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.     

Effects of Areca Nut Extract on Lipopolysaccharides‐Enhanced Adhesion and Migration of Human Mononuclear Leukocytes

Background: Areca chewers have a higher prevalence of periodontitis than non‐chewers. Cell adhesion and movement (migration) are important for leukocyte recruitment to inflammation sites. This study investigates the effects of areca nut extract (ANE) on the adhesion and migration abilities of the human immune cells, peripheral blood mononuclear cells (PBMCs). The combined effects of nicotine and lipopolysaccharides (LPS) were also analyzed. Methods: Purified PBMCs obtained from healthy adults were treated with ANE, nicotine, and/or LPS. Cell adhesion ability was examined using fibronectin‐coated microslides, Liu stain, and light microscopy. Cell migration ability was evaluated using the transwell system followed by staining and fluorescence microscopy. Statistical difference was analyzed using the Mann‐Whitney U test. Results: When compared with the media‐treated control samples, PBMCs treated with ANE for 4 hours showed a significant reduction of the adherent cells on the microslides. Interestingly, LPS treatment increased cell adhesion, which could be reduced by simultaneous ANE plus nicotine treatment. The chemotactic migration of PBMCs was reduced by ANE treatment for 1, 4, or 24 hours in a dose‐dependent manner. LPS treatment increased PBMC migration, which could be reduced by simultaneous treatment with ANE or with ANE plus nicotine. Conclusions: ANE reduced the adhesion and migration abilities of PBMC. ANEs, with or without nicotine, also attenuated the migration of LPS‐stimulated PBMCs. The results implicated that the immune cell functions were impaired in areca chewers, which might increase the host susceptibility to oral and periodontal infection.     

Effects of areca nut extract on the apoptosis pathways in human neutrophils

Ho W‐H, Lee Y‐Y, Chang L‐Y, Chen Y‐T, Liu T‐Y, Hung S‐L. Effects of areca nut extract on the apoptosis pathways in human neutrophils. J Periodont Res 2010; 45: 412–420. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: Areca nut, a major component in area quid, possesses genotoxic and carcinogenic activities. Areca nut extract (ANE) may affect the defensive functions of neutrophils. Recent studies suggest that areca nut chewing is associated with a higher prevalence of periodontal disease as a result of the detrimental effects of ANE on the host defense system. This study examined the effects of ANE on the apoptosis pathways in human neutrophils. Material and Methods: Apoptosis/necrosis of neutrophils was determined using flow cytometry. Proteins involved in the apoptosis pathway were determined using western blotting analysis. Results: The results indicated that ANE reduced early apoptosis, but increased the primary necrosis of neutrophils. ANE may arrest neutrophils in the G0/G1 phase and reduce the apoptotic hypodiploid DNA contents. The levels of cleaved forms of poly(ADP‐ribose) polymerase, and of caspase‐3 and caspase‐8 were decreased by treatment with ANE. Moreover, glycogen synthase kinase‐3α/β may be involved in the ANE‐modulated effects of neutrophils. Conclusion: Areca nut may regulate death pathways in neutrophils. This may be one mechanism by which areca nut compromises the periodontal health of areca nut chewers.     

Salivary arecoline levels during areca nut chewing in human volunteers

J Oral Pathol Med (2010) 39 : 465–469 Background: Arecoline stimulates cultured cells above 0.1 μg/ml and is cytotoxic above 10 μg/ml. Although this alkaloid seems important for areca nut induced oral carcinogenesis, little is known of the levels achieved during chewing. Materials and methods: Saliva was collected in 3‐ to 5‐min intervals over 50 min in 32 habitual chewers: before, for 25 min during, and for 20 min after chewing areca nut (0.5 g) without any other additives. Salivary arecoline was quantitated by HPLC‐MS. Controls comprised six subjects who denied areca nut use, and who were given rubber‐base material to chew during experiments instead. Results: Arecoline was detected before chewing in 22 subjects, exceeding the 0.1 μg/ml threshold in 20 cases. Salivary arecoline exceeded either the 0.1 or 10 μg/ml thresholds in all participants during chewing (P < 0.001). Maximum concentrations ranged from 5.66 to 97.39 μg/ml. All subjects reached 0.1 μg/ml salivary arecoline in at least 85% of time points studied (P < 0.0001), whereas 10 μg/ml was reached in 11 participants in at least 30% of the time points (P < 0.003). Arecoline concentrations varied greatly over time between individuals, and levels were much lower when peak concentrations were reached before 3 min, than in cases where arecoline peaked later (P < 0.02). No salivary arecoline was found in control saliva. Conclusions: Areca nut users have persistent background salivary arecoline levels long after chewing, whereas concentrations achieved are highly variable and consistent with a role in oral pre‐malignancy and malignancy.     

Areca nut extracts suppress the differentiation and functionality of human monocyte-derived dendritic cells

Wang C‐C, Chen T‐Y, Wu H‐Y, Liu T‐Y, Jan T‐R. Areca nut extracts suppress the differentiation and functionality of human monocyte‐derived dendritic cells. J Periodont Res 2012; 47: 198–203. © 2011 John Wiley & Sons A/S Background and Objective: Areca quid chewing, a major risk factor contributing to the occurrence of oral cancer and precancer, has been reported to be associated with the severity and high prevalence of periodontal diseases in areca quid chewers. As dendritic cells are critically involved in the regulation of innate and adaptive immunity in oral mucosa, the objective of the present study was to investigate the effect of areca nut extracts (ANE) on the differentiation and reactivity of dendritic cells derived from monocytes. Material and Methods: Human peripheral blood monocytes were cultured in the presence of granulocyte–monocyte colony‐stimulating factor and interleukin‐4 for 7 d to generate dendritic cells. To examine the effect of ANE on the generation of dendritic cells, the monocytes were exposed to ANE throughout the 7 d culture period. In addition, the effect of ANE on the maturation of monocyte‐derived dendritic cells induced by lipopolysaccharide (LPS) was examined. Results: Monocytes cultured in granulocyte–monocyte colony‐stimulating factor and interleukin‐4 exhibited a typical phenotype of dendritic cells, as evidenced by the heightened expression of human leukocyte antigen (HLA)‐DR, CD11c and the co‐stimulatory molecules CD40, CD80 and CD86. Exposure of the monocytes to ANE did not influence the expression of HLA‐DR and CD11c, but markedly attenuated the proportion of CD40‐positive cells and the mean fluorescence intensity of CD86. The expression of co‐stimulatory molecules in LPS‐activated dendritic cells was not affected, whereas the mRNA expression of interleukin‐12 induced by LPS was markedly suppressed by ANE treatment in a concentration‐dependent manner. Conclusion: These results suggest that ANE exposure interfered with the differentiation of dendritic cells from monocytes. Moreover, the functionality of mature monocyte‐derived dendritic cells was attenuated in the presence of ANE.     

Effects of areca nut extracts on the functions of human neutrophils in vitro

Aqueous extracts of ripe areca nut without husk (ripe ANE) and fresh and tender areca nut with husk (tender ANE) were examined for their effects on the defensive functions of human neutrophils. Exposure of peripheral blood neutrophils to ripe ANE and tender ANE inhibited their bactericidal activity against oral pathogens, including Actinobacillus actinomycetemcomitans and Streptococcus mutans, in a dose-dependent manner. At the concentrations tested, ripe and tender ANEs did not significantly affect the viability of neutrophils as verified by their ability to exclude trypan blue dye. However, both ANEs inhibited the production of bactericidal superoxide anion by neutrophils as measured by cytochrome c reduction. Moreover, the ripe ANE inhibited neutrophils more effectively than did tender ANE. Arecoline, a major alkaloid of areca nut, only exhibited an inhibitory effect on the functions of neutrophils when high concentrations were used. Therefore, arecoline could not be used to explain the inhibitory effects observed for ANEs. In conclusion, our results demonstrated that ripe and tender ANEs reduced the antibacterial activity and the superoxide anion production of neutrophils. This effect may contribute to a less efficient elimination of bacteria from the periodontal environment. Inhibition of the antimicrobial functions of neutrophils may alter the microbial ecology of the oral cavity, and this may be one possible mechanism by which areca nut compromises the oral health of users of areca nut products.     

Production of interleukin‐8 in vitro by mononuclear cells isolated from human periapical lesions

Introduction: Interleukin‐8 (IL‐8) is an important mediator of inflammation. However, little is known about its production in chronic dental periapical lesions and this was the main aim of this work. Methods: Inflammatory cells were isolated from clinically different periapical lesions and analyzed by morphological criteria. The mononuclear cells were isolated, phenotypically analyzed by immunocytochemistry and cultivated in vitro. IL‐8 was measured in culture supernatants of these periapical lesion mononuclear cells (PL‐MNC) using a microbeads fluorescence assay. Results: We found a relatively high production of IL‐8 in 19 out of 21 periapical lesions included in the study. The level of IL‐8 and the proportion of neutrophil granulocytes were significantly higher in the group of symptomatic lesions, compared to the asymptomatic lesions, but there was no statistically significant correlation between these parameters. According to the predominance of CD3⁺ T cells and Ig⁺/CD19⁺ B cells and plasma cells, lesions were divided into T‐type and B‐type lesions, respectively. The levels of IL‐8 were significantly higher in the culture supernatants of PL‐MNC in the T‐type lesions and were positively correlated with the proportion of macrophages/dendritic cells (CD11c⁺ cells) and CD4⁺ T cells. Such a correlation was not shown in B‐type lesions. Conclusion: These results suggest that PL‐MNC are a significant source of IL‐8, which is probably an important chemokine for the migration and function of different cell types at the site of chronic inflammation.     

Areca nut extract upregulates vimentin by activating PI3K/AKT signaling in oral carcinoma

J Oral Pathol Med (2011) 40 : 160–166 Background: Areca nut is a group I carcinogen. Areca nut extract (ANE) is known to activate signaling pathways in oral epithelial cells. Activation of the serine/threonine protein kinase AKT/pKB (AKT) signaling pathway is known to be important during the neoplastic process. Vimentin is a mesenchymal intermediate filament and a regulator of tumor progression. This study investigated the impact of ANE on PI3K/AKT activation during vimentin expression. Materials and methods: Oral carcinoma cells were treated with ANE to explore the signaling changes underlying vimentin expression. Oral carcinoma tissues were subjected to immunohistochemical analysis to study the implications that vimentin expression has on patient survival. Results: After ANE treatment, the OECM‐1 and Fadu cells developed a fibroblastoid morphology and there was an increase in vimentin expression. The treatment also induced the phosphorylation of AKT and glycogen synthase kinase 3β in OECM‐1 cells. Blockage of phosphatidylinositol 3‐kinase (PI3K)/AKT signaling attenuated vimentin expression when it was induced by ANE. However, it did not affect ANE‐mediated extracellular signal‐regulated kinase (ERK) activation or cyclooxygenase 2 (COX‐2) upregulation. Oral carcinoma tissue samples were found to have significantly higher levels of vimentin and pAKT expression than their controls. Tumors exhibiting no vimentin expression and weak AKT phosphorylation were found to be associated with better survival than groups with high levels of expression. Conclusion: Our results imply that PI3K/AKT activation and vimentin expression are important pathogenic cascades in areca‐associated oral carcinogenesis.     

Production of IL‐10 and IL‐12 by antigen‐presenting cells in periapical lesions

J Oral Pathol Med (2010) 39 : 690–696 Background: Interferon‐γ (IFN‐γ) plays an important role in the pathogenesis of periapical lesions. Its expression is up‐regulated by interleukin (IL)‐12) and down‐regulated by IL‐10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. Methods: Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen‐presenting cells was tested using a mixed leukocyte reaction. Results: We observed the positive correlations between the levels of IL‐12 and IFN‐γ as well as IL‐12 and IL‐10 in cultures of mononuclear cells. As IL‐10 and IL‐12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14⁺ adherent cells, produced high levels of IL‐10 and very low levels of IL‐12. In contrast, non‐adherent, strongly HLA‐DR⁺ dendritic cells, potent stimulators of the alloreactive T‐cell response, produced low levels of IL‐10 and moderate levels of IL‐12. Dendritic cells stimulated the production of IFN‐γ by allogeneic CD4⁺ T cells. In contrast, the level of IFN‐γ was significantly decreased and the production of IL‐10 was enhanced by addition of macrophages to the culture system. Conclusion: Our results suggest that a fine balance between the production of IL‐10 and IL‐12 by different antigen‐presenting cells, through IFN‐γ, may control the course of chronic inflammation in periapical lesions.     

Heat shock protein 47 expression in oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells

J Oral Pathol Med (2010) 40 : 390–396 Background: Heat shock protein 47 (HSP47) is a product of CBP2 gene located at chromosome 11q13.5, a region frequently amplified in human cancers. Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). The aim of this study was to compare HSP47 expression in normal human oral epithelium and OSCC and further to explore the potential mechanisms that may lead to induce HSP47 expression. Methods: Thirty‐two OSCC specimens and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line OC2 cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, cyclooxygenase‐2 inhibitor NS‐398, and tyrosine kinase inhibitor herbimycin A were added to find the possible regulatory mechanisms. Results: HSP47 expression was significantly higher in OSCC specimens than normal epithelium (P < 0.05). No significant difference in HSP47 expression was observed with respect to age, sex, T category, stage, and differentiation (P > 0.05). The lower HSP47 expression was associated with lymph node metastasis (P = 0.015). Arecoline was found to elevate HSP47 expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, PD98059, LY294002, NS398, and herbimycin A markedly inhibited the arecoline‐induced HSP47 expression (P < 0.05). Conclusion: Our findings demonstrated that HSP47 expression is significantly upregulated in areca quid chewing‐associated OSCCs. HSP47 could be used clinically as a marker for lymph node metastasis of oral carcinogenesis. In addition, arecoline‐induced HSP47 expression was downregulated by NAC, PD98059, LY294002, NS398, and herbimycin A.     

Heat shock protein 27 expression in areca quid chewing-associated oral squamous cell carcinomas

Oral Diseases (2012) 18 , 713–719 Objectives: Heat shock protein (HSP) 27 is a low‐molecular‐weight protein that functions as a molecular chaperone and plays a cytoprotective role through its antioxidant activity during cell stress. Areca quid chewing is associated with the high incidence of oral squamous cell carcinomas (OSCCs) in Taiwan. The aim of this study was to compare heat shock protein 27 (HSP27) expression in OSCCs and the normal oral tissues. Methods: Forty‐eight OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry for HSP27. The normal human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot for HSP27. Furthermore, epigallocatechin‐3 gallate (EGCG), glutathione precursor N‐acetyl‐l ‐cysteine (NAC), cyclooxygenase‐2 inhibitor NS‐398, HSP inhibitor quercetin, extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Heat shock protein 27 exhibited higher expression in OSCCs than normal specimens (P < 0.05). Arecoline was found to elevate HSP27 expression in a dose‐ and time‐dependent manner (P < 0.05). The additions of pharmacological agents were found to inhibit arecoline‐induced HSP27 expression (P < 0.05). Conclusions: Heat shock protein 27 expression is significantly elevated in areca quid chewing‐associated OSCCs. Arecoline‐induced HSP27 expression was downregulated by EGCG, NS398, NAC, quercetin, PD98059, and SB203580.     

Inhibitory Effects of Areca Nut Extract on Expression of Complement Receptors and Fc Receptors in Human Neutrophils

Background: Chewing of areca quid increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the phagocytic activity of human neutrophils. This in vitro study investigates the effects of ANE on complement‐ and antibody‐opsonized phagocytosis by neutrophils. Expression of complement receptors, Fc receptors, and F‐actin in ANE‐treated neutrophils is also analyzed. Methods: The viability of ANE‐treated neutrophils was determined using the propidium iodide staining method. The possible effects of ANE on the expression of complement receptors and Fc receptors were examined using an immunofluorescence staining method followed by flow cytometry and confocal laser scanning microscopy. The phagocytic activity of neutrophils against complement or immunoglobulin (Ig)G‐opsonized fluorescent beads was analyzed using flow cytometry. Expression of F‐actin was determined using confocal laser scanning microscopy. Results: ANE significantly inhibited the production of complement receptors (CR1, CR3, and CR4) and Fc receptors (FcγRII and FcγRIII) in a concentration‐dependent manner. Treatment of neutrophils with ANE significantly impaired their ability to phagocytose fluorescent beads. ANE also inhibited phagocytosis of fluorescent beads that were opsonized by complement or IgG. Moreover, expression of F‐actin was inhibited after ANE treatment. Conclusions: ANE inhibits the complement‐ and IgG‐mediated neutrophil phagocytosis that may result from reduction of the expression of complement receptors, Fc receptors, and F‐actin formation after ANE treatment. The findings suggest that areca nut chewing may jeopardize the defensive functions of neutrophils and affect periodontal health.     

Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils

BACKGROUND: Areca quid chewers have a higher prevalence of periodontal disease than non-chewers. Little is known about the influence of areca quid on the immune system. This study was to determine the possible effects of the areca nut on phagocytic activity of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE), fresh and tender areca nut with husk (tANE), a major alkaloid (arecoline), and a phenolic component ([+]-catechin) of areca nut were examined for their effects on cellular viability using trypan blue exclusion assay. The possible effects on the phagocytic activity of neutrophils against a periodontal pathogen, Actinobacillus actinomycetemcomitans ATCC 33384, were determined using flow cytometry and confocal laser scanning microscopy. RESULTS: At the concentrations tested, rANE, tANE, arecoline, and (+)-catechin did not significantly affect viability of neutrophils. However, rANE, tANE, arecoline, and (+)-catechin inhibited the phagocytic activity of neutrophils in a dose-dependent manner. Approximately 50% of the relative phagocytic activity of neutrophils was affected when 50 microg/ml of rANE, 400 microg/ml of tANE, 20,000 microg/ml of arecoline, or 2,500 microg/ml of (+)- catechin was used. Decreased levels of internalized fluorescent bacteria were also demonstrated. However, arecoline or (+)-catechin alone could not be used to explain the inhibitory effects observed for rANE and tANE. CONCLUSIONS: Components of areca nut reduced the uptake of A. actinomycetemcomitans ATCC 33384 by human neutrophils. The inhibition of areca nut on phagocytosis of neutrophils may be one possible mechanism by which the areca nut compromises the periodontal health of areca quid chewers.     

Interleukin‐2, interleukin‐6 and T regulatory cells in peripheral blood of patients with Behçet’s disease and recurrent aphthous ulcerations

J Oral Pathol Med (2012) 41 : 73–79 Background: One of the factors involved in the pathogenesis of Behçet disease (BD) and recurrent aphthous ulcerations (RAU) is a cell‐mediated immune response in which several cytokines (interleukin‐2, interleukin‐6) and T regulatory cell (T reg cell) population seem to play a major role. The aim of this study was to measured the interleukin‐2 (IL‐2), interleukin‐6 (IL‐6) levels and analysis of CD4⁺ CD25⁺ Foxp‐3⁺ Treg cells in peripheral blood from patients with BD and RAU. In addition; we also analysed peripheral blood from healthy subjects for comparison. Methods: Thirty patients (15 men and 15 women) with BD, 30 patients (12 men and 18 women) with RAU and 15 healthy control subjects (nine men and six women) participated in the study. Analysis of CD4⁺ CD25⁺ Foxp‐3⁺ Treg cells, IL‐2 and IL‐6 levels have been measured in flow cytometry. Results: No statistical differences were observed in the serum levels of IL‐2 and IL‐6 between BD and RAU patients, and healthy subjects. Although there were no statistical differences in the number of CD4⁺ CD25⁺ Foxp‐3⁺ cells between groups, there were statistically significant differences in the number of CD4⁺ CD25^bright Treg cells. CD4⁺ CD25^bright Treg cells were significantly increased in BD and RAU patients compared to healthy subjects. Statistical analysis revealed no difference according to the number of CD4⁺ CD25^bright cells between BD and RAU patients. Conclusions: These results indicate that CD4⁺ CD25^bright T regulatory cells may be contributing factor in the pathogenesis of BD and RAU.     

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.     

Identification of gingipain‐specific I‐Ab‐restricted CD4+ T cells following mucosal colonization with Porphyromonas gingivalis in C57BL/6 mice

Chronic periodontitis is associated with Porphyromonas gingivalis infection. Although virulence factors of P. gingivalis are hypothesized to contribute to the pathogenesis of periodontitis, it is unclear whether the local CD4⁺ T‐cell‐mediated response they elicit prevents or contributes to periodontal bone destruction. We hypothesize that major histocompatibility complex class II I‐A^b‐binding peptides existing in Kgp and RgpA are presented to CD4⁺ T cells during P. gingivalis oral colonization. The protein sequences of gingipains RgpA and Kgp, and OMP40 and OMP41 of P. gingivalis were scanned using an I‐A^b‐binding matrix. From this analysis we identified 53 candidate peptides that had the potential to engage the peptide‐binding groove of the I‐A^b molecule of C57BL/6 mice. An ELISpot‐based screen revealed those peptide‐primed effector/memory CD4⁺ T cells that could be re‐stimulated in vitro with P. gingivalis or the peptide itself to produce interleukin‐17A or interferon‐γ. Two immunodominant peptides, Kgp_467–477 (pKgp) and RgpA_1054–1064/Kgp_1074–1084 (pR/Kgp) were identified and engineered to be displayed on I‐A^b molecular tetramers. Peptide pR/Kgp is conserved across all sequenced P. gingivalis strains. C57BL/6 mice were orally inoculated with P. gingivalis strain 53977 and cervical lymph node cells were stained with phycoerythrin‐conjugated pKgp::I‐A^b and pR/Kgp::I‐A^b tetramers. We found that only pR/Kgp::I‐A^b bound with the desired specificity to gingipain‐specific CD4⁺ T cells. The pR/Kgp::I‐A^b tetramer complex will allow the identification of effector/memory CD4⁺ T cells specific for two virulence factors of P. gingivalis strains associated with periodontal disease.