Crevicular Porphyromonas gingivalis‐specific immunoglobulin A levels in healthy and periodontitis‐affected Thai cohorts

Abstract Aim: Immunoglobulin A is a key humoral immune component involved in defense mechanisms against infections. Periodontitis, the chronic inflammatory disease causing periodontal destruction, adversely affects adults worldwide, including Thailand. As the development of periodontitis is partly mediated by immune components, levels of total and Porphyromonas gingivalis‐specific immunoglobulin A in gingival crevicular fluid of Thai cohorts were studied. Methods: Gingival crevicular fluid was collected from 24 patients with severe generalized chronic periodontitis and 22 healthy controls. The amount and concentration of total and Porphyromonas gingivalis‐specific immunoglobulin A in each gingival crevicular fluid sample were determined by enzyme‐linked immunosorbent assay. Results: The control group contained the highest concentrations of both types of gingival crevicular fluid–immunoglobulin A, but the lowest levels of these antibodies were found in the deep sites of the periodontitis group. Moreover, the concentrations of gingival crevicular fluid–immunoglobulin A and the degree of periodontitis severity appeared to have an inverse relationship. There was no significant difference in the amounts of gingival crevicular fluid–immunoglobulin A in the control and periodontitis groups. Conclusions: This study supports the hypothesis that high concentrations of specific gingival crevicular fluid–immunoglobulin A antibodies directed against Porphyromonas gingivalis, a potent periodontic microorganism, could retard periodontitis development.     

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis

Scheres N, Laine ML, Sipos PM, Bosch‐Tijhof CJ, Crielaard W, de Vries TJ, Everts V. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction withPorphyromonas gingivalis. J Periodont Res 2011; 46: 407–416. © 2011 John Wiley & Sons A/S Background and Objective: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. Material and Methods: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n = 14) and healthy control subjects (n = 8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll‐like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor‐κB1 and its putative inhibitor NF‐κB inhibitor‐like protein 1, and of interleukin‐1β, interleukin‐6, interleukin‐8, tumour necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cel expressed, and secreted, were assessed by real‐time PCR. Results: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture‐positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis‐negative persons. Conclusion: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis‐positive donors are more responsive to an in vitro P. gingivalis challenge.     

Identification of T‐cell epitopes of Porphyromonas gingivalis heat‐shock‐protein 60 in periodontitis

The heat shock proteins (hsp) of bacterial species are considered to be involved in regulating the autoimmune mechanism in human diseases due to the considerable homology of their sequences with human hsp. To elucidate how stress proteins contribute to the immunopathogenesis of periodontitis, mononuclear cells from gingival connective tissue of 10 periodontitis patients were simulated with Porphyromonas gingivalis hsp60. T‐cell lines reactive to P. gingivalis hsp60 were established from each patient to define T‐cell epitope specificities. Anti‐P. gingivalis IgG antibody titres were elevated in all patients. We could establish P. gingivalis hsp‐reactive T‐cell lines from gingival mononuclear cells that were mixtures of CD4⁺ and CD8⁺ cells. Of 108 overlapping synthetic peptides spanning the whole P. gingivalis hsp60 molecule, 10 peptides with epitope specificities for T‐cells were identified, and were identical to those reported be B‐cell epitopes in periodontitis.     

Characterization of T lymphocyte clones derived from Porphyromonas gingivalis infected subjects

Porphyromonas gingivalis plays a major role in the pathogenesis of periodontal disease, however some individuals with P. gingivalis infection do not experience periodontal breakdown. The aim of this study was to investigate the proliferative responses of two highly defined groups of subjects and to establish and characterize peripheral blood and gingival cell T cell lines and clones from subjects from these groups, The two groups were selected on the basis of P. gingivalis in their plaque and the presence of serum anti-P. gingiralis antibodies. Both groups therefore were seen to have P. gingivalis and to have responded to it. They however differed only in their clinical susceptibility (adult periodontitis) or resistance (gingivitis) to periodontal breakdown. Dose responses of peripheral blood mononuclear cells extracted from the subjects showed a trend towards a lower response by the adult periodontitis group to P. gingivalis outer membrane (OM) antigens. Peripheral blood T cell lines and clones responsive to P. gingivalis OM were established from a high responding gingivitis subject and a low responding adult periodontitis subject. Gingival T cell lines and clones were also derived from cells extracted from the periodontal tissues of the same periodontitis subject. The majority of T cells in the peripheral blood T cell line from the gingivitis subject were CD4 while those from the adult periodontitis subject were CD8. The gingival T cell line was CD3+ve CD4-ve and CD8-ve. All lines and clones proliferated slowly to P. gingivalis OM but phytohaemagglutinin (PHA) induced an increase in DNA synthesis in those derived from the gingivitis subject with little to no effect on those established from the adult periodontitis subject. Furthermore. PHA inhibited the proliferative response of the CD8 clone derived from the adult periodontitis subject. Phenotypic analysis demonstrated that all the peripheral blood clones expressed the αβ TCR while the gingival T cell clones expressed the γδ TCR. All clones had the memory/primed CD45RO+ve phenotype and at least 80% of cells in each clone were HLA-DR + ve. A lower percent of gingival cells expressed CD45RA than the CD4 peripheral blood clones and the two CD8 clones also had a decreased CD45RA expression. The gingival T cell clones also expressed a low percent CD25 as did the CD8 clone derived from the adult periodontitis subject. The results suggest that clones derived from the gingivitis and adult periodontitis subject may be functionally different. The presence of γδ T cells in adult periodontitis remains to be confirmed and their function determined.     

Memory T cell subsets in healthy gingiva and periodontitis tissues

1 Background

In the gingival sulcus, effective and balanced innate and adaptive immune responses against subgingival plaque microbiome are crucial to maintain immune homeostasis. In this study, we investigated the memory T cell subsets in healthy gingiva and periodontitis tissues.

2 Methods

Anatomical localization of T cells (CD3⁺, CD4⁺, and CD8⁺) in healthy gingiva and periodontitis tissues were examined immunohistochemically. Subsets of memory T cells from isolated gingival cells were analyzed by flow cytometry using a cocktail of monoclonal antibodies (anti‐CD69, anti‐CD103, anti‐CD45RA, anti‐CCR7, anti‐CD28, and anti‐CD95). Intracellular cytokine staining of interleukin (IL)‐17 and interferon (IFN)‐γ expression on memory T cells in periodontitis tissues was also investigated.

3 Results

We found that healthy gingiva contains two memory T cell populations; a CD69^? recirculating population and a CD69⁺ gingiva‐resident memory T cell population. CD4⁺ T cells with transitional memory (T_TM) phenotype (CD45RA^?CCR7^?CD28⁺CD95⁺) constitute the major subset within these two populations. A significant increase in the proportion of CD4⁺CD69⁺CD103^? memory T cells was observed in periodontitis tissues compared with healthy gingiva. CD4⁺ memory T cells from periodontitis tissues produced either IL‐17 or IFN‐γ whereas CD8⁺ memory T cells produced only IFN‐γ.

4 Conclusions

Our findings suggest that recirculating and gingiva‐resident memory T cells could represent an important part of the immune surveillance network in the connective tissue, maintaining periodontal homeostasis. Imbalance of subgingival bacterial communities could damage gingival barrier allowing bacterial antigens to get access to the deeper connective tissue where they activate memory T cells leading to deleterious inflammation; a hallmark of periodontitis.     

The changes in T lymphocyte subsets following periodontal treatment in patients with chronic periodontitis

Objective: The aim of this study was to determine whether there was any change in T‐lymphocyte subsets in patients with chronic periodontitis after applying different periodontal treatment methods. Patients and methods: Twenty‐four patients with chronic periodontitis were included in the study. In every phase of the treatment (pretreatment, initial treatment, curettage and flap operations) the biopsy samples were taken from the gingival tissues at sites of chronic periodontitis. Then CD4⁺ and CD8⁺ lymphocyte and CD4⁺/CD8⁺ ratio values were determined using flow cytometry in the biopsy samples. At the same time, gingival pocket depth, Löe–Silness gingival index, and Silness–Löe plaque index scores were recorded to assess the periodontal status in patients. To determine the correlation between the clinical measurements and the laboratory results obtained before the treatment, after initial treatment, after curettage and after flap operations, we conducted an analysis using a paired t‐test. Results: Flow cytometry findings in the patients with chronic periodontitis showed that CD4⁺ and CD8⁺ lymphocyte values before treatment were under the normal value and the CD4⁺/CD8⁺ ratio was within the normal distribution interval. The CD4⁺/CD8⁺ ratio decreased postcurettage and postflap operation. This decrease was statistically significant (p < 0.001). The CD4⁺ and CD8⁺ lymphocyte values were increased postcurettage and postflap operation. This increase was also statistically significant (p < 0.001). Conclusions: These findings suggest that local immune response was poor in the patients with chronic periodontitis. CD4⁺ and CD8⁺ T‐lymphocytes could play a significant role in chronic periodontitis pathobiology.     

In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme‐labeled antigen method

Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme‐labeled antigen method was applied to detecting plasma cells producing P. gingivalis‐specific antibodies in biopsied gingival tissue of periodontitis. N‐terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg‐pro, Arg‐hgp, Lys‐pro and Lys‐hgp) were prepared by the cell‐free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme‐labeled antigen method on paraformaldehyde‐fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme‐labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis.     

Correlation of cytomegalovirus and human herpesvirus 7 with CD3+ and CD3+ CD4+ cells in chronic periodontitis patients

Thomasini RL, Bonon SH, Durante P, Costa SCB. Correlation of cytomegalovirus and human herpesvirus 7 with CD3 ⁺ and CD3 ⁺ CD4 ⁺ cells in chronic periodontitis patients. J Periodont Res 2012; 47: 114–120. © 2011 John Wiley & Sons A/S Background and Objective: Human chronic periodontitis is an inflammatory process characterized by dense accumulation of immune cells in the periodontal tissue. The periodontitis can lead to loss of teeth in the patient and the pathogenesis of this disease is not completely known. This study tested the hypothesis that chronic periodontitis‐affected sites can harbor betaherpesviruses and that viruses are linked to a profile of the inflammatory infiltrate. Material and Methods: Biopsies of periodontal tissue were taken from periodontitis‐affected patients and from healthy subjects. Immunohistochemistry was performed to count CD19⁺ B cells, CD3⁺ total T cells, T‐CD4⁺ and T‐CD8⁺ cell subsets, and PCR was performed to detect cytomegalovirus and human herpesvirus 6 and 7 in the samples. One slide of each sample was stained with Giemsa for histopathological examination and to evaluate the quality of the cellular infiltrate. Results: As expected, tissues collected from healthy subjects presented no significant level of inflammatory infiltration and were therefore excluded from immunostaining procedures. Results showed that CD19⁺ B cells were in higher number than CD3⁺ T cells in the periodontitis‐affected tissue, but this was not statistically significant. The T‐CD4⁺ lymphocyte subset was significantly higher than the T‐CD8⁺ lymphocyte subset (p = 0.004) in the samples. Cytomegalovirus and human herpesvirus 7 were found at periodontitis‐affected sites, but not in tissue collected from healthy subjects (p = 0.04 and p = 0.04, respectively). Human herpesvirus 6 was rarely detected. We found a correlation between cytomegalovirus and lower CD19⁺/CD3⁺ ratios (ratio < 0.9, p = 0.003) and between human herpesvirus 7 and lower CD19⁺/CD3⁺ ratios (ratio < 0.9, p = 0.003) and higher CD4⁺/CD8⁺ ratios (ratio > 1.1, p = 0.002). Conclusion: This study shows that cytomegalovirus and human herpesvirus 7 can be present at periodontitis‐affected sites but are uncommon at healthy periodontal sites. Moreover, our data suggest that cytomegalovirus can be related to an inflammatory infiltrate with predominance of CD3⁺ T cells, whereas human herpesvirus 7 can be associated with an infiltrate with predominance of T‐CD4⁺ cells. However, further studies are necessary to support this hypothesis. Herpesviruses could play a role in human chronic periodontitis by modulation of the T cell response.     

Protease inhibitor levels in periodontal health and disease

Kretschmar S, Yin L, Roberts F, London R, Flemmig TT, Arushanov D, Kaiyala K, Chung WO. Protease inhibitor levels in periodontal health and disease. J Periodont Res 2012; 47: 228–235. © 2011 John Wiley & Sons A/S Background and Objective: Our previous study showed that protease inhibitors were attenuated by the periodontal pathogen Porphyromonas gingivalis in cultured gingival epithelial cells. We hypothesize that fewer protease inhibitors would be present in more advanced periodontal disease sites, where the level of P. gingivalis may be high. The goal of this study was to investigate the relationship between the protease inhibitor [secretory leukocyte protease inhibitor (SLPI), elastase‐specific inhibitor (ELAFIN) and squamous cell carcinoma antigen (SCCA)] levels in gingival crevicular fluid and the number of P. gingivalis micro‐organisms in subgingival plaque. Material and Methods: Plaque samples from subjects without (n = 18) and with moderate to advanced periodontitis (n = 41) were used to quantify P. gingivalis using real‐time PCR. Protease inhibitor levels in the gingival crevicular fluid of all the subjects were determined by ELISA. Results: P. gingivalis was detected in 68.3% of patients with periodontitis, while 16.7% of subjects without periodontitis had a detectable level of P. gingivalis. Patients with periodontitis and P. gingivalis in their plaque exhibited lower SLPI and ELAFIN levels (p < 0.001) compared with control subjects without periodontitis. Secretory leukocyte protease inhibitor was also reduced (p < 0.05) in gingival crevicular fluid of periodontitis patients without a detectable level of P. gingivalis. Periodontitis patients with high vs. low levels of P. gingivalis exhibited reciprocal mean levels of SLPI and ELAFIN concentrations. Conclusion: The reduced concentrations of SLPI and ELAFIN may contribute to the loss of host protective capacity and increase susceptibility to breakdown from chronic infection. The work of this investigation may aid in finding diagnostic and prognostic markers in periodontal health and disease and may also help in finding pharmacological targets directed against periodontal inflammation.     

Evaluation of interleukin-10 producing CD19+ B cells in human gingival tissue

ObjectiveThis study aimed to evaluate IL-10 producing CD19⁺ B cells and to examine the correlation between these cells and the expression levels of IL-1β, TNF-α, RANKL, and IL-10 cytokines in the gingival tissues of individuals with and without chronic periodontitis.DesignData were obtained from 20 patients with chronic periodontitis and 10 healthy controls. The gingival samples were analyzed by immunofluorescence, while real-time PCR and enzyme-linked immunosorbent assays were performed to determine cytokine levels.ResultsThe number of IL-10 producing CD19⁺ B cells and the expression levels of IL-10 were significantly higher in the inflamed gingival tissues than in the healthy tissues. A positive correlation between the expression levels of IL-10 and the number of IL-10 producing CD19⁺ B cells were observed. IL-1β, TNF-α, and RANKL expression levels were significantly elevated in diseased gingivae compared to healthy tissues, and there was a positive correlation between the expression levels of these pro-inflammatory cytokines and the number of IL-10 producing CD19⁺ B cells.ConclusionWhile IL-10 producing CD19⁺ B cells are present in the gingival tissues of patients with periodontal disease and of those with a healthy periodontium, the diseased gingival tissues had a much greater number of these cells than the healthy. The mRNA and protein levels of IL-10, IL-1β, and RANKL, as well as mRNA levels of TNF-α, were positively correlated with the number of IL-10 producing CD19⁺ B cells, which highlights the importance of these factors in the development and progression of periodontitis.     

Soluble Triggering Receptor Expressed on Myeloid Cells 1 (sTREM‐1) in Gingival Crevicular Fluid: Association With Clinical and Microbiologic Parameters

Background: Soluble triggering receptor expressed on myeloid cells 1 (sTREM‐1) belongs to the immunoglobulin superfamily and is involved in amplification of the inflammatory response to bacterial infection. This cross‐sectional study aims to investigate the levels of sTREM‐1 in gingival crevicular fluid (GCF) of individuals without periodontitis and with chronic periodontitis (CP) or generalized aggressive periodontitis (GAgP) and their association with the levels of key periodontal pathogens in subgingival plaque. Methods: GCF and subgingival plaque samples were obtained from healthy sites of participants without periodontitis (n = 20) and periodontitis sites of patients with CP (n = 22) and GAgP (n = 20). sTREM‐1 levels in GCF were measured by enzyme‐linked immunosorbent assay. Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans levels in subgingival plaque were analyzed by quantitative real‐time polymerase chain reaction. Results: sTREM‐1 levels in GCF were higher in CP and GAgP than healthy sites by 3.6‐ and 4.4‐fold, respectively, with no significant differences between the two forms of periodontitis. Moreover, sTREM‐1 levels in GCF were positively correlated with site‐specific clinical periodontal parameters and levels of P. gingivalis, T. denticola, and T. forsythia, but not A. actinomycetemcomitans, in subgingival plaque. Conclusion: Increased GCF levels of sTREM‐1 at diseased sites and their positive correlation with clinical and microbiologic parameters strengthen the association of this inflammatory marker with periodontitis.     

Trefoil factors in saliva and gingival tissues of patients with chronic periodontitis

Background: Trefoil factors (TFFs) are secreted molecules that are involved in cytoprotection against tissue damage and the immune response. TFFs have been detected in saliva and oral tissues, but their clinical significance has never been investigated in patients with chronic periodontitis. The objective of this study is to determine whether TFF expression in saliva and gingival tissues is associated with periodontal pathology. Methods: Saliva and gingival tissue samples were collected from 25 non‐periodontitis individuals and 25 patients with chronic periodontitis (CP). Enzyme‐linked immunosorbent assay and immunohistochemical methods were used to evaluate the expression of TFFs (TFF1, TFF2, and TFF3) in saliva and gingival tissues, respectively. Periodontopathic bacteria were quantified by real‐time polymerase chain reaction. Results: Reduced salivary TFF1 and TFF3 concentrations were observed in patients with CP (P = 0.003 and P <0.001, respectively). Decreased TFF3 expression in gingival tissues of patients with CP was demonstrated (P = 0.041). Levels of salivary TFF3 concentrations were negatively correlated with periodontal pathology and number of Porphyromonas gingivalis and Tannerella forsythia (formerly known as Bacteroides forsythus). Conclusions: Altered expression of TFFs in saliva and gingival tissues was detected in patients with CP. The results suggest that TFF3 may be involved in the pathogenesis of periodontal disease.     

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.     

Serum cytokine levels in periodontitis patients in relation to the bacterial load

Background: Periodontitis is a local inflammatory disease that also has some systemic effects. We investigated the levels of interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α, and interleukin (IL)‐2, ‐4, ‐5, and ‐10 in the serum of patients with periodontitis in relation to the bacterial load in the dental plaques. Methods: Serum cytokine levels in patients with generalized periodontitis and healthy control groups were determined using the cytometric bead array kit. Bacterial load in the dental plaque was determined semiquantitatively by real‐time polymerase chain reaction. The proportions of different lymphocyte subsets were determined in the peripheral blood of patients with periodontitis by flow cytometry. Finally, relationships between the bacterial load in the subgingival plaques of patients with periodontitis and levels of cytokines and counts of lymphocyte subsets were established. Results: Serum levels of IFN‐γ, TNF‐α, and IL‐10 were significantly increased, whereas those of IL‐2 were significantly decreased in patients with periodontitis compared to healthy controls. Increased serum levels of IFN‐γ and TNF‐α in patients with periodontitis were associated with the enhanced dental plaque load with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis, respectively. Finally, as revealed by analysis of lymphocyte populations, the presence of A. actinomycetemcomitans and Trepomena denticola was associated with an increased population of CD3^?/CD16⁺ and CD3⁺/CD8⁺ cells, respectively. Conclusion: Certain periodontal pathogens could be associated with an increased level of proinflammatory cytokines in the peripheral blood and thus increased risk of systemic diseases.     

Identification of immunoreactive epitopes of the Porphyromonas gingivalis heat shock protein in periodontitis and atherosclerosis

Choi J, Lee S‐Y, Kim K, Choi B‐K. Identification of immunoreactive epitope of Porphyromonas gingivalis heat shock protein peptide in periodontitis and atherosclerosis. J Periodont Res 2011; 46: 240–245. © 2011 John Wiley & Sons A/S Background and Objective: Heat shock protein 60 (HSP60) of Porphyromonas gingivalis, a major periodontal pathogen, might be a trigger molecule linking infectious periodontitis and autoimmune atherosclerosis. The aim of this study was to identify the peptide specificity of anti‐P. gingivalis HSP60 monoclonal antibodies and their cross‐reactivity with bacterial and human HSPs. Their specific immunoreactivity to periodontal or atherosclerotic lesions was also investigated. Methods: Twenty patients with chronic periodontitis and 20 atherosclerosis patients who had undergone surgical intervention for atheromatous plaques with evidence of ongoing periodontal disease, were selected. Synthetic peptide 19 ((TLVVNRLRGSLKICAVKAPG)‐specific T‐cell lines were established from inflamed gingiva and atheromatous plaque and the phenotypes and cytokine profiles were characterized. Results: Thirty per cent of periodontitis patients and 100% of atherosclerosis patients reacted positively to cross‐reactive peptide 19 from both P. gingivalis and human HSP60. The peptide 19‐specific T‐cell lines demonstrated the phenotype characteristic of helper T cells (CD4⁺) but did not express CD25 or FOXP3. The interleukin‐10 levels were elevated significantly in the peptide 19 T‐cell line. Conclusion: Synthetic peptide 19 of P. gingivalis HSP60 is an immunoreactive epitope in the periodontitis–atherosclerosis axis.     

Characterization of progressive periodontal lesions in chronic periodontitis patients: levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells

Background and aims: Periodontitis is an infection with an episodic nature of tissue support destruction. The aim of this work was to determine the levels of chemokines, cytokines, matrix metalloproteinase‐13, periodontal pathogens and inflammatory cells in periodontal sites characterized by active periodontal connective tissue destruction. Material and Method: Fifty‐six patients with moderate or advanced severity of chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth 5 mm, clinical attachment level 3 mm and radiographic bone loss. Periodontitis progression was determined by the tolerance method. Receptor activator for nuclear factor κ B‐ligand (RANK‐L), monocyte chemoattractant protein‐1 (MCP‐1), tumour necrosis factor‐α (TNF‐α), IL‐1β, MMP‐13, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsithia and inflammatory cells levels were determined. Statistical analysis was performed using the Stata^® 7.0 software. Data were expressed as mean±SD and paired samples t‐test and χ² tests were used. Results: Higher RANK‐L, IL‐1β and MMP‐13 activity levels were observed in active sites (p<0.05). The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and the number of CD4⁺ T were higher in active than in inactive sites (p>0.05). Conclusion: The detection of periodontopathic bacteria, host matrix metalloproteinases and cytokines in periodontitis patients with lesions undergoing episodic attachment loss could partially explain the mechanisms associated with the destruction of the supporting tissues of the tooth.     

Comparison of gingival and peripheral blood T cells among patients with periodontitis suggests skewing of the gingival T cell antigen receptor V beta repertoire

The present study investigated the expression of different variable regions of T cell receptor β-chain (Vβ) among functional subsets of T cells, i.e. CD45RO⁺ (activated/memory), CD4⁺ and CD8⁺in gingiva and peripheral blood of patients with periodontitis. Gingival tissue specimens (n= 25) and peripheral blood were procured from 18 patients with periodontitis during periodontal surgery or extraction. Single-cell suspensions of gingival tissues were made by enzymatic digestion. These cells were immunofluorescently labeled with a panel of monoclonal antibodies specific for 18 TCR Vβ regions, in concert with markers for various T cell subsets. The cells were then analyzed with 3-color multivariate flow cytometry. Results demonstrated that a significantly higher proportion of T cells in gingiva expressed Vβ5.2 (0.0005), Vβ6 (0.0007) and Vβ9 (0.003) regions compared to those in peripheral blood. Comparison of CD45RO⁺ (activated/memory) and CD45RO^? (naïve) subsets of gingival T cells revealed differences in the expression of TCR Vβ regions. Vβ5.2 expression was significantly higher among CD45RO⁺ gingival T cells (p= 0.004), whereas Vβ14 expression was elevated among the CD45RO^? subset relative to peripheral blood (p= 0.008). Analysis of TCR Vβ region expression among CD4⁺ and CD8⁺ subsets did not reveal any statistically significant differences between gingiva and peripheral blood, although some Vβ regions approached significance. Collectively, these results demonstrate that the T cell repertoire in the gingival compartment differs significantly from that in the peripheral blood. Furthermore, since the skewing of TCR Vβ was observed among naive, as well as activated/memory T cells, it is likely that both developmental and environmental factors are influential in shaping the gingival TCR repertoire in patients with periodontitis. Elucidation of the cause of the skewed expression of T cell receptors in gingiva can provide insights into the specificity of T cells in periodontitis.     

Periodontitis in the absence of B cells and specific anti‐bacterial antibody

Periodontitis (PD) results from complex interactions between a dysbiotic oral microbiota and a dysregulated host immune response. The inflammatory infiltrate in the gingiva of PD patients includes an abundance of B cells, implicating these cells in the immunopathology. We sought to investigate the role of B cells in PD using a murine model. Wild‐type or B‐cell‐deficient (μMT) mice were orally infected with Porphyromonas gingivalis. One or six weeks following infection, lymphocyte populations in the gingiva and cervical draining lymph nodes (dLN) were analysed by flow cytometry; serum anti‐P. gingivalis IgG antibody titers were measured by enzyme‐linked immunosorbent assay, and alveolar bone loss was determined. In wild‐type mice, the percentage of gingival B cells expressing receptor activator of nuclear factor‐κB ligand (RANKL) was significantly increased 1 week post‐infection (5.36% control versus 11% PD, P < 0.01). The percentage of Fas⁺ GL7⁺ germinal centre B cells in the dLN was significantly increased at both 1 week (2.03% control versus 6.90% PD, P < 0.01) and 6 weeks (4.45% control versus 8.77% PD, P < 0.05) post‐infection. B‐cell‐deficient mice were protected from P. gingivalis‐induced alveolar bone loss, with a lack of B‐cell proliferation and lack of CD4⁺ CD44⁺ CD62L^? T‐cell generation in the dLN, and absence of serum anti‐P. gingivalis antibodies. Our data imply a pathological role for B cells in PD, and that selective targeting of this immune axis may have a role in treating severe periodontal disease.