Immunomodulatory Properties of Dental-Derived Mesenchymal Stem Cells

Mesenchymal stem cells are considered as an attractive tool for tissue regeneration. Almost all dental tissues contain a population of MSC-like cells, which were extensively studied within the last few years. Besides their ability to differentiate into different cell types, dental MSCs also possess strong immunomodulatory properties. Dental MSCs modulate both innate and adaptive immune response and influence the activity of almost all components of the immune system. The interaction between dental MSCs and the immune system is reciprocal because immunomodulatory activity of MSCs is strongly regulated by cytokines produced by immune cells. MSCs isolated from inflamed tissues might exhibit impaired immunomodulatory capacity, suggesting a potential role of these cells in inflammatory diseases and particularly periodontitis. Recent studies suggest that immunomodulatory properties of MSCs can also play an important role in their tissue regenerative capacity. The therapeutic effects of MSCs, including their immunomodulatory capacity, are largely explained by their tropic activity, including production of immunomodulatory proteins and growth factors. Summarizing, dental MSCs play an important role in tissue homeostasis under healthy and diseased conditions

Effect of Vitamin D3 on periodontium derived mesenchymal stem cells in health and disease

Mesenchymale Stammzellen (MSCs) des Parodonts sind an der parodontalen Gewebehomöostase beteiligt. Sie zeichnen sich durch Differenzierung in verschiedene Zelltypen wie Osteoblasten aus und besitzen auch immunmodulierende Funktionen. Studien haben gezeigt, dass diese Eigenschaften durch Vitamin D3 beeinflusst werden. Die vorliegende Dissertation beschäftigte sich mit den Effekten von Vitamin D3 auf die osteogene Differenzierung, dem Vitamin D3 Metabolismus und immunmodulierende Eigenschaften von parodontalen MSCs unter inflammatorischen Bedingungen. Dafür wurden zum einen primäre MSCs aus dem parodontalen Ligament (hPDLSCs) isoliert und mit Vitamin D3 Metaboliten unter physiologischen und inflammatorischen Bedingungen stimuliert. Letztere wurden durch die Toll-like Rezeptor Agonisten Porphromonas gingivalis Lipopolysaccharid oder Pam3CSK4 simuliert. Zudem erfolgten die Versuche unter dem Einfluss von Cytochrom P-450- und NF-B-Inhibitoren. Im Anschluss wurde die Expression von Osteogenese-assoziierten Proteinen, Enzymen des Vitamin D3 Metabolismus und dem Vitamin D Rezeptor (VDR) analysiert, sowie die Osteogenese-Induzierung untersucht. Zweitens wurden gingivale MSCs (hGMSCs) isoliert und mit Vitamin D3 Metaboliten in An- und Abwesenheit der proinflammatorischer Zytokine Interleukin 1 oder Tumornekrosefaktor stimuliert. Anschließend erfolgte die Isolierung neutrophiler Granulozyten (PMNs), die direkt mit den hGMSCs inkubiert oder mit deren konditionierten Medium stimuliert wurden. Nachfolgend wurde die Produktion reaktiver Sauerstoffspezies (ROS) und Apoptose von PMNs mittels Durchflusszytometrie gemessen. Die Effekte von Vitamin D3 auf die osteogene Kapazität von hPDLSCs wurden unter inflammatorsichen Bedingungen signifikant inhibiert, insbesondere die Expression von Osteocalcin und Osteopontin. Der Einfluss inflammatorischer Stimuli wurde teils durch Cytochrom P-450- und vollständig durch NF-B-Inhibierung umgekehrt. Während die VDR-Expression unverändert blieb, wurde das Vitamin D3-abbauende Enzym signifikant beeinflusst. hGMSCs hatten einen signifikanten Effekt auf die physiologischen Funktionen von PMNs. Diese waren abhängig von der Art der Stimulierung, den pro-inflammatorischen Zytokinen und zum Teil von Vitamin D3. Zusammenfassend konnte gezeigt werden, dass die Effekte von Vitamin D3 auf parodontale MSCs unter inflammatorsichen Bedingungen inhibiert sind und durch verschieden Faktoren reguliert werden.Periodontium derived mesenchymal stem cells (MSCs) play an important role in maintaining periodontal tissue homeostasis. Apart from their ability to differentiate into various cell types such as osteoblasts, they possess strong immunomodulatory functions. Both of these properties have been shown to be modulated by vitamin D3. This thesis aimed to investigate possible alterations of the vitamin D3mediated effects on the osteogenic capacity, vitamin D3 metabolism and immunomodulatory properties of periodontium derived MSCs under inflammatory conditions. Two different approaches were followed in order to achieve these aims. First, primary MSCs isolated from periodontal ligament (hPDLSCs) were treated with vitamin D3 metabolites under physiological and inflammatory conditions, which were simulated with Toll-like receptor agonists Porphyromonas gingivalis lipopolysaccharide or Pam3CSK4. In addition, experiments were conducted under inhibition of cytochrome P-450 or NF-B. Resulting expression of osteogenesis-related proteins, enzymes of the vitamin D3 metabolism and vitamin D receptor (VDR), as well as induction of osteogenesis were investigated. Second, primary gingival MSCs (hGMSCs) were isolated and treated with vitamin D3 metabolites in the presence and absence of pro-inflammatory cytokines interleukin-1 or tumor necrosis factor-. Subsequently, polymorphonoculear leukocytes (PMNs), the cells constituting the first line of host defense, were isolated from whole blood and incubated with pretreated hGMSCs or stimulated with their conditioned media. The effects on PMN apoptosis and their production of reactive oxygen species (ROS) were evaluated by flow cytometry. The effects of vitamin D3 on the osteogenic capacity of hPDLSCs were significantly attenuated under inflammatory conditions, particularly the expression of osteogenesis-related proteins osteocalcin and osteopontin. The effects of inflammatory stimuli were partially reversed by cytochrome P-450 and prevented by NF-B inhibition. While the expression of VDR remained unaltered, the expression of the enzyme responsible for degrading vitamin D3 was significantly affected by inflammatory conditions. hGMSCs had a significant impact on the physiological functions of PMNs, which was dependent on the treatment modality, pro-inflammatory cytokine and partially vitamin D3. In conclusion, the effects of vitamin D3 on periodontium derived MSCs have been found to be altered under inflammatory conditions and can be regulated by several factors.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftMedizinische Universität Wien, Diss., 2020(VLID)504077

The Alterations in CD14 Expression in Periodontitis: A Systematic Review

Objective: Cluster of differentiation (CD14) is an important protein involved in activating toll-like receptors by bacterial components. It exists as either a transmembrane or soluble protein, called mCD14 or sCD14, respectively. Several studies show that CD14 regulates the inflammatory response to periodontal pathogens, and its expression is altered in periodontitis, an inflammatory disease of tooth-supporting tissues. It is the intent of this review to investigate the levels of expression of mCD14 and sCD14 in peripheral blood monocytes, saliva, gingival crevicular fluid, and gingival tissue biopsies in periodontitis patients. Methods: PubMed, Scopus, Ovid/Medline, Embase, and the Cochrane Library were consulted for the online literature search. To ensure methodical quality, titles and abstracts were reviewed in accordance to the PRISMA guidelines. Data extraction and evaluation of the full texts were executed in agreement with the GRADE approach. Results: This systematic review shows that mCD14 levels are decreased in peripheral blood monocytes of periodontitis patients in comparison to healthy patients, while sCD14 levels in sera, gingival crevicular fluid (GCF), and biopsies of periodontitis patients have a tendency to be increased in comparison to healthy controls. The evaluation of CD14 in gingival biopsies and periodontal tissues elucidated the fact that interpretation of the data obtained with qPCR, ELISA, and flow cytometry is questionable

Vitamin D3 and Dental Mesenchymal Stromal Cells

Vitamin D3 is a hormone involved in the regulation of bone metabolism, mineral homeostasis, and immune response. Almost all dental tissues contain resident mesenchymal stromal cells (MSCs), which are largely similar to bone marrow-derived MSCs. In this narrative review, we summarized the current findings concerning the physiological effects of vitamin D3 on dental MSCs. The existing literature suggests that dental MSCs possess the ability to convert vitamin D3 into 25(OH)D3 and subsequently to the biologically active 1,25(OH)2D3. The vitamin D3 metabolites 25(OH)D3 and 1,25(OH)2D3 stimulate osteogenic differentiation and diminish the inflammatory response of dental MSCs. In addition, 1,25(OH)2D3 influences the immunomodulatory properties of MSCs in different dental tissues. Thus, dental MSCs are both producers and targets of 1,25(OH)2D3 and might regulate the local vitamin D3-dependent processes in an autocrine/paracrine manner. The local vitamin D3 metabolism is assumed to play an essential role in the local physiological processes, but the mechanisms of its regulation in dental MSCs are mostly unknown. The alteration of the local vitamin D3 metabolism may unravel novel therapeutic modalities for the treatment of periodontitis as well as new strategies for dental tissue regeneration

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)-1β, 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-1β- 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

Interleukin-1β Induced Matrix Metalloproteinase Expression in Human Periodontal Ligament-Derived Mesenchymal Stromal Cells under In Vitro Simulated Static Orthodontic Forces

The periodontal ligament (PDL) responds to applied orthodontic forces by extracellular matrix (ECM) remodeling, in which human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) are largely involved by producing matrix metalloproteinases (MMPs) and their local inhibitors (TIMPs). Apart from orthodontic forces, the synthesis of MMPs and TIMPs is influenced by the aseptic inflammation occurring during orthodontic treatment. Interleukin (IL)-1β is one of the most abundant inflammatory mediators in this process and crucially affects the expression of MMPs and TIMPs in the presence of cyclic low-magnitude orthodontic tensile forces. In this study we aimed to investigate, for the first time, how IL-1β induced expression of MMPs, TIMPs and how IL-1β in hPDL-MSCs was changed after applying in vitro low-magnitude orthodontic tensile strains in a static application mode. Hence, primary hPDL-MSCs were stimulated with IL-1β in combination with static tensile strains (STS) with 6% elongation. After 6- and 24 h, MMP-1, MMP-2, TIMP-1 and IL-1β expression levels were measured. STS alone had no influence on the basal expression of investigated target genes, whereas IL-1β caused increased expression of these genes. In combination, they increased the gene and protein expression of MMP-1 and the gene expression of MMP-2 after 24 h. After 6 h, STS reduced IL-1β-induced MMP-1 synthesis and MMP-2 gene expression. IL-1β-induced TIMP-1 gene expression was decreased by STS after 6- and 24-h. At both time points, the IL-1β-induced gene expression of IL-1β was increased. Additionally, this study showed that fetal bovine serum (FBS) caused an overall suppression of IL-1β-induced expression of MMP-1, MMP-2 and TIMP-1. Further, it caused lower or opposite effects of STS on IL-1β-induced expression. These observations suggest that low-magnitude orthodontic tensile strains may favor a more inflammatory and destructive response of hPDL-MSCs when using a static application form and that this response is highly influenced by the presence of FBS in vitro

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

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

Response of human periodontal ligament stem cells to IFN-γ and TLR-agonists

Abstract Periodontal ligament stem cells similarly to the mesenchymal stem cells of other tissues possess immunomodulatory properties, which are regulated by different cytokines, particularly by interferon-γ (IFN-γ). In contrast, less information is provided about the effect of toll-like receptors ligand on immunomodulatory properties of these cells. In the present study we investigated the response of human periodontal ligament stem cells (hPDLSCs) in response to simultaneous stimulation with IFN-γ and toll-like receptor (TLR) agonists. The resulting expression of indoleamine-2,3-dioxygenase-1 (IDO-1), interleukin (IL)-6, IL-8 and monocyte chemotactic protein 1 (MCP-1) was investigated. The expression of IDO-1 was upregulated by IFN-γ in both gene and protein levels. TLR2 agonists Pam3CSK4 induced gene expression of IDO-1, but had no effect on protein expression. IFN-γ induced IDO-1 protein expression was further enhanced by Pam3CSK4. TLR-4 agonist E. coli LPS has no significant effect on neither basal nor IFN-γ induced IDO-1 protein expression. The production of IL-6, IL-8, and MCP-1 was induced by TLR agonists. Neither basal nor TLR agonists induced production of these proteins was affected by IFN-γ. Our data shows potential interaction between IFN-γ and TLR2 responses in hPDLSCs, which might be involved in regulation of immune response in inflammatory diseases, and particularly periodontitis

Interleukin-1β Induced Matrix Metalloproteinase Expression in Human Periodontal Ligament-Derived Mesenchymal Stromal Cells under In Vitro Simulated Static Orthodontic Forces

The periodontal ligament (PDL) responds to applied orthodontic forces by extracellular matrix (ECM) remodeling, in which human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) are largely involved by producing matrix metalloproteinases (MMPs) and their local inhibitors (TIMPs). Apart from orthodontic forces, the synthesis of MMPs and TIMPs is influenced by the aseptic inflammation occurring during orthodontic treatment. Interleukin (IL)-1β is one of the most abundant inflammatory mediators in this process and crucially affects the expression of MMPs and TIMPs in the presence of cyclic low-magnitude orthodontic tensile forces. In this study we aimed to investigate, for the first time, how IL-1β induced expression of MMPs, TIMPs and how IL-1β in hPDL-MSCs was changed after applying in vitro low-magnitude orthodontic tensile strains in a static application mode. Hence, primary hPDL-MSCs were stimulated with IL-1β in combination with static tensile strains (STS) with 6% elongation. After 6- and 24 h, MMP-1, MMP-2, TIMP-1 and IL-1β expression levels were measured. STS alone had no influence on the basal expression of investigated target genes, whereas IL-1β caused increased expression of these genes. In combination, they increased the gene and protein expression of MMP-1 and the gene expression of MMP-2 after 24 h. After 6 h, STS reduced IL-1β-induced MMP-1 synthesis and MMP-2 gene expression. IL-1β-induced TIMP-1 gene expression was decreased by STS after 6- and 24-h. At both time points, the IL-1β-induced gene expression of IL-1β was increased. Additionally, this study showed that fetal bovine serum (FBS) caused an overall suppression of IL-1β-induced expression of MMP-1, MMP-2 and TIMP-1. Further, it caused lower or opposite effects of STS on IL-1β-induced expression. These observations suggest that low-magnitude orthodontic tensile strains may favor a more inflammatory and destructive response of hPDL-MSCs when using a static application form and that this response is highly influenced by the presence of FBS in vitro