Role of endothelial PDGFB in arterio-venous malformations pathogenesis.

Arterial-venous malformations (AVMs) are direct connections between arteries and veins without an intervening capillary bed. Either familial inherited or sporadically occurring, localized pericytes (PCs) drop is among the AVMs' hallmarks. Whether impaired PC coverage triggers AVMs or it is a secondary event is unclear. Here we evaluated the role of the master regulator of PC recruitment, Platelet derived growth factor B (PDGFB) in AVM pathogenesis. Using tamoxifen-inducible deletion of Pdgfb in endothelial cells (ECs), we show that disruption of EC Pdgfb-mediated PC recruitment and maintenance leads to capillary enlargement and organotypic AVM-like structures. These vascular lesions contain non-proliferative hyperplastic, hypertrophic and miss-oriented capillary ECs with an altered capillary EC fate identity. Mechanistically, we propose that PDGFB maintains capillary EC size and caliber to limit hemodynamic changes, thus restricting expression of Krüppel like factor 4 and activation of Bone morphogenic protein, Transforming growth factor ß and NOTCH signaling in ECs. Furthermore, our study emphasizes that inducing or activating PDGFB signaling may be a viable therapeutic approach for treating vascular malformations.

SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations.

Vascular networks form, remodel, and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with bone morphogenic proteins 9 and 10 (BMP9 and BMP10). Conversely, mutation of the BMP receptors activin-like kinase 1 (ALK1), endoglin (ENG), or the downstream effector, SMAD family member 4 (SMAD4) leads to hereditary hemorrhagic telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). How endothelial cells (ECs) integrate FSS and BMP signals in vascular development and homeostasis and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between FSS and SMAD signaling in vascular stability and how disruption of this synergy leads to AVMs. We found that loss of Smad4 increased the sensitivity of ECs to flow by lowering the FSS set point, with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling, leading to cell cycle progression-mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.

25-hydroxyvitamin D3 generates immunomodulatory plasticity in human periodontal ligament-derived mesenchymal stromal cells that is inflammatory context-dependent.

Introduction: Human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) exhibit a tight bi-directional interaction with CD4+ T lymphocytes. The hPDL-MSCs' immunomodulatory abilities are drastically enhanced by pro-inflammatory cytokines via boosting the expression of various immunomediators. 25-hydroxyvitamin D3 (25(OH)D3), the major metabolite of vitamin D3 in the blood, affects both hPDL-MSCs and CD4+ T lymphocytes, but its influence on their interaction is unknown. Methods: Therefore, primary hPDL-MSCs were stimulated in vitro with tumor necrosis factor (TNF)-α a or interleukin (IL)-1ß in the absence and presence of 25(OH)D3 followed by an indirect co-culture with phytohemagglutinin-activated CD4+ T lymphocytes. The CD4+ T lymphocyte proliferation, viability, and cytokine secretion were analyzed. Additionally, the expression of various immunomediators in hPDL-MSCs was investigated, and their implication was verified by using pharmacological inhibitors. Results: 25(OH)D3 significantly counteracted the suppressive effects of IL-1ß-treated hPDL-MSCs on CD4+ T lymphocyte proliferation, whereas no effects were observed in the presence of TNF-α. Additionally, 25(OH)D3 significantly increased the percentage of viable CD4+ T lymphocytes via TNF-α- or IL-1ß-treated hPDL-MSCs. It also caused a significant decrease in interferon-γ, IL-17A, and transforming growth factor-ß productions, which were triggered by TNF-α-treated hPDL-MSCs. 25(OH)D3 significantly decreased the production of various immunomediators in hPDL-MSCs. Inhibition of two of them, prostaglandin E2 and indoleamine-2,3-dioxygenase-1, partially abolished some of the hPDL-MSCs-mediated effects of 25(OH)D3 on CD4+ T lymphocytes. Conclusion: These data indicate that 25(OH)D3 influences the immunomodulatory activities of hPDL-MSCs. This modulatory potential seems to have high plasticity depending on the local cytokine conditions and may be involved in regulating periodontal tissue inflammatory processes.

Anti-apoptotic effects of human gingival mesenchymal stromal cells on polymorphonuclear leucocytes.

OBJECTIVES: Polymorphonuclear leucocytes (PMNs) constitute the first line of host defence and are crucial in maintaining periodontal health. Their survival and function are modulated by mesenchymal stromal cells (MSCs) from different origin. Gingival MSCs (GMSCs) play an important role in maintaining oral health and in the initial inflammatory response. The present study aimed to investigate the effects of GMSCs on PMNs apoptosis and reactive oxygen species (ROS) production. METHODS: PMNs were either directly incubated with untreated, interleukin (IL)-1ß- or tumour necrosis factor (TNF)-α-treated GMSCs or stimulated with their conditioned media. Resulting ROS production was evaluated by dichlorofluorescin diacetate staining, whereas PMNs apoptosis was assessed by Annexin V staining, followed by flow cytometry analysis. RESULTS: While conditioned media of untreated and TNF-α-treated GMSCs did not affect apoptosis of PMNs, it was significantly delayed by conditioned media of GMSCs treated with IL-1ß. In direct co-culture, GMSCs exerted anti-apoptotic effects on PMNs independently of the previous stimulation. However, the strongest impact was observed by IL-1ß-treated GMSCs. ROS production of PMNs was not influenced by GMSCs or their conditioned media. CONCLUSION: This study demonstrates for the first time the immunomodulatory properties of GMSCs towards PMNs, revealing that IL-1ß enhances anti-apoptotic effects of GMSCs.

Effect of vitamin D3 on the osteogenic differentiation of human periodontal ligament stromal cells under inflammatory conditions.

OBJECTIVES: Vitamin D3 is known to activate osteogenic differentiation of human periodontal ligament stromal cells (hPDLSCs). Recently, inflammatory stimuli were shown to inhibit the transcriptional activity of hPDLSCs, but their effect on vitamin D3 -induced osteogenic differentiation is not known. The present study aimed to investigate whether the effects of 1,25-dihydroxvitamin D3 (1,25(OH)2 D3 ) and 25-hydroxvitamin D3 (25(OH)D3 ) on the osteogenic differentiation of hPDLSCs are also altered under inflammatory conditions. Furthermore, the expression of osteogenesis-related factors by hPDLSCs under osteogenic conditions was assessed in the presence of inflammatory stimuli. MATERIALS AND METHODS: Primary hPDLSCs of six donors were cultured in osteogenic induction medium containing either 1,25(OH)2 D3 (0-10 nM) or 25(OH)D3 (0-100 nM) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) or Pam3CSK4 for 7, 14 and 21 days. Osteogenic differentiation of hPDLSCs was evaluated by analysis of mineralization as assessed by Alizarin Red S staining and gene expression levels of osteogenesis-related factors osteocalcin, osteopontin and runt-related transcription factor 2 (RUNX2) were analysed with qPCR. RESULTS: Treatment with 1,25(OH)2 D3 significantly enhanced the osteogenic differentiation of hPDLSCs and their expression of osteocalcin and osteopontin. The 1,25(OH)2 D3 -triggered expression of osteogenesis-related factors was significantly lower in the presence of Pam3CSK4, but not P. gingivalis LPS. None of the inflammatory stimuli had significant effects on the 1,25(OH)2 D3 -induced osteogenic differentiation. 25(OH)D3 neither affected gene expression levels nor osteogenic differentiation of hPDLSCs cultured in osteogenic induction medium. CONCLUSION: The results of this study indicate that inflammatory stimuli also diminish the 1,25(OH)2 D3 -induced expression of osteogenesis-related factors in hPDLSCs under osteogenic conditions, while having no effect on the osteogenic differentiation.

Transcriptional activity of vitamin D receptor in human periodontal ligament cells is diminished under inflammatory conditions.

BACKGROUND: Although vitamin D3 deficiency is considered as a risk factor for periodontitis, supplementation during periodontal treatment has not been shown to be beneficial to date. Human periodontal ligament cells (hPDLCs) are regulated by vitamin D3 and play a fundamental role in periodontal tissue homeostasis and inflammatory response in periodontitis. The aim of this study is to investigate possible alterations of the vitamin D3 activity in hPDLCs under inflammatory conditions. METHODS: Cells isolated from six different donors were treated with either 1,25(OH)2 D3 (0 to 10 nM) or 25(OH)D3 (0 to 100 nM) in the presence and absence of ultrapure or standard Porphyromonas gingivalis lipopolysaccharide (PgLPS), Pam3CSK4, or interferon-γ for 48 hours. Additionally, nuclear factor (NF)-κB inhibition was performed with BAY 11-7082. The bioactivity of vitamin D in hPDLCs was assessed based on the gene expression levels of vitamin D receptor (VDR)-regulated genes osteocalcin and osteopontin. Additionally, VDR and CYP27B1 expression levels were measured. RESULTS: The vitamin D3 -induced increase of osteocalcin and osteopontin expression was significantly decreased in the presence of standard PgLPS and Pam3CSK4, which was not observed by ultrapure PgLPS. Interferon-y had diverse effects on the response of hPDLCs to vitamin D3 metabolites. NF-kB inhibition abolished the effects of standard PgLPS and Pam3CSK4. Standard PgLPS and Pam3CSK4 increased VDR expression in the presence of vitamin D3 . CYP27B1 expression was not affected by vitamin D3 and inflammatory conditions. CONCLUSIONS: This study indicates that the transcriptional activity of VDR is diminished under inflammatory conditions, which might mitigate the effectiveness of vitamin D3 supplementation during periodontal treatment.

Continuing Effect of Cytokines and Toll-Like Receptor Agonists on Indoleamine-2,3-Dioxygenase-1 in Human Periodontal Ligament Stem/Stromal Cells.

Transplanted mesenchymal stem/stromal cells (MSCs) are a promising and innovative approach in regenerative medicine. Their regenerative potential is partly based upon their immunomodulatory activities. One of the most investigated immunomediators in MSCs, such as in periodontal ligament-derived MSCs (hPDLSCs), is indoleamine-2,3-dioxygenase-1 (IDO-1) which is upregulated by inflammatory stimuli, like cytokines. However, there are no data concerning continuing IDO-1 expression in hPDLSCs after the removal of inflammatory stimuli, such as cytokines and toll-like receptor (TLR) agonist-2 and TLR-3. Hence, primary hPDLSCs were stimulated with interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, TLR-2 agonist Pam3CSK4 or TLR-3 agonist Poly I/C. IDO-1 gene and protein expression and its enzymatic activity were measured up to five days after removing any stimuli. IL-1ß- and TNF-α-induced IDO-1 expression and enzymatic activity decreased in a time-dependent manner after cessation of stimulation. IFN-γ caused a long-lasting effect on IDO-1 up to five days after removing IFN-γ. Both, TLR-2 and TLR-3 agonists induced a significant increase in IDO-1 gene expression, but only TLR-3 agonist induced significantly higher IDO-1 protein expression and enzymatic activity in conditioned media (CM). IDO-1 activity of Poly I/C- and Pam3CSK4-treated hPDLSCs was higher at one day after removal of stimuli than immediately after stimulation and declined to basal levels after five days. Among all tested stimuli, only IFN-γ was able to induce long-lasting IDO-1 expression and activity in hPDLSCs. The high plasticity of IDO-1 expression and its enzymatic activity in hPDLSCs due to the variable cytokine and virulence factor milieu and the temporal-dependent responsiveness of hPDLSCs may cause a highly dynamic potential of hPDLSCs to modulate immune responses in periodontal tissues.

Response of Human Mesenchymal Stromal Cells from Periodontal Tissue to LPS Depends on the Purity but Not on the LPS Source.

Human periodontal ligament stromal cells (hPDLSCs) and gingival mesenchymal stromal cells (hGMSCs) are resident mesenchymal stromal cells (MSCs) of the periodontal tissue. The lipopolysaccharide (LPS) from Porphyromonas gingivalis is structurally distinct from that of other Gram-negative bacteria, and earlier studies linked this structural difference to a distinct virulence activity and the ability to activate toll-like receptor 2 (TLR-2), besides TLR-4 as commonly occurring upon LPS challenge. Later studies, in contrast, argue that TLR-2 activation by P. gingivalis LPS is due to lipoprotein contamination. In the present study, we aimed to define the influence of structure versus purity of P. gingivalis LPS on the immune response of hPDLSCs and hGMSCs. Cells were stimulated with commercially available "standard" P. gingivalis LPS, "ultrapure" P. gingivalis LPS, or "ultrapure" Escherichia coli LPS, and the expression of interleukin- (IL-) 8, IL-6, monocyte chemoattractant protein- (MCP-) 1, TLR-2, and TLR-4 was evaluated. The contribution of TLR-4 to the LPS-induced response was assessed using the specific TLR-4 inhibitor TAK-242. "Standard" P. gingivalis LPS induced significantly higher IL-8, IL-6, and MCP-1 production compared to the "ultrapure" LPS preparations, with no significant difference detectable for "ultrapure" LPS from P. gingivalis and E. coli. By using TAK-242, the response of hPDLSCs and hGMSCs to "ultrapure" LPS preparations was effectively inhibited to the levels comparable to those of nonstimulated controls. In contrast, high levels of response to "standard" LPS were observed, even in the presence of TAK-242. Our data show that the response of MSCs from periodontal tissue to LPS depends more on the purity of the LPS preparation than on the LPS source. Even a small amount of contaminating lipoproteins can drastically enhance the hPDLSCs' and hGMSCs; responsiveness to P. gingivalis LPS, which might also contribute to the progression of periodontal disease.

Cytokines Differently Define the Immunomodulation of Mesenchymal Stem Cells from the Periodontal Ligament.

Human periodontal ligament stem cells (hPDLSCs) play an important role in periodontal tissue homeostasis and regeneration. The function of these cells in vivo depends largely on their immunomodulatory ability, which is reciprocally regulated by immune cells via cytokines, particularly interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß. Different cytokines activate distinct signaling pathways and might differently affect immunomodulatory activities of hPDLSCs. This study directly compared the effect of IFN-γ, TNF-α, or IL-1ß treated primary hPDLSCs on allogenic CD4+ T lymphocyte proliferation and apoptosis in an indirect co-culture model. The effects of IFN-γ, TNF-α, and IL-1ß on the expression of specific immunomodulatory factors such as intoleamine-2,3-dioxygenase-1 (IDO-1), prostaglandin E2 (PGE2), and programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) in hPDLSCs were compared. The contribution of different immunomodulatory mediators to the immunomodulatory effects of hPDLSCs in the indirect co-culture experiments was assessed using specific inhibitors. Proliferation of CD4+ T lymphocytes was inhibited by hPDLSCs, and this effect was strongly enhanced by IFN-γ and IL-1ß but not by TNF-α. Apoptosis of CD4+ T lymphocytes was decreased by hPDLSCs per se. This effect was counteracted by IFN-γ or IL-1ß. Additionally, IFN-γ, TNF-α, and IL-1ß differently regulated all investigated immunomediators in hPDLSCs. Pharmacological inhibition of immunomediators showed that their contribution in regulating CD4+ T lymphocytes depends on the cytokine milieu. Our data indicate that inflammatory cytokines activate specific immunomodulatory mechanisms in hPDLSCs and the expression of particular immunomodulatory factors, which underlies a complex reciprocal interaction between hPDLSCs and CD4+ T lymphocytes.

Effect of implant surface material and roughness to the susceptibility of primary gingival fibroblasts to inflammatory stimuli.

OBJECTIVES: The impact of the implant surface material and roughness on inflammatory processes in peri-implantitis is not entirely clear. Hence, we investigated how titanium and zirconia surfaces with different roughness influence the susceptibility of primary human gingival fibroblasts to different inflammatory stimuli. METHODS: Primary human gingival fibroblasts were isolated from 8 healthy individuals and cultured on following surfaces: smooth titanium machined surface (TiM), smooth zirconia machined surface (ZrM), moderately rough titanium surface (SLA), or moderately rough zirconia surface (ZLA). Subsequently, stimulation with one of the following stimuli was performed: Porphyromonas gingivalis lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß. The resulting production of IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 was measured by qPCR and ELISA. RESULTS: P. gingivalis LPS induced IL-6 and MCP-1 production was slightly higher on titanium surfaces compared to zirconia surfaces. IL-1ß induced IL-6 production was not affected by any surface characteristic. The production of MCP-1 in response to IL-1ß was higher on smooth compared to rough surfaces and was not affected by the material. The production of IL-6 and MCP-1 in response to TNF-α was most strongly affected by surface characteristics. Higher production of these cytokine was observed on smooth compared to rough surfaces and on titanium compared to zirconia surfaces. Surface characteristics had only minor effects on IL-8 production. SIGNIFICANCE: The susceptibility of primary gingival fibroblasts to inflammation depends on various factors, such as surface material, surface roughness and the nature of inflammatory stimuli. All these factors might determine susceptibility to peri-implantitis.

Pleiotropic effects of vitamin D3 on CD4+ T lymphocytes mediated by human periodontal ligament cells and inflammatory environment.

AIMS: Both, vitamin D3 and human periodontal ligament cells (hPDLCs) possess immunosuppressive properties, but their combined effect on immune cells has never been investigated. Here, we analysed the impact of vitamin D3 on the immunosuppressive properties of hPDLCs towards CD4+ T lymphocytes. MATERIAL AND METHODS: Allogenic CD4+ T lymphocytes were activated by phytohemagglutinin either in monoculture or co-culture with hPDLCs, in the presence or absence of IFN-γ and 1,25(OH)2 D3 . After 5 days, CD4+ T-lymphocyte proliferation, CD4+ CD25+ FoxP3+ regulatory T lymphocytes (Tregs ) proportion and IL-10, TGF-ß1 and IL-17A production were analysed. RESULTS: In monoculture, 1,25(OH)2 D3 suppressed CD4+ T-lymphocyte proliferation, increased the percentage of CD4+ FoxP3+ CD25+ FoxP3+ Tregs and enhanced IL-10 and TGF-ß1 production. In the presence of IFN-γ treated hPDLCs, 1,25(OH)2 D3 significantly increased CD4+ T-lymphocyte proliferation and decreased the percentage of CD4+ CD25+ FoxP3+ Tregs . IL-10 and IL-17A expression was significantly diminished by 1,25(OH)2 D3 , whereas TGF-ß1 was slightly increased. The effects of 1,25(OH)2 D3 in co-culture were reversed by inhibition of indoleamine-2,3-dioxygenase-1, prostaglandin-endoperoxide synthase and programmed cell death 1 ligand 1. 1,25(OH)2 D3 also suppressed the expression of these proteins in hPDLCs. CONCLUSION: Effects of vitamin D3 on CD4+ T lymphocyte are modified by hPDLCs depending on the microenvironment.

1,25(OH)2D3 Differently Affects Immunomodulatory Activities of Mesenchymal Stem Cells Depending on the Presence of TNF-α, IL-1ß and IFN-γ.

Periodontal ligament-derived mesenchymal stem cells (hPDLSCs) possess immunomodulatory abilities which are strongly enhanced by various inflammatory cytokines. Vitamin D3 has anti-inflammatory effects on hPDLSCs and immune cells. However, no study to date has directly compared the influence of 1,25(OH)2D3 on the immunomodulatory activities of hPDLSCs in the presence of different cytokines. In the present study, the effects of hPDLSCs treated with tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, or interferon (IFN)-γ in the presence of 1,25(OH)2D3 on the proliferation of allogenic CD4+ T lymphocyte or on the functional status of primary CD68+ macrophages were analyzed in coculture models. Additionally, the effects of 1,25(OH)2D3 on TNF-α-, IL-1ß-, and IFN-γ-induced gene expression of some immunomodulatory factors in hPDLSCs were compared. Under coculture conditions, 1,25(OH)2D3 increased or decreased CD4+ T lymphocyte proliferation via hPDLSCs, depending on the cytokine. hPDLSCs primed with 1,25(OH)2D3 and different cytokines affected pro- and anti-inflammatory cytokine expression in macrophages variably, depending on the priming cytokine. With one exception, 1,25(OH)2D3 significantly reduced TNF-α-, IL-1ß-, and IFN-γ-induced expression of all the investigated immunomediators in hPDLSCs, albeit to different extents. These results suggest that 1,25(OH)2D3 influences the immunomodulatory activities of hPDLSCs depending qualitatively and quantitatively on the presence of certain inflammatory cytokines.

Soluble CD14 Enhances the Response of Periodontal Ligament Stem Cells to Toll-Like Receptor 2 Agonists.

Human periodontal ligament stem cells (hPDLSCs) do not express membrane-bound CD14, and their responsiveness to bacterial lipopolysaccharide (LPS) is drastically enhanced by soluble CD14 (sCD14), which is due to the facilitation of the interaction between LPS and Toll-like receptor- (TLR-) 4. Several studies also show that sCD14 enhances the responsiveness of different immune cells to TLR-2, but such effect in hPDLSCs has not been studied so far. In the present study, we investigated for the first time the potential effect of sCD14 on the hPDLSC response to two different TLR-2 agonists, in vitro. Primary hPDLSCs were stimulated with synthetic lipopeptide Pam3CSK4 or lipoteichoic acid (LTA) in concentrations 1-1000 ng/ml in the presence/absence of sCD14 (250 ng/ml). Additionally, the effect of different sCD14 concentrations (2.5-250 ng/ml) on the TLR-2 response was determined in Pam3CSK4- or LTA-triggered hPDLSCs. The resulting expression of interleukin- (IL-) 6, chemokine C-X-C motif ligand 8 (CXCL8), and chemokine C-C motif ligand 2 (CCL2) was measured by qPCR and ELISA. The production of IL-6, CXCL8, and CCL2 was gradually increased by both TLR-2 agonists and was significantly enhanced by sCD14. The response of hPDLSCs to low and submaximal concentrations of TLR-2 agonists (1-100 ng/ml) was most effectively enhanced by sCD14. The effect of sCD14 on TLR-2 response in hPDLSCs was concentration-dependent and was already detectable at low sCD14 levels. Our data showed that exogenous sCD14 significantly enhanced the responsiveness of hPDLSCs to TLR-2 agonists and enabled the detection of their small amounts. This effect was already detectable at low sCD14 levels, which are comparable to those in saliva and gingival crevicular fluid. Changes in the local sCD14 level may be considered as a crucial factor influencing the susceptibility of hPDLSCs to different pathogens and thus may contribute to the progression of periodontitis.

Synergistic effects triggered by simultaneous Toll-like receptor-2 and -3 activation in human periodontal ligament stem cells.

BACKGROUND: Although periodontitis is associated with disruption of the host-microbial homeostasis, viruses are currently discussed to influence disease progression. Viral pathogens are recognized by Toll-like receptor (TLR)-3, which engages a different signaling pathway than other TLRs. This study aimed to investigate the effect of TLR-3 agonist polyinosinic:polycytidylic acid (Poly I:C) on the expression of inflammatory markers and bone metabolism proteins by human periodontal ligament stem cells (hPDLSCs) compared with TLR-2 agonist Pam3CSK4, which mimics the effect of bacterial lipoproteins. To assess potential combined effects of bacterial and viral infections, hPDLSCs response to simultaneous TLR-2 and TLR-3 activation was investigated. METHODS: HPDLSCs were stimulated with Poly I:C (0.0001-1 µg/mL), Pam3CSK4 (1 µg/mL), and their combinations for 24 hours. Gene expression and protein levels of interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, and osteoprotegerin (OPG) were measured with qPCR and ELISA. RESULTS: Production of IL-6, IL-8, MCP-1, and OPG was significantly increased by Poly I:C or Pam3CSK4 to a similar extent. The levels of all inflammatory mediators induced by simultaneous stimulation with Poly I:C and Pam3CSK4 were significantly higher compared with single stimuli as well as to their summed response. Gene expression and protein levels of OPG were enhanced by Poly I:C, but by lesser extent than by Pam3CSK4. OPG levels upon simultaneous stimulation with Pam3CSK4 and Poly I:C were significantly lower compared with Pam3CSK4 stimulation alone. CONCLUSIONS: Simultaneous TLR-2 and TLR-3 activation synergistically triggers IL-6, IL-8, and MCP-1 production, which was not observed for OPG. These findings suggest that TLR-3 activation by viral infections might promote periodontitis progression.