RANKL-mediated osteoclast formation is required for bone loss in a murine model of Staphylococcus aureus osteomyelitis.

Staphylococcus aureus osteomyelitis leads to extensive bone destruction. Osteoclasts are bone resorbing cells that are often increased in bone infected with S. aureus. The cytokine RANKL is essential for osteoclast formation under physiological conditions but in vitro evidence suggests that inflammatory cytokines may by-pass the requirement for RANKL. The goal of this study was to determine whether RANKL-dependent osteoclast formation is essential for the bone loss that occurs in a murine model of S. aureus osteomyelitis. To this end, humanized-RANKL mice were infected by direct inoculation of S. aureus into a unicortical defect in the femur. Mice were treated with vehicle or denosumab, a human monoclonal antibody that inhibits RANKL, both before and during a 14-day infection period. The severe cortical bone destruction caused by infection was completely prevented by denosumab administration even though the bacterial burden in the femur was not affected. Osteoclasts were abundant near the inoculation site in vehicle-treated mice but absent in denosumab-treated mice. In situ hybridization demonstrated that S. aureus infection potently stimulated RANKL expression in bone marrow stromal cells. The extensive reactive bone formation that occurs in this osteomyelitis model was also reduced by denosumab administration. Lastly, there was a notable lack of osteoblasts near the infection site suggesting that the normal coupling of bone formation to bone resorption was disrupted by S. aureus infection. These results demonstrate that RANKL-mediated osteoclast formation is required for the bone loss that occurs in S. aureus infection and suggest that disruption of the coupling of bone formation to bone resorption may also contribute to bone loss in this condition.

Imbalanced EphB4/EphrinB2 Signaling Modulates Bone Resorption in Periodontitis Induced by Porphyromonas gingivalis.

Periodontitis, a chronic infectious disease in periodontal tissues, is characterized by an imbalance of alveolar bone resorption and remodeling, which eventually results in tooth loosening and even tooth loss. The etiology of periodontitis is polymicrobial synergy and dysbiosis, in which Porphyromonas gingivalis (P. gingivalis) is one of the primary pathogens responsible for periodontitis progression. The interplay of EphrinB2/EphB4 is crucial for osteoblast-osteoclast communication during bone remodeling and healing. This study investigates the mechanism of EphB4/EphrinB2 transduction modulating osteogenesis inhibition and bone resorption in periodontitis induced by P. gingivalis. An in vivo model of chronic periodontitis provoked by P. gingivalis was constructed, the inflammation and bone resorption were evaluated. The expression of EphB4 and EphrinB2 proteins in periodontal tissues was detected, which was also evaluated, respectively, in osteoblasts and osteoclasts infected with P. gingivalis in vitro. Then, a simulated coculture model of osteoblasts and osteoclasts was established to activate the forward and reverse pathways of EphB4/EphrinB2 with P. gingivalis infection. This study showed that P. gingivalis infection promoted alveolar bone resorption in rats and enhanced EphB4 and EphrinB2 expression in periodontal tissues. EphB4 and molecules associated with osteogenesis in osteoblasts infected with P. gingivalis were inhibited, while EphrinB2 and osteoclast differentiation-related markers in osteoclasts were activated. In conclusion, this study suggested that EphB4/EphrinB2 proteins were involved in alveolar bone remodeling in the process of periodontitis induced by P. gingivalis infection. Moreover, attenuated EphB4/EphrinB2 with P. gingivalis infection weakened osteoblast activity and enhanced osteoclast activity.

Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Clostridium butyricum Alleviates Gut Microbiota Alteration-Induced Bone Loss after Bariatric Surgery by Promoting Bone Autophagy.

Bariatric surgery is the most common and effective treatment of severe obesity; however, these bariatric procedures always result in detrimental effects on bone metabolism by underlying mechanisms. This study aims to investigate the skeletal response to bariatric surgery and to explore whether Clostridium butyricum alleviates gut microbiota alteration-induced bone loss after bariatric surgery. Consequently, male SD rats received Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) surgery, respectively, followed by body weight recording. The bone loss after bariatric surgery was further determined by dual-energy X-ray absorptiometry (DXA), micro-CT measurement, histologic analyses, and Western blot. Besides, 16S rDNA gene sequencing was performed to determine the gut microbiota alteration after surgery, and intervention with fecal microbiota from RYGB donor was conducted in obese SD rats, followed by C. butyricum administration. Accordingly, rats in the RYGB and SG groups maintained sustained weight loss, and DXA and micro-CT measurement further demonstrated significant bone loss after bariatric surgery. Besides, histologic and Western blot analyses validated enhanced osteoclastogenesis and inhibited osteoblastogenesis and defective autophagy after surgery. The 16S rDNA gene sequencing suggested a significant alteration of gut microbiota composition in the RYGB group, and intervention with fecal microbiota from RYGB donor further determined that this kind of alteration contributed to the bone loss after RYGB. Meanwhile, C. butyricum might protect against this postoperative bone loss by promoting osteoblast autophagy. In summary, this study suggests novel mechanisms to clarify the skeletal response to bariatric surgery and provides a potential candidate for the treatment of bone disorder among bariatric patients. SIGNIFICANCE STATEMENT: The significance of this study is the discovery of obvious bone loss and defective autophagy after bariatric surgery. Besides, it is revealed that gut microbiota alterations could be the reason for impaired bone mass after bariatric surgery. Furthermore, Clostridium butyricum could alleviate the gut microbiota alteration-induced bone loss after bariatric surgery by promoting osteoblast autophagy.

Root canal contamination or exposure to lipopolysaccharide differentially modulate prostaglandin E 2 and leukotriene B 4 signaling in apical periodontitis

Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.

Streptococcus gordonii induces bone resorption by increasing osteoclast differentiation and reducing osteoblast differentiation.

Streptococcus gordonii is commonly found in the periapical endodontic lesions of patients with apical periodontitis, a condition characterized by inflammation and periapical bone loss. Since bone metabolism is controlled by osteoclastic bone resorption and osteoblastic bone formation, we investigated the effects of S. gordonii on the differentiation and function of osteoclasts and osteoblasts. For the determination of bone resorption activity in vivo, collagen sheets soaked with heat-killed S. gordonii were implanted on mouse calvaria, and the calvarial bones were scanned by micro-computed tomography. Mouse bone marrow-derived macrophages (BMMs) were stimulated with M-CSF and RANKL for 2 days and then differentiated into osteoclasts in the presence or absence of heat-killed S. gordonii. Tartrate-resistant acid phosphatase staining was performed to determine osteoclast differentiation. Primary osteoblast precursors were differentiated into osteoblasts with ascorbic acid and ß-glycerophosphate in the presence or absence of heat-killed S. gordonii. Alkaline phosphatase staining and alizarin red S staining were conducted to determine osteoblast differentiation. Western blotting was performed to examine the expression of transcription factors including c-Fos, NFATc1, and Runx2. Heat-killed S. gordonii induced bone destruction in a mouse calvarial implantation model. The differentiation of RANKL-primed BMMs into osteoclasts was enhanced in the presence of heat-killed S. gordonii. Heat-killed S. gordonii increased the expression of c-Fos and NFATc1, which are essential transcription factors for osteoclast differentiation. On the other hand, heat-killed S. gordonii inhibited osteoblast differentiation and reduced the expression of Runx2, an essential transcription factor for osteoblast differentiation. S. gordonii exerts bone resorptive activity by increasing osteoclast differentiation and reducing osteoblast differentiation, which may be involved in periapical bone resorption.

A dysbiotic microbiome triggers TH17 cells to mediate oral mucosal immunopathology in mice and humans.

Periodontitis is one of the most common human inflammatory diseases, yet the mechanisms that drive immunopathology and could be therapeutically targeted are not well defined. Here, we demonstrate an expansion of resident memory T helper 17 (TH17) cells in human periodontitis. Phenocopying humans, TH17 cells expanded in murine experimental periodontitis through local proliferation. Unlike homeostatic oral TH17 cells, which accumulate in a commensal-independent and interleukin-6 (IL-6)-dependent manner, periodontitis-associated expansion of TH17 cells was dependent on the local dysbiotic microbiome and required both IL-6 and IL-23. TH17 cells and associated neutrophil accumulation were necessary for inflammatory tissue destruction in experimental periodontitis. Genetic or pharmacological inhibition of TH17 cell differentiation conferred protection from immunopathology. Studies in a unique patient population with a genetic defect in TH17 cell differentiation established human relevance for our murine experimental studies. In the oral cavity, human TH17 cell defects were associated with diminished periodontal inflammation and bone loss, despite increased prevalence of recurrent oral fungal infections. Our study highlights distinct functions of TH17 cells in oral immunity and inflammation and paves the way to a new targeted therapeutic approach for the treatment of periodontitis.

Staphylococcus Aureus Induces Osteoclastogenesis via the NF-κB Signaling Pathway.

BACKGROUND Osteomyelitis is one of the refractory diseases encountered in orthopedics, while Staphylococcus aureus (S. aureus) is the most common causative organism in osteomyelitis. However, the precise mechanisms underlying the bone loss caused by S. aureus infection have not been well defined. Here, we investigated the effect of S. aureus on osteoclast differentiation and the probable molecular mechanism. MATERIAL AND METHODS RAW 264.7 cells were treated for 5 days with live S. aureus, inactivated S. aureus, and S. aureus filtrate. Then, the formation of osteoclast-like cells and resorption pits was observed, and the expression of osteoclast-specific genes (TRAP, MMP-9, cathepsin K, CTR and Atp6v0d2) was detected by real-time PCR. Moreover, key proteins in the signaling pathway associated with osteoclast differentiation were detected with Western blot. RESULTS The data showed that live S. aureus, inactivated S. aureus, and S. aureus filtrate induced osteoclast formation, promoted bone resorption, and increased the expression of osteoclast-specific genes in a dose-dependent manner in the absence RANKL. In addition, we found that the S. aureus-induced osteoclastogenesis was related to the degradation of IκB-a, phosphorylation of NF-κB p65, and increased expression of NFATc1. Thus, we used JSH-23 to inhibit NF-κB transcriptional activity. The effect of the S. aureus-induced osteoclastogenesis and the expression of osteoclast-specific genes and NFATc1 were inhibited, which indicated that the NF-κB signaling pathway plays a role in S. aureus-induced osteoclastogenesis. CONCLUSIONS This study demonstrated that S. aureus induces osteoclastogenesis through its cell wall compound and secretion of small soluble molecules, and the NF-κB signaling pathway plays a role in this process.

Diabetes Enhances IL-17 Expression and Alters the Oral Microbiome to Increase Its Pathogenicity.

Diabetes is a risk factor for periodontitis, an inflammatory bone disorder and the greatest cause of tooth loss in adults. Diabetes has a significant impact on the gut microbiota; however, studies in the oral cavity have been inconclusive. By 16S rRNA sequencing, we show here that diabetes causes a shift in oral bacterial composition and, by transfer to germ-free mice, that the oral microbiota of diabetic mice is more pathogenic. Furthermore, treatment with IL-17 antibody decreases the pathogenicity of the oral microbiota in diabetic mice; when transferred to recipient germ-free mice, oral microbiota from IL-17-treated donors induced reduced neutrophil recruitment, reduced IL-6 and RANKL, and less bone resorption. Thus, diabetes-enhanced IL-17 alters the oral microbiota and renders it more pathogenic. Our findings provide a mechanistic basis to better understand how diabetes can increase the risk and severity of tooth loss.

Staphylococcal protein A promotes osteoclastogenesis through MAPK signaling during bone infection.

Bone infection is a common and serious complication in the orthopedics field, which often leads to excessive bone destruction and non-union. Osteoclast is the only type of cells which have the function of bone resorption. Its over activation is closely related to excessive bone loss. Staphylococcus aureus (S. aureus) is a major pathogen causing bone infection, which can produce a large number of strong pathogenic substances staphylococcal protein A (SPA). However, few studies were reported about the effects of SPA on osteoclastogenesis. In our study, we observed that S. aureus activated osteoclasts and promoted bone loss in bone infection specimens. Then, we investigated the effects of SPA on RANKL-induced osteoclastogenesis in vitro, the results revealed that SPA promoted osteoclastic differentiation and fusion, and enhanced osteoclastic bone resorption. In addition, we also showed that SPA upregulated the expression of NFATc1 and c-FOS through the activation of MAPK signaling to promote osteoclastogenesis. Our findings might help us better understand the pathogenic role of S. aureus in bone infection and develop new therapeutic strategies for infectious bone diseases.

Use of ethanol extracts of Terminalia chebula to prevent periodontal disease induced by dental plaque bacteria.

BACKGROUND: The fruit of the Terminalia chebula tree has been widely used for the treatment of various disorders. Its anti-diabetic, anti-mutagenic, anti-oxidant, anti-bacterial, anti-fungal, and anti-viral effects have been studied. Dental plaque bacteria (DPB) are intimately associated with gingivitis and periodontitis. In the quest for materials that will prove useful in the treatment and prevention of periodontal disease, we investigated the preventive effects of an ethanol extract of Terminalia chebula (EETC) on DPB-induced inflammation and bone resorption. METHODS: The anti-bacterial effect of EETC was analyzed using the disc diffusion method. The anti-inflammatory effect of EETC was determined by molecular biological analysis of the DPB-mediated culture cells. Prevention of osteoclastic bone resorption by EETC was explored using osteoclast formation and pit formation assays. RESULTS: EETC suppressed the growth of oral bacteria and reduced the induction of inflammatory cytokines and proteases, abolishing the expression of PGE2 and COX-2 and inhibiting matrix damage. By stimulating the DPB-derived lipopolysaccharides, EETC inhibited both osteoclast formation in osteoclast precursors and RANKL expression in osteoblasts, thereby contributing to the prevention of bone resorption. CONCLUSIONS: EETC may be a beneficial supplement to help prevent DPB-mediated periodontal disease.

Tannerella forsythia GroEL induces inflammatory bone resorption and synergizes with interleukin-17.

Tannerella forsythia is a major periodontal pathogen, and T. forsythia GroEL is a molecular chaperone homologous to human heat-shock protein 60. Interleukin-17 (IL-17) has been implicated in the pathogenesis of periodontitis and several systemic diseases. This study investigated the potential of T. forsythia GroEL to induce inflammatory bone resorption and examined the cooperative effect of IL-17 and T. forsythia GroEL on inflammatory responses. Human gingival fibroblasts (HGFs) and periodontal ligament (PDL) fibroblasts were stimulated with T. forsythia GroEL and/or IL-17. Gene expression of IL-6, IL-8, and cyclooxygenase-2 (COX-2) and concentrations of IL-6, IL-8, and prostaglandin E2 (PGE2 ) were measured by real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. After stimulation of MG63 cells with T. forsythia GroEL and/or IL-17, gene expression of osteoprotegerin (OPG) was examined. After subcutaneous injection of T. forsythia GroEL and/or IL-17 above the calvaria of BALB/c mice, calvarial bone resorption was assessed by micro-computed tomography and histological examination. Tannerella forsythia GroEL induced IL-6 and IL-8 production in HGFs and PDL cells, and IL-17 further promoted IL-6 and IL-8 production. Both T. forsythia GroEL and IL-17 synergistically increased PGE2 production and inhibited OPG gene expression. Calvarial bone resorption was induced by T. forsythia GroEL injection, and simultaneous injection of T. forsythia GroEL and IL-17 further increased bone resorption. These results suggest that T. forsythia GroEL is a novel virulence factor that can contribute to inflammatory bone resorption caused by T. forsythia and synergizes with IL-17 to exacerbate inflammation and bone resorption.

Inhibitory effect of 1,25-dihydroxyvitamin D3 on Porphyromonas gingivalis-induced inflammation and bone resorption in vivo.

OBJECTIVE: To investigate whether intragastric administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) could inhibit the bone resorption and inflammation in a mouse calvarial model infected by Porphyromonas gingivalis (P. gingivalis). DESIGN: Live P. gingivalis ATCC 33277 was injected once daily for 6days into the subcutaneous tissue overlying the calvaria in mice. At the same time, 1,25(OH)2D3 (50µg/kg per day) was administered by gavage for 9days, starting 3d before the infection. Mice were killed under ether anesthesia 8h after the last injection of P. gingivalis. Micro-computed tomography scanning was used to evaluate calvarial bone loss. Tartrate-resistant acid phosphatase was used to detect osteoclast activity. Real-time PCR was used to assess the mRNA expressions of OPG, RANKL, c-Fos, NFATc1, CTSK and TRAP in calvarial bone and IL-6, IL-10, IL-1ß, IL-12p40 and TNF-α in soft tissue. The levels of serum IL-6, IL-10 were determined by ELISA. RESULTS: 1,25(OH)2D3 treatment apparently attenuated bone resorption in P. gingivalis-induced mouse calvarial model and markedly reduced the number of osteoclasts. The expression levels of RANKL and osteoclast-related genes such as c-Fos, NFATc1, CTSK and TRAP were also decreased by 1,25(OH)2D3. Besides, 1,25(OH)2D3 inhibited the expression of pro-inflammatory cytokines IL-6, IL-12p40 and TNF-α and enormously elevated the expression of anti-inflammatory cytokine IL-10. CONCLUSION: 1,25(OH)2D3 may decrease bone resorption in vivo via suppressing the expression of osteoclast-related genes and its anti-inflammatory properties.

NOD1 in the modulation of host-microbe interactions and inflammatory bone resorption in the periodontal disease model.

Periodontitis is a chronic inflammatory condition characterized by destruction of non-mineralized and mineralized connective tissues. It is initiated and maintained by a dysbiosis of the bacterial biofilm adjacent to teeth with increased prevalence of Gram-negative microorganisms. Nucleotide-binding oligomerization domain containing 1 (NOD1) is a member of the Nod-like receptors (NLRs) family of proteins that participate in the activation of the innate immune system, in response to invading bacteria or to bacterial antigens present in the cytoplasm. The specific activating ligand for NOD1 is a bacterial peptidoglycan derived primarily from Gram-negative bacteria. This study assessed the role of NOD1 in inflammation-mediated tissue destruction in the context of host-microbe interactions. We used mice with whole-genome deletion of the NOD1 gene in a microbe-induced periodontitis model using direct injections of heat-killed Gram-negative or Gram-negative/Gram-positive bacteria on the gingival tissues. In vitro experiments using primary bone-marrow-derived macrophages from wild-type and NOD1 knockout mice provide insight into the role of NOD1 on the macrophage response to Gram-negative and Gram-negative/Gram-positive bacteria. Microcomputed tomography analysis indicated that deletion of NOD1 significantly aggravated bone resorption induced by Gram-negative bacteria, accompanied by an increase in the numbers of osteoclasts. This effect was significantly attenuated by the association with Gram-positive bacteria. In vitro, quantitative PCR arrays indicated that stimulation of macrophages with heat-killed Gram-negative bacteria induced the same biological processes in wild-type and NOD1-deficient cells; however, expression of pro-inflammatory mediators was increased in NOD1-deficient cells. These results suggest a bone-sparing role for NOD1 in this model.

Toll-Like Receptor 2 Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL.

Severe Staphylococcus aureus (S. aureus) infections pose an immense threat to population health and constitute a great burden for the health care worldwide. Inter alia, S. aureus septic arthritis is a disease with high mortality and morbidity caused by destruction of the infected joints and systemic bone loss, osteoporosis. Toll-Like receptors (TLRs) are innate immune cell receptors recognizing a variety of microbial molecules and structures. S. aureus recognition via TLR2 initiates a signaling cascade resulting in production of various cytokines, but the mechanisms by which S. aureus causes rapid and excessive bone loss are still unclear. We, therefore, investigated how S. aureus regulates periosteal/endosteal osteoclast formation and bone resorption. S. aureus stimulation of neonatal mouse parietal bone induced ex vivo bone resorption and osteoclastic gene expression. This effect was associated with increased mRNA and protein expression of receptor activator of NF-kB ligand (RANKL) without significant change in osteoprotegerin (OPG) expression. Bone resorption induced by S. aureus was abolished by OPG. S. aureus increased the expression of osteoclastogenic cytokines and prostaglandins in the parietal bones but the stimulatory effect of S. aureus on bone resorption and Tnfsf11 mRNA expression was independent of these cytokines and prostaglandins. Stimulation of isolated periosteal osteoblasts with S. aureus also resulted in increased expression of Tnfsf11 mRNA, an effect lost in osteoblasts from Tlr2 knockout mice. S. aureus stimulated osteoclastogenesis in isolated periosteal cells without affecting RANKL-stimulated resorption. In contrast, S. aureus inhibited RANKL-induced osteoclast formation in bone marrow macrophages. These data show that S. aureus enhances bone resorption and periosteal osteoclast formation by increasing osteoblast RANKL production through TLR2. Our study indicates the importance of using different in vitro approaches for studies of how S. aureus regulates osteoclastogenesis to obtain better understanding of the complex mechanisms of S. aureus induced bone destruction in vivo.

The combined effects of soya isoflavones and resistant starch on equol production and trabecular bone loss in ovariectomised mice.

Equol is a metabolite of the soya isoflavone (ISO) daidzein that is produced by intestinal microbiota. Equol has greater oestrogenic activity compared with other ISO, and it prevents bone loss in postmenopausal women. Resistant starch (RS), which has a prebiotic activity and is a dietary fibre, was reported to promote equol production. Conversely, the intestinal microbiota is reported to directly regulate bone health by reducing inflammatory cytokine levels and T-lymphocytes in bone. The present study evaluated the combined effects of diet supplemented with ISO and RS on intestinal microbiota, equol production, bone mineral density (BMD) and inflammatory gene expression in the bone marrow of ovariectomised (OVX) mice. Female ddY strain mice, aged 8 weeks, were either sham-operated (Sham, n 7) or OVX. OVX mice were randomly divided into the following four groups (seven per group): OVX control (OVX); OVX fed 0·05 % ISO diet (OVX+ISO); OVX fed 9 % RS diet (OVX+RS); and OVX fed 0·05 % ISO- and 9 % RS diet (OVX+ISO+RS). After 6 weeks, treatment with the combination of ISO and RS increased equol production, prevented the OVX-induced decline in trabecular BMD in the distal femur by modulating the enteric environment and altered OVX-induced inflammation-related gene expression in the bone marrow. However, there were no significant differences in bone parameters between the ISO+RS and ISO-alone groups in OVX mice. Our findings suggest that the combination of ISO and RS might alter intestinal microbiota and immune status in the bone marrow, resulting in attenuated bone resorption in OVX mice.

Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT.

The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP(+) cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP(+) cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP(+) cells with ≥3nuclei, although this difference was not significant. Levels of TGF-ß, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1ß and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis.

Role of bacterial γ-glutamyltranspeptidase as a novel virulence factor in bone-resorbing pathogenesis.

Mammalian γ-glutamyltranspeptidase (GGT) has been identified as a bone-resorbing factor. Since GGT of Bacillus subtilis exhibits similarity in their primary structure and enzymatic characteristics with mammalian GGTs, the bone-resorbing activity of bacterial GGT was examined in this study. Osteoclastogenesis was performed in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. A conditioned medium from GGT-overproducing B. subtilis culture showed significantly higher activity of osteoclast formation than a conditioned medium from wild-type B. subtilis culture. Recombinant GGT (rGGT) of wild-type B. subtilis and an enzymatic activity-defected rGGT of B. subtilis 2288 mutant were expressed in Escherichia coli and purified using His tag. Both purified rGGTs induced similar levels of osteoclastogenesis, suggesting that B. subtilis GGT possesses virulent bone-resorbing activity and its activity is probably independent of its enzymatic activity. Furthermore, a recombinant protein of B. subtilis GGT heavy subunit (Bs rGGT/H) showed strong activity of osteoclastogenesis while the light subunit failed to show strong activity, suggesting that the bone-resorbing activity is mainly located at the heavy subunit. More importantly, the GGT enzymatic activity may not be required for this virulence activity since the light subunit contains the catalytic pocket. In addition, B. subtilis rGGT stimulated mRNA expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and cyclooxygenase-2 (COX-2), while an osteoprotegerin inhibited the osteoclast formation induced by Bs rGGT/H. This is the first demonstration that bacterial GGT itself is sufficient to act as a bone-resorbing virulence factor via RANKL-dependent pathway. Therefore, it can be hypothesized that GGT of periodontopathic bacteria may play an important role as a virulence factor in bone destruction.

Flesh-eating Streptococcus pyogenes triggers the expression of receptor activator of nuclear factor-κB ligand.

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock-like syndrome. Human CD46-expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast-mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 10(5) colony-forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor-κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro-computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4(+) and CD8(+) T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte-derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast-like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.

The silencing of cathepsin K used in gene therapy for periodontal disease reveals the role of cathepsin K in chronic infection and inflammation.

BACKGROUND AND OBJECTIVE: Periodontitis is a severe chronic inflammatory disease and one of the most prevalent non-communicable chronic diseases that affects the majority of the world's adult population. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this dreadful disease. In this study, we utilized adeno-associated virus (AAV) expressing cathepsin K (Ctsk) small hairpin (sh)RNA (AAV-sh-Ctsk) to silence Ctsk in vivo and subsequently evaluated its impact in periodontitis as a potential therapeutic strategy for this disease. MATERIAL AND METHODS: We used a known mouse model of periodontitis, in which wild-type BALB/cJ mice were infected with Porphyromonas gingivalis W50 in the maxillary and mandibular periodontium to induce the disease. AAV-sh-Ctsk was then administrated locally into the periodontal tissues in vivo, followed by analyses to assess progression of the disease. RESULTS: AAV-mediated Ctsk silencing drastically protected mice (> 80%) from P. gingivalis-induced bone resorption by osteoclasts. In addition, AAV-sh-Ctsk administration drastically reduced inflammation by impacting the expression of many inflammatory cytokines as well as T-cell and dendritic cell numbers in periodontal lesions. CONCLUSION: AAV-mediated Ctsk silencing can simultaneously target both the inflammation and bone resorption associated with periodontitis through its inhibitory effect on immune cells and osteoclast function. Thereby, AAV-sh-Ctsk administration can efficiently protect against periodontal tissue damage and alveolar bone loss, establishing this AAV-mediated local silencing of Ctsk as an important therapeutic strategy for effectively treating periodontal disease.