Imbalance of Interleukin-17+ T-cell and Foxp3+ Regulatory T-cell Dynamics in Rat Periapical Lesions

Introduction

Interleukin (IL)-17⁺ T-helper (Th17) cells and Foxp3⁺ regulatory T (Treg) cells are CD4⁺ T-helper cells with reciprocal functions in immunology and bone metabolism. The present study aimed to investigate the expression dynamics of Th17 and Treg cells in rat periapical lesions as well as their correlation with bone resorption.

Methods

Experimental pulp exposures were made in the lower first molars of 28 Wistar rats to induce periapical lesions. Rats were killed on days 0, 7, 21, and 35. Mandibles were prepared for micro–computed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis.

Results

Through 3-dimensional and 2-dimensional measurements, the volume and area of periapical lesions visibly increased from day 7 to day 21 and then expanded slowly between days 21 and 35. IL-17–positive cells markedly increased from day 7 to day 35. However, Foxp3-positive cells remained at low levels until day 21 and then dramatically increased by day 35. The IL-17⁺/Foxp3⁺ ratio and number of osteoclasts simultaneously increased from day 7 to day 21 and then decreased on day 35. Finally, the distinct distribution of CD4⁺/IL-17⁺ Th17 and CD4⁺/Foxp3⁺ Treg cells was observed on days 7 and 35.

Conclusions

Our findings imply the imbalance of IL-17⁺ T cell and Foxp3⁺ Treg cell dynamics in induced periapical lesions, which may play an important role in periapical lesion progression.     

Hydrogen sulfide synergistically upregulates Porphyromonas gingivalis lipopolysaccharide-induced expression of IL-6 and IL-8 via NF-κB signalling in periodontal fibroblasts

Objectives

The periodontal pathogen Porphyromonas gingivalis produces hydrogen sulfide (H₂S). H₂S in the oral cavity is positively correlated with periodontitis but the mechanism by which H₂S contributes to periodontal diseases is obscure. We investigated the effect of H₂S in combination with P. gingivalis lipopolysaccharide (LPS) on expression of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 in periodontal fibroblasts and the underlying mechanism of action.

Material and methods

Gingival fibroblasts (GFs) and periodontal ligament cells (PDLCs) were treated with different concentrations of the H₂S donor NaHS in the presence/absence of P. gingivalis LPS for different time periods. Expression of IL-6 and IL-8 was detected by real-time PCR and ELISA. The activity of nuclear factor-kappa B (NF-κB) signalling was investigated using western blotting, EMSA and pathway blockade assays.

Results

Real-time PCR and ELISA results showed that H₂S not only upregulated expression of IL-6 and IL-8 at mRNA and protein levels in a dose- and time-dependent manner, but also aggravated P. gingivalis LPS-induced expression of IL-6 and IL-8 in GFs and PDLCs. Western blotting and EMSA showed that NF-κB signalling was activated by NaHS, P. gingivalis LPS, and both, which was in accordance with the expression levels of IL-6 and IL-8 in GFs and PDLCs. These results were confirmed using a NF-κB pathway blockade assay.

Conclusions

H₂S synergistically upregulated P. gingivalis LPS-induced expression of IL-6 and IL-8 in GFs and PDLCs via activation of NF-κB signalling, which could promote the development of periodontitis.     

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-1β 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-1β 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.     

Biologic Activity of Porphyromonas endodontalis Complex Lipids

Introduction

Periapical infections secondary to pulpal necrosis are associated with bacterial contamination of the pulp. Porphyromonas endodontalis, a gram-negative organism, is considered to be a pulpal pathogen. P. gingivalis is phylogenetically related to P. endodontalis and synthesizes several classes of novel complex lipids that possess biological activity, including the capacity to promote osteoclastogenesis and osteoclast activation. The purpose of this study was to extract and characterize constituent lipids of P. endodontalis and evaluate their capacity to promote proinflammatory secretory responses in the macrophage cell line, RAW 264.7, as well as their capacity to promote osteoclastogenesis and inhibit osteoblast activity.

Methods

Constituent lipids of both organisms were fractionated by high-performance liquid chromatography and were structurally characterized using electrospray mass spectrometry or electrospray-mass spectrometry/mass spectrometry. The virulence potential of P. endodontalis lipids was then compared with known biologically active lipids isolated from P. gingivalis.

Results

P. endodontalis total lipids were shown to promote tumor necrosis factor alpha secretion from RAW 264.7 cells, and the serine lipid fraction appeared to account for the majority of this effect. P. endodontalis lipid preparations also increased osteoclast formation from RAW 264.7 cells, but osteoblast differentiation in culture was inhibited and appeared to be dependent on Toll-like receptor 2 expression.

Conclusions

These effects underscore the importance of P. endodontalis lipids in promoting inflammatory and bone cell activation processes that could lead to periapical pathology.     

Porphyromonas gingivalis induces myocarditis and/or myocardial infarction in mice and IL-17A is involved in pathogenesis of these diseases

ObjectivesAlthough an association between periodontitis and cardiovascular diseases has been suggested, the role of Porphyromonas gingivalis in cardiovascular diseases is not clear. In this study, we examined whether experimental bacteremia of P. gingivalis causes cardiovascular diseases and investigated the mechanism of pathogenesis of cardiovascular diseases induced by P. gingivalis.DesignC57BL/6 mice were intravenously inoculated with 2.0 × 10⁸ CFU of P. gingivalis A7436 strain. Mice were sacrificed at specified days and their hearts were collected. The collected organs were divided into two halves and used for histological evaluation and cytokine analysis. IL-17A^?/?, IFN-γ^?/? and TNF-α^?/? mice were also intravenously inoculated and the histological changes of hearts in mice were examined.ResultsMyocarditis and/or myocardial infarction were observed in mice injected with P. gingivalis. The levels of IL1-β, IL-6, IL-17A, IL-18, TNF-α and IFN-γ mRNA increased significantly after P. gingivalis injection. In particular, high levels of IL-17A and IFN-γ mRNA expression were observed in hearts of mice after P. gingivalis injection in comparison with these levels before injection. Furthermore, the production of IL-17A was detected in hearts of wild-type mice after P. gingivalis injection. In wild-type, TNF-α^?/? and IFN-γ^?/? mice, moderate infiltration of neutrophils and monocytes was observed in hearts at 5 days after injection. In contrast, no inflammatory findings were observed in hearts of IL-17A^?/? mice.ConclusionWe have demonstrated that an experimental bacteremia of P. gingivalis could induce myocarditis and/or myocardial infarction in mice, and IL-17A plays an important role in the pathogenesis of these diseases.     

Porphyromonas gingivalis-induced autophagy suppresses cell proliferation through G1 arrest in oral cancer cells

Objectives

We investigated the response of oral cancer cells to intracellular invasion of Porphyromonas gingivalis to define changes in the biological characteristics of oral cancer cells evoked by the presence of oral pathogenic bacteria within a tumour microenvironment.

Designs

The proliferative activity, cell cycle, and autophagic response were evaluated in oral cancer cells infected with P. gingivalis 381. ROS generation was detected in these cells by DCFDA assay, and its role in the responses of oral cancer cells to P. gingivalis infection was further investigated.

Resutls

P. gingivalis inhibited proliferation of oral cancer cells by inducing G1 cell cycle arrest, but had no effect on apoptosis. Following infection with P. gingivalis, the expression of cyclin D1 and cdk4 was decreased in oral cancer cells, whereas p21, a Cdk inhibitor, was upregulated, in comparison with non-infected controls. Autophagy was prominently enhanced in these infected cells, presumably contributing to the suppressed proliferation. Further experiments revealed that such autophagic response was activated by the formation of reactive oxygen species, as evidenced by the lack of autophagic response and cell proliferation upon removal of reactive oxygen species.

Conclusions

These findings provide a novel insight into the mechanism by which cancer cells are influenced by tumour microenvironment including oral bacteria.     

Exposure of Porphyromonas gingivalis to cortisol increases bacterial growth

Objective

Psychological stress is considered as a risk factor for periodontal diseases. The stress-related hormone, cortisol is one of the main molecules released during human stress response and is found in plasma and gingival crevicular fluid. This hormone has been suggested to modify composition of subgingival biofilms. The aim of this study was to investigate the effect of exposure to cortisol on Porphyromonas gingivalis (P. gingivalis) growth.

Materials and methods

P. gingivalis ATCC strain 33277 was cultured under strict anaerobic conditions at 37 °C in Brain Heart Infusion medium supplemented with hemin (5 μg ml⁻¹) and menadione (1 μg ml⁻¹). Bacterial cultures were incubated with or without hydrocortisone (0.04–10 μg ml⁻¹) at 37 °C for 12, 24 and 48 h and bacterial growth was evaluated by spectrophotometric method (OD_600 nm). Cortisol consumption has been followed by HPLC.

Results

Cortisol significantly increased P. gingivalis growth in the first 24 h peaking at 12 h but this increase was not related to the concentration used. During the time period, no consumption of cortisol was observed.

Conclusions

This study provides further support for the idea that stress-induced hormone; cortisol may influence the growth of P. gingivalis. This specific effect may be involved in the relationship between stress and periodontal diseases.     

The effect of systemic anti-tumor necrosis factor-alpha treatment on Porphyromonas gingivalis infection in type 2 diabetic mice

Objective

Diabetes mellitus and periodontal disease are two common chronic diseases that have long been thought to be biologically linked. Overexpression of tumor necrosis factor alpha (TNF-α) is thought to contribute to this bidirectional inter-relationship. This study examined the effect of anti-TNF-α antibody treatment on Porphyromonas gingivalis infection in diabetic mice.

Methods

In C57BL/6 (normal) and KKAy (diabetic) mice, the area adjacent to the periosteum at a point on the skull midway between the ears was inoculated with P. gingivalis. At 24 h after the inoculation, the mice in the test group were treated with rat anti-murine TNF-α intravenously, while the control group received non-immunized rat IgG. TNF-α, IL-6, and fasting blood glucose levels in the mice were measured on day 3.

Results

Anti-TNF-α antibody treatment improved the host response to P. gingivalis and was associated with reduced serum TNF-α, IL-6, and fasting blood glucose levels in the KKAy mice. Anti-TNF-α antibody treatment also decreased the lesion size at the P. gingivalis inoculation.

Conclusions

Our results suggest that TNF-α plays a role in the two-way relationship between P. gingivalis infection and diabetes mellitus. Anti-TNF-α antibody treatment may improve the host response to P. gingivalis infection and glycemic control in diabetes mellitus.     

Ameliorating effects of Juzentaihoto on restraint stress and P. gingivalis-induced alveolar bone loss

Objective

Juzentaihoto (JTX) is a traditional Japanese medicine that consists of 10 herbs. The purpose of this study was to evaluate the efficacy of multi-herbal medicine JTX as a preventive and therapeutic drug for periodontal bone resorption and for reducing restraint stress.

Materials and methods

Porphyromonas gingivalis ATCC 33277 was used for testing the antibacterial activity of JTX and a rat experimental periodontitis model. To evaluate the effect of JTX against P. gingivalis infection, we determined the differences in alveolar bone loss among experimental groups. The concentrations of adrenocorticotropic hormones were measured as stress markers, and atrophy of the thymus and spleen was assessed.

Results

JTX had antibacterial activity against P. gingivalis ATCC 33277. JTX treatment of mouse bone marrow cells at a concentration of 0.1 μg/ml significantly inhibited osteoclast formation. Administration of JTX to rats with P. gingivalis infection and restraint stress significantly reduced alveolar bone loss compared with the case with just the combination of P. gingivalis infection and restraint stress. In the restrained groups, stress markers were elevated, and the thymus and spleen were atrophied. The groups with administration of JTX showed not only inhibition of the decrease of weight but also normalization of corticosterone and cortisol values.

Conclusion

JTX effectively inhibited restraint stress and osteoclastogenesis. It appears that the effects of JTX inhibit the destruction of periodontal tissue by suppressing stress. Our study demonstrated that JTX affects the correlation between restraint stress and periodontitis.     

An in vitro study of alginate oligomer therapies on oral biofilms

Objectives

The in vitro effect of a novel, oligosaccharide nanomedicine OligoG against oral pathogen-related biofilms, both alone and in the presence of the conventional anti-bacterial agent triclosan, was evaluated.

Methods

The effect of OligoG ± triclosan was assessed against established Streptococcus mutans and Porphyromonas gingivalis biofilms by bacterial counts and image analysis using LIVE/DEAD^® staining and atomic force microscopy (AFM). The effect of triclosan and OligoG surface pre-treatments on bacterial attachment to titanium and polymethylmethacrylate was also studied.

Results

OligoG potentiated the antimicrobial effect of triclosan, particularly when used in combination at 0.3% against S. mutans grown in artificial saliva. OligoG was less effective against established P. gingivalis biofilms. However, attachment of P. gingivalis, to titanium in particular, was significantly reduced after surface pre-treatment with OligoG and triclosan at 0.01% when compared to controls. Light microscopy and AFM showed that OligoG was biocidal to P. gingivalis, but not S. mutans.

Conclusions

OligoG and triclosan when used in combination produced an enhanced antimicrobial effect against two important oral pathogens and reduced bacterial attachment to dental materials such as titanium, even at reduced triclosan concentrations. Whilst the use of triclosan against oral bacteria has been widely documented, its synergistic use with OligoG described here, has not previously been reported. The use of lower concentrations of triclosan, if used in combination therapy with OligoG, could have environmental benefits.

Clinical importance

The potentiation of antimicrobial agents by naturally occurring oligomers such as OligoG may represent a novel, safe adjunct to conventional oral hygiene and periodontal therapy. The ability of OligoG to inhibit the growth and impair bacterial adherence highlights its potential in the management of peri-implantitis.     

Lipopolysaccharide from Escherichia coli but not from Porphyromonas gingivalis induce pro-inflammatory cytokines and alkaline phosphatase in dental follicle cells

Objectives

Dental follicle cells (DFCs) as periodontal precursor cells are the natural source for cellular therapies of periodontitis. Periodontitis is initiated after the infection of the periodontium with oral pathogens such as the Gram-negative bacteria Porphyromonas gingivalis. Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative bacteria. Previous studies have shown that especially P. gingivalis LPS induces the expression of pro-inflammatory cytokines in PDL cells and disturbs the differentiation of dental stem cells. Our study investigated the administration of LPS to DFCs for the first time.

Materials and methods

We evaluated cell proliferation (WST1 assay), expression of cytokines IL1β, IL8 and IL6 (real-time RT-PCR) and the osteogenic differentiation of DFCs (ALP-activity and Alizarin red staining) in the presence of P. gingivalis LPS and Escherichia coli LPS.

Results

All tested pro-inflammatory cytokines were highly increased after E. coli LPS treatment. P. gingivalis LPS induces only the expression of IL8, but this expression was significantly lower than that after E. coli LPS administration. The ALP activity was significantly higher in DFCs after the administration of E. coli LPS than after administration of P. gingivalis LPS or under normal cell differentiation conditions. However, the mineralization was inhibited with LPS from both bacterial species.

Conclusion

LPS disturbs osteogenic differentiation in DFCs. Moreover, the failure of pro-inflammatory cytokines induction in DFCs after the administration of P. gingivalis LPS differs greatly from that of PDL fibroblasts. These immunological properties of DFCs have to be considered for cellular therapies of periodontitis with DFCs.     

Th2 cytokines efficiently stimulate periostin production in gingival fibroblasts but periostin does not induce an inflammatory response in gingival epithelial cells

Objectives

This study aims to clarify whether gingival fibroblasts produce periostin in response to Th2 cytokines which are elevated in periodontitis lesion and, if so, whether periostin affects the inflammatory response and matrix-protein metabolism.

Design

Human gingival fibroblasts, periodontal ligament cells and the gingival epithelial cell line epi4 were stimulated with interleukin-4 (IL-4), IL-13, tumour necrosis factor-α (TNF-α) and Porphyromonas gingivalis lipopolysaccharide (LPS). Periostin expression was analysed by real-time polymerase chain-reaction (PCR) and Western blotting. The expression of the IL-4 receptor α-chain was evaluated by immunocytochemistry. The effect of periostin on the production of inflammatory cytokines and the expression of matrix protein-related genes was analysed by real-time PCR and enzyme-linked immunosorbent assay (ELISA).

Results

While IL-4 and IL-13 significantly induced periostin production in gingival fibroblasts and periodontal ligament cells, no effect was observed in epi4 cells. No stimulatory effect of TNF-α or P. gingivalis LPS on the production of periostin was observed. The effect of periostin on the production of inflammatory cytokines was weak in gingival fibroblasts; however, little or no effect was observed on periodontal ligament cells or epi4 cells. No significant effect of periostin on the expression of matrix protein-related genes was found.

Conclusion

The results suggest that gingival fibroblasts may be a source of periostin in periodontitis lesions but periostin has only a limited role either in the inflammatory response or in matrix-protein metabolism. Thus, the role of periostin in the cellular interaction between epithelial and mesenchymal cells in gingiva may be distinct from that of skin.     

Effects of Calcium Hydroxide Intracanal Medications on T Helper (Th1, Th2, Th9, Th17, and Tfh) and Regulatory T (Treg) Cell Cytokines in Apical Periodontitis: A CONSORT RCT

IntroductionThis Consolidated Standards of Reporting Trials randomized clinical trial investigated T helper (Th1, Th2, Th9, Th17, and Tfh) and regulatory T (Treg) cell–type cytokines and their networks in apical periodontitis (AP). We also assessed the effects of calcium hydroxide [Ca(OH)2] intracanal medications (ICMs) on helper T and Treg cell–type cytokines.MethodsTwenty teeth with primary endodontic infection and apical periodontitis were randomly divided into two groups: Ca(OH)2 + saline solution (n = 10) and Ca (OH)2 + 2% chlorhexidine-gel (n = 10). Samples were collected from the periradicular tissue fluid (PTF) before (PTFs1) and after 14 days of ICMs (PTFs2). The Human High Sensitivity T Cell Panel was used to quantify target T-helper (Th)1: interelukin (IL)-2, IL-12, and interferon-gamma (IFN-γ); Th2: IL-4, IL-5, and IL-13; Th9: IL-9; Th17: IL-17; T follicular helper cells (Tfh): IL-21; and Treg-cell-type cytokine: IL-10.ResultsTh1-type cytokines were higher than Th2-type ones, at PTFs1. Positive (+) associations were found among all Th1-type cytokines and all Th2-type cytokines. There were negative (-) correlations between all Th1- and Th2-type cytokines. Size of radiolucent lesions and symptoms (tenderness to percussion and/or pain on palpation) were positively correlated with Th1-type cytokines, IL-17, and IL-21 but negatively correlated with Th2-type cytokines and IL-10 (all, P < .001). Both ICMs increased Th2-type cytokines and IL-10 (P < .05) and decreased Th1-type cytokines, IL-17, and IL-21 (P < .05), with no differences among them (P > .05).ConclusionsComplex T-cell cytokine networks are involved in AP. Both Ca(OH)2 ICMs effectively increased IL-4, IL-5, IL-10, and IL-13 and lowered IL-2, IL-12, IL-17, IL-21, and IFN-γ.     

Cysteine proteases from Porphyromonas gingivalis and TLR ligands synergistically induce the synthesis of the cytokine IL-8 in human artery endothelial cells

Objective

Bacterial pathogens are frequently detected in atheromatous lesions, however, their contribution to atherosclerosis remains unknown. The present study was aimed to explore the effect of the P. gingivalis cysteine protease gingipain towards the proinflammatory response of human aortic endothelial cells (HAECs).

Design

HAECs were exposed to gingipains (Rgps) extracted from the oral pathogen P. gingivalis. In addition, HAECs were co-stimulated with the TLR ligands P. gingivalis LPS, E. coli LPS or heat-killed P. gingivalis (HKPG) in combination with gingipain-active or gingipain-inactive extracts. After stimulation, IL-8 mRNA expression and protein synthesis were analysed by RT-PCR and ELISA. Means and standard errors were computed following by statistical testing (P ≤ 0.05).

Results

In HAECs, Rgps significantly increased the IL-8 mRNA (5.8 ± 1.1-fold) and protein expression (523.0 ± 57.5 pg/ml) compared to untreated controls. Co-stimulation experiments showed a significant synergistic effect for the IL-8 mRNA expression and protein synthesis when HAECs were exposed to a combination of the purified TLR ligands (P. gingivalis or E. coli LPS) or HKPG and gingipain-active extracts.

Conclusions

These results demonstrated the synergistic effects of TLR ligands and P. gingvalis cysteine proteases for the proinflammatory responses in artery vascular endothelial cells and highlight a mechanism by which bacteria may contribute to the development of atherosclerosis.     

Association study between salivary levels of interferon (IFN)-gamma,interleukin (IL)-17, IL-21, and IL-22 with chronic periodontitis

Objective

To investigate if the salivary levels of IL-17, IL-21, IL-22, and its ratio regarding salivary IFN-γ may be linked with the periodontal clinical status.

Design

One hundred and five chronic periodontitis (CP) subjects and 44 healthy controls (HC) were recruited. Periodontal status was assessed based on full-mouth clinical periodontal measurements. Cytokine salivary levels were analyzed by ELISA. The association between the analytes with CP was analyzed using a binary logistic regression model.

Results

A statistically significant increase in salivary levels of IFN-γ and IFN-γ/IL-22 ratio in CP group could be detected, but there was no significant domination of any Th17 cytokine that could be of predictive value for health/disease status. Univariate and binary logistic regression analyses revealed a strong and independent association of IFN-γ salivary levels and IFN-γ/IL-22 ratio with disease status. An interaction effect of ageing on IFN-γ levels also could be noted.

Conclusion

While salivary levels of IFN-γ and IFN-γ/IL-22 ratio may act as strong/independent indicators of the amount and extent of periodontal breakdown, the low detection frequency of Th17 cytokines in saliva samples make these determinations useless for the detection of disease presence and/or its severity.     

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2^d), C57BL6 (H-2^b), DBA/2J (H-2^d) and CBA/CaH (H-2^k) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4⁺ and CD8⁺ T-cell subsets, CD14⁺ macrophages and CD19⁺ B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8⁺ T cells and CD19⁺ B cells were found in any of the lesions. The percentages of CD4⁺ cells, CD14⁺ cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14⁺ cells in sham-immunized mice. The percentage of CD14⁺ cells was higher than that of CD4⁺ cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4⁺ and CD14⁺ cells predominated in immunized CBA/CaH mice and CD4⁺ cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14⁺ cells and CD4⁺ cells in sham-immunized mice. IgG1⁺ plasma cells were more dominant than IgG2a⁺ cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a⁺ plasma cells were more obvious in sham-immunized mice. IgG2a⁺ plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.     

Oral immunization with Porphyromonas gingivalis outer membrane protein and CpG oligodeoxynucleotides attenuates P. gingivalis-accelerated atherosclerosis and inflammation

Objective

It has previously been shown that oral immunization with the 40-kDa outer membrane protein of Porphyromonas gingivalis (40k-OMP) and CpG oligodeoxynucleotides (ODN) as an adjuvant elicits protective antibody responses against alveolar bone loss caused by P. gingivalis infection. The objective of the present work was to assess the efficacy of this same oral vaccine on prevention of P. gingivalis-accelerated atherosclerosis.

T helper cells from aggressive periodontitis patients produce higher levels of interleukin-1 beta and interleukin-6 in interaction with Porphyromonas gingivalis

Objective

In this study, we analyzed the production of Interleukin-1 beta (IL-1β) and IL-6 by activated CD4+ cells obtained from aggressive periodontitis (AgP) patients in comparison with healthy subjects (HC).

Materials and methods

CD4+ cells were automatically separated from lymphocytes obtained from peripheral blood of patients with AgP and healthy controls. Cells were activated for 4, 8, and 24 h with three different stimuli: anti-CD3/anti-CD28, phytohemagglutinin (PHA), and Porphyromonas gingivalis (P. gingivalis) outer membrane protein (OMP). Protein levels were measured in supernatants of activated CD4+ cells by a bead-based immunoassay (CBA). In addition, serum antibodies against P. gingivalis were determined. Data were analyzed using U test (p?Results T helper cells of AgP patients activated with P. gingivalis OMP produced higher levels of IL-1β and IL-6 in comparison with healthy controls (p?P. gingivalis.

Conclusion

In view of these results, it is possible to conclude that P. gingivalis contributes to the pathogenesis of AgP by inducing high levels of pro-inflammatory cytokines such as IL-1β and IL-6 by peripheral CD4+ T helper cells.

Clinical relevance

In accordance with the clinical parameters and the immunological data, we suggest that full-mouth disinfection with adjunctive systemic antibiotics might be the anti-infectious non-surgical periodontal treatment of choice in this type of patients. Microbiological analyses at the beginning and at the end of the periodontal treatment are recommended. However, it is necessary to verify these data in longitudinal clinical studies.     

Mesenchymal Stem Cells as Active Prohealing and Immunosuppressive Agents in Periapical Environment: Evidence from Human and Experimental Periapical Lesions

Introduction

Previous studies describe contrasting molecular profiles of active and inactive periapical granulomas characterized by distinct expression of cytokines, osteoclastogenic factors, and wound healing markers. Although the molecular mechanisms underlying such a dichotomy remain unknown, in this study we investigated the potential involvement of mesenchymal stem cells (MSCs) in determining human and murine periapical lesion activity and outcomes.

Methods

Periapical granulomas (n = 83) and control samples (n = 24) were comparatively assessed for the expression levels of 11 mesenchymal stem cell (MSC) markers using real-time polymerase chain reaction. Experimental periapical lesions induced in mice were evaluated for MSC marker expression and the effects of AMD3100 treatment on lesion outcomes.

Results

MCS marker expression was prevalent in periapical granulomas compared with that in controls, whereas CD29, CD73, CD90, CD146, CD166, NANOG, Stro-1, and CXCR4 expressions were higher in inactive than in active lesions. Experimental periapical lesion inactivity was also associated with an increased expression of MSC markers. The inhibition of MSC mobilization to the periapex by AMD3100 resulted in increased lesion sizes; decreased expression of MSCs and wound healing markers; and increased expression of interleukin 1 beta (IL-17β), interleukin 17 (IL-17), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and nuclear factor kappa-B ligand (RANKL).

Conclusions

Our results show that MSC markers are overexpressed in inactive human and experimental periapical lesions and that MSC mobilization results in the attenuation of experimental lesion progression associated with immunosuppressive and prohealing mechanisms.