Internal prostaglandin synthesis augments osteoprotegerin production in human gingival fibroblasts stimulated by lipopolysaccharide

Periodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria. Osteoclast differentiation is regulated by the balance between receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG). The purpose of this study was to examine the mechanism of OPG production in human gingival fibroblasts (HGF) stimulated by lipopolysaccharide (LPS) from periodontopathic bacteria. The expressions of Toll-like receptor 2 (TLR-2) and TLR-4 in HGF were examined using flow-cytometry. HGF were stimulated with whole cell extracts or LPS from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis with or without polymyxin B, a LPS inhibitor. In addition, HGF were stimulated with LPS, prostaglandin E(2) (PGE(2)), various agonists of PGE receptors (EP1, EP2, EP3 and EP4 agonists) with or without indomethacin (IND), a prostaglandin synthesis inhibitor. OPG and PGE(2) production was measured using an enzyme-linked immunosorbent assay (ELISA). HGF expressed both TLR-2 and TLR-4. Both A. actinomycetemcomitans and P. gingivalis LPS augmented OPG expression in HGF. Whole cell extracts from A. actinomycetemcomitans and P. gingivalis augmented OPG production by HGF; the augmentation was suppressed by polymyxin B. IND suppressed OPG production in LPS-stimulated HGF. PGE(2) stimulated HGF to produce OPG. EP1 and EP2 agonists, but not EP3 and EP4 agonists, increased OPG production by HGF. These results suggest that LPS-induced OPG production by HGF is regulated via EP1 and/or EP2 receptors by endogenously generated PGE(2).     

Antigen-presenting properties of gingival fibroblasts in chronic adult periodontitis

Chronic periodontitis is characterized by dense infiltrations of T lymphocytes in the connective tissue, which consists mainly of gingival fibroblasts. It is becoming increasingly clear that T lymphocytes and gingival fibroblasts are capable of influencing each other. For example, the T cell cytokine interferon-gamma (IFN-γ) is able to induce MHC class II molecules on the surface of several cell types, including gingival fibroblasts. Histological sections of chronically inflamed gingival tissue showed a great number of CD4⁺ and CD8⁺ T cells that produced IFN-γ, and in addition showed abundant expression of MHC class II molecules on gingival fibroblasts. Therefore, we investigated whether these gingival fibroblasts acquire the capacity to carry out MHC class II-restricted functions such as antigen presentation to local T cells. In this study, we show that IFN-γ-treated gingival fibroblasts were able to function as antigen-presenting cells (APC) for superantigen-mediated T cell proliferation. However, these fibroblasts failed to present whole-cell antigens of periodontitis-associated bacteria. Moreover, gingival fibroblasts inhibited the presentation of the whole-cell antigens of these bacteria by professional APC. This inhibition could be overcome by the addition of IL-2. These results suggest that gingival fibroblasts play an important role in the local specific immune response in chronic inflammatory periodontal lesions by regulating the response of infiltrating T cells.     

CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

Chronic periodontitis is a destructive inflammatory disease linked with unbalanced production between matrix metalloproteinases (MMPs), such as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) and their endogenous tissue inhibitors of MMPs (TIMPs). In addition to aberrant MMP-1 and MMP-3 expression, periodontal lesions are characterized by dense infiltrations of activated T lymphocytes which may interact with CD40-expressing gingival fibroblasts in the connective tissue via the CD40L-CD40 pathway. In this study we investigated whether CD40 cross-linking influenced MMP production by gingival fibroblasts. Therefore, we analysed the CD40L-induced MMP production by these fibroblasts in the presence of cytokines that are increased in periodontal lesions, such as IL-1β, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We show that CD40 ligation on gingival fibroblasts resulted in a decrease of their MMP-1 and MMP-3 production, while MMP-2 and TIMP-1 production were unaffected as determined by Western blot. This down-regulatory effect of CD40 engagement on MMP-1 and MMP-3 production by gingival fibroblasts was also present when MMP production was up-regulated by IL-1β and TNF-α or down-regulated by IFN-γ. These results suggest that CD40 ligation on gingival fibroblasts leads to a restraining of MMP-1 and MMP-3 production by gingival fibroblasts and thereby may be an important mechanism in the retardation of further periodontal tissue damage.     

IFN-gamma-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL

The current study explored our hypothesis that IFN-gamma-producing human T cells inhibit human osteoclast formation. Activated T cells derived from human PBMC were divided into IFN-gamma-producing T cells (IFN-gamma(+) T cells) and IFN-gamma-non-producing T cells (IFN-gamma(-) T cells). IFN-gamma(+) T cells were cultured with human monocytes in the presence of macrophage-CSF alone. The concentration of soluble receptor activator of NF-kappaB ligand (RANKL) and IFN-gamma, and the amount of membrane type RANKL expressed on T cells, were measured by ELISA. In the patients with early rheumatoid arthritis (RA) treated with non-steroidal anti-inflammatory drugs alone, CD4+ T cells expressing both IFN-gamma and RANKL were detected by flow cytometry. Surprisingly, IFN-gamma(+) T cells, but not IFN-gamma(-) T cells, induced osteoclastogenesis from monocytes, which was completely inhibited by adding osteoprotegerin and increased by adding anti-IFN-gamma antibodies. The levels of both soluble and membrane type RANKL were elevated in IFN-gamma(+) T cells. The ratio of CD4+ T cells expressing both IFN-gamma and RANKL in total CD4+ T cells from PBMC was elevated in RA patients. Contrary to our hypothesis, IFN-gamma(+) human T cells induced osteoclastogenesis through the expression of RANKL, suggesting that Th1 cells play a direct role in bone resorption in Th1 dominant diseases such as RA.     

Isolation of the multipotent MSC subpopulation from human gingival fibroblasts by culturing on chitosan membranes

Literature has different opinions regarding the percentage of mesenchymal stem cell (MSC)-like population in human gingival tissue. Isolation of these cells is thus important for clinical applications. In this study, two typical but distinct types of gingival fibroblasts (GF), GF-A and GF-B, were grown from human gingival biopsies. They were characterized for surface markers by flow cytometry as well as the expressions of stemness and neural crest marker genes by RT-PCR. The two types of GF were slightly different in their surface markers; however, they had dramatic difference in the expression levels of stemness marker genes and neural crest marker genes. They also demonstrated distinct differentiation capacity. Upon the appropriate induction, GF-A were capable of osteogenic, adipogenic, chondrogenic, and neurogenic differentiation while GF-B only underwent osteogenic differentiation. By culturing either type of GF on chitosan membranes for 24 h, we were able to isolate two distinct subpopulations in each type of GF, i.e. cells with spheroid-forming ability (GF-AS and GF-BS) or those remained flat and attached (GF-AN and GF-BN). We further characterized these cells, and determined the common properties shared by the spheroid-forming subpopulation “S”, as well as those shared by the non-spheroid-forming subpopulation “N”. The subpopulation “S” was capable of the multilineage differentiation, while the subpopulation “N” was only efficient in osteogenic differentiation. GF-A and GF-B had different proportions of subpopulations. Chitosan as the cell culture substratum up-regulated the N-cadherin expression of the “S” but not “N” subpopulation, which may account for the cell sorting effect. This study showed that chitosan membranes could be used for isolation of the spheroid forming subpopulation in human GF that contained multipotent adult stem cells of which the number varied among donors and sites.     

A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells

BACKGROUND: Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells. METHODS: We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment. RESULTS: Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24-36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing. CONCLUSIONS: We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.     

Cytolethal distending toxin upregulates RANKL expression in Jurkat T-cells

Cytolethal distending toxin, a bacterial exotoxin produced by a number of Gram-negative species, causes growth arrest and morphological alterations in host cells. Among these species are Haemophilus ducreyi, the etiological agent of chancroid, and the periodontal pathogen Aggregatibacter actinomycetemcomitans, highly implicated in localized aggressive periodontitis. CDT induces receptor activator of NF-kappaB ligand (RANKL) expression in periodontal fibroblasts, the key bone-resorbing cytokine. T-cells are actively involved in localized inflammation-induced bone destruction, including periodontitis. The aim of this study was to investigate the effects of purified CDT on the expression of RANKL and its decoy receptor osteoprotegerin (OPG), in the Jurkat T-cell line. Quantitative real-time PCR indicated that 100 pg/ml of purified H. ducreyi CDT upregulated RANKL mRNA expression by 2.2-fold, after 24 h of exposure. This increase was corroborated by a 2.0-fold increase in RANKL protein release, as determined by ELISA. OPG was not detected in this experimental system. In conclusion, CDT enhances RANKL expression in T-cells, denoting that these cells are a potential target for the toxin and strengthening the potential link between this virulence factor and mechanisms associated with localized bone resorption.     

Adenosine regulates the IL-1 beta-induced cellular functions of human gingival fibroblasts.

In this study we examined the influence of adenosine on the cellular functions of human gingival fibroblasts (HGF), such as the production of inflammatory cytokines and extracellular matrices (ECM), and the expression and function of adhesion molecules. Concerning the expression of adenosine receptors, RT-PCR analysis revealed that HGF expressed adenosine receptor A1, A2a and A2b, but not A3 mRNA. Ligation of adenosine receptors by adenosine or its related analogue, 2-chloroadenosine (2-CADO), N(6)-cyclopentyladenosine (CPA) or CGS21680 synergistically increased IL-1beta-induced IL-6 and IL-8 production. In terms of ECM expression, adenosine and the adenosine receptor agonists, 2-CADO and CPA, enhanced constitutive and IL-1beta-induced expression of hyaluronate synthase mRNA, but not the mRNA levels of other ECM, such as collagen type I, III and fibronectin. Moreover, the adherence of IL-1beta-stimulated HGF to activated lymphocytes was also inhibited by adenosine, which is in part explained by the fact that adenosine down-regulated the IL-1beta-induced expression of ICAM-1 on HGF. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses in periodontal tissues.     

Regulation of RANKL and OPG gene expression in human gingival fibroblasts and periodontal ligament cells by Porphyromonas gingivalis: a putative role of the Arg-gingipains

Porphyromonas gingivalis is highly implicated in the pathogenesis of periodontitis, which is characterized by the destruction of periodontal connective tissues and the supporting alveolar bone. Receptor Activator of NF-kappaB Ligand (RANKL) stimulates bone resorption, whereas osteoprotegerin (OPG) blocks its action, and this bi-molecular system is implicated in periodontitis. The aim of this work was (a) to investigate the regulation of RANKL and OPG gene expression in human periodontal ligament (PDL) cells and gingival fibroblasts (GF), in response to P. gingivalis culture supernatants, by quantitative real-time PCR and (b) to attempt to identify putative virulence factors involved in this process. The results indicated that P. gingivalis induced RANKL and reduced OPG mRNA expression by the studied cells, resulting in an increased RANKL/OPG expression ratio. Heat-inactivation of P. gingivalis resulted in significant reduction of RANKL mRNA expression. A Lys-gingipain mutant strain did not affect, whereas an Arg-gingipain mutant strain further enhanced RANKL mRNA expression, compared to their parental wild-type strain. In conclusion, P. gingivalis up-regulates the RANKL/OPG expression ratio in GF and PDL cells, denoting an enhanced osteoclastogenic potential by the cells. The component mainly responsible for RANKL induction appears to be proteinaceous, and it may be regulated by the Arg-gingipains.     

Insights into the role of fibroblasts in human autoimmune diseases

Traditional wisdom has considered fibroblasts as contributing to the structural integrity of tissues rather than playing a dynamic role in physiological or pathological processes. It is only recently that they have been recognized as comprising diverse populations of cells exhibiting complex patterns of biosynthetic activity. They represent determinants that react to stimuli and help define tissue remodelling through the expression of molecules imposing constraints on their cellular neighbourhood. Moreover, fibroblasts can initiate the earliest molecular events leading to inflammatory responses. Thus they must now be viewed as active participants in tissue reactivity. In this short review, I will provide an overview of contemporary thought about the contribution of fibroblasts to the pathogenesis of autoimmune processes through their expression of, and responses to, mediators of inflammation and tissue remodelling.     

成纤维细胞的生物学特性及分化潜能

文题释义：多向分化潜能：指细胞具有自我更新、持续增殖分裂并可以在不同微环境影响下向相应方向分化为多种组织的能力,即经过不同细胞因子的诱导,能够分化为脂肪、骨、软骨、肌肉、血管内皮、肝、神经等细胞系类型,一般为干细胞或类干细胞所具备的能力。组织工程种子细胞：应用组织工程方法再造组织和器官所用的各类细胞,其中包括分化成熟的成体细胞和具有分化潜能的干细胞,应满足以下几点：采用微创或无创手段即可获取的组织,分裂增殖能力强,功能旺盛,无或仅有极微弱的免疫排斥反应,能够连续传代,且传代培养后不发生形态、功能及遗传物质的改变。背景：组织工程技术的出现可以从根本上解决组织、器官的修复与重建问题,其中种子细胞的选择和应用则成为当前研究的热难点,成纤维细胞是各组织工程研究的热门选择。目的：总结分析成纤维细胞的生物学特性、自身分化潜能以及对干细胞分化增殖的影响。方法：运用计算机检索中国期刊全文数据库(2015至2019年)和PubMed数据库(2005至2019年)中有关于成纤维细胞生物学特性以及多向分化潜能的相关文献,并进行系统的归纳、总结和分析,对成纤维“干性”的研究新进展进行全面阐述。结果与结论：成纤维细胞代谢旺盛、增殖能力强,具有合成和分泌蛋白质的功能,在不同微环境中可分化为不同细胞,拥有与干细胞一样强大的多向分化潜能,因此常运用于转分化、细胞培养、损伤修复以及组织工程,其中促进干细胞增殖及诱导分化的作用尤为显著,今后应最大化利用成纤维细胞生物学优势与干细胞共培养,为组织工程学提供种子细胞,为临床中创伤的修复提供更佳思路。ORCID: 0000-0002-4261-361X(杨桂然)中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Streptococcus mitis/human gingival fibroblasts co-culture: the best natural association in answer to the 2-hydroxyethyl methacrylate release

One of the major components of dental polymerized resin-based restorative materials is 2-hydroxyethyl methacrylate (HEMA) and its release in monomeric form interferes with the oral cavity environment. This study aimed to evaluate HEMA monomeric effects on the co-culture of Streptococcus mitis and human gingival fibroblasts (HGFs). Streptococcus mitis DS12 and S. mitis ATCC 6249 were co-cultivated with HGF in the presence of HEMA (3 mM), for 48 and 72 h; the amount of sessile and planktonic cells, as well as the prokaryotic and eukaryotic cell viability were analyzed in treated and untreated samples. The treatment of S. mitis/HGFs with HEMA did not produce significant effects on the bacterial adhesion and induced an increase in planktonic S. mitis ATCC 6249 population after 48 and 72 h. HEMA increased significantly the planktonic S. mitis ATCC 6249 viability when co-cultured with HGFs, while a cytotoxic effect on HGFs, without bacteria, was recorded. An increase of bacterial aggregation on HGFs was also detected with HEMA. Data obtained in this study suggest that HEMA exhibits a toxic effect mainly on eukaryotic cells and this effect can be modulated by co-cultivation with the S. mitis cells which, in the presence of the monomer, enhance their aggregation rate on HGFs.     

Capability of human mesenchymal cells isolated from different sources to differentiation into tissues of mesodermal origin

We compared the capacity of cultured human skin fibroblasts, human umbilical cord cells obtained after normal delivery on gestation week 38–40, and mesenchymal bone marrow stem cells to differentiation into adipocytes, osteoblasts, and chondrocytes. Our findings suggest that mesenchymal stem cells are multipotent cells and can differentiate into adipose, cartilaginous, and bone tissue. Umbilical cord fibroblast-like cells can differentiate into adipocytes and chondrocytes, and only few cells in this culture can differentiate into osteoblasts. Skin fibroblasts differentiate only into adipocytes. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 1, pp. 3–10, January, 2007     

Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1–3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic–osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic–osteogenic coupling.     

Accelerated in vitro differentiation of blood monocytes into dendritic cells in human sepsis

Summary Sepsis-induced immune depression is characterized by infection susceptibility and monocyte early deactivation. Because monocytes are precursors for dendritic cells (DC), alterations in their differentiation into DC may contribute to defective immune responses in septic patients. We therefore investigated the ability of monocytes to differentiate into functional DC in vitro in patients undergoing surgery for peritonitis. Monocytes from 20 patients collected immediately after surgery (D0), at week 1 and at weeks 3-4 and from 11 control donors were differentiated into immature DC. We determined the phenotype of monocytes and derived DC, and analysed the ability of DC to respond to microbial products and to elicit T cell responses in a mixed leucocyte reaction (MLR). We show that, although monocytes from septic patients were deactivated with decreased responses to lipopolysaccharide (LPS) and peptidoglycan and low human leucocyte antigen D-related (HLA-DR) expression, they expressed the co-stimulatory molecule CD80, CD40 and CCR7. Monocytes collected from patients at D0 and week 1 differentiated faster into DC with early loss of CD14 expression. Expression of HLA-DR increased dramatically in culture to reach control levels, as did responses of DC to LPS and peptidoglycan. However, although patient and control immature DC had similar abilities to induce T cell proliferation in MLR, maturation of DC derived from patients did not increase T cell responses. These results show that circulating monocytes from septic patients express markers of activation and/or differentiation despite functional deactivation, and differentiate rapidly into phenotypically normal DC. These DC fail, however, to increase their T cell activation abilities upon maturation.     

Increased expression of receptor activator of NF-kappaB ligand (RANKL), its receptor RANK and its decoy receptor osteoprotegerin in the colon of Crohn's disease patients

Crohn's disease (CD) is associated with low bone mass due to chronic inflammation and other factors. Receptor activator of NF-kappaB ligand (RANKL), its receptor RANK and its decoy receptor osteoprotegerin (OPG) are potentially involved in this process as they regulate osteoclastogenesis and are influenced by pro-inflammatory cytokines. The aim of this study was to determine the levels of soluble RANKL (sRANKL), RANK and OPG expression both in the serum and in the colon of CD patients. Levels of sRANKL and OPG were assessed in the serum and the supernatants of cultured colonic biopsies in patients with CD and controls by ELISA. RANK expression was explored by immunostaining and immunofluorescence of fixed colonic samples. OPG and sRANKL levels were higher in the serum of CD patients as compared to age- and sex-matched controls. Levels of sRANKL and OPG were significantly enhanced in cultured colonic biopsies from CD, and OPG levels correlated with histological inflammation, and pro- and anti-inflammatory cytokine levels. No significant correlation was found for sRANKL. RANK+ cells were increased in the colon of CD, particularly in inflamed areas. These cells were positive for CD68 or S100 protein. We conclude that serum and local levels of sRANKL and OPG are increased in CD. Moreover, RANK is expressed in the colonic mucosa by subpopulations of activated macrophages or dendritic cells at higher levels in CD compared to normal colon.     

Bee venom enhances the differentiation of human regulatory T cells

Venom‐specific immunotherapy (VIT) is well recognized by its efficacy, and compelling evidence implicates regulatory T cells (Tregs) in the underlying tolerogenic mechanisms. Additionally, hymenoptera venom has for a long time been claimed to modulate immunity. Here, we investigated the putative role of bee venom (Bv) in human FOXP3‐expressing Treg homeostasis and differentiation, irrespective of the donors' allergic status. We found that Bv significantly enhanced the differentiation of FOXP3‐expressing cells both from conventional naïve CD4 T cells and mature CD4 thymocytes, a property that may contribute to the VIT′s capacity to expand circulating Tregs in allergic individuals. We expect that our data enlightening the Treg‐mediated immunomodulatory properties of Bv regardless of TCR specificity, to have application in other allergies, as well as in other clinical settings, such as autoimmunity and transplantation.