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.

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.

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.

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.

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.

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.

Immunomodulatory Properties of Induced Pluripotent Stem Cell-Derived Mesenchymal Cells.

MSC-like populations derived from induced pluripotent stem cells (iPSC-MSC) serve as an alternative stem cell source due to their high proliferative capacity. In this study, we assessed the immunomodulatory potential of iPSC-MSC generated from periodontal ligament (PDL) and gingival (GF) tissue. The iPSC-MSC lines exhibited a similar level of suppression of mitogen-stimulated peripheral blood mononuclear cells (PBMNC) proliferation compared to their respective parental fibroblast populations in vitro. Moreover, iPSC-MSC demonstrated the ability to suppress T-cells effector cells, Th1/Th2/Th17 populations, and increase levels of Treg cells. In order to investigate the mechanisms involved, expression of common MSC-derived soluble factors known to supress lymphocyte proliferation were assessed in iPSC-MSC cultured with PBMNC with direct cell-cell contact or separated in transwells. Real-time PCR analysis of factors known to be involved in MSC mediated immune regulation, found a general trend of elevated IDO1 and IL6 transcript levels in iPSC-MSC lines and their respective primary cells co-cultured with activated PBMNC, with a wide range of gene expression levels between the different mesenchymal cell types. The results suggest that different iPSC-MSC may be useful as a potential alternative source of cells for future clinical use in therapeutic applications because of their potent immunosuppressive properties. J. Cell. Biochem. 117: 2844-2853, 2016. © 2016 Wiley Periodicals, Inc.

Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.

Combined effects of proinflammatory cytokines and intermittent cyclic mechanical strain in inhibiting osteogenicity in human periodontal ligament cells.

Mechanical strain plays an important role in bone formation and resorption during orthodontic tooth movement. The mechanism has not been fully studied, and the process becomes complex with increased amounts of periodontal patients seeking orthodontic care. Our aims were to elucidate the combined effects of proinflammatory cytokines and intermittent cyclic strain (ICS) on the osteogenic capacity of human periodontal ligament cells. Cultured human periodontal ligament cells were exposed to proinflammatory cytokines (interleukin-1ß 5 ng/mL and tumor necrosis factor-α 10 ng/mL) for 1 and 5 days, and ICS (0.5 Hz, 12% elongation) was applied for 4 h per day. The autocrine of inflammatory cytokines was measured by enzyme-linked immunosorbent assay. The expression of osteoblast markers runt-related transcription factor 2 and rabbit collagen type I was determined using real-time polymerase chain reaction and Western blot. The osteogenic capacity was also detected by alkaline phosphatase (ALP) staining, ALP activity, and alizarin red staining. We demonstrated that ICS impaired the osteogenic capacity of human periodontal ligament cells when incubated with proinflammatory cytokines, as evidenced by the low expression of ALP staining, low ALP activity, reduced alizarin red staining, and reduced osteoblast markers. These data, for the first time, suggest that ICS has a negative effect on the inductive inhibition of osteogenicity in human PDL cells mediated by proinflammatory cytokines.

S100A4 upregulation suppresses tissue ossification and enhances matrix degradation in experimental periodontitis models.

AIM: S100A4, also known as fibroblast-specific protein 1 or metastasin 1, is not only highly expressed in growth-stimulated cultured cells and metastatic tumor cells, but also in the periodontal ligament. The aim of this study was to investigate the roles of S100A4 in the pathogenesis of periodontitis and its regulatory mechanisms in inflammatory milieu. METHODS: Experimental periodontitis was induced in rats by submarginal silk ligatures. TRAP activity and S100A4 expression in periodontal ligaments were examined using immunohistochemistry and immunofluorescence methods. IL-1ß-treated human periodontal ligament cells (hPDLCs) were used as in vitro model of experimental periodontitis. S100A4 mRNA and protein were assessed using qRT-PCR and Western blot, respectively. hPDLCs were transfected with either S100A4 overexpression plasmids or shRNAs plasmids. The mineralization in hPDLCs was evaluated with a 12-d osteogenic induction assay, and the expression of ALP, OCN, MMP-2 and MMP-13 was analyzed by qRT-PCR. RESULTS: In the periodontal ligaments of rats with experimental periodontitis, TRAP activity and S100A4 protein staining were considerably more intense compared with those in the control rats. Treatment of hPDLCs with IL-1ß (10, 50 and 100 ng/mL) dose-dependently increased the mRNA and protein levels of S100A4. Transfection with shRNAs markedly increased mineralized nodule formation and the osteogenic-related markers ALP and OCN levels in hPDLCs, whereas the overexpression of S100A4 significantly reduced mineralized nodule formation, and increased the matrix degradation enzymes MMP-2 and MMP-13 levels in hPDLCs. CONCLUSION: S100A4 is upregulated in the experimental rat periodontitis and in IL-1ß-treated hPDLCs, where S100A4 suppresses osteogenic differentiation and enhances matrix degradation. Thus, S100A4 is a potential target for the treatment of periodontitis.

Cytokine measurements in gingival crevicular fluid and periodontal ligament: Are they correlated?

INTRODUCTION: Mechanical stress can induce molecular changes in gingival crevicular fluid (GCF) and the periodontal ligament (PDL). It is still not clear whether changes in the PDL and GCF are linked. In this study, we aimed to analyze the expression of cytokines in GCF and PDL after mechanical stress. METHODS: Twenty-three healthy patients were included. The experimental group consisted of premolars subjected to a force of 0.980 N for 1, 3, 7, 14, 21, or 28 days. The contralateral teeth were the controls. GCF and PDL samples were collected at the same time points for analysis of cytokines using the cytometric bead array. RESULTS: Interleukin (IL)-6 (IL-6) production was significantly elevated in the PDL on day 1 after force application. Significantly strong positive correlations between GCF and PDL in experimental group were seen on days 3 (interferon-gamma), 7 (IL-10), 14 (IL-17A), and 28 (IL-17A, tumor necrosis factor-alpha), and significantly strong negative correlation were seen on days 14 (interferon-gamma) and 21 (IL-2, IL-10). CONCLUSIONS: Different patterns of IL-6 expression were seen in the PDL and GCF after mechanical stress. Despite occasional correlations between GCF and PDL, the molecular contributions of the PDL to the GCF changes could not be clearly defined by our model.

Periodontal ligament stem cells regulate B lymphocyte function via programmed cell death protein 1.

Periodontal ligament stem cells (PDLSCs) have provided novel cell sources for tooth and periodontal tissue regeneration. Allogeneic PDLSCs can reconstruct periodontal ligament tissue that has been damaged by periodontal diseases and regulate T-cell immunity. However, the effect of PDLSCs on B cells remains unknown. Here, we treated periodontitis in a miniature pig model using allogeneic PDLSCs and showed a reduction in humoral immunity in the animals. When cocultured with normal B cells, human PDLSCs (hPDLSCs) had similar effects as bone marrow mesenchymal stem cells in suppressing B cell proliferation, differentiation, and migration, while intriguingly, hPDLSCs increased B cell viability by secreting interleukin-6. Mechanistically, hPDLSCs suppressed B cell activation through cell-to-cell contact mostly mediated by programmed cell death protein 1 and programmed cell death 1 ligand 1. Our data revealed a previously unrecognized function of PDLSCs in regulating humoral immune responses, which may represent a novel therapeutic strategy for immune-related disorders.

P2X7 receptor-Pannexin1 interaction mediates stress-induced interleukin-1 beta expression in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Pannexin 1 (Panx1) has been found to form nonjunctional hemichannels. It is also proposed to combine with the P2X7 receptor, forming a complex involved in adenosine triphosphate (ATP)-induced interleukin-1beta (IL-1ß) release in macrophages. Previously, we reported that mechanical stress induced IL-1ß expression via the ATP/P2X7 receptor-dependent pathway in human periodontal ligament (HPDL) cells and that ATP was released through the connexin 43 (Cx43) hemichannel. In the present work, we examined the role of Panx1 in stress-induced IL-1ß induction in HPDL cells. MATERIAL AND METHODS: Cultured HPDL cells were treated with compressive loading or ATP to stimulate IL-1ß expression. Inhibitors, antagonists and the small interfering RNA technique were used to investigate the involvement of Panx1 in IL-1ß induction. Co-immunoprecipitation (Co-IP) and immunostaining were used to determine the association of Panx1 with the P2X7 receptor. The IL-1ß release mechanism was analyzed using inhibitors. RESULTS: Blocking Panx1 significantly decreased ATP release, as well as IL-1ß up-regulation, upon stimulation with stress or ATP. Co-IP revealed the association of Panx1 and the P2X7 receptor in HPDL cells, which was increased in response to mechanical loading. Pretreatment with vesicular trafficking inhibitors significantly reduced the amount of IL-1ß released from stimulated cells, suggesting that IL-1ß might be released through vesicles. CONCLUSION: We clearly illustrated the contribution of Panx1 in ATP release, as well as in IL-1ß induction in HPDL cells. The association of Panx1 and the P2X7 receptor might be required for IL-1ß induction, and their possible novel role in IL-1ß vesicular release was indicated.

Exploring the role of innate lymphoid cells in the periodontium: insights into immunological dynamics during orthodontic tooth movement.

Background: The periodontal ligament (PDL) experiences considerable mechanical stresses between teeth and bone, vital for tissue adaptation, especially in orthodontic tooth movement (OTM). While recent research emphasizes the role of innate lymphoid cells (ILCs) in regulating sterile inflammation, their involvement in periodontal tissues during OTM remains largely unexplored. Methods: In this study, PDL tissues from orthodontic patients (n = 8) were examined using flow cytometry to detect ILC subtypes. Transwell co-culture systems were used to expose PDL cells to mechanical strain, followed by measuring migration and ratios of sorted ILC subtypes. Statistical analyses were conducted using paired Student's t-test, Kruskal-Wallis test, Dunn's post-test and one-way/two-way ANOVA with Tukey's post-test (p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001). Results: Our findings demonstrate a significant increase in CD127+ CD161+ ILC frequencies in PDL tissues during OTM, indicating ILC involvement in sterile inflammation induced by orthodontic forces. Co-culture assays show directed migration of ILC subsets towards PDL cells and substantial proliferation and expansion of ILCs. Conclusions: This study is the first to comprehensively investigate the role of ILCs in sterile inflammation during OTM, revealing their presence and distribution within PDL tissues' innate immune response in vivo, and exploring their migratory and proliferative behavior in vitro. The results suggest a crosstalk between ILCs and PDL cells, potentially influencing the inflammatory response and tissue remodeling processes associated with OTM.

Glycated matrix up-regulates inflammatory signaling similarly to Porphyromonas gingivalis lipopolysaccharide.

BACKGROUND AND OBJECTIVE: Hyperglycemia and advanced glycation end-products (AGEs) have been hypothesized as the etiologic factors of diabetic periodontitis. The aim of this study was to clarify in greater detail the patterns of AGE-mediated periodontal inflammation under various physiological conditions. MATERIAL AND METHODS: The deposition of AGEs and expression of the receptor for AGEs (RAGE) were identified by immunohistochemistry in Sprague-Dawley rats with experimentally induced periodontitis or diabetes. Human periodontal ligament cells (PDLCs) and mesenchymal stem cells (MSCs) were cultured under simulated conditions of hyperglycemia, Porphyromonas gingivalis lipopolysaccharide (LPS) stimulation and matrix glycation. Cell viability and expression of toll-like receptors (TLRs), Rage, an inflammatory signaling initiator (nuclear factor kappa light chain enhancer of activator ß cells), an oxidative stressor (heme oxygenase-1) and collagen synthesis (type I and type IV) genes were evaluated. RESULTS: The deposition of AGEs and the expression of Rage were evident in the inflamed periodontal tissues in all rats and appeared to be enhanced in rats with diabetes. Matrix glycation augmented cytotoxicity, up-regulated RAGE and TLRs in both PDLCs and MSCs, and significantly activated downstream inflammatory signaling in MSCs. Oxidative stress was significantly increased under matrix glycation in both PDLCs and MSCs and was significantly increased at a high-glucose concentration in MSCs. A consistent decrease in expression of type I and type IV collagens was observed in MSCs, but a delayed reduction was noted in PDLCs. CONCLUSIONS: Matrix glycation modulated cell behavior to induce inflammation equivalent to that produced by incubation with P. gingivalis LPS. Periodontal inflammation also led to matrix glycation, thus demonstrating a definite interaction between diabetes and periodontitis.

Th17-cells in atopic dermatitis stimulate orthodontic root resorption.

OBJECTIVE: The aim of this study was to investigate how atopic dermatitis (AD) contributes to root resorption during orthodontic tooth movement. MATERIALS AND METHODS: Atopic dermatitis model mice and wild-type mice were subjected to an excessive orthodontic force (OF) to induce movement of the upper first molars. The expression levels of the tartrate-resistant acid phosphatase (TRAP), IL-17, IL-6, and RANKL proteins were determined in the periodontal ligament (PDL) by an immunohistochemical analysis. Furthermore, the effects of the compression force on co-cultures of CD4(+) cells from AD patients or healthy individuals and human PDL cells were investigated with regard to the levels of secretion and mRNA expression of IL-17, IL-6, RANKL, and osteoprotegerin. RESULTS: The immunoreactivities for TRAP, IL-17, IL-6, and RANKL in the AD group were found to be significantly increased. The double immunofluorescence analysis for IL-17/CD4 detected immunoreaction. The secretion of IL-17, IL-6, and RANKL, and the mRNA levels of IL-6 and RANKL in the AD patients were increased compared with those in healthy individuals. CONCLUSION: Th17 cells may therefore be associated with the deterioration of root resorption of AD mice, and may explain why AD patients are more susceptible to root resorption than healthy individuals when an excessive OF is applied.

Mechanical stress-activated immune response genes via Sirtuin 1 expression in human periodontal ligament cells.

Recently, Sirtuin 1 (SIRT1) has been implicated in the molecular control of ageing and immune response. Although the remodelling of periodontal ligament (PDL) in response to mechanical stress (MS) is mediated by several host factors, including cytokines and chemokines, the transmission of mechanical stimuli into specific cellular activity is still not understood fully. This study aimed to investigate the effects of MS, particularly cyclic strain, on immune response genes, as well as SIRT1 and its signal transduction pathways, in human PDL cells. MS up-regulated the expression of SIRT1 and immune response genes encoding cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-1ß], chemokines [IL-8, monocyte cheoattractant protein (CCL)-20], defensins [human ß-defensin (hBD)-2, hBD-3] and Toll-like receptors (TLR-2 and TLR-4) in a force- and time-dependent manner. The SIRT1 inducers resveratrol and isonicotinamide attenuated MS-induced cytokine and chemokine expression, but enhanced the expression of defensins and TLRs. Blockade of SIRT1 activity by the SIRT1 inhibitors sirtinol and nicotinamide and down-regulation of SIRT1 expression by SIRT1 siRNA reduced the stimulatory effects of MS on defensins and TLRs, but increased its effects on cytokines and chemokines. MS induced activation of protein kinase B (Akt), protein kinase C (PKC), nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Treatment with the anti-oxidants N-acetylcysteine and glutathione inhibited MS-induced reactive oxygen species production and expression of cytokines, chemokines, defensins and TLRs. These results suggest that MS activates human PDL cells to express immune/defence genes encoding cytokines, chemokines, defensins and TLRs via a SIRT1 pathway.

Human periodontal ligament cells facilitate leukocyte recruitment and are influenced in their immunomodulatory function by Th17 cytokine release.

The objective of this in vitro study was to examine the immunomodulatory impact of human periodontal ligament (PDL) cells on the nature and magnitude of the leukocyte infiltrate in periodontal inflammation, particularly with regard to Th17 cells. PDL cells were challenged with pro-inflammatory cytokines (IL-1ß, IL-17A, and IFN-γ) and analyzed for the expression of cytokines involved in periodontal immunoinflammatory processes (IL-6, MIP-3 alpha, IL-23A, TGFß1, IDO, and CD274). In order to further investigate a direct involvement of PDL cells in leukocyte function, co-culture experiments were conducted. The expression of the immunomodulatory cytokines studied was significantly increased under pro-inflammatory conditions in PDL cells. Although PDL cells did not stimulate leukocyte proliferation or Th17 differentiation, these cells induced the recruitment of leukocytes. The results of our study suggest that PDL cells might be involved in chronic inflammatory mechanisms in periodontal tissues and thus in the transition to an adaptive immune response in periodontitis.

Antigen-presenting cell marker expression and phagocytotic activity in periodontal ligament cells.

BACKGROUND: Periodontal ligament (PDL) cells are the main cellular constituents of the periodontium, maintain the integrity of the connective tissue, and impact pathology in periodontitis. The aim of this study was to analyze whether PDL cells recognize foreign particles and participate in the immune response to periodontal pathogens. METHODS: Expression of surface proteins characteristic of antigen-presenting cells (APCs) (major histocompatibility complex [MHC] class II, CD40, CD80, CD86) was analyzed in PDL cells after challenge with the cytokines interleukin (IL)-1ß, IL-17A, and interferon-gamma (IFN-γ) or with heat-killed Aggregatibacter actinomycetemcomitans using real-time PCR and flow cytometry. Confocal laser scanning microscopy, transmitted light microscopy, flow cytometry, and time-lapse microscopy were applied to analyze their phagocytotic capacity of collagen (carboxylate-modified microspheres), non-periodontal (Escherichia coli) and periodontal (Aggregatibacter actinomycetemcomitans) pathogens. Furthermore, it was examined whether cytokine activation of PDL cells affects the phagocytosis of collagen or bacteria. RESULTS: PDL cells upregulated MHC class II after cytokine stimulation on transcriptional level, whereas co-stimulatory molecules characteristic of professional APCs were not induced. Analyses on protein level revealed that MHC class II was not constitutively expressed in all PDL cell lines used. PDL cells phagocytosed both collagen and bacteria via acidic vesicles, suggesting the formation of phagosomes. Phagocytosis could be partially inhibited by inhibitors of phagocytosis, i.e., dynasore and wortmannin. Pre-incubation with cytokines did not further enhance the phagocytosis rate of collagen or bacteria. CONCLUSIONS: These results suggest that PDL cells do not only represent bystanders in periodontal infections, but display non-professional APC characteristics, suggesting possible participation in immune reactions of the oral cavity.

Mesenchymal stem cells derived from inflamed periodontal ligaments exhibit impaired immunomodulation.

AIM: The purpose of this study was to investigate the immunomodulatory properties of periodontal ligament stem cells derived from inflamed periodontal ligament tissues. MATERIAL AND METHODS: Periodontal ligament stem cells were identified and isolated from healthy or inflamed periodontal ligament tissues. Peripheral blood mononuclear cells were cocultured with inflamed or healthy periodontal ligament stem cells, and T-lymphocyte proliferation was determined by incubation with carboxyfluorescein succinimidyl ester. T-helper cells (Th1/Th2, Th17) and regulatory T cells were analysed by flow cytometry. Cytokine profiles in supernatants were tested with a cytometric bead array. RESULTS: Compared to healthy cells, inflamed periodontal ligament stem cells showed significantly diminished inhibition of T-cell proliferation. In cocultures, stimulated peripheral blood mononuclear cells showed significantly less induction of CD4+CD25+FOXP3+ regulatory T cells and IL-10 secretion in the presence of inflamed compared with healthy periodontal ligament stem cells. Furthermore, suppression of Th17 differentiation and IL-17 production by inflamed periodontal ligament stem cells was significantly lesser than by healthy cells. CONCLUSION: This study demonstrated that inflamed periodontal ligament stem cells had markedly dysfunctional immunomodulatory properties; this may contribute to an imbalanced immune response, acceleration of osteoclastogenesis and inflammatory alveolar bone loss in periodontitis.