T helper 1 and 2 stimuli induce distinct phenotypes in gingival fibroblasts.

OBJECTIVE: Gingival fibroblasts (GFs) can participate in the immune response and play an immune-regulatory role. This study was to evaluate whether GFs can be differently activated and polarized into distinct functional subtypes in response to T helper type 1 (Th1), or Th2 stimuli. DESIGN: Human GFs were stimulated by the Th1 cytokine, IFN-γ, and lipopolysaccharide (LPS), separately or in combination (Th1 stimuli), or by Th2 cytokines, IL-4 and IL-13, separately or in combination (Th2 stimuli). mRNA expression of pro-inflammatory cytokines (IL-1ß, IL-6, IL-12), anti-inflammatory cytokines (IL-4, IL-10, IL-13), chemokines ((C-X-C motif chemokine (CXCL)10, CXCL11, C-C motif ligand 20), molecules involved in antigen presentation ((human leukocyte antigen (HLA)-ABC, HLA-DP, CD74, CD40)), LPS-recognizing components (Toll-like receptor 4, CD14), collagen type 1 (COL1), matrix metalloproteinase (MMP)-1, and inducible nitric oxide synthase (iNOS) and the protein levels of IL-6, CXCL11, CD14, and COL1 accumulated in supernatants were analyzed using real-time PCR and ELISA, respectively. The cell surface levels of the molecules involved in antigen presentation were detected by flow cytometry. RESULTS: Th1 stimuli strongly upregulated the expression of diverse cytokines, chemokines, molecules involved in antigen presentation, LPS-recognizing components, MMP-1, and iNOS, whereas Th1 stimuli inhibited the expression of COLI. In contrast, Th2 stimuli strongly upregulated the levels of COL1 and IL-4, while the expression levels of the other factors were minimally affected or even inhibited. CONCLUSION: These results show that GFs can be polarized into functionally distinct subtypes, immune-activating but tissue-destructive GF1 or tissue-reparative GF2, in response to Th1 and Th2 stimuli, respectively.

Relatively low invasive capacity of Porphyromonas gingivalis strains into human gingival fibroblasts in vitro.

OBJECTIVE: Bacterial invasion into host cells is a common strategy to escape the host immune system. Gingival fibroblasts (GFs) are the most predominant non-phagocytic cell type in gingival connective tissue. Therefore, invasion into GFs was thought to be the first strategy for the survival of Porphyromonas gingivalis. The present study compared the invasive ability of P. gingivalis into GFs with those of other red-complex and relatively less pathogenic bacterial strains, especially Fusobacterium nucleatum. MATERIALS AND METHODS: Invasive ability of bacterial strains into GFs was measured using a flow cytometric invasion assay at a multiplicity of infection of 1000. The effect of dual infection with F. nucleatum CCUG 37843T on P. gingivalis ATCC 49417 invasion was investigated. The invasive ability of F. nucleatum and P. gingivalis was confirmed using confocal microscopy. RESULTS: The invasive ability of red-complex bacteria was markedly lower than that of F. nucleatum or Campylobacter gracilis. The invasive ability of 4 types and 10 clinical strains of P. gingivalis was less than 6%, and that of F. nucleatum strains was greater than 45%. Confocal analysis revealed that the percentage of bacteria invading GFs in the cell-treated P. gingivalis and F. nucleatum were 0.0068% and 1.22%, respectively. Dual infection with F. nucleatum increased the invasive ability of P. gingivalis. CONCLUSION: The invasive capacities of P. gingivalis into GFs were comparatively lower than those of relatively less pathogenic bacteria. Invasion into GFs cannot be the first strategy for survival of P. gingivalis in gingival connective tissue.

The presence of bacteria within tissue provides insights into the pathogenesis of oral lichen planus.

Oral lichen planus (OLP) is a chronic T cell-mediated mucocutaneous disease of unknown etiopathogenesis. Although various antigens have been considered, what actually triggers the inflammatory response of T cells is unknown. In the present study, we propose that intracellular bacteria present within tissues trigger T cell infiltration and provide target antigens. Sections of OLP (n = 36) and normal (n = 10) oral mucosal tissues were subjected to in situ hybridization using a universal probe targeting the bacterial 16S rRNA gene and immunohistochemistry with anti-CD3, anti-CD4, anti-CD8, and anti-macrophage-specific antibodies. Bacteria were abundant throughout the epithelium and the lamina propria of OLP tissues, which exhibited positive correlations with the levels of infiltrated CD3(+), CD4(+), and CD8(+) cells. Furthermore, bacteria were detected within the infiltrated T cells. Pyrosequencing analysis of the mucosal microbiota from OLP patients (n = 13) and control subjects (n = 11) revealed a decrease in Streptococcus and increases in gingivitis/periodontitis-associated bacteria in OLP lesions. Using the selected bacterial species, we demonstrated that certain oral bacteria damage the epithelial physical barrier, are internalized into epithelial cells or T cells, and induce production of T cell chemokines CXCL10 and CCL5. Our findings provide insights into the pathogenesis of OLP.

Mucosal and salivary microbiota associated with recurrent aphthous stomatitis.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is a common oral mucosal disorder of unclear etiopathogenesis. Although recent studies of the oral microbiota by high-throughput sequencing of 16S rRNA genes have suggested that imbalances in the oral microbiota may contribute to the etiopathogenesis of RAS, no specific bacterial species associated with RAS have been identified. The present study aimed to characterize the microbiota in the oral mucosa and saliva of RAS patients in comparison with control subjects at the species level. RESULTS: The bacterial communities of the oral mucosa and saliva from RAS patients with active lesions (RAS, n = 18 for mucosa and n = 8 for saliva) and control subjects (n = 18 for mucosa and n = 7 for saliva) were analyzed by pyrosequencing of the 16S rRNA genes. There were no significant differences in the alpha diversity between the controls and the RAS, but the mucosal microbiota of the RAS patients showed increased inter-subject variability. A comparison of the relative abundance of each taxon revealed decreases in the members of healthy core microbiota but increases of rare species in the mucosal and salivary microbiota of RAS patients. Particularly, decreased Streptococcus salivarius and increased Acinetobacter johnsonii in the mucosa were associated with RAS risk. A dysbiosis index, which was developed using the relative abundance of A. johnsonii and S. salivarius and the regression coefficients, correctly predicted 83 % of the total cases for the absence or presence of RAS. Interestingly, A. johnsonii substantially inhibited the proliferation of gingival epithelial cells and showed greater cytotoxicity against the gingival epithelial cells than S. salivarius. CONCLUSION: RAS is associated with dysbiosis of the mucosal and salivary microbiota, and two species associated with RAS have been identified. This knowledge may provide a diagnostic tool and new targets for therapeutics for RAS.

Induction of proteinase 3-anti-neutrophil cytoplasmic autoantibodies by proteinase 3-homologous bacterial protease in mice.

Proteinase 3 (PR3) is the principal target of antineutrophil cytoplasmic autoantibodies (ANCA) associated with granulomatosis with polyangiitis. The aim of this study was to investigate whether bacterial PR3-homologous protease can induce autoantibodies to PR3 and ANCA-associated pathology in mice. Among the bacterial proteases that have greater than 30 % identity with PR3, a trypsin-like serine protease of Saccharomonospora viridis, a bacterium that causes hypersensitivity pneumonitis, was chosen. When the mice were immunized with the recombinant protease of S. viridis (SvPR), 75 % of NZBWF1 and 100 % of C57BL/6 mice developed high levels of autoantibodies to mouse PR3 (mPR3). The levels of antibodies to mPR3 had a strong positive correlation with those to SvPR. In addition, more than half of the mPR3-reactive sera (63 %) reacted to purified human PR3 (hPR3), and the levels of antibodies to hPR3 had a positive correlation with those to mPR3. The sera from the immunized mice strongly stained murine neutrophils in a C-ANCA pattern. Although granulomatous inflammation and signs of vasculitis were observed in several mice, they were attributable to the use of complete Freund's adjuvant in the immunization. Collectively, exposure to PR3-homologous bacterial protease could induce ANCA in mice, and this finding may provide a new insight into the triggering mechanisms for the production of PR3-ANCA.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA.

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

Association of the invasion ability of Porphyromonas gingivalis with the severity of periodontitis.

Porphyromonas gingivalis is one of the well-characterized periodontal pathogens involved in periodontitis. The invasive and proteolytic activities of P. gingivalis clinical isolates have been shown to be associated with heterogenic virulence, as determined in a mouse abscess model. The aims of the present study were to identify a P. gingivalis strain with a low virulence among clinical isolates, based on its invasive ability and cytokine proteolytic activities, and to explore the preferential degradation of a certain cytokine by P. gingivalis. P. gingivalis ATCC 33277, W50, and 10 clinical isolates were used. After incubating bacteria with IL-4, IL-6, IL-10, IL-17A, TNFα, IFNγ, and IL-1α, the amounts of remaining cytokines were determined by ELISA. Invasion ability was measured by a flow cytometric invasion assay. There was inter-strain variability both in the cytokine proteolytic activities and invasion ability. In addition, differential degradation of cytokines by P. gingivalis was observed: while IFNγ and IL-17A were almost completely degraded, inflammatory cytokines TNFα and IL-1α were less susceptible to degradation. Interestingly, the invasion index, but not cytokine proteolytic activities, of P. gingivalis had strong positive correlations with clinical parameters of subjects who harbored the isolates. Therefore, the invasive ability of P. gingivalis is an important virulence factor, and the bacterial invasion step may be a good target for new therapeutics of periodontitis.

N-acetylcysteine and the human serum components that inhibit bacterial invasion of gingival epithelial cells prevent experimental periodontitis in mice.

PURPOSE: We previously reported that human serum significantly reduces the invasion of various oral bacterial species into gingival epithelial cells in vitro. The aims of the present study were to characterize the serum component(s) responsible for the inhibition of bacterial invasion of epithelial cells and to examine their effect on periodontitis induced in mice. METHODS: Immortalized human gingival epithelial (HOK-16B) cells were infected with various 5- (and 6-) carboxy-fluorescein diacetate succinimidyl ester-labeled oral bacteria, including Fusobacterium nucleatum, Provetella intermedia, Porphyromonas gingivalis, and Treponiema denticola, in the absence or presence of three major serum components (human serum albumin [HSA], pooled human IgG [phIgG] and α1-antitrypsin). Bacterial adhesion and invasion were determined by flow cytometry. The levels of intracellular reactive oxygen species (ROS) and activation of small GTPases were examined. Experimental periodontitis was induced by oral inoculation of P. gingivalis and T. denticola in Balb/c mice. RESULTS: HSA and phIgG, but not α1-antitrypsin, efficiently inhibited the invasion of various oral bacterial species into HOK-16B cells. HSA but not phIgG decreased the adhesion of F. nucleatum onto host cells and the levels of intracellular ROS in HOK-16B cells. N-acetylcysteine (NAC), a ROS scavenger, decreased both the levels of intracellular ROS and invasion of F. nucleatum into HOK-16B cells, confirming the role of ROS in bacterial invasion. Infection with F. nucleatum activated Rac1, a regulator of actin cytoskeleton dynamics. Not only HSA and NAC but also phIgG decreased the F. nucleatum-induced activation of Rac1. Furthermore, both HSA plus phIgG and NAC significantly reduced the alveolar bone loss in the experimental periodontitis induced by P. gingivalis and T. denticola in mice. CONCLUSIONS: NAC and the serum components HSA and phIgG, which inhibit bacterial invasion of oral epithelial cells in vitro, can successfully prevent experimental periodontitis.

Mechanisms of IL-8 suppression by Treponema denticola in gingival epithelial cells.

The purpose of this study was to investigate the mechanism(s) of interleukin (IL)-8 suppression by Treponema denticola, one of the major periodontal pathogens, in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with wild-type (WT), dentilisin-deficient (K1) or flagellin-deficient (flgE) T. denticola in the presence or absence of 2% human serum for 24 h. The levels of IL-8 expression were measured with real-time reverse transcription PCR and ELISA. In the absence of human serum, the WT and flgE, but not K1, substantially reduced not only the levels of IL-8 protein but also of IL-8 mRNA. Such downregulation of IL-8 mRNA was independent of bacterial invasion. Degradation of cytokine mixture by the WT, K1 and flgE revealed dentilisin-dependent preferential degradation of tumor necrosis factor (TNF)-α, an IL-8-inducing cytokine. WT and flgE significantly decreased the levels of TNFα secreted by HOK-16B cells, suggesting modulation of IL-8 through dentilisin-mediated degradation of TNFα. The addition of human serum to the culture potentiated the suppressive effect of T. denticola, resulting in substantial reductions of IL-8 and TNFα levels, even by K1. The serum-dependent effects of T. denticola were attributed to its ability to suppress the accumulation of intracellular reactive-oxygen species (ROS), a group of ubiquitous signaling molecules. Pretreatment with an antioxidant suppressed TNFα-induced IL-8 expression, confirming the role of ROS in TNFα signaling. Collectively, T. denticola targeted a key inflammatory cytokine and its signaling molecule to modulate the host innate immune response, which provides a new insight into modulation of host immunity by a periodontal pathogen.

Gingival fibroblasts from periodontitis patients exhibit inflammatory characteristics in vitro.

OBJECTIVE: Gingival fibroblasts (GFs) are an important regulatory cell type in the progression of periodontitis. This study aimed to compare the expression levels of genes associated with inflammation, extracellular matrix degradation and bone destruction in GFs isolated from healthy and periodontitis subjects in the absence and presence of Porphyromonas gingivalis. DESIGNS: Primary GFs from healthy (n=10) and periodontitis subjects (n=10) were stimulated in vitro with viable P. gingivalis ATCC 49417 and 3 clinical isolates of P. gingivalis with type II fimbriae from one healthy subject (KUMC-H1) and two periodontitis patients (KUMC-P1, -P2). The mRNA expression of proinflammatory cytokines (interleukin (IL)-6, IL-8, IL-1B), anti-inflammatory cytokines (IL-4, IL-10), matrix metalloproteinase (MMP)-1 and 2, tissue inhibitor matrix metalloproteinase (TIMP)-3 and osteoprotegerin (OPG) were assessed using real-time PCR. The levels of IL-6, IL-1ß and TIMP-3 protein were measured by an enzyme-linked immunosorbent assay. RESULTS: The mRNA expression of IL-6, IL-1B and TIMP-3 was higher in the periodontitis group compared with the healthy group, whereas IL-4 expression was higher in the healthy group both in the absence and presence of the P. gingivalis strains. The expression levels of IL-6, IL-1ß and TIMP-3 protein were also higher in the periodontitis group in the absence and/or presence of the P. gingivalis strains. There was inter-strain variability among P. gingivalis strains in the ability to induce expression of the proinflammatory cytokines, MMPs and OPG and in the ability to degrade IL-6 protein. CONCLUSION: High expression of proinflammatory cytokines and TIMP-3 and low expression of IL-4 can be a signature of GFs associated with periodontitis.

A periodontal pathogen Treponema denticola hijacks the Fusobacterium nucleatum-driven host response.

Periodontitis is a polymicrobial disease that arises from the dysbiosis of the plaque biofilm. To study polymicrobial interactions with gingival epithelial cells, the oral commensal Fusobacterium nucleatum and the periodontal pathogen Treponema denticola were chosen due to their opposing effects on the expression of human beta-defensins (HBDs) and interleukin (IL)-8 in gingival epithelial cells. Immortalized gingival epithelial HOK-16B cells were infected with either F. nucleatum or T. denticola alone or together, and the expression of HBDs and IL-8 was investigated. Coinfection with F. nucleatum and T. denticola neutralized the stimulatory and suppressive effects on the expression of HBD-2 and -3, but the suppressive effect of T. denticola on IL-8 expression remained. In CHO/CD14/TLR2 reporter cells, T. denticola attenuated F. nucleatum-induced activation of TLR2, a receptor that mediates HBD induction. Although F. nucleatum facilitated the invasion of T. denticola into host cells, T. denticola interfered with the fusion of internalized F. nucleatum with lysosomes, which may avert TLR9-dependent IL-8 induction. Furthermore, T. denticola suppressed the F. nucleatum-stimulated accumulation of intracellular reactive oxygen species (ROS), a group of essential signaling molecules for the TLR2 and TLR9 pathways. The elimination of ROS using N-acetyl cysteine completely blocked the inductions of HBD-3 and IL-8 and significantly reduced HBD-2 induction by F. nucleatum, confirming the importance of ROS in the host response. In sum, T. denticola incapacitates the F. nucleatum-induced expression of HBDs and IL-8 in gingival epithelial cells by interrupting endo-lysosomal maturation and ROS-dependent TLR activation. These results may provide new insights into polymicrobial interactions in the gingival sulcus.