Jianxia Hou

The pro-apoptotic and pro-inflammatory effects of calprotectin on human periodontal ligament cells.

Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is associated with inflammatory disorders such as rheumatoid arthritis and cystic fibrosis. Although calprotectin levels are increased significantly in the gingival crevicular fluid (GCF) of periodontitis patients, its effects on periodontal ligament cells (PDLCs) remain largely unknown. The aim of this study was to evaluate calprotectin levels in the GCF of generalized aggressive periodontitis (AgP) patients and to investigate the effects of recombinant human calprotectin (rhS100A8/A9) and its subunits (rhS100A8 and rhS100A9) in PDLCs. Both the concentration and amount of crevicular calprotectin were significantly higher in the AgP group compared with healthy controls. In addition, the GCF calprotectin levels were correlated positively with clinical periodontal parameters including bleeding index, probing depth, and clinical attachment loss. rhS100A8/A9 promoted cell apoptosis, whereas rhS100A8 and rhS100A9 individually exerted little effect on apoptosis in PDLCs. rhS100A9 and rhS100A8/A9 increased the activation of nuclear factor-κB (NF-κB) by promoting the nuclear translocation of p65 in PDLCs, subsequently inducing expression of the pro-inflammatory cytokines IL-6, IL-8, TNFα, and COX2. Treatment with an NF-κB inhibitor partially reversed the rhS100A9- and rhS100A8/A9-induced upregulation of the pro-inflammatory cytokines. rhS100A9, and not rhS100A8, was mainly responsible for the pro-inflammatory role of calprotectin. Collectively, our results suggest that calprotectin promotes apoptosis and the inflammatory response in PDLCs via rhS100A9. These findings might help identify novel treatments for periodontitis.

Characterization of the Autocrine/Paracrine Function of Vitamin D in Human Gingival Fibroblasts and Periodontal Ligament Cells

Background We previously demonstrated that 25-hydroxyvitamin D3, the precursor of 1α,25-dihydroxyvitamin D3, is abundant around periodontal soft tissues. Here we investigate whether 25-hydroxyvitamin D3 is converted to 1α,25-dihydroxyvitamin D3 in periodontal soft tissue cells and explore the possibility of an autocrine/paracrine function of 1α,25-dihydroxyvitamin D3 in periodontal soft tissue cells. Methodology/Principal Findings We established primary cultures of human gingival fibroblasts and human periodontal ligament cells from 5 individual donors. We demonstrated that 1α-hydroxylase was expressed in human gingival fibroblasts and periodontal ligament cells, as was cubilin. After incubation with the 1α-hydroxylase substrate 25-hydroxyvitamin D3, human gingival fibroblasts and periodontal ligament cells generated detectable 1α,25-dihydroxyvitamin D3 that resulted in an up-regulation of CYP24A1 and RANKL mRNA. A specific knockdown of 1α-hydroxylase in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 1α,25-dihydroxyvitamin D3 production and mRNA expression of CYP24A1 and RANKL. The classical renal regulators of 1α-hydroxylase (parathyroid hormone, calcium and 1α,25-dihydroxyvitamin D3) and Porphyromonas gingivalis lipopolysaccharide did not influence 1α-hydroxylase expression significantly, however, interleukin-1β and sodium butyrate strongly induced 1α-hydroxylase expression in human gingival fibroblasts and periodontal ligament cells. Conclusions/Significance In this study, the expression, activity and functionality of 1α-hydroxylase were detected in human gingival fibroblasts and periodontal ligament cells, raising the possibility that vitamin D acts in an autocrine/paracrine manner in these cells.

Activity of 25-hydroxylase in human gingival fibroblasts and periodontal ligament cells.

Background We previously demonstrated that 25-hydroxyvitamin D3 concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D3 can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells. Methodology/Principal Findings Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D3, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D3 that resulted in the production of 1α,25-dihydroxyvitamin D3. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D3 synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells. Conclusions The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.

S100A9-induced release of interleukin (IL)-6 and IL-8 through toll-like receptor 4 (TLR4) in human periodontal ligament cells

S100A8, S100A9, and calprotectin (the S100A8/S100A9 complex) are calcium-binding proteins that promote extracellular pro-inflammatory functions and may play an important role in periodontal disease. Both toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE) are thought to be important receptors for S100A8, S100A9, and calprotectin, but the specific pathways in periodontal ligament (PDL) cells are not yet clear. Our study was designed to identify the specific receptors for S100A9 in human PDL cells. Additionally, we investigated the specific pathways that activate the secretion of pro-inflammatory cytokines interleukins (IL)-6 and IL-8 in PDL cells. The role of nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) in S100A9-induced pro-inflammatory cytokines were investigated through western blot analysis, dichlorodihydrofluorescein diacetate (H2DCFDA) probe and the application of specific pathway inhibitors. Our results suggest that the S100A9-induced release of IL-6 and IL-8 from human PDL cells is dependent on TLR4, but not RAGE. We provide evidence that S100A9 promotes the secretion of IL-6 and IL-8 through different pathways. Specifically, S100A9 up-regulates the secretion of IL-6 from human PDL cells through NF-κB and p38 pathways and up-regulates the release of IL-8 from human PDL cells through the NF-κB, extracellular-regulated kinase (ERK) 1/2, c-Jun amino-terminal kinase (JNK) 1/2, and p38 signaling pathways. In addition, the release of both cytokines depends on ROS production. The release of both cytokines depends on ROS production. These results suggest that S100A9 promotes pro-inflammatory responses in PDL cells through the TLR4-mediated NF-κB and MAPK signaling pathways.

Proinflammatory effects and mechanisms of calprotectin on human gingival fibroblasts

BACKGROUND AND OBJECTIVE Calprotectin (S100A8/A9) is a heterodimer of S100A8 and S100A9 and is associated with multiple inflammatory diseases, including Crohns disease, rheumatoid arthritis and periodontitis. Levels of calprotectin are elevated in the gingival crevicular fluid of patients with periodontitis; however, the effects of calprotectin on human gingival fibroblasts (HGFs) remain unknown. This study investigated the proinflammatory activity of calprotectin on HGFs and the functional receptors and signaling pathways engaged by calprotectin. MATERIAL AND METHODS HGFs were stimulated by equimolar concentrations of S100A8 and/or S100A9, and the expression levels of interleukin (IL)-6 and IL-8 were detected using real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. The calprotectin receptors were identified by pre-incubating HGFs with the toll-like receptor (TLR) 4 inhibitor or the antibody targeting the advanced glycation end product receptor (RAGE). The involvement of reactive oxygen species (ROS) and signaling pathways were also investigated by treating HGFs with ROS inhibitor or specific pathway inhibitors, respectively. RESULTS S100A9 and S100A8/A9 significantly upregulated IL-6 and IL-8 expression, which was inhibited upon treatment with the TLR4 inhibitor TAK242. Pretreatment with RAGE-blocking antibodies did not affect cytokine expression. Additionally, S100A9 promoted the production of IL-6 and IL-8 from HGFs via different signaling pathways. IL-6 expression was upregulated via the NF-κB, c-Jun amino-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK) pathways, and IL-8 expression was upregulated via NF-κB, p38, JNK1/2 and extracellular-regulated kinase 1/2 MAPK pathways. The release of both cytokines was dependent upon the production of ROS. CONCLUSION Our findings suggest that calprotectin exerts proinflammatory effects on HGFs via the S100A9 subunit and TLR4-mediated NF-κB and MAPK signaling pathways.

Leptin and its receptor expression in dental and periodontal tissues of primates

Leptin and its receptor (OBR) have attracted much attention since their discovery. They have been reported to play central roles in energy balance, the immune-inflammatory response and bone metabolism. Evidence indicates that leptin and OBR are associated with inflammatory diseases of dental and periodontal tissues. The first step for establishing this is to determine the expression of leptin and OBR in these tissues. Our study is the first to examine systematically the expression of leptin and OBR in dental and periodontal tissues of monkeys (Macaca fascicularis) by immunohistochemistry and in primary cultured cells, isolated from human dental and periodontal tissues, by reverse transcription plus the polymerase chain reaction and immunocytochemistry. Our results show that leptin and OBR are constitutively expressed and widely distributed in dental and periodontal tissues of primates. Their immunoreaction is especially strong in junctional epithelium, a unique front-line defense around teeth and in mineralizing areas of the dental pulp and periodontal ligament. The expression of the long and also functional form of OBR (OBRb) indicates that leptin has a direct effect on these cells. Thus, we can reasonably infer that leptin and OBR exert effects on defense, mineralization and angiogenesis in dental and periodontal tissues of primates.

Upregulated Leptin in Periodontitis Promotes Inflammatory Cytokine Expression in Periodontal Ligament Cells.

BACKGROUND Imbalance or disruption in the expression of inflammatory mediators contributes greatly to the breakdown of the periodontal supporting tissues. Leptin, through binding to its receptor (obesity-related leptin and leptin receptor [OBR]), has potent effects on immunity and inflammation. However, to date, researchers only indicated a role of leptin in periodontitis. No direct or valid evidence exists about how leptin and its receptor are regulated by local inflammation, what effects they have, and the underlying mechanisms. METHODS Experimental periodontitis was induced by ligation of mandibular second molars in beagle dogs. The expression of leptin, OBR, and interleukin (IL)-1β was examined by immunohistochemistry. Meanwhile, recombinant human IL-1β was used to stimulate human periodontal ligament cells (hPDLCs) in vitro, and mRNA and protein levels of leptin were measured using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Then, mRNA and protein levels of IL-6 and IL-8 were measured using real-time PCR and ELISA, after stimulation with various concentrations of leptin, knocking down all or only the long form of OBR (OBRb) by small interfering RNA and incubation with multiple intracellular signaling pathway inhibitors, respectively. RESULTS Leptin and OBR increased substantially in inflammatory periodontal tissues, which correlated well with the extent of inflammatory infiltration, and was a result of the upregulation in resident cells themselves. A high dose of leptin could induce the expression of mRNA and protein of IL-6 and IL-8 in hPDLCs through binding with OBRb and activating different intracellular signaling pathways. CONCLUSION Upregulated leptin and OBR in periodontitis stimulated proinflammatory cytokine expression in PDL cells to additionally promote local inflammation.

Platelet activation and platelet-leukocyte interaction in generalized aggressive periodontitis.

Generalized aggressive periodontitis (GAgP) is an inflammatory disease of host response to bacterial challenge. To explore the role of platelets in host–microbial interactions in patients with periodontitis, 124 patients with GAgP and 57 healthy subjects were enrolled. Reliable indicators of subclinical platelet functional status, platelet count (PLT), platelet large cell ratio (PLCR), and mean platelet volume (MPV), were significantly lower in the GAgP group than in the control group and were negatively correlated with clinical periodontal parameters. The levels of important cytosolic protein in neutrophils, calprotectin (S100A8/A9) in plasma, and gingival crevicular fluid (GCF) were significantly higher in patients with GAgP compared with healthy subjects. Moreover, the GCF calprotectin level was negatively correlated with PLCR and MPV values. To explore the possible mechanisms of changes in platelet indices in periodontitis, flow cytometry analysis was performed, and patients with GAgP were found to have a higher status of platelet activation compared with healthy controls. Porphyromonas gingivalis (P. gingivalis) and recombinant human S100A8/A9 (rhS100A8/A9) induced platelet activation and facilitated platelet–leukocyte aggregate formation in whole blood of healthy subjects. In response to P. gingivalis and rhS100A8/A9, platelets from patients with GAgP increased activation and increased formation of platelet–leukocyte aggregates compared with those from healthy subjects. Platelet aggregates and platelets attached to leukocytes were found on gingival tissues from patients with GAgP, suggesting that decreased platelet size and count in the circulation might be related to consumption of large, activated platelets at inflamed gingiva. Platelets may have a previously unrecognized role in host response to periodontal infection.

Influence of rs2228570 on Transcriptional Activation by the Vitamin D Receptor in Human Gingival Fibroblasts and Periodontal Ligament Cells

BACKGROUND rs2228570 is the only known single nucleotide polymorphism of the vitamin D receptor (VDR) that alters the protein structure. VDRs can be distinguished using the restriction endonuclease FokI and accordingly divided into three genotypes: FF, Ff, and ff. Influence of rs2228570 on transcriptional activation by VDRs in human gingival fibroblasts (hGFs) and periodontal ligament cells (hPDLCs) is investigated in this study. METHODS From 15 donors, hGFs and hPDLCs were cultured, genomic DNA was extracted, and genotypes were determined using the polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Cells were stimulated with calcitriol with or without VDR antagonist ZK159222 or osteogenic induction. Alkaline phosphatase, osteocalcin, and VDR messenger RNA (mRNA) expression were detected using real-time PCR. Alkaline phosphatase and osteocalcin protein expression were detected by enzyme activity assays with p-nitrophenyl phosphate substrate and enzyme-linked immunosorbent assay, respectively. RESULTS Among the 15 donor cell cultures, the number of FF, ff, and Ff genotypes were 5, 3, and 7, respectively. There were no significant differences in expression of alkaline phosphatase or osteocalcin among the three genotypes in hGFs. However, after stimulation with calcitriol, alkaline phosphatase and osteocalcin mRNA levels in FF-hPDLCs were significantly higher than in other hPDLCs genotypes, as was osteocalcin protein expression. Furthermore, when ZK159222 was included, this difference disappeared, and when osteogenic induction was performed, alkaline phosphatase and osteocalcin mRNA and protein levels were higher in FF-hPDLCs than in the other hPDLCs genotypes. CONCLUSION The FF-VDR genotype is associated with the most remarkable upregulation of alkaline phosphatase and osteocalcin in hPDLCs.

Vitamin D‐binding protein expression in healthy tooth and periodontium: an experimental study both in monkeys in vivo and in humans in vitro

BACKGROUND AND OBJECTIVE Vitamin D-binding protein (DBP) is a highly expressed plasma protein with many important functions, including transport of vitamin D metabolites, sequestration of actin, control of bone metabolism and modulation of immune and inflammatory responses. Previous results of our study indicated an association between DBP and periodontitis. We hypothesized that periodontium might be another source of DBP in gingival crevicular fluid other than serum. MATERIAL AND METHODS DBP expression was examined in dental and periodontal tissues of monkeys by immunohistochemistry, and in primary cells isolated from human dental and periodontal tissues by reverse transcription plus the polymerase chain reaction and immunocytochemistry. RESULTS DBP was constitutively expressed and widely distributed in dental and periodontal tissues of primates. Their immunoreaction was evident in gingival epithelium, particularly in junctional epithelium, and in mineralizing areas of the dental pulp, periodontal ligament and bone marrow. Correspondingly, mRNA and protein expression were detected in primary human gingival epithelial cells, dental pulp cells and periodontal ligament cells. CONCLUSION DBP is highly expressed and widely distributed in dental and periodontal tissues, which may take an active part in local host defense and hard tissue metabolism.