Human T-cell responses to oral streptococci in human PBMC-NOD/SCID mice

We investigated cellular and humoral immune responses to oral biofilm bacteria, including Streptococcus mutans, Streptococcus anginosus, Streptococcus sobrinus, and Streptococcus sanguinis, in NOD/SCID mice immunized with human peripheral blood mononuclear cells (hu-PBMC-NOD/SCID mice) to explore the pathogenicity of each of those organisms in dental and oral inflammatory diseases. hu-PBMC-NOD/SCID mice were immunized by intraperitoneal injections with the whole cells of the streptococci once a week for 3 weeks. FACS analyses were used to determine the percentages of various hu-T cell types, as well as intracellular cytokine production of interleukin-4 and interferon-gamma. Serum IgG and IgM antibody levels in response to the streptococci were also determined by enzyme-linked immunosorbent assay. S. anginosus induced a significant amount of the proinflammatory cytokine interferon-gamma in CD4(+) and CD8(+) T cells in comparison with the other streptococci. However, there was no significant differences between the streptococci in interleukin-4 production by CD4(+) and CD8(+) T cells after inoculation. Further, S. mutans significantly induced human anti-S. mutans IgG, IgG(1), IgG(2), and IgM antibodies in comparison with the other organisms. In conclusion, S. anginosus up-regulated Th1 and Tc1 cells, and S. mutans led to increasing levels of their antibodies, which was associated with the induction of Th2 cells. These results may contribute to a better understanding of human lymphocyte interactions to biofilm bacteria, along with their impact on dental and mucosal inflammatory diseases, as well as endocarditis.     

Utilization and acid production of β‐galactosyllactose by oral streptococci and human dental plaque

β‐Galactosyllactose is a trisaccharide containing the β‐galactosidic linkage at the nonreducing end. The purpose of this study was to determine whether certain oral streptococci could utilize four kinds of β‐galactosyllactoses. Three of four β‐galactosyllactoses were unable to support growth of the oral streptococci and to be a substrate for producing acid from the cell suspensions and dental plaque. 4′‐β‐Galactosyllactose supported growth of Streptococcus sanguis ATCC 35105, ATCC 49298, Streptococcus mitis ATCC 15914, Streptococcus oralis ATCC 35037, ATCC 10557 and Streptococcus milleri 10707 and produced acid from dental plaque. Although β‐galactosidase activities were observed in all the strains, 4′‐β‐galactosyllactose could not be used as a carbon source for the growth of mutans streptococci. Enzymes metabolizing 4′‐β‐galactosyllactose were induced when S. oralis ATCC 10557 was cultured in medium containing galactose. These results suggested that 4′‐β‐galactosyllactose could be as cariogenic as lactose if it is consumed frequently and retained for a long period in the mouth.     

Toll‐like receptor 2‐mediated modulation of growth and functions of regulatory T cells by oral streptococci

This study was designed to determine whether oral streptococci modulate the growth and functions of regulatory T cells. Heat‐killed cells of wild‐type strains of Streptococcus gordonii and Streptococcus mutans induced the Toll‐like receptor 2 (TLR2) ‐mediated nuclear factor‐κB (NF‐κB) activation, but their lipoprotein‐deficient strains did not. Stimulation with these streptococci resulted in a significant increase in the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in splenocytes derived from both TLR2^+/+ and TLR2^?/? mice, but the level of increase in TLR2^+/+ splenocytes was stronger than that in TLR2^?/? splenocytes. Both strains of S. gordonii enhanced the proliferation of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells isolated from TLR2^+/+ mice at the same level as those from TLR2^?/? mice in an interleukin‐2‐independent manner. However, wild‐type and lipoprotein‐deficient strains of both streptococci did not enhance the suppressive activity of the isolated regulatory T cells in vitro, but rather inhibited it. TLR ligands also inhibited the suppressive activity of the regulatory T cells. Inhibition of the suppressive activity was recovered by the addition of anti‐IL‐6 antibody. Pretreatment of antigen‐presenting cells with the NF‐κB inhibitor BAY11‐7082 enhanced the suppressive activity of the regulatory T cells. These results suggested that interleukin‐6 produced by antigen‐presenting cells inhibits the suppressive activity of the regulatory T cells. Wild‐type strain, but not lipoprotein‐deficient strain, of S. gordonii reduced the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in the acute infection model, whereas both strains of S. gordonii increased it in the chronic infection model mice. Hence, this study suggests that oral streptococci are capable of modulating the growth and functions of regulatory T cells in vitro and in vivo.     

Immunization by Arg‐gingipain A DNA vaccine protects mice against an invasive Porphyromonas gingivalis infection through regulation of interferon‐γ production

We previously demonstrated that a Porphyromonas gingivalis rgpA DNA vaccine induced protective immune responses against P. gingivalis infection in mice (Yonezawa et al. Infect Immun 2001: 69: 2858–2864). In the present study, reduction in lethality against infection by lethal doses of P. gingivalis was observed in the rgpA DNA vaccine‐immunized mice. Cytokine levels in the mouse model with nonlethal doses of infection by P. gingivalis were evaluated to analyze the mechanism of protection by immunization with the rgpA DNA vaccine. After nonlethal challenge with invasive P. gingivalis W50, production of interleukin (IL)‐2, IL‐4, IL‐5 and IL‐12 was elevated; however, interferon (IFN)‐γ was lower in the serum of the DNA vaccine‐immunized mice than in the serum of nonimmunized mice. The regulation of IFN‐γ production elicited by immunization with the rgpA DNA vaccine may play a significant role in protection against P. gingivalis infection in mice.     

T‐cell lymphoma of the oral cavity: a case report

Lymphomas are defined as heterogeneous malignancies of the lymphatic system characterized by a proliferation of lymphoid cells or their precursors. Malignant lymphoma of the oral cavity has been described previously although reports on the occurrence of intraoral extranodal T‐cell lymphomas are scarce. Oral lesions may appear as a painless enlargement, erythematous, often with surface ulceration secondary to trauma. This is a report of a rare case in which a specific subtype of T‐cell lymphoma appeared in the oral cavity.     

HLA‐DR4 and salivary immunoglobulin A reactions to oral streptococci

The aim of this study was to describe and compare salivary immunoglobulin A (IgA) antibody reactions to extracts of strains of three oral streptococci in human leukocyte antigen (HLA)‐DR4‐positive and ‐DR4‐negative subjects. Whole paraffin‐stimulated saliva samples were collected from 27 apparently healthy subjects. Previous HLA typing showed that 20 subjects were DR4 positive and 7 were DR4 negative. HLA‐DRB1*04 subtyping was performed among the DR4‐positive subjects. Whole‐cell antigen extracts from Streptococcus mutans (KPSK 2), Streptococcus sobrinus (OMZ 65) and Streptococcus parasanguis (Nt 62) were separated in SDS‐PAGE. The antigens were immunoblotted with diluted saliva (Western blot), scanned and analyzed in a computer system. All immunoblot bands were recorded in DR4‐positive and DR4‐negative saliva pools, and bands with an optical density ≥0.1 were selected for analysis in individual salivas. The DR4‐negative subjects in general had more immunoblot bands and more distinct bands than did the DR4‐positive subjects. A higher concentration of total IgA in saliva was correlated with more bands, especially to antigens separated from S. mutans. When the number of bands was calculated per IgA unit, significant differences were observed between DR4‐positive and DR4‐negative salivas. This was particularly seen for S. mutans and S. parasanguis. As the number of bands was analyzed in relation to DR4 subgroups, DRB1*04, there was a lower salivary IgA activity to S. mutans in the DRB1*0401 and *0404. The variable level of correlation previously demonstrated for S. mutans colonisation and serologically defined DR4 positive subjects might be explained by the heterogeneity in this group, and the relation should be sought on a subgroup level.     

Epithelial Cells Secrete Interferon‐γ Which Suppresses Expression of Receptor Activator of Nuclear Factor Kappa‐B Ligand in Human Mandibular Osteoblast‐Like Cells

Background: Prostaglandin (PG)E₂ accumulates in inflamed periodontal tissue and induces receptor activator of nuclear factor kappa‐B ligand (RANKL)‐RANK‐osteoprotegerin (OPG) signaling associated with bone resorption. Although oral epithelial cells maintain tissue homeostasis, the role of these cells in RANKL regulation remains unknown. Methods: To mimic an inflamed condition, RANKL upregulation in human mandibular osteoblast‐like cells (HMOBs) were stimulated with PGE₂. Effect of recombinant human interferon (IFN)‐γ or epithelial‐derived IFN‐γ in constitutively released or Porphyromonas gingivalis lipopolysaccharide (PgLPS)‐stimulated epithelial supernatant was investigated in HMOBs. Some HMOBs were pretreated with an anti‐IFN‐γ antibody before PGE₂ stimulation. THP‐1 human monocytes and HMOBs were cocultured in a transwell system to investigate RANKL‐driven THP‐1 osteoclastic activity. Results: PGE₂ significantly increased RANKL messenger RNA (mRNA) and protein in HMOBs in a dose‐dependent manner, while OPG protein remained similar to baseline. Epithelial cells constitutively released IFN‐γ, which was substantially increased by PgLPS. HMOBs treated with epithelial supernatant or recombinant IFN‐γ, concurrently with PGE₂ stimulation, reduced RANKL, but not OPG, expression. In contrast, anti‐IFN‐γ antibody reversed the effect of epithelial mediators on RANKL expression. When cocultured with THP‐1, RANKL released by PGE₂‐stimulated HMOBs is adequate to drive THP‐1 differentiation as osteoclastogenic gene expression and bone resorption pit are increased. However, recombinant IFN‐γ, or IFN‐γ derived from oral epithelial cells, suppressed RANKL expression at both the mRNA and protein level, resulting in decreased THP‐1‐derived osteoclastic activity. Conclusion: Oral epithelial cells interact with HMOBs by releasing IFN‐γ to regulate RANKL expression and contribute to osteoclastogenesis.     

Salivary IgA reactions to cell‐surface antigens of oral streptococci

Background: In the immunoblot technique, using whole bacteria cell extracts as antigens, both intra‐ and extracellular antigens are detected, which gives a large number of immunoglobulin A (IgA) reactions (immunoblot bands) when incubated with saliva. It is important to distinguish which immunoblot bands represent bacterial cell‐surface antigens, since these antigens could be involved in adhesion mechanisms and be available for blocking in vivo. Methods: Bacterial extracts of Streptococcus mutans, Streptococcus sobrinus, Streptococcus parasanguis and the streptococcal antigen I/II were separated using SDS–PAGE. The antigens were detected with saliva in Western blot. Untreated saliva and saliva in which cell‐surface reactive IgA had been absorbed with whole bacteria cells were analyzed. Results: Approximately half the number of the bands were absent for saliva absorbed with homologous cells, compared to untreated saliva. The absorption pattern was almost identical for S. mutans and S. sobrinus but not for S. parasanguis. Salivary IgA reactive against streptococcal antigen I/II was absorbed by S. mutans cells, to a lesser extent by S. sobrinus cells, and not at all by S. parasanguis cells. Conclusion: It is likely that the bands that were absent after absorption represented cell‐surface antigens. For S. mutans and S. sobrinus, these bands were probably the streptococcal antigen I/II.     

Anti‐phosphorylcholine‐opsonized low‐density lipoprotein promotes rapid production of proinflammatory cytokines by dendritic cells and natural killer cells

Kikuchi T, El Shikh MM, El Sayed RM, Purkall DB, Elaasser MM, Sarraf A, Barbour SE, Schenkein HA, Tew JG. Anti‐phosphorylcholine opsonized low‐density lipoprotein promotes rapid production of proinflammatory cytokines by dendritic cells and natural killer cells. J Periodont Res 2010; 45: 720–730. © 2010 John Wiley & Sons A/S Background and Objective: Epidemiological and animal studies suggest that periodontal infections increase atherosclerosis risk. Periodontitis patients have elevated levels of anti‐phosphorylcholine (anti‐PC) reactive not only with numerous periodontal organisms but also with minimally modified low‐density lipoprotein (mmLDL). Dendritic cells (DCs) reside in arterial walls and accumulate in atherosclerotic lesions. The ability of anti‐PC to bind mmLDL prompted the hypothesis that opsonized mmLDL would stimulate DCs and enhance the production of proinflammatory cytokines that promote atherogenic plaque development. Material and Methods: Monocyte‐derived DCs (mDCs) were generated using granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4, then stimulated with mmLDL or with anti‐PC‐opsonized mmLDL. The anti‐PC effect was determined using flow cytometry, cofocal microscopy and cytokine assays. The production of CD83, IL‐12p35 mRNA, IL‐12p40 mRNA, IL‐12p70 and IL‐10 by DCs was monitored. Results: Dendritic cells stimulated with mmLDL expressed little CD83 and produced little IL‐12p70. However, anti‐PC‐opsonized mmLDL enhanced DC maturation, as indicated by upregulated CD83 and rapid (≤ 48 h) production of IL‐12p70 if a source of interferon‐γ (IFN‐γ) was available. In leukocyte cultures, natural killer (NK) cells rapidly produced IFN‐γ (≤ 48 h) when interacting with IL‐12‐producing DCs activated by anti‐PC‐opsonized mmLDL. Moreover, IFN‐γ promoted DC IL‐12 responses that were further augmented when mmLDL was opsonized with anti‐PC. Conclusion: Minimally modified LDL‐stimulated DCs and NK cells were mutually stimulatory, with DC IL‐12p70 needed by NK cells and with NK cell IFN‐γ needed by DCs. Moreover, production of these proinflammatory cytokines was markedly enhanced when LDL was opsonized by anti‐PC. In short, the data suggest that the elevated anti‐PC levels in periodontitis patients could promote a mechanism that facilitates atherosclerosis.     

Expression of CD163, interleukin‐10, and interferon‐gamma in oral squamous cell carcinoma: mutual relationships and prognostic implications

Tumor‐associated macrophages (TAMs) and their associated inflammatory cytokines represent the major inflammatory component of the stroma of many tumors and can affect prognosis in the case of neoplasms. The objective of this study was to determine the prognostic significance of CD163⁺ cells, interleukin‐10 (IL‐10), and interferon‐gamma (IFN‐γ) in oral lesions associated with oral squamous cell carcinoma (OSCC). The levels of CD163, IFN‐γ, and IL‐10 in the tissue samples of 240 patients with OSCC and 58 patients with other oral lesions were assessed by immunohistochemistry. Individuals with low IFN‐γ levels, high IL‐10 levels, and low CD163 levels were of special concern with respect to OSCC progression. We found that high levels of CD163, or a combination of low IFN‐γ levels, high IL‐10 levels, and low CD163 levels, were associated with poorer overall survival (OS). CD163⁺ cells provide better predictive power for OS in comparison with traditional markers, such as clinical stage and lymph node metastasis. Therefore, CD163⁺ cells may be effective prognostic predictors of OSCC. IL‐10 may also indicate poor outcomes when IFN‐γ secretion is low and the cells are CD163^?.     

Lethal outcome caused by Porphyromonas gingivalis A7436 in a mouse chamber model is associated with elevated titers of host serum interferon‐γ

Background/aims: Septic shock caused by gram‐negative bacteria has been associated with cytokines produced by hosts. Porphyromonas gingivalis A7436, a disseminating strain, caused septic shock‐like symptoms and even animal death in a mouse chamber model. However, P. gingivalis exhibits lower endotoxin activities in its lipopolysaccharide than other typical gram‐negative bacteria. In this study, we examined the effects of P. gingivalis lethal infection on host pro‐inflammatory cytokines production. Methods: Nude and normal BALB/c mice were infected with a lethal dose of P. gingivalis A7436 using a mouse chamber model. Serum levels of tumor necrosis factor, interleukin (IL)‐1β, IL‐12 and interferon‐γ were evaluated. The effects of tumor necrosis factor inhibitor (thalidomide) and anti‐interferon‐γ antibody on infection outcomes were examined. Results: All nude mice survived infectious challenge, whereas 100% of normal mice died with abdominal lesions. Bacterial cultures indicated P. gingivalis dissemination to the circulation. Serum levels of tumor necrosis factor, IL‐1β and IL‐12 showed no significant differences between nude and normal mice. Thalidomide treatment did not protect normal mice from death but decreased remote lesion occurrence, with concurrent reduced bacterial counts recoverable from blood. There was a 3.5‐fold elevation in normal mice serum interferon‐γ titers compared to those of nude mice and anti‐interferon‐γ antibody treatment resulted in 100% protection from lethal outcome. Conclusion: Lethal outcome following P. gingivalis A7436 infection is T‐lymphocyte dependent and involves an increase in systemic interferon‐γ levels. The data further indicate that P. gingivalis transvascular dissemination (bacteremia) alone is not sufficient for lethal outcome.     

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.