Neutrophil extracellular traps mediate bone erosion in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. EXPERIMENTAL APPROACH: The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. KEY RESULTS: Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.

Neutrophil Extracellular Traps Aggravate Apical Periodontitis by Stimulating Osteoclast Formation.

INTRODUCTION: Neutrophil extracellular traps (NETs) have been described as structures composed of DNA and proteins, such as elastase and myeloperoxidase, that are able to kill bacteria extracellularly. The aim of the present study was to evaluate the role of NETs in bone resorption observed in pulp infection-induced apical periodontitis in mice. METHODS: Apical periodontitis was experimentally induced by exposing the dental pulp of the mandibular first molar of mice to the oral microenvironment. The expression of NETs was evaluated by immunofluorescence in mice and biopsies of apical periodontitis. Mice were treated with vehicle or DNase I to degrade NETs, and the samples were collected after 7 days. The size of the apical lesion and the osteoclast number were determined in hematoxylin-eosin- and tartrate-resistant acid phosphatase-stained sections, respectively. Osteoclast differentiation and function markers were evaluated by quantitative polymerase chain reaction. The level of NETs in the serum was determined by the myeloperoxidase-DNA PicoGreen assay. RESULTS: We first confirmed the presence of neutrophils and NETs at the site of the lesion in mice and in biopsies of patients with apical periodontitis. The treatment of mice with DNase I reduced the level of NETs in the serum and led to a reduction in apical lesion size and alveolar bone resorption. This effect was associated with a reduction of local inflammatory infiltrate and a reduced number of osteoclasts. We found that the increased expression of Acp5, Ctsk, and Rankl genes associated with osteoclast formation and function were abrogated by the absence of NETs. CONCLUSIONS: Our data highlight NETs as an important player in the pathogenesis of apical periodontitis with regard to the local inflammation and consequent bone resorption after pulp infection.

PI3Kγ controls IL-17A expression and attenuates alveolar bone loss in an experimental periodontitis model.

OBJECTIVE: In this study, we investigated the modulatory effects of PI3Kγ on IL-17A expression and the progression of experimental periodontitis in vivo. METHODS: Ligature-induced periodontitis was developed around the first molar of mice. Animals were treated with anti-mouse IL-17A or IPI-549 (PI3Kγ inhibitor). In addition, PI3Kγ-deficient mice (PI3Kγ-/-) were used in the study. Alveolar bone loss was measured and real-time PCR of Il17a and Rankl genes was performed. A bioinformatics analysis was carried out using the Gene Set Enrichment Analysis computational tool. RESULTS: Nine days after ligature placement, alveolar bone loss scores were significantly increased, with upregulation of Il17a and Rankl genes in the gingival tissues. Treatment with anti-mouse IL-17A (100 µg/mice) significantly attenuated alveolar bone loss. Mice with ligature-induced periodontitis treated with IPI-549 (3 mg/kg) or PI3Kγ-/- mice showed reduced alveolar bone loss and downregulation of Il17a and Rankl gene expression in the gingival tissues. Consistent with this, the bioinformatics analysis showed upregulation of IL17F, IL17A, IL17D, and STAT3 genes, as well as greater activation of IL-17 and PI3KCI pathways (upregulation of PIK3CG gene) in the gingival tissue of patients with periodontitis. CONCLUSION: PI3Kγ plays an important role in modulating IL-17A expression and alveolar bone loss in vivo and can be considered a promising pathway for the management of periodontal disease and the development of new therapies.

Gene expression data of inflammatory mediators in apical periodontitis in 129 (wild type) and 5-lipoxygenase knockout mice.

Apical periodontitis is an immune inflammatory response around periapical tissues as a result of pathogens invasion into the root canal. The host immunoinflammatory response could determine the progression of this disease, which involves the recruitment of immune cells, and the release of several cytokines in the lesion site. The 5-lipoxygenase pathway has been activated in some osteolytic diseases due to its capacity to interfere in the proliferation and differentiation of bone cells, including the osteoclasts. As mean to understand the inflammatory genes regulation in the apical periodontitis progression, we evaluated the network of 66 genes related to cytokines, chemokines and other inflammatory mediators and receptors in the wild-type (WT) and 5-lipoxygenase enzyme genetically deficient mice (KO). This article presents data not published but related to the research article "Effects of 5-lipoxygenase gene disruption on inflammation, osteoclastogenesis and bone resorption in polymicrobial apical periodontitis" .

Probiotic Bifidobacterium animalis subsp. lactis consumption slows down orthodontic tooth movement in mice.

INTRODUCTION: Probiotics are live microorganisms that, when consumed in appropriate amount, can provide health benefits. Although many studies have shown positive results with the use of probiotics in bone loss control, as in periodontal disease, the effect of probiotics on a mechanical force-induced alveolar bone resorption is still unknown. Therefore, this study aimed to investigate the impact of the specific probiotic Bifidobacterium animalis subsp. lactis on bone remodeling induced by orthodontic tooth movement. METHODS: For this study, thirty C57BL6/J male mice were used and divided into two groups: 1- Mice were orally treated with the probiotic; 2- Mice were treated with vehicle. All mice were submitted to the experimental model of orthodontic tooth movement (OTM). Bone parameters and OTM was evaluated by MicroCT. OTM and TRAP positive cells were analyzed by histomorphometric analysis. Osteoclasts markers were evaluated by qPCR and short chain fatty acids were measured in feces. RESULTS: Micro-CT analysis showed that probiotic treatment did not modify the alveolar bone parameters. However, supplementation with probiotics restrained the tooth movement, as demonstrated by the reduced distance of OTM. Probiotic-treated mice presented down-regulation of Trap expression and reduced osteoclast numbers compared to the control. Accordingly, probiotics supplemented mice exhibited a higher concentration of short-chain fatty acid in their feces. CONCLUSIONS: The supplementation with Bifidobacterium animalis subsp. lactis impaired tooth movement without altering the alveolar bone microarchitecture. The effect on bone remodeling induced by Bifidobacterium animalis subsp. lactis may be associated with the short-chain fatty acids' production.

Uncaria tomentosa reduces osteoclastic bone loss in vivo.

BACKGROUND: The genus Uncaria (Rubiaceae) has several biological properties significant to human health. However, the mechanisms underlying the protective effect of this plant on bone diseases are uncertain. PURPOSE: The present study investigated the role of Uncaria tomentosa extract (UTE) on alveolar bone loss in rats and on osteoclastogenesis in vitro. MATERIALS: UTE was characterized by an Acquity UPLC (Waters) system, coupled to an Electrospray Ionization (ESI) interface and Quadrupole/Flight Time (QTOF, Waters) Mass Spectrometry system (MS). The effect of UTE treatment for 11 days on the ligature-induced bone loss was assessed focusing on several aspects: macroscopic and histological analysis of bone loss, neutrophil and osteoclast infiltration, and anabolic effect. The effect of UTE on bone marrow cell differentiation to osteoclasts was assessed in vitro. RESULTS: The analysis of UTE by UPLC-ESI-QTOF-MS/MS identified 24 compounds, among pentacyclic or tetracyclic oxindole alkaloids and phenols. The administration of UTE for 11 days on ligature-induced rat attenuated the periodontal attachment loss and alveolar bone resorption. It also diminished neutrophil migration to the gingiva tissue, demonstrated by a lower level of MPO. UTE treatment also decreased the level of RANKL/OPG ratio, the main osteoclast differentiation-related genes, followed by reduced TRAP-positive cell number lining the alveolar bone. Additionally, the level of bone-specific alkaline phosphatase, an anabolic bone marker, was elevated in the plasma of UTE treated rats. Next, we determined a possible direct effect of UTE on osteoclast differentiation in vitro. The incubation of primary osteoclast with UTE decreased RANKL-induced osteoclast differentiation without affecting cell viability. This effect was supported by downregulation of the nuclear factor activated T-cells, cytoplasmic 1 expression, a master regulator of osteoclast differentiation, and other osteoclast-specific activity markers, such as cathepsin K and TRAP. CONCLUSION: UTE exhibited an effective anti-resorptive and anabolic effects, which highlight it as a potential natural product for the treatment of certain osteolytic diseases, such as periodontitis.

Cardiovascular and Autonomic Dysfunction in Murine Ligature-Induced Periodontitis.

The present study examined the hemodynamics [arterial pressure (AP), AP variability (APV), heart rate (HR), and heart rate variability (HRV)], cardiac function (echocardiographycally), and myocardial inflammation in Balb/c mice submitted to Periodontitis, through the ligation of the left first molar, or Sham surgical procedure. The first protocol indicated that the AP was similar (136 ± 2 vs. 132 ± 3 mmHg in Sham), while the HR was higher in mice with Periodontitis (475 ± 20 vs. 412 ± 18 bpm in Sham), compared to their Sham counterparts. The APV was higher in mice with Periodontitis when evaluated in the time domain (4.5 ± 0.3 vs. 3.4 ± 0.2 mmHg in Sham), frequency domain (power of the LF band of systolic AP), or through symbolic analysis (patterns 0V + 1V), indicating a sympathetic overactivity. The HRV was similar in the mice with Periodontitis, as compared to their Sham counterparts. In the second protocol, the mice with Periodontitis showed decreased cardiac output (10 ± 0.8 vs. 15 ± 1.4 mL/min in Sham) and ejection fraction (37 ± 3 vs. 47 ± 2% in Sham) associated with increased myocardial cytokines (Interleukin-17, Interleukin-6, and Interleukin-4). This study shows that experimental Periodontitis caused cardiac dysfunction, increased heart cytokines, and sympathetic overactivity, in line with epidemiological studies indicating an increased risk of cardiovascular events in clinical Periodontitis.

Inhibitory effects of dabigatran etexilate, a direct thrombin inhibitor, on osteoclasts and osteoblasts.

INTRODUCTION: Anticoagulants are widely used in orthopedic surgery to decrease the risk of deep vein thrombosis. While significant bone impairment is induced by long-term heparin therapy, little is known about the effects of direct oral anticoagulants (DOACs). Herein, we investigated the effects of dabigatran etexilate (Pradaxa®), a DOAC inhibitor of thrombin, on bone cells using in vitro and ex vivo cell culture models. MATERIALS AND METHODS: Osteoblasts and osteoclasts exposed to different concentrations of dabigatran etexilate and untreated cells were assayed for cell differentiation and activity. Favorable osteogenic conditions for osteoblasts were tested using titanium with nanotopography (Ti-Nano). In addition, mice treated with a dabigatran etexilate solution had bone marrow cells analyzed for the ability to generate osteoclasts. RESULTS: Dabigatran etexilate at concentrations of 1 µg/mL and 2 µg/mL did not impact osteoclast or osteoblast viability. The drug inhibited osteoclast differentiation and activity as observed by the reduction of TRAP+ cells, resorption pits and gene and protein expression of cathepsin K. Consistently, osteoclasts from mice treated with dabigatran showed decreased area, resorptive activity, as well as gene and protein expression of cathepsin K. In osteoblast cultures, grown both on polystyrene and Ti-Nano, dabigatran etexilate reduced alkaline phosphatase (ALP) activity, matrix mineralization, gene expression of ALP and osteocalcin. CONCLUSIONS: Dabigatran etexilate inhibited osteoclast differentiation in ex vivo and in vitro models in a dose-dependent manner. Moreover, the drug reduced osteoblast activity even under optimal osteogenic conditions. This study provides new evidence regarding the negative overall impact of DOACs on bone cells.

Effects of Cinnamoyloxy-mammeisin from Geopropolis on Osteoclast Differentiation and Porphyromonas gingivalis-Induced Periodontitis.

Bone-loss-related diseases such as rheumatoid arthritis, osteomyelitis, osteoporosis, and periodontitis are associated with high rates of morbidity worldwide. These disorders are characterized by an imbalance between the formation and activity of osteoblasts and osteoclasts, leading to bone loss. In this context, we evaluated the effect of cinnamoyloxy-mammeisin (CNM), an anti-inflammatory coumarin found in Melipona scutellaris geopropolis, on key targets related to bone remodeling. In the present study we investigated the in vitro effects of CNM on osteoclast differentiation and M-CSF+RANKL-induced osteoclastogenic marker expression. Additionally, the interference of CNM treatment on osteoclast activity was evaluated by zymography and resorption area. Finally, we assessed the capacity of the compound to mitigate alveolar bone loss in vivo in experimental murine periodontitis induced by Porphyromonas gingivalis. We observed that treatment with CNM impaired osteoclast differentiation, as evidenced by a reduced number of tartrate-resistant acid-phosphatase-positive multinucleated cells (TRAP+) as well as the expression of osteoclastogenic markers upon M-CSF+RANKL-induced stimulation. Similarly, we observed reduced gelatinolytic and resorption capacity in M-CSF+RANKL-induced cells in vitro. Lastly, CNM attenuated alveolar bone loss in an experimental murine periodontitis model. These findings indicate that CNM may be considered a promising treatment for bone loss diseases.