interleukin-8 and Bacteroidaceae-Infections

Interleukin-34 (IL-34) is a cytokine that supports the viability and differentiation of macrophages. An important cytokine for the development of epidermal immunity, IL-34, is present and plays a role in the immunity of the oral environment. IL-34 has been linked to inflammatory periodontal diseases, which involve innate phagocytes, including macrophages. Whether IL-34 can alter the ability of macrophages to effectively interact with oral microbes is currently unclear. Using macrophages derived from human blood monocytes with either the canonical cytokine colony-stimulating factor (CSF)1 or IL-34, we compared the ability of the macrophages to phagocytose, kill, and respond through the production of cytokines to the periodontal keystone pathogen Porphyromonas gingivalis. While macrophages derived from both cytokines were able to engulf the bacterium equally, IL-34-derived macrophages were much less capable of killing internalized P. gingivalis. Of the macrophage cell surface receptors known to interact with P. gingivalis, dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin was found to have the largest variation between IL-34- and CSF1-derived macrophages. We also found that upon interaction with P. gingivalis, IL-34-derived macrophages produced significantly less of the neutrophil chemotactic factor IL-8 than macrophages derived in the presence of CSF1. Mechanistically, we identified that the levels of IL-8 corresponded with P. gingivalis survival and dephosphorylation of the major transcription factor NF-κB p65. Overall, we found that macrophages differentiated in the presence of IL-34, a dominant cytokine in the oral gingiva, have a reduced ability to kill the keystone pathogen P. gingivalis and may be susceptible to specific bacteria-mediated cytokine modification.

Topics: Bacteroidaceae Infections; Gingiva; Gingival Diseases; Humans; Interleukin-8; Interleukins; Macrophages; NF-kappa B; Porphyromonas gingivalis

Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs.. Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human β-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR.. Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum.. Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.

Topics: Bacteroidaceae Infections; Caspases, Initiator; Cells, Cultured; Epithelium; Gingiva; Humans; Interleukin-18; Interleukin-8; Periodontitis; Porphyromonas gingivalis; RNA, Messenger

Topics: Apoptosis; Bacteroidaceae Infections; Cell Line; Gingiva; Humans; Inflammation; Interleukin-6; Interleukin-8; Keratinocytes; NF-kappa B; Periodontitis; Porphyromonas gingivalis; RNA, Small Interfering; Signal Transduction; Tumor Necrosis Factor alpha-Induced Protein 3

The development of pharmaceutical agents possessing anti‑invasive and anti‑metastatic abilities, as well as apoptotic activity, is important in decreasing the incidence and recurrence of oral cancer. Cancer cells are known to acquire invasiveness not only through epigenetic changes, but also from inflammatory stimuli within the tumor microenvironment. Accordingly, the identification of agents that can suppress the inflammation‑promoted invasiveness of cancer cells may be important in treating cancer and improving the prognosis of patients with cancer. Acetylshikonin, a flavonoid with anti‑inflammatory activity, inhibits proliferation and induces apoptosis of oral cancer cells. In the present study, the anti‑invasive effect of acetylshikonin on YD10B oral cancer cells infected with Porphyromonas gingivalis, a major pathogen of chronic periodontitis, and the mechanisms involved were investigated. Firstly, we examined whether P. gingivalis infection increased the invasiveness of YD10B cells. Results suggested that YD10B oral cancer cells become more aggressive when they are infected with P. gingivalis. Secondly, acetylshikonin significantly inhibited the invasion of P. gingivalis‑infected YD10B cells by suppressing IL‑8 release and IL‑8‑dependent MMP release. These data suggest that acetylshikonin may be a useful preventive and therapeutic candidate for oral cancer that is chronically infected with periodontal pathogens.

Topics: Anthraquinones; Bacteroidaceae Infections; Cell Line, Tumor; Cells, Cultured; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Interleukin-8; Matrix Metalloproteinases; Mouth Neoplasms; Porphyromonas gingivalis

Genetic factors play a role in periodontitis. Here we examined whether the risk haplotype of MHC class III region BAT1-NFKBIL1-LTA and lymphotoxin-α polymorphisms associate with salivary biomarkers of periodontal disease. A total of 455 individuals with detailed clinical and radiographic periodontal health data were included in the study. A 610 K genotyping chip and a Sequenom platform were used in genotyping analyses. Phospholipid transfer protein activity, concentrations of lymphotoxin-α, IL-8 and myeloperoxidase, and a cumulative risk score (combining Porphyromonas gingivalis, IL-1β and matrix metalloproteinase-8) were examined in saliva samples. Elevated IL-8 and myeloperoxidase concentrations and cumulative risk scores associated with advanced tooth loss, deepened periodontal pockets and signs of periodontal inflammation. In multiple logistic regression models adjusted for periodontal parameters and risk factors, myeloperoxidase concentration (odds ratio (OR); 1.37, P = 0.007) associated with increased odds for having the risk haplotype and lymphotoxin-α concentration with its genetic variants rs2857708, rs2009658 and rs2844482. In conclusion, salivary levels of IL-8, myeloperoxidase and cumulative risk scores associate with periodontal inflammation and tissue destruction, while those of myeloperoxidase and lymphotoxin-α associate with genetic factors as well.

Topics: Adaptor Proteins, Signal Transducing; Aged; Bacteroidaceae Infections; DEAD-box RNA Helicases; Female; Genetic Predisposition to Disease; Genotype; Haplotypes; Histocompatibility Antigens Class II; Humans; Interleukin-8; Lymphotoxin-alpha; Male; Matrix Metalloproteinases; Middle Aged; Periodontitis; Polymorphism, Single Nucleotide; Porphyromonas gingivalis; Risk; Saliva; Salivary Glands

Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of bacteria, and at the same time activates immune response in eukaryotic organisms. We recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the four species in the Prevotella intermedia group. Here, we examined the combined effects of estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK) against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella pallens, and 2) biofilm formation of these three Prevotella species. All experiments were performed in the presence or absence of estradiol, and with different QS molecules: DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-1β, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was quantitatively evaluated by measuring protein concentration via the Bradford method, and the microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging. Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced regulation of HMK interleukin expression and, at the same time, inhibited the biofilm formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in biofilm mass after application of butyl-DPD, which was most detectable among the P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine response and, at the same time, to inhibit the biofilm formation of P. intermedia and P. nigrescens.

Topics: Bacteroidaceae Infections; Biofilms; Epithelial Cells; Gingiva; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Keratinocytes; Prevotella; Prevotella intermedia; Prevotella nigrescens; Quorum Sensing

Porphyromonas gingivalis produces different LPS isoforms with significant structural variations of their lipid A and O-antigen moieties that can affect its pro-inflammatory and bone-resorbing potential. We show here, for the first time, that P. gingivalis LPS isolated from W83 strain is highly sialylated and possesses significantly reduced inflammatory potential compared with less sialylated ATCC 33277 strain LPS. Nevertheless, the reduction in the endotoxin activity is not mediated by the presence of sialic acid LPS moieties as the sialic acid-free LPS produced by the mutant W83 strain exhibits a similar inflammatory potential to the wild type strain. Furthermore, our findings suggest that the interaction between the sialic acid LPS moieties and the inhibitory CD33 receptor is prevented by endogenously expressed sialic acid on the surface of THP-1 cells that cannot be out-competed by sialic acid containing P. gingivalis LPS. The present study also highlights the importance of endogenous sialic acid as a 'self-associated molecular pattern' and CD33 receptors in modulation of innate immune response as human gingival fibroblasts, which do not express CD33 receptors, and desialylated THP-1 cells have both been found to have much higher spontaneous IL-8 production than naïve THP-1 cells.

Topics: Bacteroidaceae Infections; Cell Line; Fibroblasts; Gingiva; Host-Pathogen Interactions; Humans; Immunity, Innate; Interleukin-8; Lipid A; Lipopolysaccharides; Monocytes; Mutation; N-Acetylneuraminic Acid; O Antigens; Periodontal Diseases; Porphyromonas gingivalis; Protein Processing, Post-Translational; Sialic Acid Binding Ig-like Lectin 3

Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW.. To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy.. Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion.. Our novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

Topics: Animals; Bacteroidaceae Infections; Caspase 3; Chronic Periodontitis; Cyclooxygenase 2; Disease Models, Animal; Female; Infant, Low Birth Weight; Inflammation; Interleukin-17; Interleukin-6; Interleukin-8; Mice; NF-kappa B; Placenta; Porphyromonas gingivalis; Pregnancy; Premature Birth; Tumor Necrosis Factor-alpha

Osteoclasts are bone-specific multinucleated cells generated by the differentiation of monocyte/macrophage lineage precursors. Regulation of osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone-lytic diseases. Periodontitis is an inflammatory disease characterized by extensive bone resorption. In this study, we investigated the effects of sodium fluoride (NaF) on osteoclastogenesis induced by Porphyromonas gingivalis, an important colonizer of the oral cavity that has been implicated in periodontitis. NaF strongly inhibited the P. gingivalis-induced alveolar bone loss. That effect was accompanied by decreased levels of cathepsin K, interleukin (IL)-1β, matrix metalloproteinase 9 (MMP9), and tartrate-resistant acid phosphatase, which were up-regulated during P. gingivalis-induced osteoclastogenesis. Consistent with the in vivo anti-osteoclastogenic effect, NaF inhibited osteoclast formation caused by the differentiation factor RANKL (receptor activator of nuclear factor κB ligand) and macrophage colony-stimulating factor (M-CSF). The RANKL-stimulated induction of the transcription factor nuclear factor of activated T cells (NFAT) c1 was also abrogated by NaF. Taken together, our data demonstrate that NaF inhibits RANKL-induced osteoclastogenesis by reducing the induction of NFATc1, ultimately leading to the suppressed expression of cathepsin K and MMP9. The in vivo effect of NaF on the inhibition of P. gingivalis-induced osteoclastogenesis strengthens the potential usefulness of NaF for treating periodontal diseases.

Topics: Acid Phosphatase; Alveolar Bone Loss; Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Bacteroidaceae Infections; Bone Density Conservation Agents; Cathepsin K; Interleukin-1beta; Interleukin-6; Interleukin-8; Isoenzymes; Macrophage Colony-Stimulating Factor; Male; Matrix Metalloproteinase 9; Osteoclasts; Periodontitis; Porphyromonas gingivalis; RANK Ligand; Rats; Rats, Sprague-Dawley; Sodium Fluoride; Tartrate-Resistant Acid Phosphatase; Transcription Factors; X-Ray Microtomography

Periodontitis is an inflammatory disease of polymicrobial origin affecting the tissues supporting the tooth. The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, triggers a series of host inflammatory responses that promote the destruction of periodontal tissues. Among the virulence factors of P. gingivalis, hemoglobin receptor protein (HbR) is a major protein found in culture supernatants. In this study, we investigated the roles of HbR in the production of inflammatory mediators. We found that HbR induced interleukin-8 (IL-8) production in the human gingival epithelial cell line Ca9-22. p38 mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (Erk1/2) were activated in HbR-stimulated Ca9-22 cells. Inhibitors of p38 MAPK (SB203580) and Erk1/2 (PD98059) blocked HbR-induced IL-8 production. Additionally, HbR stimulated the translocation of NF-κB-p65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-κB pathway. In addition, small interfering RNA (siRNA) targeting activating transcription factor 2 (ATF-2) or cyclic AMP-response element-binding protein (CREB) inhibited HbR-induced IL-8 production. Moreover, pretreatment with SB203580 and PD98059 reduced HbR-induced phosphorylation of CREB and ATF-2, respectively. Combined pretreatment with an inhibitor of NF-κB (BAY11-7082) and SB203580 was more efficient in inhibiting the ability of HbR to induce IL-8 production than pretreatment with either BAY11-7082 or SB203580 alone. Thus, in Ca9-22 cells, the direct activation of p38 MAPK and Erk1/2 by HbR caused the activation of the transcription factors ATF-2, CREB, and NF-κB, thus resulting in the induction of IL-8 production.

Topics: Bacterial Proteins; Bacteroidaceae Infections; Cells, Cultured; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Gingiva; Humans; Interleukin-8; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Periodontitis; Porphyromonas gingivalis; Protein Kinase Inhibitors; Receptors, Immunologic; Signal Transduction

To observe if Porphyromonas gingivalis (Pg) infection could enhance the adhesion of human monocytic cell line (THP-1) to human umbilical vein endothelial cells (HUVEC).. PgATCC33277 was cultured in anaerobic jar, and THP-1 was infected with various concentrations of PgATCC33277 at multiplicity of infection (MOI) of 1: 100 for 8 and 24 hours, respectively. After removal of the free Pg, THP-1 cells were cocultured with HUVEC for 1 hour to observe the adhesion of THP-1 to HUVEC.HUVEC with adhesive THP-1 cells were co-cultured for additional 23 hours. The medium and cells were separately collected. The expression of related chemotactic cytokine[monocyte chemotactic protein 1(MCP-1) and interleukin 8(IL-8)] and intercellular adhesion molecule-1(ICAM-1) were detected with enzyme-linked immunosorbent assay.. The adhesion of THP-1 to HUVEC was enhanced (13.8%-35.2%, P = 0.006) and the expression of ICAM-1 of HUVEC was increased from (132.5 ± 7.7) to (164.9 ± 9.1) ng/L (P = 0.005) after infection for 24 hours by Pg. Both of the secreted MCP-1 and IL-8 elevated after infection of Pg for 24 hours from (183.2 ± 3.1) to (221.0 ± 4.9) ng/L (P = 0.012) and from (587.2 ± 5.1) to (787.2 ± 10.3) ng/L (P = 0.002), respectively.. Pg could enhance the adhesion of monocytes to endothelial cells and stimulate the inflammation, suggesting that Pg infection may be one of the risk factors in promoting the development of atherosclerosis.

Topics: Bacteroidaceae Infections; Cell Line; Cells, Cultured; Chemokine CCL2; Coculture Techniques; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-8; Monocytes; Porphyromonas gingivalis; Umbilical Veins

Synthetic studies of lipid A and LPS partial structures have been performed to investigate the relationship between structures and functions of LPS. Recent studies have suggested several pathological implications of LPS from parasitic bacteria due to its influence on the host immune responses. To address this issue, we established an efficient synthetic strategy that is widely applicable to the synthesis of various lipid As by using a key disaccharide intermediate with selectively cleavable protecting groups. Porphyromonas gingivalis and Helicobacter pylori lipid As were synthesized and their biological activities were evaluated. All synthetic lipid As did not induce strong inflammatory responses: some are very weak cytokine inducers and others are antagonistic in IL-6 and IL-8 induction with E. coli LPS. On the other hand, P. gingivalis lipid As showed potent IL-18 inducing activity. Since IL-18 has been shown to correlate with chronic inflammation, P. gingivalis LPS may be implicated in the chronic inflammatory responses.

Topics: Bacteroidaceae Infections; Chemistry Techniques, Synthetic; Enzyme-Linked Immunosorbent Assay; Helicobacter Infections; Helicobacter pylori; Humans; Immunity, Innate; Immunomodulation; Interleukin-6; Interleukin-8; Lipid A; Lipopolysaccharides; Models, Chemical; Molecular Structure; Porphyromonas gingivalis

Chronic periodontal diseases are characterised by a dysregulated and exaggerated inflammatory/immune response to plaque bacteria. We have demonstrated previously that oral keratinocytes up-regulate key molecular markers of inflammation, including NF-κB and cytokine signalling, when exposed to the periodontal bacteria Porphyromonas gingivalis and Fusobacterium nucleatum in vitro. The purpose of the current study was to investigate whether α-lipoic acid was able to abrogate bacterially-induced pro-inflammatory changes in the H400 oral epithelial cell line. Initial studies indicated that α-lipoic acid supplementation (1-4 mM) significantly reduced cell attachment; lower concentrations (<0.5 mM) enabled >85% cell adhesion at 24 h. While a pro-inflammatory response, demonstrable by NF-κB translocation, gene expression and protein production was evident in H400 cells following exposure to P. gingivalis and F. nucleatum, pre-incubation of cells with 0.5 mM α-lipoic acid modulated this response. α-Lipoic acid pre-treatment significantly decreased levels of bacterially-induced NF-κB activation and IL-8 protein production, and differentially modulated transcript levels for IL-8, IL-1β, TNF-α and GM-CSF, TLR2, 4, 9, S100A8, S100A9, lysyl oxidase, NF-κB1, HMOX, and SOD2. Overall, the data indicate that α-lipoic acid exerts an anti-inflammatory effect on oral epithelial cells exposed to periodontal bacteria and thus may provide a novel adjunctive treatment for periodontal diseases.

Topics: Bacteroidaceae Infections; Cell Line; Fusobacterium Infections; Fusobacterium nucleatum; Humans; Inflammation Mediators; Interleukin-8; Keratinocytes; Mouth; NF-kappa B; Porphyromonas gingivalis; Signal Transduction; Thioctic Acid; Transcription Factors; Transcriptional Activation

Gingival epithelia utilize multiple signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. In this study we report that DNA methyltransferase (DNMT1) and histone deacetylase expression were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and nonpathogen Fusobacterium nucleatum. Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5'-azacytidine increased hBD2 and CCL20 expression in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.

Topics: Bacteroidaceae Infections; beta-Defensins; Chemokine CCL20; Chromatin; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Epigenomics; Epithelial Cells; Fusobacterium nucleatum; Gene Expression Regulation; Gingiva; Histone Deacetylase 1; Histone Deacetylase 2; Histones; Humans; Immunity, Innate; Inflammation; Interleukin-8; Methylation; Porphyromonas gingivalis; Promoter Regions, Genetic; RNA, Messenger

A-type cranberry proanthocyanidins (AC-PACs) have recently been reported to be beneficial for human health, especially urinary tract health. The effect of these proanthocyanidins on periodontitis, a destructive disease of tooth-supporting tissues, needs to be investigated. The purpose of this study was to investigate the effects of AC-PACs on various virulence determinants of Porphyromonas gingivalis as well as on the inflammatory response of oral epithelial cells stimulated by this periodontopathogen. We examined the effects of AC-PACs on P. gingivalis growth and biofilm formation, adherence to human oral epithelial cells and protein-coated surfaces, collagenase activity, and invasiveness. We also tested the ability of AC-PACs to modulate the P. gingivalis-induced inflammatory response by human oral epithelial cells. Our results showed that while AC-PACs neutralized all the virulence properties of P. gingivalis in a dose-dependent fashion, they did not interfere with growth. They also inhibited the secretion of interleukin-8 (IL-8) and chemokine (C-C motif) ligand 5 (CCL5) but did not affect the secretion of IL-6 by epithelial cells stimulated with P. gingivalis. This anti-inflammatory effect was associated with reduced activation of the nuclear factor-kappaB (NF-kappaB) p65 pathway. AC-PACs may be potentially valuable bioactive molecules for the development of new strategies to treat and prevent P. gingivalis-associated periodontal diseases.

Topics: Anti-Inflammatory Agents; Bacterial Adhesion; Bacteroidaceae Infections; Biofilms; Cells, Cultured; Chemokine CCL5; Epithelial Cells; Extracellular Matrix Proteins; Humans; Interleukin-6; Interleukin-8; Mouth Mucosa; Nuclear Magnetic Resonance, Biomolecular; Periodontal Diseases; Porphyromonas gingivalis; Proanthocyanidins; Vaccinium macrocarpon

Porphyromonas gingivalis is a periodontal pathogen that is also associated with preterm low-birthweight delivery. We investigated the transcriptional responses of human extravillous trophoblasts (HTR-8) to infection with P. gingivalis. Over 2000 genes were differentially regulated in HTR-8 cells by P. gingivalis. In ontology analyses of regulated genes, overpopulated biological pathways included mitogen-activated protein (MAP) kinase signaling and cytokine production. Immunoblots confirmed overexpression of the MAP kinase pathway components MEK3, p38 and Max. Furthermore, P. gingivalis infection induced phosphorylation and activation of MEK3 and p38. Increased production of interleukin (IL)-1beta and IL-8 by HTR-8 cells was demonstrated phenotypically by enzyme-linked immunosorbent assay of HTR-8 cell lysates and culture supernatants. Hence, infection of trophoblasts by P. gingivalis can impact signal transduction pathways and modulate cytokine expression, outcomes that could disrupt the maintenance of pregnancy.

Topics: Bacteroidaceae Infections; Basic-Leucine Zipper Transcription Factors; Cell Line; Coculture Techniques; Female; Humans; Interleukin-1beta; Interleukin-8; Interleukins; MAP Kinase Kinase 3; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Porphyromonas gingivalis; Pregnancy; Premature Birth; Transcriptional Activation; Trophoblasts

CXCL8 (interlukin 8, IL-8) has a diverse spectrum of biological activities including T cell, neutrophil and basophil chemotactic properties. It is produced by a wide variety of cell types and plays a significant role in the initiation of the acute inflammatory response. During inflammation, CXCL8 attracts and activates leukocytes at the site of infection leading to leukocyte infiltration, which can lead to tissue damage. Porphyromonas gingivalis, an aetiological agent of periodontitis, induces production of CXCL8 from several types of cells via its LPS and outer membrane proteins. Bacterial chaperones elicit a strong pro-inflammatory response in cells of the innate immune system. In P. gingivalis the htpG gene codes for the homologue of human Hsp90, a chaperone that associates with transcription factors, hormone receptors and protein kinases, affecting signal transduction pathways. CXCL8 mRNA and CXCL8 protein production was induced in monocytic/human microvascular vein endothelial cells treated with P. gingivalis cells or rHtpG protein. Blocking of receptors CD91 and TLR4 reduced the production of CXCL8 by rHtpG either using receptor-specific antibody or by siRNA silencing. Pre-incubation of P. gingivalis rHtpG preparations with human anti-HtpG significantly inhibited CXCL8 production. A P. gingivalis HtpG disruption mutant also induced less CXCL8 mRNA and protein. These results suggest that P. gingivalis HtpG might be involved in CXCL8-mediated immunopathogenesis.

Topics: Antibodies, Bacterial; Bacterial Proteins; Bacteroidaceae Infections; Cell Line, Tumor; Endothelial Cells; HSP90 Heat-Shock Proteins; Humans; Immunoglobulin G; Interleukin-8; Lipopolysaccharides; Monocytes; Porphyromonas gingivalis; RNA Interference; RNA, Small Interfering; Veins

Periodontitis is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. Leukocytes play a major role in the host response to Porphyromonas gingivalis, a major aetiological agent of chronic periodontitis. Secretion of high levels of inflammatory mediators, including cytokines and prostaglandins, by leucocytes is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of an ex vivo whole blood model to P. gingivalis stimulation. The production of interleukin-1 beta (IL-1beta), IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), IFN-gamma-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), Regulated on Activation Normal T cell Expressed and Secreted (RANTES) and prostaglandin E2 (PGE2) were quantified by enzyme-linked immunosorbent assays. P. gingivalis induced the secretion of the pro-inflammatory cytokines IL-1beta, TNF-alpha, IL-6 and IFN-gamma, the chemokines IL-8, RANTES and MCP-1 and the inflammatory mediator PGE2 in an ex vivo human whole blood model. The secretion levels were dependent on the strain and the infectious dose used. While the mediator profiles were comparable between six healthy subjects, a high interindividual variability in the levels of secreted mediators was observed. This study supports the view that P. gingivalis, by inducing high levels of inflammatory mediators from a mixed leucocyte population, can contribute to the progression of periodontitis.

Topics: Analysis of Variance; Bacteriological Techniques; Bacteroidaceae Infections; Cells, Cultured; Chemokine CCL2; Chemokine CCL5; Cytokines; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Humans; Interferon-gamma; Interleukin-1; Interleukin-6; Interleukin-8; Periodontitis; Porphyromonas gingivalis; Serum Albumin; Species Specificity; Tumor Necrosis Factor-alpha

Epidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice, while an isogenic fimbria-deficient (FimA-) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzyme-linked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)-1beta, IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and E-selectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL-1beta production. In addition, the major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1beta. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1beta appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.

Topics: Aorta; Bacteroidaceae Infections; Cells, Cultured; Chemokine CCL2; E-Selectin; Endothelial Cells; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fimbriae Proteins; Fimbriae, Bacterial; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-8; Mutation; Porphyromonas gingivalis; Vascular Cell Adhesion Molecule-1

Bacteremia frequently occurs after dental treatment. Periodontal inflammation may influence the incidence, magnitude and duration of bacteremia. The presence of circulating oral bacteria or bacterial components may induce cytokine synthesis in blood cells, which may contribute to the development or exacerbation of atherosclerosis. The present study tested the hypothesis that bacteremia occurring after scaling in periodontitis patients results in altered plasma levels of cytokines. Twenty periodontitis patients were subjected to scaling. Blood samples at baseline and at 0.5, 10 and 30 minutes postscaling were examined for bacteremia whereas baseline and eight-hour postscaling blood samples were examined for the levels of IL-1beta, TNF-alpha, IL-6, IL-8, IL-10 and IL-12p70. IL-6 levels were significantly increased eight hours after scaling, while IL-8 was significantly decreased. No systematic changes occurred in the levels of IL-1beta, TNF-alpha, IL-10 and IL-2p70. IL-6 levels may be increased while IL-8 may be decreased due to scaling, which may have implications for general health.

Topics: Adult; Bacteremia; Bacteroidaceae Infections; Dental Plaque Index; Dental Scaling; Female; Follow-Up Studies; Gingival Hemorrhage; Humans; Interleukin-10; Interleukin-12; Interleukin-6; Interleukin-8; Male; Middle Aged; Periodontal Attachment Loss; Periodontal Index; Periodontal Pocket; Periodontitis; Prevotella; Streptococcal Infections; Streptococcus; Time Factors; Tumor Necrosis Factor-alpha

Porphyromonas gingivalis (Pg) is a major etiologic agent for chronic periodontitis. Tissue destruction by Pg results partly from induction of host inflammatory responses through TLR2 signaling. This work examines the role of apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), an adaptor molecule important for TLR-mediated caspase-1 activation. Results demonstrate that ASC levels are stable upon infection of human THP1 monocytic cells with Pg but decrease after cytokine induction. Using short hairpin RNA, we demonstrate an essential role for ASC in induction of IL-1beta by TLR2, 4, and 5 agonists, live Escherichia coli, and Pg. Induction of IL-6, IL-8, IL-10, and TNF also requires ASC, but this induction is not inhibited by IL-1 receptor antagonist or caspase-1 inhibitor. Similar results in U937 indicate broad applicability of these findings. Pg-infected ASC knockdown THP1 cells exhibit reduced transcript levels and NF-kappaB activation. These results suggest a role for ASC in cytokine induction by Pg involving both caspase-1-dependent and -independent mechanisms.

Topics: Bacteroidaceae Infections; Blotting, Western; CARD Signaling Adaptor Proteins; Caspase 1; Cytokines; Cytoskeletal Proteins; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Humans; Interleukin-1; Interleukin-10; Interleukin-6; Interleukin-8; NF-kappa B; Porphyromonas gingivalis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 5; Tumor Necrosis Factor-alpha; U937 Cells

Viable and inactivated Porphyromonas gingivalis dose-dependently induced interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) secretion in human umbilical vein endothelial cells (HUVECs). The inactivated P. gingivalis, in comparison with viable bacteria, tended to enhance the production of both chemokines more strongly. The production of MCP-1 protein began increasing immediately after stimulation by P. gingivalis, and there was a nearly linear increase from 0 to 8 h of incubation, whereas IL-8 production showed a linear increase between 4 and 12 h of incubation. The IL-8 and MCP-1 mRNA expressions in HUVECs as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) or Quantikine mRNA colorimetric quantification kits were found to be enhanced by P. gingivalis. Furthermore, the time courses of IL-8 and MCP-1 mRNA expressions were in accordance with those of protein production. Addition of polymyxin B or boiling did not weaken the stimulatory effect of P. gingivalis, which inhibited the effect of Escherichia coli lipopolysaccharide (E. coli LPS) and tumour necrosis factor-alpha (TNF-alpha), respectively. In contrast, the induction of IL-8 and MCP-1 by P. gingivalis was significantly reduced by anti-CD14 antibody. Our results suggest that some heat-stable component of P. gingivalis, including LPS, may be responsible for the induction of IL-8 and MCP-1 in HUVECs by a CD14-dependent mechanism. These effects might be involved in the accumulation and activation of neutrophils and monocytes at an early stage of the periodontal pathogenesis.

Topics: Bacteroidaceae Infections; Base Sequence; Cells, Cultured; Chemokine CCL2; Endothelium, Vascular; Gene Expression; Hot Temperature; Humans; Interleukin-8; Kinetics; Lipopolysaccharide Receptors; Periodontitis; Polymyxin B; Porphyromonas gingivalis; RNA, Messenger

The objective was to evaluate the pro-inflammatory response of bovine macrophages towards Porphyromonas levii, an etiologic agent of acute interdigital phlegmon, by evaluating the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interleukin 8 (IL-8). Bovine macrophages detect the presence of bacteria, such as P. levii, and respond by upregulating transcription of the genes for pro-inflammatory cytokines TNF-alpha and IL-1beta in addition to the neutrophil chemoattractant IL-8. Monocytes were isolated from blood obtained from Holstein steers. These cells were cultured and differentiated into macrophages over 7 d, followed by exposure to P. levii, Escherichia coli lipopolysaccharide (LPS), or tissue culture medium for 1, 1.5, 2, 4, 6, 8,12, or 24 h. Total RNA was extracted, and reverse transcriptase polymerase chain reaction was conducted to examine the presence of TNF-alpha, IL-1beta, or IL-8 mRNA. Products were visualized on agarose gels to determine the presence or absence of cytokine mRNA amplified DNA. Bovine macrophages, when exposed to P. levii or E. coli LPS, produced mRNA for TNF-alpha, IL-1beta, and IL-8. Expression of all 3 cytokines was higher in the P. levii and LPS-exposed macrophages at all time points examined, compared with tissue culture medium-treated cells. Expression of these cytokines by macrophages is likely directly involved in orchestration of the immune response, and particularly in neutrophil recruitment to affected tissues in acute interdigital phlegmon.

Topics: Animals; Bacteroidaceae Infections; Cattle; Cattle Diseases; Cells, Cultured; Cytokines; Interleukin-1; Interleukin-8; Lipopolysaccharides; Macrophage Activation; Macrophages; Male; Porphyromonas; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.

Topics: Bacteroidaceae Infections; Cells, Cultured; Coculture Techniques; Down-Regulation; Fusobacterium nucleatum; Gingiva; Intercellular Adhesion Molecule-1; Interleukin-8; Mouth Mucosa; Porphyromonas gingivalis; RNA, Messenger

Periodontopathic bacteria induce inflammation of periodontal tissues. The cytokines and nitric oxide released in periodontal lesions have been reported to play a protective role in bacterial infection and to relate to the process of inflammation. To clarify the relationship between colonization of periodontopathic bacteria and cytokines, we evaluated profiles of inflammatory cytokines, chemokine, anti-inflammatory cytokines, and inducible nitric oxide synthase (iNOS) and colonization by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, which are major pathogens of periodontitis.. mRNA expression of cytokines and iNOS in inflamed and healthy gingival tissue was determined using reverse transcription-polymerase chain reaction (RT-PCR), and the relationship between their profiles and the detection of specific bacteria was analyzed.. The relative expression of interleukin (IL)-6 and iNOS mRNAs in periodontal lesions was significantly higher than those in healthy individuals. IL-6 mRNA expression was also significantly higher at bleeding on probing (BOP)-positive sites than at BOP-negative sites. The expressions of IL-1alpha and IL-8 increased, but IL-10 expression decreased at sites where A. actinomycetemcomitans was detected. We found no correlation between the expression of cytokine and iNOS mRNA and infection by P. gingivalis.. The expression of IL-6 may reflect inflammation in gingival tissue, and iNOS may be involved in the inflammatory process in periodontitis. The presence of A. actinomycetemcomitans or P. gingivalis might relate to the different cytokine profiles of IL-1alpha, IL-8, and IL-10.

Topics: Actinobacillus Infections; Adolescent; Adult; Aged; Aggregatibacter actinomycetemcomitans; Bacteroidaceae Infections; Chemokines; Child; Cytokines; Female; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Gingival Hemorrhage; Gingivitis; Humans; Inflammation Mediators; Interleukin-1; Interleukin-10; Interleukin-6; Interleukin-8; Male; Middle Aged; Nitric Oxide Synthase; Periodontal Pocket; Periodontitis; Porphyromonas gingivalis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics as Topic; Statistics, Nonparametric

This study aimed to determine the relationships among interleukin (IL)-8 and granulocyte elastase levels in gingival crevicular fluid (GCF) and the concomitant presence of periodontopathogens in untreated adult periodontitis.. GCF and subgingival plaque samples were collected from 16 patients with untreated adult periodontitis and 10 healthy control subjects. IL-8 levels were determined by enzyme-linked immunosorbent assay (ELISA). Granulocyte elastase was analyzed with a neutrophilic granulocyte-specific, low molecular weight and chromogenic substrate, L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide, and the maximal rate of elastase activity (MR-EA) was calculated. Five DNA probes were used to detect the presence of A. actinomycetemcomitans (A.a.), B. forsythus (B.f.), P. gingivalis (P.g.), P. intermedia (P.i.), and T. denticola (T.d.).. Lower IL-8 concentrations and higher granulocyte elastase activities were found in patients than in healthy controls as well as in diseased conditions co-infected with B.f., P.g., P.i., and T.d. as compared to healthy conditions without the target species (P <0.05). IL-8 concentrations were positively correlated with MR-EA levels in the periodontitis conditions co-infected with B.f., P.g., P.i., and T.d. (P <0.05). A wide range of IL-8 concentrations was found among 15 patients when the periodontitis condition was characterized by co-infection with B.f., P.g., P.i., and T.d. MR-EA levels in the high IL-8 group of subjects were significantly higher than those in the low IL-8 group of subjects (P <0.01).. The present study shows that the local host-bacteria interactions in untreated periodontitis are diverse in terms of the intensity of inflammatory responses measured by IL-8-related granulocyte elastase activity in GCF. This might reflect different phases of the inflammatory response due to shifts in host-bacteria interactions and therefore be indicative of a range of periodontal disease activity levels.

Topics: Actinobacillus Infections; Adult; Aggregatibacter actinomycetemcomitans; Analysis of Variance; Bacteria; Bacteroidaceae Infections; Bacteroides; Bacteroides Infections; Dental Plaque; Gingival Crevicular Fluid; Humans; Interleukin-8; Leukocyte Elastase; Linear Models; Middle Aged; Periodontitis; Porphyromonas gingivalis; Prevotella intermedia; Statistics, Nonparametric; Treponema; Treponemal Infections

Periodontal diseases are inflammatory disorders caused by microorganisms of dental plaque that colonize the gingival sulcus and, subsequently, the periodontal pocket. As in other mucosal infections, the host response to plaque bacteria is characterized by an influx of polymorphonuclear leukocytes (PMNs) to the gingival crevice. Neutrophil migration through the epithelial lining of the gingival pocket is thought to be the first line of defense against plaque bacteria. In order to model this phenomenon in vitro, we used the oral epithelial cell line KB and human PMNs in the Transwell system and examined the impact of Porphyromonas gingivalis-epithelial cell interactions on subsequent PMN transepithelial migration. We demonstrate here that P. gingivalis infection of oral epithelial cells failed to trigger transmigration of PMNs. Furthermore, it significantly inhibited neutrophil transmigration actively induced by stimuli such as N-formylmethionyl leucyl phenylalanine, interleukin-8 (IL-8), and the intestinal pathogen enterotoxigenic Escherichia coli. The ability of P. gingivalis to block PMN transmigration was strongly positively correlated with the ability to adhere to and invade epithelial cells. In addition, P. gingivalis attenuated the production of IL-8 and the expression of intercellular adhesion molecule 1 by epithelial cells. The ability of P. gingivalis to block neutrophil migration across an intact epithelial barrier may critically impair the potential of the host to confront the bacterial challenge and thus may play an important role in the pathogenesis of periodontal disease.

Topics: Bacterial Adhesion; Bacteroidaceae Infections; CD18 Antigens; Cell Communication; Chemotaxis, Leukocyte; Diffusion Chambers, Culture; Down-Regulation; Epidermal Cells; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Mouth Mucosa; Neutrophils; Periodontal Diseases; Porphyromonas gingivalis; Tumor Cells, Cultured