Silencing of Ac45 Simultaneously Inhibits Osteoclast-Mediated Bone Resorption and Attenuates Dendritic Cell-Mediated Inflammation through Impairing Acidification and Cathepsin K Secretion.

Endodontic disease is characterized by inflammation and destruction of periapical tissues, leading to severe bone resorption and tooth loss. ATP6AP1 (Ac45) has been implicated in human immune diseases, yet the mechanism underlying how Ac45 regulates immune response and reaction in inflammatory diseases remains unknown. We generated endodontic disease mice through bacterial infection as an inflammatory disease model and used adeno-associated virus (AAV)-mediated Ac45 RNA interference knockdown to study the function of Ac45 in periapical inflammation and bone resorption. We demonstrated that the AAV small hairpin RNA targeting Ac45 (AAV-sh-Ac45) impaired cellular acidification, extracellular acidification, and bone resorption. Our results showed that local delivery of AAV-sh-Ac45 in periapical tissues in bacterium-induced inflammatory lesions largely reduced bone destruction, inhibited inflammation, and dramatically reduced mononuclear immune cells. T-cell, macrophage, and dendritic cell infiltration in the periapical lesion was dramatically reduced, and the periodontal ligament was protected from inflammation-induced destruction. Furthermore, AAV-sh-Ac45 significantly reduced osteoclast formation and the expression of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-10 (IL-10), IL-12, IL-1α, IL-6, and IL-17. Interestingly, AAV-sh-Ac45 impaired mature cathepsin K secretion more significantly than that by AAV-sh-C1 and AAV-sh-CtsK Unbiased genome-wide transcriptome sequencing analysis of Ctsk-/- dendritic cells stimulated with lipopolysaccharide demonstrated that the ablation of Ctsk dramatically reduced dendritic cell-mediated inflammatory signaling. Taken together, our results indicated that AAV-sh-Ac45 simultaneously inhibits osteoclast-mediated bone resorption and attenuates dendritic cell-mediated inflammation through impairing acidification and cathepsin K secretion. Thus, Ac45 may be a novel target for therapeutic approaches to attenuate inflammation and bone erosion in endodontic disease and other inflammation-related osteolytic diseases.

A new multifunctional hydroxytyrosol-clofibrate with hypolipidemic, antioxidant, and hepatoprotective effects.

Oxidative stress has been regarded as the leading mechanism of the hepatotoxicity of clofibrate (CF). To achieve multifunctional novel hypolipidemic agents with hypolipidemia, antioxidant, and ameliorating liver injury, clofibric acid derivative hydroxytyrosol-clofibrate (CF-HT) was synthesized by molecular hybridization. CF-HT exhibited significant hypolipidemia, reducing serum triglyceride (TG), total cholesterol (TC), and malonaldehyde (MDA) by 30%, 33%, and 29% in hyperlipidemic mice induced by Triton WR 1339. CF-HT also shown hepatoprotective effect, a significant decrease in hepatic indices toxicity was observed, i.e. aspartate and lactate transaminases (AST and ALT) activities, alkalines phosphatases (ALP), and total bilirubin (TBIL) levels. The liver weight and liver coefficient were also ameliorated. Serum superoxide dismutase (SOD) was significantly elevated, and serum catalase (CAT) and malondialdehyde (MDA) content were remarkably restored. The hepatic glutathione (GSH) content was obviously increased and hepatic oxidized glutathione (GSSG) content was reduced dramatically by CF-HT, as compared to the CF treated mice (p < 0.05). Moreover, the histopathological damage that hepatocyte hyperplasia and hypertrophy was also significantly ameliorated by treatment with CF-HT. Therefore, the results indicated that CF-HT exerted more potent hypolipidemic activity and definite hepatoprotective effect which may mainly be associated with its antioxidative property in mice.

Cbfß deletion in mice recapitulates cleidocranial dysplasia and reveals multiple functions of Cbfß required for skeletal development.

The pathogenesis of cleidocranial dysplasia (CCD) as well as the specific role of core binding factor ß (Cbfß) and the Runt-related transcription factor (RUNX)/Cbfß complex in postnatal skeletogenesis remain unclear. We demonstrate that Cbfß ablation in osteoblast precursors, differentiating chondrocytes, osteoblasts, and odontoblasts via Osterix-Cre, results in severe craniofacial dysplasia, skeletal dysplasia, abnormal teeth, and a phenotype recapitulating the clinical features of CCD. Cbfß(f/f)Osterix-Cre mice have fewer proliferative and hypertrophic chondrocytes, fewer osteoblasts, and almost absent trabecular bone, indicating that Cbfß may maintain trabecular bone formation through its function in hypertrophic chondrocytes and osteoblasts. Cbfß(f/f)Collagen, type 1, alpha 1 (Col1α1)-Cre mice show decreased bone mineralization and skeletal deformities, but no radical deformities in teeth, mandibles, or cartilage, indicating that osteoblast lineage-specific ablation of Cbfß results in milder bone defects and less resemblance to CCD. Activating transcription factor 4 (Atf4) and Osterix protein levels in both mutant mice are dramatically reduced. ChIP assays show that Cbfß directly associates with the promoter regions of Atf4 and Osterix. Our data further demonstrate that Cbfß highly up-regulates the expression of Atf4 at the transcriptional regulation level. Overall, our genetic dissection approach revealed that Cbfß plays an indispensable role in postnatal skeletal development and homeostasis in various skeletal cell types, at least partially by up-regulating the expression of Atf4 and Osterix. It also revealed that CCD may result from functional defects of the Runx2/Cbfß heterodimeric complex in various skeletal cells. These insights into the role of Cbfß in postnatal skeletogenesis and CCD pathogenesis may assist in the development of new therapies for CCD and osteoporosis.

A small molecule, odanacatib, inhibits inflammation and bone loss caused by endodontic disease.

Periapical disease, an inflammatory disease mainly caused by dental caries, is one of the most prevalent infectious diseases of humans, affecting both children and adults. The infection travels through the root, leading to inflammation, bone destruction, and severe pain for the patient. Therefore, the development of a new class of anti-periapical disease therapies is necessary and critical for treatment and prevention. A small molecule, odanacatib (ODN), which is a cathepsin K (Ctsk) inhibitor, was investigated to determine its ability to treat this disease in a mouse model of periapical disease. While Ctsk was originally found in osteoclasts as an osteoclast-specific lysosomal protease, we were surprised to find that ODN can suppress the bacterium-induced immune response as well as bone destruction in the lesion area. X rays and microcomputed tomography (micro-CT) showed that ODN treatment had significant bone protection effects at different time points. Immunohistochemical and immunofluorescent staining show that ODN treatment dramatically decreased F4/80+ macrophages and CD3+ T cells in the lesion areas 42 days after infection. Consistent with these findings, quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) analysis showed low levels of proinflammatory mRNAs (for tumor necrosis factor alpha, interleukin 6, and interleukin 23α) and corresponding cytokine expression in the ODN-treated disease group. The levels of mRNA for Toll-like receptors 4, 5, and 9 also largely decreased in the ODN-treated disease group. Our results demonstrated that ODN can inhibit endodontic disease development, bone erosion, and immune response. These results indicate that application of this small molecule offers a new opportunity to design effective therapies that could prevent periapical inflammation and revolutionize current treatment options.

Ac45 silencing mediated by AAV-sh-Ac45-RNAi prevents both bone loss and inflammation caused by periodontitis.

AIM: Periodontitis induced by oral pathogens leads to severe periodontal tissue damage and osteoclast-mediated bone resorption caused by inflammation. On the basis of the importance of Ac45 in osteoclast formation and function, we performed this study to evaluate the therapeutic potential of periodontitis by local adeno-associated virus (AAV)-mediated Ac45 gene knockdown. MATERIAL AND METHODS: We used AAV-mediated short hairpin RNAi knockdown of Ac45 gene expression (AAV-sh-Ac45) to inhibit bone erosion and gingival inflammation simultaneously in a well-established periodontitis mouse model induced by Porphyromonas gingivalis W50. Histological studies were performed to evaluate the bone protection of AAV-sh-Ac45. Immunochemistry, ELISA and qRT-PCR were performed to reveal the role of Ac45 knockdown on inflammation, immune response and expression of cytokine. RESULTS: We found that Ac45 knockdown impaired osteoclast-mediated extracellular acidification and bone resorption in vitro and in vivo. Furthermore, local administration of AAV-sh-Ac45 protected mice from bone erosion by >85% and attenuated inflammation and decreased infiltration of T cells, dendritic cells and macrophages in the periodontal lesion. Notably, the expression of pro-inflammatory cytokines was also reduced. CONCLUSIONS: Local AAV-sh-Ac45 gene therapy efficiently protects against periodontal tissue damage and bone erosion through both inhibition of osteoclast function and attenuating inflammation, and may represent a powerful new treatment strategy for periodontitis.

The significance of Notch ligand expression in the peripheral blood of children with hand, foot and mouth disease (HFMD).

BACKGROUND: Hand, foot and mouth disease (HFMD), a virus-induced infectious disease that usually affects infants and children, has an increased incidence in China in recent years. This study attempted to investigate the role of the Notch signaling pathway in the pathogenesis of HFMD. METHODS: Eighty-two children diagnosed with HFMD were enrolled into this study. The HFMD group was further divided into the uncomplicated HFMD and HFMD with encephalitis groups. The control group included 40 children who underwent elective surgery for treatment of inguinal hernias. RESULTS: Children with HFMD displayed significantly reduced CD3+, CD3+CD4+ and CD3+CD8+ cell subsets, but substantially enhanced CD3-CD19+ cell subset (p<0.05 versus control subjects). The expression levels of Notch ligands Dll1 and Dll4 in the peripheral blood of the HFMD group were significantly higher than those in the control group (p<0.05). There were statistically significant differences in CD3+, CD3+CD4+ and CD3-CD19+ cell subsets, but not in Notch ligand expression, between the uncomplicated HFMD and HFMD with encephalitis groups. Dll4 expression in HFMD subjects correlated negatively with the CD3+ and CD3+CD8+ cell subsets (p<0.05), but positively with the CD3-CD19+ cell subset (p<0.05). Furthermore, Dll4 expression in HFMD with encephalitis subjects correlated positively with total white blood cell (WBC) counts and total protein contents in cerebrospinal fluid (CSF) (p<0.05). CONCLUSIONS: The Notch ligand Dll4 exhibits a strong correlation with the CD3+, CD3+CD8+ and CD3-CD19+ cell subsets in children with HFMD, indicating that the Notch signaling may be involved in the development of HFMD by affecting the number and status of peripheral lymphocytes.

RNA interference-mediated silencing of Atp6i prevents both periapical bone erosion and inflammation in the mouse model of endodontic disease.

Dental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7 gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6i inhibition impaired osteoclast function in vitro and in vivo and decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7 knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i(+/-) mice with haploinsufficiency of Atp6i exhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7 knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease.

Atp6i deficient mouse model uncovers transforming growth factor-ß1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation.

The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood. We found that Atp6i deficient mice (Atp6i-/-) arrested tooth root formation, indicated by truncated Hertwig's epithelial root sheath (HERS) progression. Furthermore, Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation. Atp6i-/- mice had largely decreased expression of odontoblast differentiation marker gene expression profiles (Col1a1, Nfic, Dspp, and Osx) in the alveolar bone. Atp6i-/- mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers. Additionally, there was a significant reduction in Smad2/3 activation, inhibiting transforming growth factor-ß (TGF-ß) signaling in Atp6i-/- odontoblasts. Through treating pulp precursor cells with Atp6i-/- or wild-type OC bone resorption-conditioned medium, we found the latter medium to promote odontoblast differentiation, as shown by increased odontoblast differentiation marker genes expression (Nfic, Dspp, Osx, and Runx2). This increased expression was significantly blocked by anti-TGF-ß1 antibody neutralization, whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i-/- OC conditioned medium. Importantly, ectopic TGF-ß1 partially rescued root development and root dentin deposition of Atp6i-/- mice tooth germs were transplanted under mouse kidney capsules. Collectively, our novel data shows that the prevention of TGF-ß1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation. As such, this study uncovers TGF-ß1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.

Trio cooperates with Myh9 to regulate neural crest-derived craniofacial development.

Trio is a unique member of the Rho-GEF family that has three catalytic domains and is vital for various cellular processes in both physiological and developmental settings. TRIO mutations in humans are involved in craniofacial abnormalities, in which patients present with mandibular retrusion. However, little is known about the molecular mechanisms of Trio in neural crest cell (NCC)-derived craniofacial development, and there is still a lack of direct evidence to assign a functional role to Trio in NCC-induced craniofacial abnormalities. Methods:In vivo, we used zebrafish and NCC-specific knockout mouse models to investigate the phenotype and dynamics of NCC development in Trio morphants. In vitro, iTRAQ, GST pull-down assays, and proximity ligation assay (PLA) were used to explore the role of Trio and its potential downstream mediators in NCC migration and differentiation. Results: In zebrafish and mouse models, disruption of Trio elicited a migration deficit and impaired the differentiation of NCC derivatives, leading to craniofacial growth deficiency and mandibular retrusion. Moreover, Trio positively regulated Myh9 expression and directly interacted with Myh9 to coregulate downstream cellular signaling in NCCs. We further demonstrated that disruption of Trio or Myh9 inhibited Rac1 and Cdc42 activity, specifically affecting the nuclear export of ß-catenin and NCC polarization. Remarkably, craniofacial abnormalities caused by trio deficiency in zebrafish could be partially rescued by the injection of mRNA encoding myh9, ca-Rac1, or ca-Cdc42. Conclusions: Here, we identified that Trio, interacting mostly with Myh9, acts as a key regulator of NCC migration and differentiation during craniofacial development. Our results indicate that trio morphant zebrafish and Wnt1-cre;Triofl/fl mice offer potential model systems to facilitate the study of the pathogenic mechanisms of Trio mutations causing craniofacial abnormalities.

Developments in Antibacterial Therapy: Focus on Physical Stimuli Approaches.

At present, various antibacterial therapeutic modalities are available in the clinic. However, due to the rampant abuse of antibiotics over the past few decades and the consequent emergence of innumerable drug-resistant strains of bacteria, it is imperative to develop new and effective antibacterial therapeutic strategies. In recent years, the physical stimuli-based approach to antibacterial therapy has aroused much interest as an alternative to antibiotics and has become a major focus of antibacterial research. In this review, the application of different physical stimuli, including electricity, magnetism, light, ultrasound and thermal stimulation, in antibacterial research is critically examined in order to provide new ideas and directions for the further development of antibacterial therapy in clinical dentistry.

C1 Silencing Attenuates Inflammation and Alveolar Bone Resorption in Endodontic Disease.

INTRODUCTION: Endodontic disease, 1 of the most prevalent chronic infectious diseases worldwide, occurs when the dental pulp becomes infected and inflamed, leading to bone destruction around the tooth root, severe pain, and tooth loss. Although many studies have tried to develop therapies to alleviate the bone erosion and inflammation associated with endodontic disease, there is an urgent need for an effective treatment. METHODS: In this study, we used a gene-based therapy approach by administering recombinant adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown to target both periapical bone resorption and inflammation in the mouse model of endodontic disease. RESULTS: The results showed that Atp6v1c1 knockdown is simultaneously capable of reducing bone resorption by 70% through impaired osteoclast activation, inhibiting inflammation by decreasing T-cell infiltration in the periapical lesion by 75%, and protecting the periodontal ligament from destruction caused by inflammation. Notably, AAV-mediated gene therapy of Atp6v1c1 knockdown significantly reduced proinflammatory cytokine expression, including tumor necrosis factor α, interleukin 1α, interleukin 17, interleukin 12, and interleukin 6 levels in periapical tissues caused by bacterial infection. Quantitative real-time polymerase chain reaction revealed that Atp6v1c1 knockdown reduced osteoclast-specific functional genes (ie, Ctsk) in periapical tissues. CONCLUSIONS: Our results showed that AAV-mediated Atp6v1c1 knockdown in periapical tissues slowed endodontic disease progression, prevented bone erosion, and alleviated inflammation in the periapical tissues and periodontal ligament potentially through regulation of toll-like receptor signaling, indicating that targeting Atp6v1c1 may facilitate the design of novel therapeutic approaches to reduce inflammation and bone erosion in endodontic disease.

GATA4 as a novel regulator involved in the development of the neural crest and craniofacial skeleton via Barx1.

The role of GATA-binding protein 4 (GATA4) in neural crest cells (NCCs) is poorly defined. Here we showed that mouse NCCs lacking GATA4 exhibited developmental defects in craniofacial bone, teeth, and heart. The defects likely occurred due to decreased cell proliferation at the developmental stage. The in vitro results were consistent with the mouse model. The isobaric tags for relative and absolute quantitation assay revealed that BARX1 is one of the differentially expressed proteins after GATA4 knockdown in NCCs. On the basis of the results of dual-luciferase, electro-mobility shift, and chromatin immunoprecipitation assays, Barx1 expression is directly regulated by GATA4 in NCCs. In zebrafish, gata4 knockdown affects the development of NCCs derivatives. However, the phenotype in zebrafish could be partly rescued by co-injection of gata4 morpholino oligomers and barx1 mRNA. This study identified new downstream targets of GATA4 in NCCs and uncovered additional evidence of the complex regulatory functions of GATA4 in NCC development.

Bone resorption deficiency affects tooth root development in RANKL mutant mice due to attenuated IGF-1 signaling in radicular odontoblasts.

The tooth root is essential for normal tooth physiological function. Studies on mice with mutations or targeted gene deletions revealed that osteoclasts (OCs) play an important role in tooth root development. However, knowledge on the cellular and molecular mechanism underlying how OCs mediate root formation is limited. During bone formation, growth factors (e.g. Insulin-like growth factor-1, IGF-1) liberated from bone matrix by osteoclastic bone resorption stimulate osteoblast differentiation. Thus, we hypothesize that OC-osteoblast coupling may also apply to OC-odontoblast coupling; therefore OCs may have a direct impact on odontoblast differentiation through the release of growth factor(s) from bone matrix, and consequently regulate tooth root formation. To test this hypothesis, we used a receptor activator of NF-κB ligand (RANKL) knockout mouse model in which OC differentiation and function was entirely blocked. We found that molar root formation and tooth eruption were defective in RANKL-/- mice. Disrupted elongation and disorganization of Hertwig's epithelial root sheath (HERS) was observed in RANKL-/- mice. Reduced expression of nuclear factor I C (NFIC), osterix, and dentin sialoprotein, markers essential for radicular (root) odontogenic cell differentiation indicated that odontoblast differentiation was disrupted in RANKL deficient mice likely contributing to the defect in root formation. Moreover, down-regulation of IGF/AKT/mTOR activity in odontoblast indicated that IGF signaling transduction in odontoblasts of the mutant mice was impaired. Treating odontoblast cells in vitro with conditioned medium from RANKL-/- OCs cultured on bone slices resulted in inhibition of odontoblast differentiation. Moreover, depletion of IGF-1 in bone resorption-conditioned medium (BRCM) from wild-type (WT) OC significantly compromised the ability of WT osteoclastic BRCM to induce odontoblast differentiation while addition of IGF-1 into RANKL-/- osteoclastic BRCM rescued impaired odontoblast differentiation, confirming that root and eruption defect in RANKL deficiency mice may result from failure of releasing of IGF-1 from bone matrix through OC bone resorption. These results suggest that OCs are important for odontoblast differentiation and tooth root formation, possibly through IGF/AKT/mTOR signaling mediated by cell-bone matrix interaction. These findings provide significant insights into regulatory mechanism of tooth root development, and also lay the foundation for root regeneration studies.

The Triple Functions of D2 Silencing in Treatment of Periapical Disease.

INTRODUCTION: Dental caries is the most widespread chronic infectious disease. Inflammation in pulp tissues caused by dental caries will lead to periapical granulomas, bone erosion, loss of the tooth, and severe pain. Despite numerous efforts in recent studies to develop effective treatments for dental caries, the need for a potent therapy is still urgent. METHODS: In this study, we applied a gene-based therapy approach by administering recombinant adeno-associated virus (AAV)-mediated Atp6v0d2 (d2) RNA interference knockdown of d2 gene expression to prevent periapical bone loss and suppress periapical inflammation simultaneously. RESULTS: The results showed that d2 depletion is simultaneously capable of reducing bone resorption with 75% protection through reducing osteoclasts, enhancing bone formation by increasing osterix expression, and inhibiting inflammation by decreasing T-cell infiltration. Notably, AAV-mediated gene therapy of d2 knockdown significantly reduced proinflammatory cytokine expression, including tumor necrosis factor α, interferon-γ, interleukin-1α, and interleukin 6 levels in periapical diseases caused by bacterial infection. Quantitative real-time polymerase chain reaction revealed that d2 knockdown reduced osteoclast-specific functional genes (ie, Acp5 and Ctsk) and increased osteoblast marker genes (ie, Osx and Opg) in periapical tissues. CONCLUSIONS: Collectively, our results showed that AAV-mediated d2 depletion in the periapical lesion area can prevent the progression of endodontic disease and bone erosion while significantly reducing the inflammatory over-response. These findings show that the depletion of d2 simultaneously reduces bone resorption, enhances bone formation, and inhibits inflammation caused by periapical diseases and provide significant insights into the potential effectiveness of AAV-sh-d2-mediated d2 silencing gene therapy as a major endodontic treatment.

Odanacatib, A Cathepsin K-Specific Inhibitor, Inhibits Inflammation and Bone Loss Caused by Periodontal Diseases.

BACKGROUND: Periodontitis is a bacteria-induced inflammatory disease mainly affecting periodontal tissues, leading to periodontal inflammation, bone breakdown, and loss of the tooth. The main obstacle for treating periodontitis effectively is the difficulty in finding a target that can inhibit bone loss and inflammation simultaneously. Recent studies showed that cathepsin K (CTSK) might have functions in the immune system besides its role in osteoclasts. Thus, targeting CTSK would have a potential therapeutic effect in both the bone system and the immune system during the progression of periodontitis. METHODS: In the current study, a small molecular inhibitor (odanacatib [ODN]) is explored to inhibit the function of CTSK in a bacteria-induced periodontitis mouse model. RESULTS: The application of ODN decreased the number of osteoclasts, macrophages, and T cells, as well as the expression of Toll-like receptors (TLRs) in the periodontitis lesion area. Furthermore, lack of CTSK inhibited the expression of TLR4, TLR5, and TLR9 and their downstream cytokine signaling in the gingival epithelial cells in periodontitis lesions, demonstrating that the innate immune response was inhibited in periodontitis. CONCLUSION: The present results show that inhibition of CTSK can prevent bone loss and the immune response during the progression of periodontitis, indicating that CTSK is a promising target for treating inflammatory diseases such as periodontitis by affecting both osteoclasts and the immune system.

RNAi-mediated silencing of Atp6i and Atp6i haploinsufficiency prevents both bone loss and inflammation in a mouse model of periodontal disease.

Periodontal disease affects about 80% of adults in America, and is characterized by oral bacterial infection-induced gingival inflammation, oral bone resorption, and tooth loss. Periodontitis is also associated with other diseases such as rheumatoid arthritis, diabetes, and heart disease. Although many efforts have been made to develop effective therapies for this disease, none have been very effective and there is still an urgent need for better treatments and preventative strategies. Herein we explored for the first time the possibility that adeno-associated virus (AAV)-mediated RNAi knockdown could be used to treat periodontal disease with improved efficacy. For this purpose, we used AAV-mediated RNAi knockdown of Atp6i/TIRC7 gene expression to target bone resorption and gingival inflammation simultaneously. Mice were infected with the oral pathogen Porphyromonas gingivalis W50 (P. gingivalis) in the maxillary periodontium to induce periodontitis. We found that Atp6i depletion impaired extracellular acidification and osteoclast-mediated bone resorption. Furthermore, local injection of AAV-shRNA-Atp6i/TIRC7 into the periodontal tissues in vivo protected mice from P. gingivalis infection-stimulated bone resorption by >85% and decreased the T-cell number in periodontal tissues. Notably, AAV-mediated Atp6i/TIRC7 knockdown also reduced the expression of osteoclast marker genes and inflammation-induced cytokine genes. Atp6i(+/-) mice with haploinsufficiency were similarly protected from P. gingivalis infection-stimulated bone loss and gingival inflammation. This suggests that AAV-shRNA-Atp6i/TIRC7 therapeutic treatment may significantly improve the health of millions who suffer from P. gingivalis-mediated periodontal disease.

B and T lymphocytes are the primary sources of RANKL in the bone resorptive lesion of periodontal disease.

Receptor activator of nuclear factor-kappaB (RANKL)-mediated osteoclastogenesis plays a pivotal role in inflammatory bone resorption. The aim of this study was to identify the cellular source of RANKL in the bone resorptive lesions of periodontal disease. The concentrations of soluble RANKL, but not its decoy receptor osteoprotegerin, measured in diseased tissue homogenates were significantly higher in diseased gingival tissues than in healthy tissues. Double-color confocal microscopic analyses demonstrated less than 20% of both B cells and T cells expressing RANKL in healthy gingival tissues. By contrast, in the abundant mononuclear cells composed of 45% T cells, 50% B cells, and 5% monocytes in diseased gingival tissues, more than 50 and 90% of T cells and B cells, respectively, expressed RANKL. RANKL production by nonlymphoid cells was not distinctly identified. Lymphocytes isolated from gingival tissues of patients induced differentiation of mature osteoclast cells in a RANKL-dependent manner in vitro. However, similarly isolated peripheral blood B and T cells did not induce osteoclast differentiation, unless they were activated in vitro to express RANKL; emphasizing the osteoclastogenic potential of activated RANKL-expressing lymphocytes in periodontal disease tissue. These results suggest that activated T and B cells can be the cellular source of RANKL for bone resorption in periodontal diseased gingival tissue.