Innate immune response of human periodontal ligament fibroblasts via the Dectin-1/Syk pathway.

Introduction. A diverse microbiota including fungi exists in the subgingival sites of patients with chronic periodontitis. The cell wall of Candida albicans, the most abundant fungal species, contains ß-glucan. Dectin-1 binds ß-glucan and participates in fungal recognition.Gap statement. Human periodontal ligament fibroblasts (PDLFs) are present in the periodontal ligament and synthesize immunomodulatory cytokines that influence the local response to infections. However, the expression and role of Dectin-1 in PDLFs have not been explored.Aim. This study aimed to determine if PDLFs express Dectin-1 and induce innate immune responses through Dectin-1 and the signalling molecule Syk.Methodology. The expression of Dectin-1 in PDLFs was determined by flow cytometry, western blotting and confocal microscopy. Real-time PCR and Western blotting were used to determine the immune response of PDLFs stimulated with ß-glucan-rich zymosan and C. albicans.Results. Dectin-1 was constitutively expressed in PDLFs. Zymosan induced the expression of cytokines, including IL6, IL1B and IL17A, and the chemokine IL8. Zymosan also induced the expression of the antimicrobial peptide ß-defensin-1 (DEFB1). Further, the phosphorylation of Syk and NF-κB occurred upon Dectin-1 activation. Notably, heat-killed C. albicans induced the expression of IL6, IL17A, IL8 and DEFB1, and this activation was suppressed by the Syk inhibitor, R406.Conclusion. These findings indicate that the Dectin-1/Syk pathway induces an innate immune response of PDLFs, which may facilitate the control of oral infections such as candidiasis and periodontitis.

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression.

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.

STAT1/SOCS1/3 Are Involved in the Inflammation-Regulating Effect of GAS6/AXL in Periodontal Ligament Cells Induced by Porphyromonas gingivalis Lipopolysaccharide In Vitro.

Periodontitis involves chronic inflammation of the tissues around the teeth caused by plaque and the corresponding immune response. Growth arrest-specific protein 6 (GAS6) and AXL receptor tyrosine kinase (AXL) are known to be involved in inflammatory diseases, while signal transducer and activator of transcription-1 (STAT1) and suppressor of cytokine signaling (SOCS) are related to inflammatory processes. Moreover, miRNA34a directly targets AXL to regulate the AXL expression. However, the specific roles of GAS6 and AXL in periodontitis remain unclear. This study was designed to explore the effect and mechanism of AXL on the expression of inflammatory cytokines induced by Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) in human periodontal ligament cells (hPDLCs). The effects of different concentrations of P. gingivalis LPS on the expression of GAS6/AXL in hPDLCs were observed. Additionally, the effect of LPS on AXL was investigated by transfection of the miRNA34a inhibitor. AXL was knocked down or overexpressed to observe the release of inflammatory cytokines interleukin- (IL-) 8 and IL-6. The results showed that the expression levels of GAS6 and AXL decreased after P. gingivalis LPS infection. Transfection of a miR-34a inhibitor to hPDLCs demonstrated a role of miR-34a in the downregulation of AXL expression induced by LPS. Moreover, AXL knockdown or overexpression influencing the expression of IL-8 and IL-6 was investigated under LPS stimulation. AXL knockdown decreased the expression of STAT1 and SOCS1/3. Overall, these results demonstrate that AXL inhibits the expression of LPS-induced inflammatory cytokines in hPDLCs and that STAT1 and SOCS1/3 are involved in the regulation of inflammation by GAS6/AXL.

ILC1s and ILC3s Exhibit Inflammatory Phenotype in Periodontal Ligament of Periodontitis Patients.

Innate lymphoid cells (ILCs) are emerging as important players in inflammatory diseases. The oral mucosal barrier harbors all ILC subsets, but how these cells regulate the immune responses in periodontal ligament tissue during periodontitis remains undefined. Here, we show that total ILCs are markedly increased in periodontal ligament of periodontitis patients compared with healthy controls. Among them, ILC1s and ILC3s, particularly NKp44+ILC3 subset, are the predominant subsets accumulated in the periodontal ligament. Remarkably, ILC1s and ILC3s from periodontitis patients produce more IL-17A and IFN-γ than that from healthy controls. Collectively, our results highlight the role of ILCs in regulating oral immunity and periodontal ligament inflammation and provide insights into targeting ILCs for the treatment of periodontitis.

Hyperlipidemic Conditions Impact Force-Induced Inflammatory Response of Human Periodontal Ligament Fibroblasts Concomitantly Challenged with P. gingivalis-LPS.

In obese patients, enhanced serum levels of free fatty acids (FFA), such as palmitate (PA) or oleate (OA), are associated with an increase in systemic inflammatory markers. Bacterial infection during periodontal disease also promotes local and systemic low-grade inflammation. How both conditions concomitantly impact tooth movement is largely unknown. Thus, the aim of this study was to address the changes in cytokine expression and the secretion of human periodontal ligament fibroblasts (HPdLF) due to hyperlipidemic conditions, when additionally stressed by bacterial and mechanical stimuli. To investigate the impact of obesity-related hyperlipidemic FFA levels on HPdLF, cells were treated with 200 µM PA or OA prior to the application of 2 g/cm2 compressive force. To further determine the additive impact of bacterial infection, HPdLF were stimulated with lipopolysaccharides (LPS) obtained from Porphyromonas gingivalis. In mechanically compressed HPdLF, PA enhanced COX2 expression and PGE2 secretion. When mechanically stressed HPdLF were additionally stimulated with LPS, the PGE2 and IL6 secretion, as well as monocyte adhesion, were further increased in PA-treated cultures. Our data emphasize that a hyperlipidemic condition enhances the susceptibility of HPdLF to an excessive inflammatory response to compressive forces, when cells are concomitantly exposed to bacterial components.

Baicalein inhibits inflammatory response and promotes osteogenic activity in periodontal ligament cells challenged with lipopolysaccharides.

BACKGROUND: Periodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS). METHODS: Human PDLCs were incubated with baicalein (0-100 µM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/ß-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot. RESULTS: MTT results showed that baicalein up to 100 µM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/ß-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs. CONCLUSIONS: With the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

Mesenchymal stem cell markers in periodontal tissues and periapical lesions.

INTRODUCTION: Mesenchymal stem cells (MSCs) are characterized by the potential to differentiate into multiple cell lineages, high proliferation rates, and self-renewal capacity, in addition to the ability to maintain their undifferentiated state. These cells have been identified in physiological oral tissues such as pulp tissue, dental follicle, apical papilla and periodontal ligament, as well as in pathological situations such as chronic periapical lesions (CPLs). The criteria used for the identification of MSCs include the positive expression of specific surface antigens, with CD73, CD90, CD105, CD44, CD146, STRO-1, CD166, NANOG and OCT4 being the most specific for these cells. AIM: The aim of this review was to explore the literature on markers able to identify MSCs as well as the presence of these cells in the healthy periodontal ligament and CPLs, highlighting their role in regenerative medicine and implications in the progression of these lesions. METHODS: Narrative literature review searching the PubMed and Medline databases. Articles published in English between 1974 and 2020 were retrieved. CONCLUSION: The included studies confirmed the presence of MSCs in the healthy periodontal ligament and in CPLs. Several surface markers are used for the characterization of these cells which, although not specific, are effective in cell recognition. Mesenchymal stem cells participate in tissue repair, exerting anti- inflammatory, immunosuppressive and proangiogenic effects, and are therefore involved in the progression and attenuation of CPLs or even in the persistence of these lesions.

In Silico Selection and In Vitro Evaluation of New Molecules That Inhibit the Adhesion of Streptococcus mutants through Antigen I/II.

Streptococcus mutans is the main early colonizing cariogenic bacteria because it recognizes salivary pellicle receptors. The Antigen I/II (Ag I/II) of S. mutans is among the most important adhesins in this process, and is involved in the adhesion to the tooth surface and the bacterial co-aggregation in the early stage of biofilm formation. However, this protein has not been used as a target in a virtual strategy search for inhibitors. Based on the predicted binding affinities, drug-like properties and toxicity, molecules were selected and evaluated for their ability to reduce S. mutans adhesion. A virtual screening of 883,551 molecules was conducted; cytotoxicity analysis on fibroblast cells, S. mutans adhesion studies, scanning electron microscopy analysis for bacterial integrity and molecular dynamics simulation were also performed. We found three molecules ZINC19835187 (ZI-187), ZINC19924939 (ZI-939) and ZINC19924906 (ZI-906) without cytotoxic activity, which inhibited about 90% the adhesion of S. mutans to polystyrene microplates. Molecular dynamic simulation by 300 nanoseconds showed stability of the interaction between ZI-187 and Ag I/II (PDB: 3IPK). This work provides new molecules that targets Ag I/II and have the capacity to inhibit in vitro the S. mutans adhesion on polystyrene microplates.

Mesenchymal Stem/Stromal Cells Derived from Dental Tissues: A Comparative In Vitro Evaluation of Their Immunoregulatory Properties Against T cells.

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) have immunoregulatory properties and have been used as immune regulators for the treatment of graft-versus-host disease (GVHD). Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to BM-MSCs for potential clinical applications because of their accessibility and easy preparation. The aim of this in vitro study was to compare MSCs from dental pulp (DP-MSCs), gingival tissue (G-MSCs), and periodontal ligament (PDL-MSCs) in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3+ T cells to determine which MSCs would be the most appropriate for in vivo immunoregulatory applications. BM-MSCs were included as the gold standard. Our results demonstrated, in vitro, that MSCs from DP, G, and PDL showed immunoregulatory properties similar to those from BM, in terms of the cellular proliferation inhibition of both CD4+- and CD8+-activated T-cells. This reduced proliferation in cell co-cultures correlated with the production of interferon-γ and tumor necrosis factor alpha (TNF-α) and the upregulation of programmed death ligand 1 (PD-L1) in MSCs and cytotoxic T-cell-associated Ag-4 (CTLA-4) in T-cells and increased interleukin-10 and prostaglandin E2 production. Interestingly, we observed differences in the production of cytokines and surface and secreted molecules that may participate in T-cell immunosuppression in co-cultures in the presence of DT-MSCs compared with BM-MSCs. Importantly, MSCs from four sources favored the generation of T-cell subsets displaying the regulatory phenotypes CD4+CD25+Foxp3+ and CD4+CD25+CTLA-4+. Our results in vitro indicate that, in addition to BM-MSCs, MSCs from all of the dental sources analyzed in this study might be candidates for future therapeutic applications.

Cyclic Stretch Force Induces Periodontal Ligament Cells to Secrete Exosomes That Suppress IL-1ß Production Through the Inhibition of the NF-κB Signaling Pathway in Macrophages.

In the oral mechanical environment, periodontal ligament cells (PDL cells) contribute to maintaining periodontal tissue homeostasis. Recent studies showed that exosomes, which are small vesicles secreted by various types of cells, play a pivotal role in cell-to-cell communication in biological processes. We examined the secretion of exosomes from PDL cells stimulated with cyclic stretch and their role in the inflammatory response of macrophages using the human macrophage cell line THP-1 and human primary monocytes/macrophages. We prepared supernatants from human PDL cells (PDL-sup) stimulated with cyclic stretch. The treatment of macrophages with PDL-sup, but not PDL-sup from unstimulated PDL cells, inhibited the production of IL-1ß in LPS/nigericin-stimulated macrophages. The pretreatment of PDL cells with GW4869, an inhibitor of exosome secretion, or siRNA for Rab27B, which controls exosome secretion, abrogated the inhibitory effects of PDL-sup. A transmission electron microscopy analysis demonstrated the existence of exosomes with diameters ranging between 30 and 100 nm in PDL-sup, suggesting that exosomes in PDL-sup contribute to this inhibition. An immunofluorescence microscopy analysis revealed that exosomes labeled with PKH67, a fluorescent dye, were incorporated by macrophages as early as 2 h after the addition of exosomes. Purified exosomes inhibited IL-1ß production in LPS/nigericin-stimulated macrophages and the nuclear translocation of NF-κB as well as NF-κB p65 DNA-binding activity in LPS-stimulated macrophages, suggesting that exosomes suppress IL-1ß production by inhibiting the NF-κB signaling pathway. Our results indicate that PDL cells in mechanical environments contribute to the maintenance of periodontal immune/inflammatory homeostasis by releasing exosomes.

LPS­induced upregulation of the TLR4 signaling pathway inhibits osteogenic differentiation of human periodontal ligament stem cells under inflammatory conditions.

Toll­like receptor 4 (TLR4) is a transmembrane receptor responsible for the activation of a number of signal transduction pathways. Despite its involvement in inflammatory processes, the regulation of TLR4 signaling in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions remains to be fully elucidated. The present study aimed to clarify the regulatory mechanisms of the TLR4 signaling pathway and its role in the differentiation of hPDLSCs under inflammatory conditions. hPDLSCs from the periodontal tissues of healthy subjects and patients with periodontitis were identified by analyzing their cell surface marker molecules, and their osteogenic and adipogenic differentiation abilities. To determine the effect of TLR4 signaling on osteogenic and adipogenic differentiation under inflammatory conditions, cells were challenged with TLR4 agonist and antagonist under pluripotent differentiation conditions. Cell proliferation, apoptosis and migration were then determined using appropriate methods. The alkaline phosphatase (ALP) activity, Alizarin Red staining, Oil red O staining and relative gene and protein levels expression were also determined. The results showed that lipopolysaccharide (LPS)­induced inflammation inhibited cell proliferation and migration, promoted cell apoptosis and affected the cell cycle. Under inflammatory conditions, the activation of TLR4 decreased the activity of ALP and the expression of osteogenic markers, including osteocalcin, Runt­related transcription factor 2 and collagen I, compared with the control group, but increased the expression of adipogenesis­related genes poly (ADP­ribose) polymerase Î³ and lipoprotein lipase. The activation of TLR4 also induced the expression of proinflammatory cytokines interleukin­1ß, tumor necrosis factor­α, nuclear factor­κBP65 and TLR4, compared with that in the control group and the TLR4 antagonist group. The findings showed that LPS­induced upregulation of the TLR4 signaling pathway inhibited osteogenic differentiation and induced adipogenesis of the hPDLSCs under inflammatory conditions. The present study provided a novel understanding of the physiopathology of periodontitis, and a novel avenue for targeted treatments based on stem cell regeneration.

Characterization of the Cellular Responses of Dental Mesenchymal Stem Cells to the Immune System.

INTRODUCTION: Dental stem cells have gained importance recently and are being used for various purposes in regenerative medicine and dentistry. Although much research has been done to show the various properties of these dental stem cells, the immunomodulatory properties of some of these stem cells are still unknown. This is important considering these cells are being used routinely. Therefore, the aim of this study was to investigate the interactions between the activated immune cells and 3 types of dental-derived mesenchymal stem cells: dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and stem cells of the apical papilla (SCAP). METHODS: SCAP, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and periodontal ligament fibroblasts were cultured, and various assays were performed including a proliferation assay, flow cytometric analysis, lactate dehydrogenase and chromium-51 cytotoxicity assays, and an enzyme-linked immunosorbent assay to evaluate the interactions of these dental stem cells when cocultured with either peripheral blood mononuclear cells or natural killer cells. RESULTS: SCAP were less resistant to immune cell-mediated cytotoxicity as seen from the results obtained from the LDH and chromium-51 cytotoxicity assays. The flow cytometric analysis showed a lower resilience of SCAP to cytotoxic compounds. The enzyme-linked immunosorbent assay results demonstrated that the SCAP induced high levels of proinflammatory cytokine secretion compared with the other dental stem cells. CONCLUSIONS: SCAP did not perform as well as the other dental stem cells. This could in turn affect their survival and differentiation abilities as well as their functionality. This may be an important aspect to consider when selecting dental stem cells for various regenerative procedures.

Comparative study on metabolite level in tissue-specific human mesenchymal stem cells by an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry.

Mesenchymal stem cells (MSCs) are a promising therapeutic option for cell-based therapy due to their immunomodulatory and regenerative properties. They can be isolated from various adult tissues, including bone marrow, fat, dental tissue, and glandular tissue. Although they share common characteristics, little is known about the biological differences between MSC populations derived from different tissues. In this study, we used MS to compare the endogenous metabolite level in the human MSCs originating from the bone marrow, adipose tissue, periodontal ligaments, and salivary glands. Using an optimized metabolomics technique, we verified that human MSCs exhibit differences in the endogenous metabolite level depending on their source material, while the multivariate analysis showed that 5 lysophosphatidylcholines and 3 lysophosphatidylethanolamines can serve as markers for the discrimination between MSC sources and may be related to differences in their differentiation capacity. These results may significantly contribute to further mechanistic studies on the MSCs and provide novel insights into the properties and optimal usage of MSCs from different tissues.

Mice dental pulp and periodontal ligament endothelial cells exhibit different proangiogenic properties.

Dental pulp is a highly vascularized tissue with a high regenerative capacity. This is attributed to its unique blood supply and the presence of progenitor or postnatal dental pulp stem cells. Here we aimed to isolate and compare the angiogenic properties of endothelial cells (EC) prepared from mouse dental pulp and periodontal ligament (PDL). EC were isolated from 4-week-old wild type immorto mice. Mice were sacrificed and after mandible isolation, the molar and incisor teeth and the PDL from molar teeth were dissected. EC were prepared by collagenase digestion of tissues and affinity purification using magnetic beads coated with platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) antibody. EC prepared from incisor and molar pulps and PDL were examined for expression of appropriate markers by fluorescence-activated cell sorting (FACS) analysis. The proliferation, migration, and capillary morphogenesis of EC were evaluated. Ex vivo sprouting angiogenesis from various tissues was also compared. Data were analyzed at the level of significance of P<0.05. Pulp EC prepared from incisors proliferated and migrated significantly faster than molar and PDL EC (P<0.05). In addition, molar and PDL EC formed a more extensive capillary network when plated on Matrigel. This is consistent with the lower proliferative and migratory characteristics of these cells compared with incisor EC (P<0.05). However, PDL tissue showed significantly more sprouting area than molar and incisor pulp tissues (P<0.05). Thus, pulp EC from molar and incisor and PDL EC present different proangiogenic properties. Collectively our results suggest that EC from different tooth tissue have unique characteristics related to their target tissue and function.

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling.

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast-like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF-ß, fibronectin (FN), α-SMA, and NG2. LPS also increased protein and gene expression levels of anti-inflammatory COX-2 and pro-inflammatory IL-6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS-treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen-stimulated proliferation of CD4+ and the ratio of CD4+ CD25high /CD4+ CD25low lymphocytes. LPS-treated PDLSCs did not change the frequency of CD34+ and CD45+ cells, but decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU-GM number. The results indicated that LPS-activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.

Streptococcus gordonii lipoproteins induce IL-8 in human periodontal ligament cells.

Streptococcus gordonii, a Gram-positive oral bacterium, is a life-threatening pathogen that causes infective endocarditis. It is frequently isolated from the periapical lesions of patients with apical periodontitis and has thus been implicated in inflammatory responses. However, little is known about the virulence factors of S. gordonii responsible for the induction of inflammatory responses in the periapical areas. Here, we investigated the role of S. gordonii cell wall-associated virulence factors on interleukin (IL)-8 induction in human periodontal ligament (PDL) cells using ethanol-inactivated wild-type S. gordonii, a lipoteichoic acid (LTA)-deficient mutant (ΔltaS), and a lipoprotein-deficient mutant (Δlgt). Wild-type S. gordonii induced IL-8 expression at both the protein and mRNA levels in human PDL cells in a dose- and time-dependent manner. A transient transfection and reporter gene assay demonstrated that wild-type S. gordonii activated Toll-like receptor 2 (TLR2). Additionally, IL-8 production induced by wild-type S. gordonii was substantially inhibited by anti-TLR2-neutralizing antibodies. Both wild-type S. gordonii and the ΔltaS mutant induced IL-8 production; however, this response was not observed when cells were stimulated with the Δlgt mutant. Interestingly, lipoproteins purified from S. gordonii induced IL-8 production, whereas purified LTA did not. In addition, purified lipoproteins stimulated TLR2 more potently than LTA. Furthermore, S. gordonii-induced IL-8 expression was specifically inhibited by blocking p38 kinase, while lipoprotein-induced IL-8 expression was inhibited by blocking p38 kinase, ERK, or JNK. Of particular note, exogenous addition of purified S. gordonii lipoproteins enhanced Δlgt-induced IL-8 production in human PDL cells to an extent similar to that induced by the wild-type strain. Collectively, these results suggest that lipoproteins are an important component of S. gordonii for the induction of IL-8 production in human PDL cells through TLR2 activation. Therefore, lipoproteins potentially contribute to inflammatory apical periodontitis.

The innate host response in caries and periodontitis.

INTRODUCTION: Innate immunity rapidly defends the host against infectious insults. These reactions are of limited specificity and exhaust without providing long-term protection. Functional fluids and effector molecules contribute to the defence against infectious agents, drive the immune response, and direct the cellular players. AIM: To review the literature and present a summary of current knowledge about the function of tissues, cellular players and soluble mediators of innate immunity relevant to caries and periodontitis. METHODS: Historical and recent literature was critically reviewed based on publications in peer-reviewed scientific journals. RESULTS: The innate immune response is vital to resistance against caries and periodontitis and rapidly attempts to protect against infectious agents in the dental hard and soft tissues. Soluble mediators include specialized proteins and lipids. They function to signal to immune and inflammatory cells, provide antimicrobial resistance, and also induce mechanisms for potential repair of damaged tissues. CONCLUSIONS: Far less investigated than adaptive immunity, innate immune responses are an emerging scientific and therapeutic frontier. Soluble mediators of the innate response provide a network of signals to organize the near immediate molecular and cellular response to infection, including direct and immediate antimicrobial activity. Further studies in human disease and animal models are generally needed.

Vitamin D reduces the inflammatory response by Porphyromonas gingivalis infection by modulating human ß-defensin-3 in human gingival epithelium and periodontal ligament cells.

Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process. Porphyromonas gingivalis, a Gram-negative black-pigmented anaerobe, is a major pathogen in the initiation and progression of periodontitis; it produces several virulence factors that stimulate human gingival epithelium (HGE) cells and human periodontal ligament (HPL) cells to produce various inflammatory mediators. A variety of substances, such as vitamin D, have growth-inhibitory effects on some bacterial pathogens and have shown chemo-preventive and anti-inflammatory activity. We used a model with HGE and HPL cells infected with P. gingivalis to determine the influence of vitamin D on P. gingivalis growth and adhesion and the immunomodulatory effect on TNF-α, IL-8, IL-12 and human-ß-defensin 3 production. Our results demonstrated, firstly, the lack of any cytotoxic effect on the HGE and HPL cells when treated with vitamin D; in addition, vitamin D inhibited P. gingivalis adhesion and infectivity in HGE and HPL cells. Our study then showed that vitamin D reduced TNF-α, IL-8, IL-12 production in P. gingivalis-infected HGE and HPL cells. In contrast, a significant upregulation of the human-ß-defensin 3 expression in HGE and HPL cells induced by P. gingivalis was demonstrated. Our results indicate that vitamin D specifically enhances the production of the human-ß-defensin 3 antimicrobial peptide and exerts an inhibitory effect on the pro-inflammatory cytokines, thus suggesting that vitamin D may offer possible therapeutic applications for periodontitis.

Interleukin-12 modulates the immunomodulatory properties of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: The cytokine interleukin 12 (IL-12) has been implicated as a potent stimulator of tissue degradation in the pathogenesis of several inflammatory diseases, including periodontitis. In patients with periodontitis, an increased level of IL-12 is found in serum and gingival crevicular fluid. As inflammatory cytokines have been demonstrated to induce activation of the immunomodulatory properties of mesenchymal stem cells (MSCs), this study aimed to investigate the influence of IL-12 on these properties in human periodontal ligament (hPDL) cells. MATERIAL AND METHODS: Human PDL cells were isolated from periodontal tissue and incubated with 0-10 ng/mL of IL-12 for 24 h. The levels of expression of interferon gamma (IFN-γ), indoleamine 2,3-dioxygenase (IDO) and human leukocyte antigen G (HLA-G), as well as of the stem cell markers, CD73, CD90 and CD105, were assessed by quantitative PCR. The level of IFN-γ protein was measured by ELISA, and IDO activity was measured by activity assay. The participation of IFN-γ in the expression of IDO and HLA-G was analyzed using neutralizing antibody against IFN-γ. RESULTS: IL-12 upregulated the expression of IFN-γ in a dose-dependent manner. Moreover, IL-12 induced the expression of the immunomodulatory proteins IDO and HLA-G via an IFN-γ-dependent pathway, as indicated by experiments using an IFN-γ neutralizing antibody. Addition of exogenous IFN-γ upregulated the expression of HLA-G and IDO. Expression of the stem cell markers CD73, CD90 and CD105, as well as the pluripotent markers Nanog homeobox, octamer-binding transcription factor 4 and SRY-box 2, were also upregulated in IL-12-treated hPDL cells. Finally, IL-12 inhibited osteogenic differentiation of the hPDL cells and preserved the self-clonal expansion property of these cells, as assessed by Alizarin Red S staining and the colony-forming unit assay. CONCLUSION: Expression of IL-12 during periodontitis may play an important role in the control of the inflammatory response via the induction of immunosuppressive molecules by hPDL cells. We hypothesize that this immunomodulatory property of IL-12 will serve as a protective mechanism to preserve a population of stem cells under inflammatory conditions.

E-cigarettes and flavorings induce inflammatory and pro-senescence responses in oral epithelial cells and periodontal fibroblasts.

Electronic-cigarettes (e-cigs) represent a significant and increasing proportion of tobacco product consumption, which may pose an oral health concern. Oxidative/carbonyl stress via protein carbonylation is an important factor in causing inflammation and DNA damage. This results in stress-induced premature senescence (a state of irreversible growth arrest which re-enforces chronic inflammation) in gingival epithelium, which may contribute to the pathogenesis of oral diseases. We show that e-cigs with flavorings cause increased oxidative/carbonyl stress and inflammatory cytokine release in human periodontal ligament fibroblasts, Human Gingival Epithelium Progenitors pooled (HGEPp), and epigingival 3D epithelium. We further show increased levels of prostaglandin-E2 and cycloxygenase-2 are associated with upregulation of the receptor for advanced glycation end products (RAGE) by e-cig exposure-mediated carbonyl stress in gingival epithelium/tissue. Further, e-cigs cause increased oxidative/carbonyl and inflammatory responses, and DNA damage along with histone deacetylase 2 (HDAC2) reduction via RAGE-dependent mechanisms in gingival epithelium. A greater response is elicited by flavored e-cigs. Increased oxidative stress, pro-inflammatory and pro-senescence responses (DNA damage and HDAC2 reduction) can result in dysregulated repair due to proinflammatory and pro-senescence responses in periodontal cells. These data highlight the pathologic role of e-cig aerosol and its flavoring to cells and tissues of the oral cavity in compromised oral health.