05) fold, respectively, in IL-1Ra KO mice stimulated with A.actinomycetemcomitans LPS compared with WT mice. IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Different expression of cytokines induced by Actinobacillus actinomycetemcomitans lipopolysaccharide in monocytes/macrophages from different organs of rabbits

To investigate the expression of cytokines induced by Actinobacillus actinomycetemcomitans lipopolysaccharide (Aa-LPS) in monocytes/macrophages from different organs of rabbits. The peripheral mononuclear cells (Mo), alveolar macrophages (AM), peritoneal macrophages (PM) and Kupffer cells (KC) from five New Zealand rabbits were isolated respectively. Then the cells from different organs were stimulated with Escherichia coli (Ec)-LPS or Aa-LPS at the dose of 1 mg/L. After culture for 24 hours, the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)6, IL-1β, IL-8 mRNA and protein were determined by real-time PCR and enzyme-linked immunosorbent assay respectively. The monocytes/macrophages challenged by Ec-LPS or Aa-LPS expressed more cytokines both in mRNA and protein levels compared with the controls (P < 0.05). Among them, AM displayed the highest respond when encount with Aa-LPS, with the TNF-α, IL-6, IL-1β, IL-8 mRNA relative levels were (0.4719 ± 0.0171), (2.7895 ± 0.0669), (5.1527 ± 0.1190), (3.6785 ± 0.1836) and the proteins concentrations were (82.2 ± 5.4), (40.2 ± 2.0), (50 308.3 ± 445.0), (35 305.3 ± 1480.9) ng/L respectively. And the inducibility of Aa-LPS was stronger than that of Ec-LPS (P < 0.05). Meanwhile the cells from different organs showed discrepant response when exposed to Aa-LPS (P < 0.05). The results showed their abilities to secrete cytokines were in the sequence of AM > Mo > KC > PM. Aa-LPS influenced the expression of cytokines in monocytes/macrophages from different organs of rabbits.

A. actinomycetemcomitans LPS Enhances Foam Cell Formation Induced by LDL

The objective of this study was to examine whether native low-density lipoprotein (LDL) induces foam cell formation by macrophages and to examine the effect of lipopolysaccharide (LPS) on native LDL-induced foam cell formation by macrophages in vitro. RAW 264.7 cells were cultured with LDL or high-density lipoprotein (HDL) in the presence of LPS derived from Aggregatibacter actinomycetemcomitans. Foam cell formation was determined by staining with Oil-red-O to visualize cytoplasmic lipid droplet accumulation. The expression of LDL-receptor and the degree of internalization of FITC-conjugated LDL in RAW 264.7 cells were examined by immunofluorescence microscopy. The images were digitally recorded and analyzed with Image J software. Statistical analysis was performed by JMP software. Foam cell formation was induced by the addition of native LDL in dose- and time-dependent manners, whereas HDL showed no effect. LPS enhanced the foam cell formation induced by native LDL. In addition, LPS stimulated the expression of LDL-receptor protein on RAW 264.7 cells and enhanced the internalization of LDL. The enhancement of foam cell formation induced by LPS and LDL was inhibited by the depolymerizing agent nocodazole and amiloride analog 5-(N-ethyl-N-isoprophyl) amiloride (EIPA). Our findings indicate that LPS plays an important role in foam cell formation by LDL-stimulated macrophages.

Alpha‐amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS-binding proteins (Aa.LPS-BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS-BPs in human saliva using a LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Aa.LPS-beads) activated Toll-like receptor 4 and produced nitric oxide and Interferon gamma-inducible protein-10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS-BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS-beads followed by identification with the mass spectrometry. Aa.LPS-BPs include α-amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen-regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin-inducible protein, lipocalin-1, and basic salivary proline-rich protein 2. Specific binding was validated using a pull-down assay with α-amylase which was captured at the highest frequency. Alpha-amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat-inactivated α-amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS-BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.

Aggregatibacter actinomycetemcomitans lipopolysaccharide regulates bone sialoprotein gene transcription

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is believed to be associated with aggressive periodontitis characterized by a rapid bone loss. A. actinomycetemcomitans lipopolysaccharide (LPS) has a similar structure to Escherichia coli LPS, and they are Toll-like receptor 4 agonists. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of A. actinomycetemcomitans LPS on bone formation, we targeted BSP as a marker for osteogenic differentiation and bone formation. BSP mRNA levels were decreased by 0.1 µg/ml and increased by 0.01 µg/ml A. actinomycetemcomitans LPS at 6 h in osteoblast-like ROS17/2.8 cells. In transient transfection analyses, 0.1 µg/ml decreased and 0.01 µg/ml A. actinomycetemcomitans LPS increased luciferase activities of the construct (-116 to +60). Introduction of 2 bp mutations to the constructs showed that the effects of A. actinomycetemcomitans LPS were mediated by a cAMP response element (CRE), a FGF2 response element (FRE), and a homeodomain protein-binding site (HOX). Tyrosine kinase, ERK1/2, and PI3-kinase/Akt participated in the effects of both 0.1 and 0.01 µg/ml A. actinomycetemcomitans LPS. The results of gel shift showed that 0.1 µg/ml decreased while 0.01 µg/ml A. actinomycetemcomitans LPS increased CRE-, FRE-, and HOX-binding protein complexes formation at 6 h, and revealed that 0.01 µg/ml A. actinomycetemcomitans LPS induced BSP transcription through CREB1, JunD, Fra2, c-Fos, Runx2, Dlx5, and Smad1 targeting those response elements. These studies therefore indicated that 0.1 µg/ml suppressed and 0.01 µg/ml A. actinomycetemcomitans LPS increased BSP gene transcription mediated through CRE, FRE, and HOX elements in the rat BSP gene promoter.

Neutralizing effect of green tea epigallocatechin‐3‐gallate on nicotine‐induced toxicity and chemokine (C‐C motif) ligand 5 secretion in human oral epithelial cells and fibroblasts

Tobacco use has been identified as the most important environmental risk factor for periodontitis. The aim of this study was to investigate the effect of green tea epigallocatechin-3-gallate on the nicotine-induced toxic and inflammatory responses in oral epithelial cells and gingival fibroblasts. The effect of nicotine, alone and in combination with the lipopolysaccharide of Aggregatibacter actinomycetemcomitans, on the viability of oral epithelial cells and fibroblasts was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. The ability of epigallocatechin-3-gallate to neutralize the nicotine-induced cytotoxicity was then investigated. The nicotine-induced cytokine secretion in epithelial cells and the inhibitory effect of epigallocatechin-3-gallate were determined by enzyme-linked immunosorbent assay. Our results indicated that nicotine caused a dose-dependent loss of viability in both epithelial cells and fibroblasts. A mixture of nicotine and A. actinomycetemcomitans lipopolysaccharide demonstrated additive instead of synergistic effects on loss of cell viability. Pretreatment of cells with epigallocatechin-3-gallate efficiently neutralized the nicotine-induced toxic effects in epithelial cells and fibroblasts. It also dose dependently inhibited the nicotine-induced secretion of chemokine (C-C motif) ligand 5 by epithelial cells. The present study suggests that epigallocatechin-3-gallate, the major polyphenol in green tea, may represent a novel preventive/therapeutic agent for smoking-related periodontitis.

Comparative Evaluation of Two Structurally Related Flavonoids, Isoliquiritigenin and Liquiritigenin, for Their Oral Infection Therapeutic Potential

Isoliquiritigenin (1) and liquiritigenin (2) are structurally related flavonoids found in a variety of plants. The purpose of this study was to perform a comparative analysis of biological properties of these compounds in regard to their therapeutic potential for oral infections. Compound 1 demonstrated significant antibacterial activity against three major periodontopathogens, Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia. In contrast, 2 exerted less pronounced effects on the above bacterial species. Neither compound was effective against cariogenic bacteria (Streptococcus mutans and Streptococcus sobrinus). Furthermore, 1 exhibited a stronger inhibitory activity than 2 toward P. gingivalis collagenase and human matrix metalloproteinase 9. Finally, the capacity of 1 to attenuate the inflammatory response of macrophages induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) was much higher when compared to 2. The activation of transcriptional factors nuclear factor-κB (NF-κB) p65 and activator protein-1 (AP-1) associated with the LPS-induced inflammatory response in macrophages was inhibited strongly by 1, but less affected by 2.

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

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). 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. 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. 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.