Complement Is Required for Microbe-Driven Induction of Th17 and Periodontitis.

In both mice and humans, complement and Th17 cells have been implicated in periodontitis, an oral microbiota-driven inflammatory disease associated with systemic disorders. A recent clinical trial showed that a complement C3 inhibitor (AMY-101) causes sustainable resolution of periodontal inflammation, the main effector of tissue destruction in this oral disease. Although both complement and Th17 are required for periodontitis, it is uncertain how these immune components cooperate in disease development. In this study, we dissected the complement-Th17 relationship in the setting of ligature-induced periodontitis (LIP), a model that previously established that microbial dysbiosis drives Th17 cell expansion and periodontal bone loss. Complement was readily activated in the periodontal tissue of LIP-subjected mice but not when the mice were placed on broad-spectrum antibiotics. Microbiota-induced complement activation generated critical cytokines, IL-6 and IL-23, which are required for Th17 cell expansion. These cytokines as well as Th17 accumulation and IL-17 expression were significantly suppressed in LIP-subjected C3-deficient mice relative to wild-type controls. As IL-23 has been extensively studied in periodontitis, we focused on IL-6 and showed that LIP-induced IL-17 and bone loss required intact IL-6 receptor signaling in the periodontium. LIP-induced IL-6 was predominantly produced by gingival epithelial cells that upregulated C3a receptor upon LIP challenge. Experiments in human gingival epithelial cells showed that C3a upregulated IL-6 production in cooperation with microbial stimuli that upregulated C3a receptor expression in ERK1/2- and JNK-dependent manner. In conclusion, complement links the periodontal microbiota challenge to Th17 cell accumulation and thus integrates complement- and Th17-driven immunopathology in periodontitis.

Ligneous periodontitis exacerbated by Behçet's disease in a patient with plasminogen deficiency and a stop-gained variant PLG c.1468C > T: a case report.

BACKGROUND: Plasminogen serves as the precursor to plasmin, an essential element in the fibrinolytic process, and is synthesized primarily in the liver. Plasminogen activation occurs through the action of plasminogen activator, converting it into plasmin. This conversion greatly enhances the fibrinolytic system within tissues and blood vessels, facilitating the dissolution of fibrin clots. Consequently, congenital deficiency of plasminogen results in impaired fibrin degradation. Patients with plasminogen deficiency typically exhibit fibrin deposits in various mucosal sites throughout the body, including the oral cavity, eyes, vagina, and digestive organs. Behcet's disease is a chronic recurrent systemic inflammatory disease with four main symptoms: aphthous ulcers of the oral mucosa, vulvar ulcers, skin symptoms, and eye symptoms, and has been reported worldwide. This disease is highly prevalent around the Silk Road from the Mediterranean to East Asia. We report a case of periodontitis in a patient with these two rare diseases that worsened quickly, leading to alveolar bone destruction. Genetic testing revealed a novel variant characterized by a stop-gain mutation, which may be a previously unidentified etiologic gene associated with decreased plasminogen activity. CASE PRESENTATION: This case report depicts a patient diagnosed with ligneous gingivitis during childhood, originating from plasminogen deficiency and progressing to periodontitis. Genetic testing revealed a suspected association with the PLG c.1468C > T (p.Arg490*) stop-gain mutation. The patient's periodontal condition remained stable with brief intervals of supportive periodontal therapy. However, the emergence of Behçet's disease induced acute systemic inflammation, necessitating hospitalization and treatment with steroids. During hospitalization, the dental approach focused on maintaining oral hygiene and alleviating contact-related pain. The patient's overall health improved with inpatient care and the periodontal tissues deteriorated. CONCLUSIONS: Collaborative efforts between medical and dental professionals are paramount in comprehensively evaluating and treating patients with intricate complications from rare diseases. Furthermore, the PLG c.1468C > T (p.Arg490*) stop-gain mutation could contribute to the association between plasminogen deficiency and related conditions.

An injectable hydrogel-formulated inhibitor of prolyl-4-hydroxylase promotes T regulatory cell recruitment and enhances alveolar bone regeneration during resolution of experimental periodontitis.

The hypoxia-inducible factor 1α (HIF-1α) is critically involved in tissue regeneration. Hence, the pharmacological prevention of HIF-1α degradation by prolyl hydroxylase (PHD) under normoxic conditions is emerging as a promising option in regenerative medicine. Using a mouse model of ligature-induced periodontitis and resolution, we tested the ability of an injectable hydrogel-formulated PHD inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA/hydrogel), to promote regeneration of alveolar bone lost owing to experimental periodontitis. Mice injected subcutaneously with 1,4-DPCA/hydrogel at the onset of periodontitis resolution displayed significantly increased gingival HIF-1α protein levels and bone regeneration, as compared to mice treated with vehicle control. The 1,4-DPCA/hydrogel-induced increase in bone regeneration was associated with elevated expression of osteogenic genes, decreased expression of pro-inflammatory cytokine genes, and increased abundance of FOXP3+ T regulatory (Treg) cells in the periodontal tissue. The enhancing effect of 1,4-DPCA/hydrogel on Treg cell accumulation and bone regeneration was reversed by AMD3100, an antagonist of the chemokine receptor CXCR4 that mediates Treg cell recruitment. In conclusion, the administration of 1,4-DPCA/hydrogel at the onset of periodontitis resolution promotes CXCR4-dependent accumulation of Treg cells and alveolar bone regeneration, suggesting a novel approach for regaining bone lost due to periodontitis.

Induction of migration of periodontal ligament cells by selective regulation of integrin subunits.

The recruitment of tissue-resident stem cells is important for wound regeneration. Periodontal ligament cells (PDL cells) are heterogeneous cell populations with stemness features that migrate into wound sites to regenerate periodontal fibres and neighbouring hard tissues. Cell migration is regulated by the local microenvironment, coordinated by growth factors and the extracellular matrix (ECM). Integrin-mediated cell adhesion to the ECM provides essential signals for migration. We hypothesized that PDL cell migration could be enhanced by selective expression of integrins. The migration of primary cultured PDL cells was induced by platelet-derived growth factor-BB (PDGF-BB). The effects of blocking specific integrins on migration and ECM adhesion were investigated based on the integrin expression profiles observed during migration. Up-regulation of integrins α3, α5, and fibronectin was identified at distinct localizations in migrating PDL cells. Treatment with anti-integrin α5 antibodies inhibited PDL cell migration. Treatment with anti-integrin α3, α3-blocking peptide, and α3 siRNA significantly enhanced cell migration, comparable to treatment with PDGF-BB. Furthermore, integrin α3 inhibition preferentially enhanced adhesion to fibronectin via integrin α5. These findings indicate that PDL cell migration is reciprocally regulated by integrin α3-mediated inhibition and α5-mediated promotion. Thus, targeting integrin expression is a possible therapeutic strategy for periodontal regeneration.

Molecular imaging assessment of periodontitis lesions in an experimental mouse model.

OBJECTIVE: We aimed to evaluate molecular imaging as a novel diagnostic tool for mice periodontitis model induced by ligature and Porphyromonas gingivalis (Pg) inoculation. MATERIALS AND METHODS: Twelve female mice were assigned to the following groups: no treatment as control group (n = 4); periodontitis group induced by ligature and Pg as Pg group (n = 4); and Pg group treated with glycyrrhizinic acid (GA) as Pg + GA group (n = 4). All mice were administered a myeloperoxidase (MPO) activity-specific luminescent probe and observed using a charge-coupled device camera on day 14. Image analysis on all mice was conducted using software to determine the signal intensity of inflammation. Additionally, histological and radiographic evaluation for periodontal inflammation and bone resorption at the site of periodontitis, and quantitative enzyme-linked immunosorbent assay (ELISA) were conducted on three mice for each group. Each experiment was performed three times. RESULTS: Levels of serum IgG antibody against P. gingivalis were significantly higher in the Pg than in the Pg + GA group. Histological analyses indicated that the number of osteoclasts and neutrophils were significantly lower in the Pg + GA than in the Pg group. Micro-CT image analysis indicated no difference in bone resorption between the Pg and Pg + GA groups. The signal intensity of MPO activity was detected on the complete craniofacial image; moreover, strong signal intensity was localized specifically at the periodontitis site in the ex vivo palate, with group-wise differences. CONCLUSIONS: Molecular imaging analysis based on MPO activity showed high sensitivity of detection of periodontal inflammation in mice. CLINICAL RELEVANCE: Molecular imaging analysis based on MPO activity has potential as a diagnostic tool for periodontitis.

Anti-HMGB1 Neutralizing Antibody Attenuates Periodontal Inflammation and Bone Resorption in a Murine Periodontitis Model.

High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that is secreted into the extracellular milieu in response to inflammatory stimuli. The secreted HMGB1 mediates various inflammatory diseases, including periodontitis; however, the underlying mechanisms of HMGB1-induced periodontal inflammation are not completely understood. Here, we examined whether anti-HMGB1 neutralizing antibody inhibits periodontal progression and investigated the molecular pathology of HMGB1 in vitro and in vivo. In vitro analysis indicated that HMGB1, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-1ß (IL-1ß) were secreted in response to tumor necrosis factor-α (TNF-α) stimuli in human gingival epithelial cells (HGECs) and human monocytic leukemia cells (THP-1) treated with phorbol myristate acetate. Increased levels of GM-CSF and IL-1ß were observed in the conditioned media from TNF-α-stimulated HGECs and THP-1 in vitro Simultaneous stimulation with TNF-α and anti-HMGB1 antibody significantly decreased TNF-α-induced inflammatory cytokine secretion. Experimental periodontitis was induced in mice using Porphyromonas gingivalis-soaked ligatures. The extracellular translocation was confirmed in gingival epithelia in the periodontitis model mice by immunofluorescence analysis. Systemic administration of anti-HMGB1 neutralizing antibody significantly inhibited translocation of HMGB1. The anti-HMGB1 antibody inhibited periodontal inflammation, expression of IL-1ß and C-X-C motif chemokine ligand 1 (CXCL1), migration of neutrophils, and bone resorption, shown by bioluminescence imaging of myeloperoxidase activity, quantitative reverse transcription-PCR (RT-PCR), and micro-computed tomography analysis. These findings indicate that HMGB1 is secreted in response to inflammatory stimuli caused by periodontal infection, which is crucial for the initiation of periodontitis, and the anti-HMGB1 antibody attenuates the secretion of a series of inflammatory cytokines, consequently suppressing the progression of periodontitis.

HMGB1-induced inflammatory response promotes bone healing in murine tooth extraction socket.

High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that is secreted into the extracellular milieu in response to inflammatory stimuli. The secreted HMGB1 has been suggested to mediate various inflammatory diseases. However, it is still unknown whether HMGB1 is involved in a healing process in the tooth extraction socket, the tissue containing gingival epithelium, and alveolar bone that is exposed to oral bacteria. In this study, we constructed a murine tooth extraction model with anti-HMGB1 neutralization antibody administration and observed the inflammatory response and bone healing process in tooth extraction sockets by molecular imaging of myeloperoxidase (MPO) activity, histological analysis, and quantitative RT-PCR. The translocation of HMGB1 from the nucleus to the cytoplasm in gingival epithelial cells and inflammatory cells was inhibited by anti-HMGB1 antibody administration. The MPO activity around the tooth extraction socket was significantly reduced, and the numbers of CD31- and CD68-positive cells were significantly lower in the anti-HMGB1 antibody treatment samples than in the control samples. The TRAP-positive cells, osteocalcin positive cells, and the neoplastic bone area were significantly lower in anti-HMGB1 antibody treatment samples than in control samples. The expression levels of IL-1ß and VEGF-A were also decreased in anti-HMGB1 antibody treatment samples compared to that in control samples. Secreted HMGB1 induced initial acute inflammation and inflammatory cells recruitment after tooth extraction. HMGB1 was associated with angiogenesis and bone remodeling by osteoclast and osteoblast activation and promoted bone healing in the tooth extraction socket.

Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells.

Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, ß4, and ß6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, ß4, and ß6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells.

The periodontal ligament is a multifunctional soft connective tissue, which functions not only as a cushion supporting the teeth against occlusal force, but is also a source of osteogenic cells that can regenerate neighboring hard tissues. Periodontal ligament cells (PDL cells) contain heterogeneous cell populations, including osteogenic cell progenitors. However, the precise mechanism underlying the differentiation process remains elusive. Cell differentiation is regulated by the local biochemical and mechanical microenvironment that can modulate gene expression and cell morphology by altering actin cytoskeletal organization mediated by Rho-associated, coiled-coil containing protein kinase (ROCK). To determine its role in PDL cell differentiation, we examined the effects of ROCK on cytoskeletal changes and kinetics of gene expression during osteogenic differentiation. PDL cells were isolated from human periodontal ligament on extracted teeth and cultured in osteogenic medium for 14 days. Y-27632 was used for ROCK inhibition assay. Osteogenic phenotype was determined by monitoring alkaline phosphatase (ALP) activity and calcium deposition by Alizarin Red staining. ROCK-induced cytoskeletal changes were examined by immunofluorescence analysis of F-actin and myosin light chain 2 (MLC2) expression. Real-time PCR was performed to examine the kinetics of osteogenic gene expression. F-actin and phospho-MLC2 were markedly induced during osteogenic differentiation, which coincided with upregulation of ALP activity and mineralization. Subsequent inhibition assay indicated that Y-27632 significantly inhibited F-actin and phospho-MLC2 expression in a dose-dependent manner with concomitant partial reversal of the PDL cell osteogenic phenotype. PCR array analysis of osteogenic gene expression indicated that extracellular matrix genes, such as fibronectin 1, collagen type I and III, and biglycan, were significantly downregulated by Y27632. These findings indicated crucial effects of ROCK in cytoskeletal reorganization and differentiation of PDL cells toward osteogenic cells. ROCK contributes to induction of osteogenic differentiation by synergistic increases in extracellular matrix gene expression in PDL cells.

Resolvin D2-induced reparative dentin and pulp stem cells after pulpotomy in a rat model.

Introduction: Vital pulp therapy (VPT) is performed to preserve dental pulp. However, the biocompatibility of the existing materials is of concern. Therefore, novel materials that can induce pulp healing without adverse effects need to be developed. Resolvin D2 (RvD2), one of specialized pro-resolving mediators, can resolve inflammation and promote the healing of periapical lesions. Therefore, RvD2 may be suitable for use in VPT. In the present study, we evaluated the efficacy of RvD2 against VPT using in vivo and in vitro models. Methods: First molars of eight-week-old male Sprague-Dawley rats were used for pulpotomy. They were then divided into three treatment groups: RvD2, phosphate-buffered saline, and calcium hydroxide groups. Treatment results were assessed using radiological, histological, and immunohistochemical (GPR18, TNF-α, Ki67, VEGF, TGF-ß, CD44, CD90, and TRPA1) analyses. Dental pulp-derived cells were treated with RvD2 in vitro and analyzed using cell-proliferation and cell-migration assays, real-time PCR (Gpr18, Tnf-α, Il-1ß, Tgf-ß, Vegf, Nanog, and Trpa1), ELISA (VEGF and TGF-ß), immunocytochemistry (TRPA1), and flow cytometry (dental pulp stem cells: DPSCs). Results: The formation of calcified tissue in the pulp was observed in the RvD2 and calcium hydroxide groups. RvD2 inhibited inflammation in dental pulp cells. RvD2 promoted cell proliferation and migration and the expression of TGF-ß and VEGF in vitro and in vivo. RvD2 increased the number of DPSCs. In addition, RvD2 suppressed TRPA1 expression as a pain receptor. Conclusion: RvD2 induced the formation of reparative dentin, anti-inflammatory effects, and decreased pain, along with the proliferation of DPSCs via the expression of VEGF and TGF-ß, on the pulp surface in pulpotomy models.