Porphyromonas gingivalis under palmitate-induced obesogenic microenvironment modulates the inflammatory transcriptional signature of macrophage-like cells.

Metabolic diseases and low-grade chronic inflammation are interconnected: obese persons are at higher risk of developing periodontitis. However, the molecular mechanisms involved in the development and progression of periodontitis in an obesogenic microenvironment in response to periodontopathogens are still lacking. This study aims to investigate the combined effects of palmitate and Porphyromonas gingivalis on the secretion of pro-inflammatory cytokines and on transcriptional landscape modifications in macrophage-like cells. U937 macrophage-like cells were treated with palmitate and stimulated with P. gingivalis for 24h. Cytokines IL-1ß, TNF-α and IL-6 were measured by ELISA in the culture medium and cell extracted RNA was submitted to a microarray analysis followed by Gene Ontology analyses. P. gingivalis, in presence of palmitate, potentiated IL-1ß and TNF-α secretion in comparison to palmitate alone. Gene Ontology analyses also revealed that the combination palmitate-P. gingivalis potentiated the number of gene molecular functions implicated in the regulation of immune and inflammatory pathways compared to macrophages treated with palmitate alone. Our results provide the first comprehensive mapping of gene interconnections between palmitate and P. gingivalis during inflammatory responses in macrophage-like cells. These data highlight the importance of considering systemic conditions, specifically obesogenic microenvironment, in the management of periodontal disease in obese patients.

The curcumin analog (PAC) suppressed cell survival and induced apoptosis and autophagy in oral cancer cells.

PAC (3,5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone), a novel bioactive curcumin analog, has been reported to have anticancer properties against various tumors. However, the anti-cancer effects of PAC on oral cavity squamous cell carcinoma were not studied yet. Our aim is to investigate the anti-oral cancer properties of PAC in vitro, and determine the molecular mechanisms underlying these effects. Viability assays including MTT and LDH were conducted to measure cell proliferation. Flow cytometry-based cytotoxicity assay was performed to detect autophagic cell death and oxidative stress markers. Western blotting was used for measuring protein expression/activation in apoptotic, autophagic and pro-carcinogenic cellular signaling pathways. We demonstrated that PAC preferentially and, in a dose, -dependent way kills oral cancer cells, but was not toxic to normal human gingival cells. PAC destabilizes cell-cycle distributions, inhibits the expression of oncogenes (cyclin D1) and that of cyclin-dependent kinase inhibitor (p21WAF1) is upregulated, increases the expression of p53 gene, and inhibits epithelial-mesenchymal transition markers in oral cancer cells. The PAC effect involve various signaling pathways including NF-κB, MAPK, Wnt, caspase-3/9 and PARP1. Finally, PAC demonstrated ability to induce autophagy, decrease production of reactive oxygen species, increase intracellular glutathione (GSH) activity, and reduce mitochondrial membrane potential in oral cancer cells. In conclusion, PAC inhibits the proliferation and increases the apoptosis and autophagy and oxidative stress of oral cancer cells. These effects involve ERK1/2, p38/JNK, NF-κB and Wnt cellular signaling pathways. Overall, our study suggests the potential use of PAC to treat oral cancer.

Anethole induces anti-oral cancer activity by triggering apoptosis, autophagy and oxidative stress and by modulation of multiple signaling pathways.

Oral cancer is one of the major public health problems. The aim of this study was to evaluate the effects of anethole, 1-methoxy-4-[(E)-1-propenyl]-benzene, on growth and apoptosis of oral tumor cells, and to identify the signaling pathways involved in its interaction with these cancer cells. Cancer gingival cells (Ca9-22) were treated with different concentrations of anethole. Cell proliferation and cytotoxic effects were measured by MTT and LDH assays. Cell death, autophagy and oxidative stress markers were assessed by flow cytometry while cell migration was determined by a healing capacity assay. The effect of anethole on apoptotic and pro-carcinogenic signaling pathways proteins was assessed by immunoblotting. Our results showed that anethole selectively and in a dose-dependent manner decreases the cell proliferation rate, and conversely induces toxicity and apoptosis in oral cancer cells. This killing effect was mediated mainly through NF-κB, MAPKinases, Wnt, caspase 3, 9 and PARP1 pathways. Anethole showed an ability to induce autophagy, decrease reactive oxygen species (ROS) production and increased intracellular glutathione (GSH) activity. Finally, anethole treatment inhibits the expression of oncogenes (cyclin D1) and up-regulated cyclin-dependent kinase inhibitor (p21WAF1), increases the expression of p53 gene, but inhibits the epithelial-mesenchymal transition markers. These results indicate that anethole could be a potential molecule for the therapy of oral cancer.

Biochemical parameters and oxidative stress markers in Tunisian patients with periodontal disease.

BACKGROUND: Oxidative stress is involved in many diseases including diabetes and cancer. Numbers of studies have suggested its involvement in the pathogenesis of periodontal diseases. The aim of this study was to evaluate the levels of biochemical parameters and oxidative stress markers in plasma of healthy and chronic periodontitis patients. METHODS: One hundred thirty subjects were divided into two groups; patients (mean age = 42 ± 13.6 y.o) and control (mean age = 44.8 ± 12.6 y.o). Patients and healthy subjects were free from any infection, coronary or heart disease, diabetes or liver failure. Total cholesterol, LDLc, HDLc, Triglycerides (TG), creatinine, uric acid (UA), glucose and urea levels as well as the activities of enzymatic antioxidants such as catalase, glutathione reductase (GR) and total antioxidant capacity (TAOC), were measured in plasma samples using colorimetric assays. Statistical differences between groups were determined by Student's t-test and p ≤ 0.05 was considered as significant. RESULTS: Periodontitis patients exhibited significant decrease in the activities of catalase, TAOC, GR and TG, cholesterol, LDLc, glucose, HDLc, uric acid levels in plasma samples in comparison with healthy subjects. However, no statistically significant differences in the levels of creatinine and urea were observed between the two groups. CONCLUSION: The reduction of plasma antioxidant activities (Catalase, TAOC, GR) may have a role in the pathogenesis of periodontal diseases. Our findings suggest a decrease in the host capacity to control the damage caused by oxidative stress. Therefore, therapeutic strategies, aiming at modulating the oxidative stress could be considered as potential tools for the prevention or treatment of periodontal diseases and their potential systemic effects on the general health.

Piezocorticision-assisted orthodontics: Efficiency, safety, and long-term evaluation of the inflammatory process.

OBJECTIVES: The aim of this work was to compare the duration of treatment between orthodontic treatment combined with piezocorticision (OT-PC) and conventional orthodontic treatment (COT), as well as to evaluate the safety, inflammatory process, periodontal health, and soft tissue healing in the OT-PC group. METHODS: Twelve patients were included in each group, and their treatment times were compared for preliminary bracket alignment (PBA) and for overall treatment. In the OT-PC group, the inflammatory process was evaluated by quantifying cytokines in the gingival crevicular fluid. A calibrated examiner measured the probing depth (PD), the distance between the gingival margin and the cementoenamel junction (GM-CEJ), and the clinical attachment level (CAL), before and after OT-PC. The presence of gingival scars was evaluated. Bone and root injuries were assessed with the use of cone-beam computed tomography. RESULTS: The treatment time was significantly reduced in the OT-PC group for PBA in both maxilla (45%; P = 0.001) and mandible (31%; P = 0.023), as well as for overall treatment (52%; P < 0.0001). Although not statistically significant, several inflammatory mediators demonstrated peaks at 3-5 and 16 weeks. There were not significant changes in PD and GM-CEJ after OT-PC treatment, unlike CAL (0.09 ± 0.12 mm; P = 0.024); 47.5% of piezocorticisions caused gingival scars. Only one patient showed no scars. Four cortical bones did not heal completely, and 2 roots had piezoelectric injuries. CONCLUSION: OT-PC was effective at reducing the orthodontic treatment time.

Silver-zeolite combined to polyphenol-rich extracts of Ascophyllum nodosum: potential active role in prevention of periodontal diseases.

The purpose of this study was to evaluate various biological effects of silver-zeolite and a polyphenol-rich extract of A. nodosum (ASCOP) to prevent and/or treat biofilm-related oral diseases. Porphyromonas gingivalis and Streptococcus gordonii contribute to the biofilm formation associated with chronic periodontitis. In this study, we evaluated in vitro antibacterial and anti-biofilm effects of silver-zeolite (Ag-zeolite) combined to ASCOP on P. gingivalis and S. gordonii growth and biofilm formation capacity. We also studied the anti-inflammatory and antioxidant capacities of ASCOP in cell culture models. While Ag-zeolite combined with ASCOP was ineffective against the growth of S. gordonii, it showed a strong bactericidal effect on P. gingivalis growth. Ag-zeolite combined with ASCOP was able to completely inhibit S. gordonii monospecies biofilm formation as well as to reduce the formation of a bi-species S. gordonii/P. gingivalis biofilm. ASCOP alone was ineffective towards the growth and/or biofilm formation of S. gordonii and P. gingivalis while it significantly reduced the secretion of inflammatory cytokines (TNFα and IL-6) by LPS-stimulated human like-macrophages. It also exhibited antioxidant properties and decreased LPS induced lipid peroxidation in gingival epithelial cells. These findings support promising use of these products in future preventive or therapeutic strategies against periodontal diseases.

Periodontitis and Porphyromonas gingivalis in patients with rheumatoid arthritis.

OBJECTIVE: To examine the degree to which shared risk factors explain the relationship of periodontitis (PD) to rheumatoid arthritis (RA) and to determine the associations of PD and Porphyromonas gingivalis with pathologic and clinical features of RA. METHODS: Patients with RA (n = 287) and patients with osteoarthritis as disease controls (n = 330) underwent a standardized periodontal examination. The HLA-DRB1 status of all participants was imputed using single-nucleotide polymorphisms from the extended major histocompatibility complex. Circulating anti-P gingivalis antibodies were measured using an enzyme-linked immunosorbent assay, and subgingival plaque was assessed for the presence of P gingivalis using polymerase chain reaction (PCR). Associations of PD with RA were examined using multivariable regression. RESULTS: Presence of PD was more common in patients with RA and patients with anti-citrullinated protein antibody (ACPA)-positive RA (n = 240; determined using the anti-cyclic citrullinated peptide 2 [anti-CCP-2] test) than in controls (35% and 37%, respectively, versus 26%; P = 0.022 and P = 0.006, respectively). There were no differences between RA patients and controls in the levels of anti-P gingivalis or the frequency of P gingivalis positivity by PCR. The anti-P gingivalis findings showed a weak, but statistically significant, association with the findings for both anti-CCP-2 (r = 0.14, P = 0.022) and rheumatoid factor (RF) (r = 0.19, P = 0.001). Presence of PD was associated with increased swollen joint counts (P = 0.004), greater disease activity according to the 28-joint Disease Activity Score using C-reactive protein level (P = 0.045), and higher total Sharp scores of radiographic damage (P = 0.015), as well as with the presence and levels of anti-CCP-2 (P = 0.011) and RF (P < 0.001). The expression levels of select ACPAs (including antibodies to citrullinated filaggrin) were higher in patients with subgingival P gingivalis and in those with higher levels of anti-P gingivalis antibodies, irrespective of smoking status. Associations of PD with established seropositive RA were independent of all covariates examined, including evidence of P gingivalis infection. CONCLUSION: Both PD and P gingivalis appear to shape the autoreactivity of RA. In addition, these results demonstrate an independent relationship between PD and established seropositive RA.

Effect of periodontopathogen lipopolysaccharides and proinflammatory cytokines on CD46, CD55, and CD59 gene/protein expression by oral epithelial cells.

Membrane-anchored complement regulatory proteins (CRPs), including CD46, CD55, and CD59, protect host cells from complement attack. In the present study, we investigated whether periodontopathogen lipopolysaccharide and proinflammatory cytokines modulate CRP gene/protein expression in human oral epithelial cells. The lipopolysaccharide of Treponema denticola and Tannerella forsythia were the most potent for increasing the gene expression of CD55 and CD59, and to a lesser extent CD46, after a 48-h stimulation. An lipopolysaccharide-induced upregulation of epithelial cell-surface CRP was also demonstrated. The stimulation of epithelial cells with lipopolysaccharide was associated with interleukin-6 (IL-6) and IL-8 secretion. Although these two cytokines had no effect on CD46 and CD55 gene expression in epithelial cells, IL-1ß and tumor necrosis factor-α induced a significant upregulation. The cell-surface expression of CRP was also increased by the stimulation of epithelial cells with cytokines. The CD46, CD55, and CD59 gene/protein expression was upregulated by periodontopathogen lipopolysaccharide and proinflammatory cytokines. It can be hypothesized that, when faced with bacterial challenges and inflammatory conditions associated with active periodontal sites, oral epithelial cells may respond by increasing CRP gene/protein expression to avoid cell lysis by the complement system, which is activated during periodontitis.

Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

BACKGROUND: Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. METHODS: Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. RESULTS: Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. CONCLUSIONS: The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis.

Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.

Antibodies to porphyromonas gingivalis are associated with anticitrullinated protein antibodies in patients with rheumatoid arthritis and their relatives.

OBJECTIVE: Anticitrullinated protein antibodies (ACPA) are relatively specific for rheumatoid arthritis (RA), and predate disease. The oral pathogen Porphyromonas gingivalis may play a role in breaking immune tolerance to citrullinated antigens. We studied a cohort of patients with RA and their relatives looking for associations between anti-P. gingivalis antibodies and ACPA. METHODS: Patients with RA (n = 82) and their relatives (n = 205) from a North American Native (NAN) population were studied, along with 47 NAN and 60 non-NAN controls. IgM and IgA rheumatoid factor (RF) were tested by nephelometry and ELISA. Second-generation anticyclic citrullinated peptide (anti-CCP2) isotypes and IgG anti-P. gingivalis lipopolysaccharides were tested by ELISA. HLA-DRB1 typing was performed by sequencing. Oral hygiene and smoking habits were assessed by questionnaires. RESULTS: Autoantibody frequency in patients with RA and relatives: ACPA 91% vs 19%, respectively; IgM RF 82% vs 17%; IgA RF 48% vs 22%. Anti-P. gingivalis levels were higher in patients with RA compared to relatives and controls (p = 0.005) and higher in ACPA-positive patients with RA than in ACPA-negative patients with RA (p = 0.04) and relatives (p < 0.001), but comparable in RF-positive and RF-negative patients and relatives. Poor oral hygiene and smoking were prevalent, but with no clear association with autoantibodies. Relatives with 2 shared-epitope alleles were more likely to be ACPA-positive (OR 2.5, p = 0.02). CONCLUSION: In a genetically predisposed population of NAN patients with RA and their relatives, anti-P. gingivalis antibodies were associated with ACPA. These findings suggest that immune responses to P. gingivalis may be involved in breaking immune tolerance to citrullinated antigens.

Putative respiratory chain of Porphyromonas gingivalis.

The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

Cranberry components inhibit interleukin-6, interleukin-8, and prostaglandin E production by lipopolysaccharide-activated gingival fibroblasts.

Periodontitis is a chronic inflammatory disease that affects the tooth supporting tissues. Gingival fibroblasts are the most abundant cells in periodontal tissues and participate actively in the host inflammatory response to periodontopathogens, which is known to mediate local tissue destruction in periodontitis. The aim of this study was to investigate the effect of a proanthocyanidin-enriched cranberry fraction, prepared from cranberry juice concentrate, on inflammatory mediator production by gingival fibroblasts stimulated by the lipopolysaccharide (LPS) of Aggregatibacter actinomycetemcomitans. Interleukin (IL)-6, IL-8, and prostaglandin E(2) (PGE(2)) production by fibroblasts treated with the cranberry fraction and stimulated by A. actinomycetemcomitans LPS was evaluated by enzyme-linked immunosorbent assay. Changes induced by A. actinomycetemcomitans LPS and the cranberry fraction in the expression and phosphorylation state of fibroblast intracellular signaling proteins were characterized by antibody microarrays. The LPS-induced IL-6, IL-8, and PGE(2) responses of gingival fibroblasts were inhibited by treatment with the cranberry fraction. This fraction was found to inhibit fibroblast intracellular signaling proteins, a phenomenon that may lead to a down-regulation of activating protein-1 activity. Cranberry components also reduced cyclooxygenase 2 expression. This study suggests that cranberry juice contains molecules with interesting properties for the development of new host-modulating therapeutic strategies in the adjunctive treatment of periodontitis.

Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens.

BACKGROUND: During phagocytosis or stimulation with bacterial components, macrophages activate various cell processes, including the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are critical for successful defense against invading organisms. Increased levels of ROS/RNS create oxidative stress that results in tissue and bone destruction. Grape seed proanthocyanidins have been reported to possess a wide range of biologic properties against oxidative stress. In the present study, we investigated the effects of a grape seed proanthocyanidin extract (GSE) and commercial polyphenols on the production of ROS and RNS and on the protein expression of inducible nitric oxide synthase (iNOS) by murine macrophages stimulated with lipopolysaccharides (LPS) of periodontopathogens. METHODS: Macrophages (RAW 264.7) were treated with non-toxic concentrations of either GSE or commercial polyphenols (gallic acid [GA] and [-]-epigallocatechin-3-gallate [EGCG]) and stimulated with LPS of Actinobacillus actinomycetemcomitans or Fusobacterium nucleatum, and iNOS expression was evaluated by immunoblotting. Nitric oxide (NO) production was quantified using the colorimetric Griess assay, whereas ROS production was measured with the fluorescent 123-dihydrorhodamine dye. RESULTS: GSE strongly decreased NO and ROS production and iNOS expression by LPS-stimulated macrophages. GA also revealed a strong inhibitory effect on NO production without affecting iNOS expression but slightly increasing ROS production. EGCG showed an inhibitory effect on NO and ROS production and on iNOS expression by macrophages. CONCLUSION: Our findings demonstrate that proanthocyanidins have potent antioxidant properties and should be considered a potential agent in the prevention of periodontal diseases.

Inflammatory responses of a macrophage/epithelial cell co-culture model to mono and mixed infections with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia.

Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.

Modulation of cytokine production by Porphyromonas gingivalis in a macrophage and epithelial cell co-culture model.

Epithelial cells and macrophages play a major role in the host response to Porphyromonas gingivalis, a major etiologic agent of chronic periodontitis. Secretion of high levels of cytokines by these cells is believed to contribute to periodontal tissue destruction. To investigate the interactions between P. gingivalis and these two major cell types, we characterized the production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and regulated on activation normal T cell expressed and secreted (RANTES) by an in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 cell line) following a challenge with different strains of P. gingivalis. P. gingivalis cells stimulated the secretion of pro-inflammatory cytokines (IL-1beta and IL-6) and chemokines (IL-8 and RANTES) in the co-culture model. Responses to P. gingivalis infection were influenced by the macrophage/epithelial cell ratios of the cultures. In addition, the level of secretion of these inflammatory mediators was dependent on the bacterial strain and the multiplicity of infection (MOI) used. The use of a gingipain-deficient mutant of P. gingivalis or the addition of a cysteine protease inhibitor suggested that the level of cytokines secreted by the co-culture model was underestimated due to an extensive proteolytic degradation. This study showed that P. gingivalis can modulate the levels of inflammatory mediators, which may contribute to the progression of periodontitis.

Loss of lipopolysaccharide receptor CD14 from the surface of human macrophage-like cells mediated by Porphyromonas gingivalis outer membrane vesicles.

Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles. In this study, we investigated the capacity of P. gingivalis vesicles to promote the shedding or cleavage of the lipopolysaccharide (LPS) receptor CD14 from the surface of human U937 macrophage-like cells. SDS-PAGE/Western immunoblotting analysis of gingival crevicular fluid samples from patients affected by moderate or advanced periodontitis revealed the presence of soluble CD14 and CD14 fragments, thus supporting the hypothesis of an in vivo shedding and cleavage of CD14 receptors. Flow cytometry analysis of macrophage-like cells treated with a vesicle-containing culture supernatant of P. gingivalis showed a significant decrease in the binding of anti-human CD14 to the cell surface. However, no accumulation of soluble CD14 or immunoreactive CD14 fragments in the assay supernatant could be demonstrated by ELISA. Treatment of macrophage-like cells with various concentrations of P. gingivalis vesicles substantially suppressed TNF-alpha production triggered by Escherichia coli LPS. This suppressive effect was much less important using heat-treated vesicles or in the presence of leupeptin, a gingipain inhibitor, during the treatment. Recombinant human CD14 receptors were found to be susceptible to proteolytic degradation by P. gingivalis vesicles. A purified Arg-gingipain preparation produced much more degradation than a Lys-gingipain preparation. This study provides evidence that P. gingivalis outer membrane vesicles contribute to the loss of membrane-bound CD14 receptors and that gingipains degrade this LPS receptor. Such a phenomenon, which results in an hyporesponsiveness of macrophages to LPS stimulation, may contribute to an increased capacity of P. gingivalis, and other periodontopathogens, to evade the host immune system mechanisms.

Effect of inactivation of the Arg- and/or Lys-gingipain gene on selected virulence and physiological properties of Porphyromonas gingivalis.

Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis. Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis, inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis, including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects.