Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models.

The mechanical property, microhardness, is evaluated in dental enamel, dentin, and bone in oral disease models, including dental fluorosis and periodontitis. Micro-CT (µCT) provides 3D imaging information (volume and mineral density) and scanning electron microscopy (SEM) produces microstructure images (enamel prism and bone lacuna-canalicular). Complementarily to structural analysis by µCT and SEM, microhardness is one of the informative parameters to evaluate how structural changes alter mechanical properties. Despite being a useful parameter, studies on microhardness of alveolar bone in oral diseases are limited. To date, divergent microhardness measurement methods have been reported. Since microhardness values vary depending on the sample preparation (polishing and flat surface) and indentation sites, diverse protocols can cause discrepancies among studies. Standardization of the microhardness protocol is essential for consistent and accurate evaluation in oral disease models. In the present study, we demonstrate a standardized protocol for microhardness analysis in tooth and alveolar bone. Specimens used are as follows: for the dental fluorosis model, incisors were collected from mice treated with/without fluoride-containing water for 6 weeks; for ligature-induced periodontal bone resorption (L-PBR) model, alveolar bones with periodontal bone resorption were collected from mice ligated on the maxillary 2nd molar. At 2 weeks after the ligation, the maxilla was collected. Vickers hardness was analyzed in these specimens according to the standardized protocol. The protocol provides detailed materials and methods for resin embedding, serial polishing, and indentation sites for incisors and alveolar. To the best of our knowledge, this is the first standardized microhardness protocol to evaluate the mechanical properties of tooth and alveolar bone in rodent oral disease models.

Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome.

The COVID-19 pandemic persists despite the availability of vaccines, and it is therefore crucial to develop new therapeutic and preventive approaches. In this study, we investigated the potential role of the oral microbiome in SARS-CoV-2 infection. Using an in vitro SARS-CoV-2 pseudovirus infection assay, we found a potent inhibitory effect exerted by Porphyromonas gingivalis on SARS-CoV-2 infection mediated by known P. gingivalis compounds such as phosphoglycerol dihydroceramide (PGDHC) and gingipains as well as by unknown bacterial factors. We found that the gingipain-mediated inhibition of infection is likely due to cytotoxicity, while PGDHC inhibited virus infection by an unknown mechanism. Unidentified factors present in P. gingivalis supernatant inhibited SARS-CoV-2 likely via the fusion step of the virus life cycle. We addressed the role of other oral bacteria and found certain periodontal pathogens capable of inhibiting SARS-CoV-2 pseudovirus infection by inducing cytotoxicity on target cells. In the human oral cavity, we observed the modulatory activity of oral microbial communities varied among individuals in that some saliva-based cultures were capable of inhibiting while others were enhancing infection. These findings contribute to our understanding of the complex relationship between the oral microbiome and viral infections, offering potential avenues for innovative therapeutic strategies in combating COVID-19.

Extracellular Release of Citrullinated Vimentin Directly Acts on Osteoclasts to Promote Bone Resorption in a Mouse Model of Periodontitis.

Elevated osteoclast (OC)-mediated bone resorption, a common pathological feature between periodontitis and rheumatoid arthritis (RA), implicates a possible mutually shared pathogenesis. The autoantibody to citrullinated vimentin (CV), a representative biomarker of RA, is reported to promote osteoclastogenesis (OC-genesis). However, its effect on OC-genesis in the context of periodontitis remains to be elucidated. In an in vitro experiment, the addition of exogenous CV upregulated the development of Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear OCs from mouse bone marrow cells and increased the formation of resorption pits. However, Cl-amidine, an irreversible pan-peptidyl arginine deiminase (PAD) inhibitor, suppressed the production and secretion of CV from RANKL-stimulated OC precursors, suggesting that the citrullination of vimentin occurs in OC precursors. On the other hand, the anti-vimentin neutralizing antibody suppressed in vitro Receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced OC-genesis. The CV-induced upregulation of OC-genesis was abrogated by the Protein kinase C (PKC)-δ inhibitor Rottlerin, accompanied by the downmodulation of OC-genesis-related genes, including Osteoclast stimulatory transmembrane protein (OC-STAMP), TRAP and Matrix Metallopeptidase 9 (MMP9) as well as extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP)-kinase phosphorylation. Elevated levels of soluble CV and vimentin-bearing mononuclear cells were found in the bone resorption lesions of periodontitis induced in mice in the absence of an anti-CV antibody. Finally, local injection of anti-vimentin neutralizing antibody suppressed the periodontal bone loss induced in mice. Collectively, these results indicated that the extracellular release of CV promoted OC-genesis and bone resorption in periodontitis.

Porphyromonas gingivalis outer membrane vesicles exacerbate retinal microvascular endothelial cell dysfunction in diabetic retinopathy.

Diabetic retinopathy (DR) is one of the leading causes of blindness. Periodontitis is one of the highest oral incidences and has been closely related to various systemic conditions through Porphyromonas gingivalis (P. gingivalis). P. gingivalis OMVs, derived from P. gingivalis, can cause endothelial dysfunction and potentially affect microvascular diseases. Current epidemiological studies provide limited evidence suggesting that periodontitis is associated with DR. However, there is a lack of basic research elucidating how periodontitis affects the severity of DR. This study aimed to explore the potential of P. gingivalis OMVs to contribute to the pathogenesis of DR and explore how it affect the retinal microvascular endothelium. The results demonstrated that P. gingivalis OMVs accelerated the blood-retinal barrier damage in DR mice. In vitro studies showed that the expression of inflammatory factors in human retinal microvascular endothelial cells (HRMECs) was increased after P. gingivalis OMVs stimulation, and the increased reactive oxygen species production, mitochondrial dysfunction, apoptosis, and altered endothelial permeability were observed in HRMECs under P. gingivalis OMVs stimulation. In addition, we found that protease-activated receptor-2 (PAR-2) regulated OMVs-induced TNF-α, MMP-9 mRNA expression, cell death, and endothelial permeability. Overall, we suggested that P. gingivalis OMVs induced mitochondria-related cell death of HRMECs and accelerated endothelial dysfunction, thus aggravating DR, in which PAR-2 plays a potential role. This study is the first research report to delineate the potential molecular mechanism of P. gingivalis OMVs on DR pathogenesis, which uniquely focused on elucidating the possible impact of periodontal pathogen derivatives on DR progression.

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis.

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.

Oxidative stress impairs the calcification ability of human dental pulp cells.

BACKGROUND: The relationship between internal root resorption and oxidative stress has not yet been reported. This study aimed to add molecular insight into internal root resorption. The present study was conducted to investigate the effect of hydrogen peroxide (H2O2) as an inducer of oxidative stress on the calcification ability of human dental pulp cells (hDPCs) and the involvement of inositol 1, 4, 5-trisphosphate (IP3). MATERIAL AND METHODS: hDPCs (Lonza, Basel, Switzerland) were exposed to H2O2. Cell viability and reactive oxygen species (ROS) production were then evaluated. To investigate the effect of H2O2 on the calcification ability of hDPCs, real-time PCR for alkaline phosphatase (ALP) mRNA expression, ALP staining, and Alizarin red staining were performed. Data were compared with those of hDPCs pretreated with 2-aminoethyldiphenylborate (2-APB), which is an IP3 receptor inhibitor. RESULTS: H2O2 at concentrations above 250 µM significantly reduced cell viability (P < 0.01). More ROS production occurred in 100 µM H2O2-treated hDPCs than in control cells (P < 0.01). 2-APB significantly decreased the production (P < 0.05). H2O2-treated hDPCs showed significant reductions in ALP mRNA expression (P < 0.01), ALP activity (P < 0.01), and mineralized nodule deposition compared with negative control cells (P < 0.01). 2-APB significantly inhibited these reductions (P < 0.01, P < 0.05 and P < 0.01, respectively). Data are representative of three independent experiments with three replicates for each treatment and values are expressed as means ± SD. CONCLUSION: To the best of our knowledge, this is the first study documenting the involvement of IP3 signaling in the calcification ability of human dental pulp cells impaired by H2O2.

Oxytocin suppresses CXCL10 production in TNF-α-stimulated human dental pulp stem cells.

Oxytocin (OX) is a posterior pituitary hormone secreted into the blood from axon terminals projecting from the posterior pituitary. Recent reports indicate OX plays an important role in the progression of inflammatory diseases such as rheumatoid arthritis. Pulpitis is caused by the activation of the biological defense mechanism of the dental pulp against cariogenic bacteria. However, the role of OX in the pathogenesis of pulpitis remains unknown. The aim of this study was to examine the effect of OX on CXC chemokine ligand 10 (CXCL10) production in human dental pulp stem cells (HDPSCs). Expression of the oxytocin receptor (OXR) on HDPSCs was detected by Western blot analysis and immunofluorescence. CXCL10 production in HDPSCs was measured using an enzyme-linked immunosorbent assay kit. Western blot analysis was performed to determine the phosphorylation levels of signal transduction molecules, including nuclear factor kappa B, mitogen-activated protein kinases (MAPKs), and Akt in HDPSCs. HDPSCs expressed OXR. OX significantly decreased CXCL10 production in tumor necrosis factor (TNF)-α-stimulated HDPSCs. The p38 MAPK and Akt pathways were related to the OX-suppressed CXCL10 production in TNF-α-stimulated HDPSCs. These results indicate that OX appears to modulate the immune response in pulpitis via suppression of CXCL10 production by HDPSCs.

Locally Secreted Semaphorin 4D Is Engaged in Both Pathogenic Bone Resorption and Retarded Bone Regeneration in a Ligature-Induced Mouse Model of Periodontitis.

It is well known that Semaphorin 4D (Sema4D) inhibits IGF-1-mediated osteogenesis by binding with PlexinB1 expressed on osteoblasts. However, its elevated level in the gingival crevice fluid of periodontitis patients and the broader scope of its activities in the context of potential upregulation of osteoclast-mediated periodontal bone-resorption suggest the need for further investigation of this multifaceted molecule. In short, the pathophysiological role of Sema4D in periodontitis requires further study. Accordingly, attachment of the ligature to the maxillary molar of mice for 7 days induced alveolar bone-resorption accompanied by locally elevated, soluble Sema4D (sSema4D), TNF-α and RANKL. Removal of the ligature induced spontaneous bone regeneration during the following 14 days, which was significantly promoted by anti-Sema4D-mAb administration. Anti-Sema4D-mAb was also suppressed in vitro osteoclastogenesis and pit formation by RANKL-stimulated BMMCs. While anti-Sema4D-mAb downmodulated the bone-resorption induced in mouse periodontitis, it neither affected local production of TNF-α and RANKL nor systemic skeletal bone remodeling. RANKL-induced osteoclastogenesis and resorptive activity were also suppressed by blocking of CD72, but not Plexin B2, suggesting that sSema4D released by osteoclasts promotes osteoclastogenesis via ligation to CD72 receptor. Overall, our data indicated that ssSema4D released by osteoclasts may play a dual function by decreasing bone formation, while upregulating bone-resorption.

Dual-Function Semaphorin 4D Released by Platelets: Suppression of Osteoblastogenesis and Promotion of Osteoclastogenesis.

Effects of the antiosteoblastogenesis factor Semaphorin 4D (Sema4D), expressed by thrombin-activated platelets (TPs), on osteoblastogenesis, as well as osteoclastogenesis, were investigated in vitro. Intact platelets released both Sema4D and IGF-1. However, in response to stimulation with thrombin, platelets upregulated the release of Sema4D, but not IGF-1. Anti-Sema4D-neutralizing monoclonal antibody (mAb) upregulated TP-mediated osteoblastogenesis in MC3T3-E1 osteoblast precursors. MC3T3-E1 cells exposed to TPs induced phosphorylation of Akt and ERK further upregulated by the addition of anti-sema4D-mAb, suggesting the suppressive effects of TP-expressing Sema4D on osteoblastogenesis. On the other hand, TPs promoted RANKL-mediated osteoclastogenesis in the primary culture of bone-marrow-derived mononuclear cells (BMMCs). Among the known three receptors of Sema4D, including Plexin B1, Plexin B2 and CD72, little Plexin B2 was detected, and no Plexin B1 was detected, but a high level of CD72 mRNA was detected in RANKL-stimulated BMMCs by qPCR. Both anti-Sema4D-mAb and anti-CD72-mAb suppressed RANKL-induced osteoclast formation and bone resorptive activity, suggesting that Sema4D released by TPs promotes osteoclastogenesis via ligation to a CD72 receptor. This study demonstrated that Sema4D released by TPs suppresses osteogenic activity and promotes osteoclastogenesis, suggesting the novel property of platelets in bone-remodeling processes.

Semaphorin3A released from human dental pulp cells inhibits the increase in interleukin-6 and CXC chemokine ligand 10 production induced by tumor necrosis factor-α through suppression of nuclear factor-κB activation.

Human dental pulp cells (HDPCs) play an important role in pulpitis. Semaphorin3A (Sema3A), which is an axon guidance molecule, is a member of the secretory semaphorin family. Recently, Sema3A has been reported to be an osteoprotective factor and to be involved in the immune response. However, the role of Sema3A in dental pulp inflammation remains unknown. The aim of this study was to reveal the existence of Sema3A in human dental pulp tissue and the effect of Sema3A which is released from tumor necrosis factor (TNF)-α-stimulated HDPCs on production of proinflammatory cytokines, such as interleukin (IL)-6 and CXC chemokine ligand 10 (CXCL10), from HDPCs stimulated with TNF-α. Sema3A was detected in inflamed pulp as compared to normal pulp. HDPCs expressed Neuropilin-1(Nrp1) which is Sema3A receptor. TNF-α increased the levels of IL-6 and CXCL10 in HDPCs in time-dependent manner. Sema3A inhibited production of these two cytokines from TNF-α-stimulated HDPCs. TNF-α induced soluble Sema3A production from HDPCs. Moreover, antibody-based neutralization of Sema3A further promoted production of IL-6 and CXCL10 from TNF-α-stimulated HDPCs. Sema3A inhibited nuclear factor (NF)-κB P65 phosphorylation and inhibitor κBα degradation in TNF-α-stimulated HDPCs. These results indicated that Sema3A is induced in human dental pulp, and TNF-α acts on HDPCs to produce Sema3A, which partially inhibits the increase in IL-6 and CXCL10 production induced by TNF-α, and that the inhibition leads to suppression of NF-κB activation. Therefore, it is suggested that Sema3A may regulate inflammation in dental pulp and be novel antiinflammatory target molecule for pulpitis.

Interleukin (IL)-35 Suppresses IL-6 and IL-8 Production in IL-17A-Stimulated Human Periodontal Ligament Cells.

Interleukin (IL)-35 is a novel anti-inflammatory cytokine that is produced by regulatory T cells. IL-35 is reported to suppress IL-17A-producing helper T (Th17) cell activation. IL-17A is related to progression of periodontitis. Furthermore, IL-35 and IL-17A are detected in human gingival crevicular fluid. However, the effect of IL-35 and interaction between IL-35 and IL-17A on pro-inflammatory cytokine production in human periodontal resident cells are still unclear. The aim of this study was to clarify the effect of IL-35 on IL-6 and IL-8 production in human periodontal ligament cells (HPDLCs) stimulated with IL-17A. IL-35 inhibited IL-6 and IL-8 production in IL-17A-stimulated HPDLCs. Moreover, western blot analysis showed that IL-35 suppressed extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB p65 phosphorylation in IL-17A-stimulated HPDLCs. Our findings suggested that IL-35 produced from regulatory T cells might inhibit progression of periodontitis by decreasing IL-17A-induced levels of IL-6 and IL-8.

OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

Cell fusion-mediated formation of multinuclear osteoclasts (OCs) plays a key role in bone resorption. It is reported that 2 unique OC-specific fusogens [ i.e., OC-stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP)], and permissive fusogen CD9, are involved in OC fusion. In contrast to DC-STAMP-knockout (KO) mice, which show the osteopetrotic phenotype, OC-STAMP-KO mice show no difference in systemic bone mineral density. Nonetheless, according to the ligature-induced periodontitis model, significantly lower level of bone resorption was found in OC-STAMP-KO mice compared to WT mice. Anti-OC-STAMP-neutralizing mAb down-modulated in vitro: 1) the emergence of large multinuclear tartrate-resistant acid phosphatase-positive cells, 2) pit formation, and 3) mRNA and protein expression of CD9, but not DC-STAMP, in receptor activator of NF-κB ligand (RANKL)-stimulated OC precursor cells (OCps). While anti-DC-STAMP-mAb also down-regulated RANKL-induced osteoclastogenesis in vitro, it had no effect on CD9 expression. In our mouse model, systemic administration of anti-OC-STAMP-mAb suppressed the expression of CD9 mRNA, but not DC-STAMP mRNA, in periodontal tissue, along with diminished alveolar bone loss and reduced emergence of CD9+ OCps and tartrate-resistant acid phosphatase-positive multinuclear OCs. The present study demonstrated that OC-STAMP partners CD9 to promote periodontal bone destruction by up-regulation of fusion during osteoclastogenesis, suggesting that anti-OC-STAMP-mAb may lead to the development of a novel therapeutic regimen for periodontitis.-Ishii, T., Ruiz-Torruella, M., Ikeda, A., Shindo, S., Movila, A., Mawardi, H., Albassam, A., Kayal, R. A., Al-Dharrab, A. A., Egashira, K., Wisitrasameewong, W., Yamamoto, K., Mira, A. I., Sueishi, K., Han, X., Taubman, M. A., Miyamoto, T., Kawai, T. OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

IL-29 Enhances CXCL10 Production in TNF-α-stimulated Human Oral Epithelial Cells.

Interleukin-29 (IL-29) is a cytokine belonging to the Type III interferon family. It was recently detected in the gingival crevicular fluid of periodontitis patients. However, the role of IL-29 in the pathogenesis of periodontal disease remains unknown. The aim of this study was to examine the effects of IL-29 on C-X-C motif chemokine ligand 10 (CXCL10) production in human oral epithelial cells. We measured CXCL10 production in TR146 cells, which is a human oral epithelial cell line, using an enzyme-linked immunosorbent assay. We used a Western blot analysis to detect IL-29 receptor expression and the phosphorylation levels of signal transduction molecules, including p38 mitogen-activated protein kinases (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor (NF)- κB p65, in the TR146 cells. The TR146 cells expressed the IL-29 receptor. IL-29 induced CXCL10 production in the TR146 cells. IL-29 significantly enhanced CXCL10 production in tumor necrosis factor (TNF)-α-stimulated TR146 cells. The p38 MAPK, STAT3, and NF-κB pathways were found to be related to the IL-29-induced enhancement of CXCL10 production in TNF-α-stimulated TR146 cells. IL-29 promotes T helper 1-cell accumulation in periodontal lesions by inducing CXCL10 production in oral epithelial cells.

Gomisin N Decreases Inflammatory Cytokine Production in Human Periodontal Ligament Cells.

Gomisin N, which is a lignan isolated from Schisandra chinensis, has some pharmacological effects. However, the anti-inflammatory effects of gomisin N on periodontal disease are uncertain. The aim of this study was to examine the effect of gomisin N on inflammatory mediator production in tumor necrosis factor (TNF)-α-stimulated human periodontal ligament cells (HPDLC). Gomisin N inhibited interleukin (IL)-6, IL-8, CC chemokine ligand (CCL) 2, and CCL20 production in TNF-α-stimulated HPDLC in a dose-dependent manner. Moreover, we revealed that gomisin N could suppress extracellular signal-regulated kinase (ERK) and c-Jun N terminal kinase (JNK) phosphorylation in TNF-α-stimulated HPDLC though protein kinase B (Akt) phosphorylation was not suppressed by gomisin N treatment. In summary, gomisin N might exert anti-inflammatory effects by attenuating cytokine production in periodontal ligament cells via inhibiting the TNF-α-stimulated ERK and JNK pathways activation.

Alkannin inhibits CCL3 and CCL5 production in human periodontal ligament cells.

Alkannin, which is found in Alkanna tinctoria, a member of the borage family, is used as a food coloring. Alkannin has recently been reported to have certain biological functions, such as anti-microbial and anti-oxidant effects. It is known that CC chemokine receptor (CCR) 5-positive leukocytes contribute to alveolar bone resorption in periodontal lesions. The aim of this study was to examine whether alkannin inhibits the production of CC chemokine ligand (CCL) 3 and CCL5, which are CCR5 ligands, in human periodontal ligament cells (HPDLC). Interleukin (IL)-1ß induced CCL3 and CCL5 production in HPDLC. Alkannin inhibited IL-1ß-mediated CCL3 and CCL5 production in HPDLC in a dose-dependent manner. Moreover, we revealed that alkannin suppressed inhibitor of kappa B-α degradation in IL-1ß-stimulated HPDLC. In addition, a nuclear factor (NF)-κB inhibitor significantly inhibited CCL3 and CCL5 production in IL-1ß-stimulated HPDLC. These results demonstrate that alkannin inhibits CCR5 ligand production in IL-1ß-stimulated HPDLC by attenuating the NF-κB signaling pathway.

Melatonin Inhibits CXCL10 and MMP-1 Production in IL-1ß-Stimulated Human Periodontal Ligament Cells.

Melatonin is a hormone that is mainly secreted by the pineal gland and exhibits a wide spectrum of activities, including antioxidant functions. Melatonin has been detected in gingival crevicular fluid. However, the role of melatonin in periodontal tissue is still uncertain. The aim of this study was to examine the effects of melatonin on inflammatory mediator expression in human periodontal ligament cells (HPDLC). Interleukin (IL)-1ß induced CXC chemokine ligand (CXCL)10, matrix metalloproteinase (MMP)-1, and tissue inhibitors of metalloproteinase (TIMP)-1 production in HPDLC. Melatonin decreased CXCL10 and MMP-1 production and increased TIMP-1 production in IL-1ß-stimulated HPDLC. Western blot analysis showed that melatonin inhibited p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK) phosphorylation, and IkB-α degradation and phosphorylation in IL-1ß-stimulated HPDLC. These results suggest that melatonin might inhibit Th1 cell migration by reducing CXCL10 production. Moreover, melatonin might inhibit soft tissue destruction by decreasing MMP-1 production in periodontal lesions.

TRAP-positive osteoclast precursors mediate ROS/NO-dependent bactericidal activity via TLR4.

Osteoclastogenesis was induced by RANKL stimulation in mouse monocytes to examine the possible bactericidal function of osteoclast precursors (OCp) and mature osteoclasts (OCm) relative to their production of NO and ROS. Tartrate-resistant acid phosphatase (TRAP)-positive OCp, but few or no OCm, phagocytized and killed Escherichia coli in association with the production of reactive oxygen species (ROS) and nitric oxide (NO). Phagocytosis of E. coli and production of ROS and NO were significantly lower in TRAP+ OCp derived from Toll-like receptor (TLR)-4 KO mice than that derived from wild-type (WT) or TLR2-KO mice. Interestingly, after phagocytosis, TRAP+ OCp derived from wild-type and TLR2-KO mice did not differentiate into OCm, even with continuous exposure to RANKL. In contrast, E. coli-phagocytized TRAP+ OCp from TLR4-KO mice could differentiate into OCm. Importantly, neither NO nor ROS produced by TRAP+ OCp appeared to be engaged in phagocytosis-induced suppression of osteoclastogenesis. These results suggested that TLR4 signaling not only induces ROS and NO production to kill phagocytized bacteria, but also interrupts OCm differentiation. Thus, it can be concluded that TRAP+ OCp, but not OCm, can mediate bactericidal activity via phagocytosis accompanied by the production of ROS and NO via TLR4-associated reprograming toward phagocytic cell type.

Shikonin Inhibits Inflammatory Cytokine Production in Human Periodontal Ligament Cells.

Shikonin, which is derived from Lithospermum erythrorhizon, a herb used in traditional medicine, has long been considered to be a useful treatment for various diseases in traditional oriental medicine. Shikonin has recently been reported to have several pharmacological properties, e.g., it has anti-microbial, anti-tumor, and anti-inflammatory effects. The aim of this study was to examine whether shikonin is able to influence the production of interleukin (IL)-6, IL-8, and/or chemokine C-C motif ligand (CCL)20, which contribute to the pathogenesis of periodontal disease, in human periodontal ligament cells (HPDLC). The production levels of IL-6, IL-8, and CCL20 in HPDLC were determined using an ELISA. Western blot analysis was used to detect nuclear factor kappa B (NF-κB) pathway activation in HPDLC. Shikonin prevented IL-1ß- or tumor necrosis factor (TNF)-α-mediated IL-6, IL-8, and CCL20 production in HPDLC. Moreover, we found that shikonin suppressed the phosphorylation and degradation of inhibitor of kappa B-alpha (IκB-α) in IL-1ß- or TNF-α-stimulated HPDLC. These findings suggest that shikonin could have direct beneficial effects against periodontal disease by reducing IL-6, IL-8, and CCL20 production in periodontal lesions.

IL-4 Modulates CCL11 and CCL20 Productions from IL-1ß-Stimulated Human Periodontal Ligament Cells.

BACKGROUND/AIMS: IL-4 is a multifunctional cytokine that is related with the pathological conditions of periodontal disease. However, it is uncertain whether IL-4 could control T cells migration in periodontal lesions. The aim of this study was to examine the effects of IL-4 on CCL11, which is a Th2-type chemokine, and CCL20, which is related with Th17 cells migration, productions from human periodontal ligament cells (HPDLCs). METHODS: CCL20 and CCL11 productions from HPDLCs were monitored by ELISA. Western blot analysis was performed to detect phosphorylations of signal transduction molecules in HPDLCs. RESULTS: IL-1ß could induce both CCL11 and CCL20 productions in HPDLCs. IL-4 enhanced CCL11 productions from IL-1ß-stimulated HPDLCs, though IL-4 inhibited CCL20 production. Western blot analysis showed that protein kinase B (Akt) and signal transducer and activator of transcription (STAT)6 pathways were highly activated in IL-4/IL-1ß-stimulated HPDLCs. Akt and STAT6 inhibitors decreased CCL11 production, but enhanced CCL20 production in HPDLCs stimulated with IL-4 and IL-1ß. CONCLUSIONS: These results mean that IL-4 enhanced Th2 cells migration in periodontal lesion to induce CCL11 production from HPDLCs. On the other hand, IL-4 inhibits Th17 cells accumulation in periodontally diseased tissues to inhibit CCL20 production. Therefore, IL-4 is positively related with the pathogenesis of periodontal disease to control chemokine productions in periodontal lesions.

Calcitriol Suppressed Inflammatory Reactions in IL-1ß-Stimulated Human Periodontal Ligament Cells.

Vitamin D has important roles on control of calcium and phosphate levels in the body. However, the role of vitamin D on the pathogenesis of periodontal disease is still uncertain. Therefore, we examined the effect of the hormonal form of vitamin D, calcitriol, on inflammatory responses of human periodontal ligament cells (HPDLC). We detected vitamin D receptor expression in non-stimulated HPDLC. Calcitriol inhibited interleukin (IL)-6, IL-8, CC chemokine ligand (CCL) 20, CXC chemokine ligand (CXCL) 10, and matrix metalloproteinase (MMP)-3 release from IL-1ß-stimulated HPDLC. Tissue inhibitor of metalloproteinase (TIMP)-1 production did not change by calcitriol. Moreover, we found c-jun N-terminal kinase (JNK) phosphorylation and IκB-α degradation in IL-1ß-stimulated HPDLC were inhibited by calcitriol, and JNK and nuclear factor (NF)-κB inhibitors could decrease IL-6, IL-8, CCL20, CXCL10, and MMP-3 productions in IL-1ß-treated HPDLC. These findings suggest that vitamin D could modulate inflammatory response in periodontal tissues.