Oral Microbially-Induced Small Extracellular Vesicles Cross the Blood-Brain Barrier.

Porphyromonas gingivalis (Pg) and its gingipain proteases contribute to Alzheimer's disease (AD) pathogenesis through yet unclear mechanisms. Cellular secretion of small extracellular vesicles or exosomes (EXO) increases with aging as part of the senescence-associated secretory phenotype (SASP). We have shown that EXO isolated from Pg-infected dendritic cells contain gingipains and other Pg antigens and transmit senescence to bystander gingival cells, inducing alveolar bone loss in mice in vivo. Here, EXO were isolated from the gingiva of mice and humans with/without periodontitis (PD) to determine their ability to penetrate the blood-brain barrier (BBB) in vitro and in vivo. PD was induced by Pg oral gavage for 6 weeks in C57B6 mice. EXO isolated from the gingiva or brain of donor Pg-infected (PD EXO) or control animals (Con EXO) were characterized by NTA, Western blot, and TEM. Gingival PD EXO or Con EXO were labeled and injected into the gingiva of uninfected WT mouse model. EXO biodistribution in brains was tracked by an in vivo imaging system (IVIS) and confocal microscopy. The effect of human PD EXO on BBB integrity and permeability was examined using TEER and FITC dextran assays in a human in vitro 3D model of the BBB. Pg antigens (RGP and Mfa-1) were detected in EXO derived from gingival and brain tissues of donor Pg-infected mice. Orally injected PD EXO from donor mice penetrated the brains of recipient uninfected mice and colocalized with hippocampal microglial cells. IL-1ß and IL-6 were expressed in human PD EXO and not in Con EXO. Human PD EXO promoted BBB permeability and penetrated the BBB in vitro. This is the first demonstration that microbial-induced EXO in the oral cavity can disseminate, cross the BBB, and may contribute to AD pathogenesis.

Engineered Human Dendritic Cell Exosomes as Effective Delivery System for Immune Modulation.

Exosomes (exos) contain molecular cargo of therapeutic and diagnostic value for cancers and other inflammatory diseases, but their therapeutic potential for periodontitis (PD) remains unclear. Dendritic cells (DCs) are the directors of immune response and have been extensively used in immune therapy. We previously reported in a mouse model of PD that custom murine DC-derived exo subtypes could reprogram the immune response toward a bone-sparing or bone-loss phenotype, depending on immune profile. Further advancement of this technology requires the testing of human DC-based exos with human target cells. Our main objective in this study is to test the hypothesis that human monocyte-derived dendritic cell (MoDC)-derived exos constitute a well-tolerated and effective immune therapeutic approach to modulate human target DC and T cell immune responses in vitro. MoDC subtypes were generated with TGFb/IL-10 (regulatory (reg) MoDCs, CD86lowHLA-DRlowPDL1high), E. coli LPS (stimulatory (stim) MoDCs, CD86highHLA-DRhighPDL1low) and buffer (immature (i) MoDCs, CD86lowHLA-DRmedPDL1low). Exosomes were isolated from different MoDC subtypes and characterized. Once released from the secreting cell into the surrounding environment, exosomes protect their prepackaged molecular cargo and deliver it to bystander cells. This modulates the functions of these cells, depending on the cargo content. RegMoDCexos were internalized by recipient MoDCs and induced upregulation of PDL1 and downregulation of costimulatory molecules CD86, HLADR, and CD80, while stimMoDCexos had the opposite influence. RegMoDCexos induced CD25+Foxp3+ Tregs, which expressed CTLA4 and PD1 but not IL-17A. In contrast, T cells treated with stimMoDCexos induced IL-17A+ Th17 T cells, which were negative for immunoregulatory CTLA4 and PD1. T cells and DCs treated with iMoDCexos were immune 'neutral', equivalent to controls. In conclusion, human DC exos present an effective delivery system to modulate human DC and T cell immune responses in vitro. Thus, MoDC exos may present a viable immunotherapeutic agent for modulating immune response in the gingival tissue to inhibit bone loss in periodontal disease.

Role of dendritic cell-mediated immune response in oral homeostasis: A new mechanism of osteonecrosis of the jaw.

Dendritic cells are an important link between innate and adaptive immune response. The role of dendritic cells in bone homeostasis, however, is not understood. Osteoporosis medications that inhibit osteoclasts have been associated with osteonecrosis, a condition limited to the jawbone, thus called medication-related osteonecrosis of the jaw. We propose that disruption of the local immune response renders the oral microenvironment conducive to osteonecrosis. We tested whether zoledronate (Zol) treatment impaired dendritic cell (DC) functions and increased bacterial load in alveolar bone in vivo and whether DC inhibition alone predisposed the animals to osteonecrosis. We also analyzed the role of Zol in impairment of differentiation and function of migratory and tissue-resident DCs, promoting disruption of T-cell activation in vitro. Results demonstrated a Zol induced impairment in DC functions and an increased bacterial load in the oral cavity. DC-deficient mice were predisposed to osteonecrosis following dental extraction. Zol treatment of DCs in vitro caused an impairment in immune functions including differentiation, maturation, migration, antigen presentation, and T-cell activation. We conclude that the mechanism of Zol-induced osteonecrosis of the jaw involves disruption of DC immune functions required to clear bacterial infection and activate T cell effector response.

Enterococcus faecalis Induces Differentiation of Immune-Aberrant Dendritic Cells from Murine Bone Marrow-Derived Stem Cells.

Enterococcus faecalis, long implicated in serious systemic infections and failure of root canal treatment, is a persistent inhabitant of oral periapical lesions. Dendritic cells (DCs) and other innate immune cells patrol the oral mucosa for infecting microbes. Dendritic cells are efficient at capturing microbes when immature, whereupon they can transform into potent antigen-presenting cells upon full maturation. Autophagy, a sophisticated intracellular process first described for elimination of damaged organelles, regulates DC maturation and other important immune functions of DCs. The present study examined how E. faecalis influences the differentiation of murine bone marrow-derived stem cells (BMSCs) into functional DCs in the presence of the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Although the viability and differentiation of DCs were not affected by E. faecalis, expression of the autophagy-related proteins ATG7, Beclin1, and LC3bI/II were significantly suppressed in an mTOR-dependent manner. Ultrastructurally, E. faecalis was identified in single-membrane vacuoles, some of which were in the process of binary fission. Bacterium-containing autophagosomes were absent within the cytoplasm. Accessory molecules (major histocompatibility complex class II [MHC-II], CD80, and CD86) and anti-inflammatory cytokine (transforming growth factor ß1 [TGF-ß1]) were suppressed in E. faecalis-induced DCs, while IL-1ß, tumor necrosis factor alpha (TNF-α), and IL-12 levels were upregulated. When pulsed with ovalbumin (OVA), the E. faecalis-induced DCs showed reduction in CD4+ OVA-specific OT-II T cell proliferation. It is concluded that E. faecalis promotes the differentiation of bone marrow stem cells into CD11c-positive DCs with aberrant immune functions while retaining the capability of proinflammatory cytokine induction.

Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice.

Epigenetic mechanisms control skeletal development and osteoblast differentiation. Pharmacological inhibition of the histone 3 Lys-27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) in WT mice enhances osteogenesis and stimulates bone formation. However, conditional genetic loss of Ezh2 early in the mesenchymal lineage (i.e. through excision via Prrx1 promoter-driven Cre) causes skeletal abnormalities due to patterning defects. Here, we addressed the key question of whether Ezh2 controls osteoblastogenesis at later developmental stages beyond patterning. We show that Ezh2 loss in committed pre-osteoblasts by Cre expression via the osterix/Sp7 promoter yields phenotypically normal mice. These Ezh2 conditional knock-out mice (Ezh2 cKO) have normal skull bones, clavicles, and long bones but exhibit increased bone marrow adiposity and reduced male body weight. Remarkably, in vivo Ezh2 loss results in a low trabecular bone phenotype in young mice as measured by micro-computed tomography and histomorphometry. Thus, Ezh2 affects bone formation stage-dependently. We further show that Ezh2 loss in bone marrow-derived mesenchymal cells suppresses osteogenic differentiation and impedes cell cycle progression as reflected by decreased metabolic activity, reduced cell numbers, and changes in cell cycle distribution and in expression of cell cycle markers. RNA-Seq analysis of Ezh2 cKO calvaria revealed that the cyclin-dependent kinase inhibitor Cdkn2a is the most prominent cell cycle target of Ezh2 Hence, genetic loss of Ezh2 in mouse pre-osteoblasts inhibits osteogenesis in part by inducing cell cycle changes. Our results suggest that Ezh2 serves a bifunctional role during bone formation by suppressing osteogenic lineage commitment while simultaneously facilitating proliferative expansion of osteoprogenitor cells.

Inhibition of Osteocyte Membrane Repair Activity via Dietary Vitamin E Deprivation Impairs Osteocyte Survival.

Osteocytes experience plasma membrane disruptions (PMD) that initiate mechanotransduction both in vitro and in vivo in response to mechanical loading, suggesting that osteocytes use PMD to sense and adapt to mechanical stimuli. PMD repair is crucial for cell survival; antioxidants (e.g., alpha-tocopherol, also known as Vitamin E) promote repair while reactive oxygen species (ROS), which can accumulate during exercise, inhibit repair. The goal of this study was to determine whether depleting Vitamin E in the diet would impact osteocyte survival and bone adaptation with loading. Male CD-1 mice (3 weeks old) were fed either a regular diet (RD) or Vitamin E-deficient diet (VEDD) for up to 11 weeks. Mice from each dietary group either served as sedentary controls with normal cage activity, or were subjected to treadmill exercise (one bout of exercise or daily exercise for 5 weeks). VEDD-fed mice showed more PMD-affected osteocytes (+ 50%) after a single exercise bout suggesting impaired PMD repair following Vitamin E deprivation. After 5 weeks of daily exercise, VEDD mice failed to show an exercise-induced increase in osteocyte PMD formation, and showed signs of increased osteocytic oxidative stress and impaired osteocyte survival. Surprisingly, exercise-induced increases in cortical bone formation rate were only significant for VEDD-fed mice. This result may be consistent with previous studies in skeletal muscle, where myocyte PMD repair failure (e.g., with muscular dystrophy) initially triggers hypertrophy but later leads to widespread degeneration. In vitro, mechanically wounded MLO-Y4 cells displayed increased post-wounding necrosis (+ 40-fold) in the presence of H2O2, which could be prevented by Vitamin E pre-treatment. Taken together, our data support the idea that antioxidant-influenced osteocyte membrane repair is a vital aspect of bone mechanosensation in the osteocytic control of PMD-driven bone adaptation.

Heat generation during removal of an abutment screw fragment from dental implants.

STATEMENT OF PROBLEM: Little information is available on the effect of drilling speed on surrounding bone during the removal of an abutment screw fragment. PURPOSE: The purpose of this in vitro study was to compare, in vitro, the peak temperature increase during the removal of fractured abutment screws from implants placed in a porcine mandible, using drilling speeds of 600 or 2000 rpm. MATERIAL AND METHODS: Twenty 4.3×13-mm dental implants were placed in 10 dissected porcine mandibles: 2 implants per mandible, 1 on each side. Localized defects were created in 20 surface-treated abutment screws, which were then tightened into each implant until a reproducible fracture occurred in each screw. The fractured screws were removed with a handpiece removal kit and irrigated with room-temperature water at either 600 or 2000 rpm. The temperature rise at the implant surface was measured at 3 levels with 3 type-K thermocouples. Repeated measure ANOVA was performed with the Tukey-Kramer post hoc test for mean pair-wise comparisons (α=.05 for all tests). RESULTS: Mean peak temperatures were significantly higher at 2000 rpm than at 600 rpm in the mid-body (P<.001) and crestal (P=.003) regions but not in the apical (P=.225) implant locations. No significant differences in mean peak temperatures were found among the 3 locations using 600 rpm (P=.179). In the 2000-rpm group, mean peak temperature in the mid-body area was consistently higher than that in the apical (P<.001) area, and more instances of temperature rise above 56°C and 60°C were observed. In 1 implant from this group, the estimated peak temperature exceeded the bone damage threshold value (50°C for 30 seconds). CONCLUSIONS: A drilling speed of 2000 rpm during the removal of abutment screw fragments caused overheating of the outer surface of the implant which may damage the surrounding bone; a speed of 600 rpm appears to be safe.

Microbially-Induced Exosomes from Dendritic Cells Promote Paracrine Immune Senescence: Novel Mechanism of Bone Degenerative Disease in Mice.

As the aging population grows, chronic age-related bone degenerative diseases become more prevalent and severe. One such disease, periodontitis (PD), rises to 70.1% prevalence in Americans 65 years and older. PD has been linked to increased risk of other age-related diseases with more serious mortality and morbidity profiles such as Alzheimer's disease and cardiovascular disease, but the cellular and biological mechanisms remain unclear. Recent in vitro studies from our group indicate that murine dendritic cells (DCs) and T cells are vulnerable to immune senescence. This occurs through a distinct process involving invasion of DCs by dysbiotic pathogen Porphyromonas gingivalis (Pg) activating the senescence associated secretory phenotype (SASP). Exosomes of the Pg-induced SASP transmit senescence to normal bystander DC and T cells, ablating antigen presentation. The biological significance of these findings in vivo and the mechanisms involved were examined in the present study using young (4-5mo) or old (22-24mo) mice subjected to ligature-induced PD, with or without dysbiotic oral pathogen and injection of Pg-induced DC exosomes. Senescence profiling of gingiva and draining lymph nodes (LN) corroborates role of advanced age and PD in elevation of senescence biomarkers beta galactosidase (SA-ß-Gal), p16 INK4A p21Waf1/Clip1, IL6, TNFα, and IL1ß, with attendant increase in alveolar bone loss, reversed by senolytic agent rapamycin. Immunophenotyping of gingiva and LN revealed that myeloid CD11c+ DCs and T cells are particularly vulnerable to senescence in vivo under these conditions. Moreover, Pg-induced DC exosomes were the most potent inducers of alveolar bone loss and immune senescence, and capable of overcoming senescence resistance of LN T cells in young mice. We conclude that immune senescence, compounded by advanced age, and accelerated by oral dysbiosis and its induced SASP exosomes, plays a pivotal role in the pathophysiology of experimental periodontitis.

Exogenous and Endogenous Dendritic Cell-Derived Exosomes: Lessons Learned for Immunotherapy and Disease Pathogenesis.

Immune therapeutic exosomes, derived exogenously from dendritic cells (DCs), the 'directors' of the immune response, are receiving favorable safety and tolerance profiles in phase I and II clinical trials for a growing number of inflammatory and neoplastic diseases. DC-derived exosomes (EXO), the focus of this review, can be custom tailored with immunoregulatory or immunostimulatory molecules for specific immune cell targeting. Moreover, the relative stability, small size and rapid uptake of EXO by recipient immune cells offer intriguing options for therapeutic purposes. This necessitates an in-depth understanding of mechanisms of EXO biogenesis, uptake and routing by recipient immune cells, as well as their in vivo biodistribution. Against this backdrop is recognition of endogenous exosomes, secreted by all cells, the molecular content of which is reflective of the metabolic state of these cells. In this regard, exosome biogenesis and secretion is regulated by cell stressors of chronic inflammation and tumorigenesis, including dysbiotic microbes, reactive oxygen species and DNA damage. Such cell stressors can promote premature senescence in young cells through the senescence associated secretory phenotype (SASP). Pathological exosomes of the SASP amplify inflammatory signaling in stressed cells in an autocrine fashion or promote inflammatory signaling to normal neighboring cells in paracrine, without the requirement of cell-to-cell contact. In summary, we review relevant lessons learned from the use of exogenous DC exosomes for immune therapy, as well as the pathogenic potential of endogenous DC exosomes.

Porphyromonas gingivalis Provokes Exosome Secretion and Paracrine Immune Senescence in Bystander Dendritic Cells.

Periodontitis is a disease of ageing or inflammaging, and is comorbid with other more severe age-related chronic diseases. With advanced age comes an increase in accumulation of senescent cells that release soluble and insoluble pro-inflammatory factors collectively termed the senescence associated secretory phenotype (SASP). In the present report, we examined whether immune cells typical of those at the oral mucosa-microbe interface, are vulnerable to cellular senescence (CS) and the role of dysbiotic oral pathogen Porphyromonas gingivalis. Bone marrow-derived dendritic cells (DCs) from young (yDCs) and old (oDCs) mice were co-cultured in vitro with CS inducer doxorubicin or P.gingivalis (Pg), plus or minus senolytic agent rapamycin. CS profiling revealed elevated CS mediators SA-ß-Gal, p16 INK4A, p53, and p21Waf1/Clip1 in oDCs, or yDCs in response to doxorubicin or P. gingivalis, reversible with rapamycin. Functional studies indicate impaired maturation function of oDCs, and yDC exposed to P. gingivalis; moreover, OVA-driven proliferation of CD4+ T cells from young OTII transgenic mice was impaired by oDCs or yDCs+Pg. The SASP of DCs, consisting of secreted exosomes and inflammasome-related cytokines was further analyzed. Exosomes of DCs cocultured with P. gingivalis (PgDCexo) were purified, quantitated and characterized. Though typical in terms of size, shape and phenotype, PgDCexo were 2-fold greater in number than control DCs, with several important distinctions. Namely, PgDCexo were enriched in age-related miRNAs, and miRNAs reported to disrupt immune homeostasis through negative regulation of apoptosis and autophagy functions. We further show that PgDCexo were enriched in P. gingivalis fimbrial adhesin protein mfa1 and in inflammasome related cytokines IL-1ß, TNFα and IL-6. Functionally PgDCexo were readily endocytosed by recipient yDCs, amplifying functional impairment in maturation and ability to promote Ova-driven proliferation of OTII CD4+ T cells from young mice. In conclusion P. gingivalis induces premature (autocrine) senescence in DCs by direct cellular invasion and greatly amplifies senescence, in paracrine, of bystander DCs by secretion of inflammatory exosomes. The implications of this pathological pathway for periodontal disease in vivo is under investigation in mouse models.

Matrix-Bound Zolzoledronate Enhances the Biofilm Colonization of Hydroxyapatite: Effects on Osteonecrosis.

(1) Background: The aim of this study was to test whether matrix-bound zoledronate (zol) molecules enhanced the oral biofilm colonization of a mineralized matrix, rendering the alveolar bone more susceptible to medication-related osteonecrosis of the jaw (MRONJ) following invasive dental procedures. (2) Methods: We tested the effect of matrix-bound zol on the growth and attachment of Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn) and Actinomyces israelii (Ai), and whether the nitrogen-containing component of zol contributed to such effect. The role of oral bacteria in the induction of osteonecrosis was then tested using an extra-oral bone defect model. (3) Results: The attachment of biofilm to hydroxyapatite discs increased when the discs were pre-treated with zol. Bacterial proliferation was not affected. Matrix-bound zol was more potent than non-nitrogen-containing etidronate in enhancing the colonization. Stimulation was dampened by pre-treating the bacteria with histidine. The delivery of oral biofilm to a tibial defect caused osteonecrosis in zol-treated rats. (4) Conclusions: We conclude that matrix-bound zol enhances the oral biofilm colonization of hydroxyapatite. This enhancement depended on the presence of the nitrogen-containing group. The oral biofilm rendered the extra-oral bone susceptible to medication-related osteonecrosis, suggesting that it has an important role in the induction of MRONJ.

Enterococcus faecalis shifts macrophage polarization toward M1-like phenotype with an altered cytokine profile.

Background: The macrophage is an innate immune defense cell involved in pathogen recognition and clearance. Aim: In view of the diversity of the macrophage phenotype and function, the present study investigated how Enterococcus faecalis infection affects the differentiation, phenotype and cytokine profile of macrophages. Methods: Murine bone marrow-derived stem cells were co-cultured with E. faecalis before and after differentiation. Macrophage M0 polarization towards M1 or M2 was initiated at day 6 by addition of LPS and INF-γ, or IL-4 and IL-13, respectively. Results: E. faecalis did not inhibit macrophage differentiation and were identified within macrophages. Viability of the macrophages infected with E. faecalis prior to differentiation was enhanced, evidenced by apoptosis inhibition, as was expression of CD38 and IRF5 proteins, indicators of M1-like polarization. These M1-like macrophages expressed an aberrant cytokine mRNA profile, with reduction in inflammatory cytokines IL-1ß and IL-12 and increase in regulatory cytokine IL-10. No changes in TNF-α or TGF-ß1 were detected, compared with the control groups. This atypical M1-like phenotype was retained even upon stimulation with growth factors that normally trigger their development into M2 macrophages. Conclusions: These findings suggested that E. faecalis infection of bone marrow-derived stem cells during differentiation into macrophages induces an atypical M1-like phenotype associated with intracellular bacterial survival.

Proteomic Characterization, Biodistribution, and Functional Studies of Immune-Therapeutic Exosomes: Implications for Inflammatory Lung Diseases.

Dendritic cell (DC)-derived exosomes (DC EXO), natural nanoparticles of endosomal origin, are under intense scrutiny in clinical trials for various inflammatory diseases. DC EXO are eobiotic, meaning they are well-tolerated by the host; moreover, they can be custom-tailored for immune-regulatory or -stimulatory functions, thus presenting attractive opportunities for immune therapy. Previously we documented the efficacy of immunoregulatory DCs EXO (regDCs EXO) as immunotherapy for inflammatory bone disease, in an in-vivo model. We showed a key role for encapsulated TGFß1 in promoting a bone sparing immune response. However, the on- and off-target effects of these therapeutic regDC EXO and how target signaling in acceptor cells is activated is unclear. In the present report, therapeutic regDC EXO were analyzed by high throughput proteomics, with non-therapeutic EXO from immature DCs and mature DCs as controls, to identify shared and distinct proteins and potential off-target proteins, as corroborated by immunoblot. The predominant expression in regDC EXO of immunoregulatory proteins as well as proteins involved in trafficking from the circulation to peripheral tissues, cell surface binding, and transmigration, prompted us to investigate how these DC EXO are biodistributed to major organs after intravenous injection. Live animal imaging showed preferential accumulation of regDCs EXO in the lungs, followed by spleen and liver tissue. In addition, TGFß1 in regDCs EXO sustained downstream signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFß1 signaling require initial interaction of regDCs EXO with TGFß1R followed by internalization of regDCs EXO with TGFß1-TGFß1R complex. Finally, these regDCs EXO that contain immunoregulatory cargo and showed biodistribution to lungs could downregulate the main severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target receptor, ACE2 on recipient lung parenchymal cells via TGFß1 in-vitro. In conclusion, these results in mice may have important immunotherapeutic implications for lung inflammatory disorders.

Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo.

Chronic bone degenerative diseases represent a major threat to the health and well-being of the population, particularly those with advanced age. This study isolated exosomes (EXO), natural nano-particles, from dendritic cells, the "directors" of the immune response, to examine the immunobiology of DC EXO in mice, and their ability to reprogram immune cells responsible for experimental alveolar bone loss in vivo. Distinct DC EXO subtypes including immune-regulatory (regDC EXO), loaded with TGFB1 and IL10 after purification, along with immune stimulatory (stimDC EXO) and immune "null" immature (iDCs EXO) unmodified after purification, were delivered via I.V. route or locally into the soft tissues overlying the alveolar bone. Locally administrated regDC EXO showed high affinity for inflamed sites, and were taken up by both DCs and T cells in situ. RegDC EXO-encapsulated immunoregulatory cargo (TGFB1 and IL10) was protected from proteolytic degradation. Moreover, maturation of recipient DCs and induction of Th17 effectors was suppressed by regDC EXO, while T-regulatory cell recruitment was promoted, resulting in inhibition of bone resorptive cytokines and reduction in osteoclastic bone loss. This work is the first demonstration of DC exosome-based therapy for a degenerative alveolar bone disease and provides the basis for a novel treatment strategy.

Bone Marrow Derived Extracellular Vesicles Activate Osteoclast Differentiation in Traumatic Brain Injury Induced Bone Loss.

Traumatic brain injury (TBI) is a major source of worldwide morbidity and mortality. Patients suffering from TBI exhibit a higher susceptibility to bone loss and an increased rate of bone fractures; however, the underlying mechanisms remain poorly defined. Herein, we observed significantly lower bone quality and elevated levels of inflammation in bone and bone marrow niche after controlled cortical impact-induced TBI in in vivo CD-1 mice. Further, we identified dysregulated NF-κB signaling, an established mediator of osteoclast differentiation and bone loss, within the bone marrow niche of TBI mice. Ex vivo studies revealed increased osteoclast differentiation in bone marrow-derived cells from TBI mice, as compared to sham injured mice. We also found bone marrow derived extracellular vesicles (EVs) from TBI mice enhanced the colony forming ability and osteoclast differentiation efficacy and activated NF-κB signaling genes in bone marrow-derived cells. Additionally, we showed that miRNA-1224 up-regulated in bone marrow-derived EVs cargo of TBI. Taken together, we provide evidence that TBI-induced inflammatory stress on bone and the bone marrow niche may activate NF-κB leading to accelerated bone loss. Targeted inhibition of these signaling pathways may reverse TBI-induced bone loss and reduce fracture rates.

Removal of matrix-bound zoledronate prevents post-extraction osteonecrosis of the jaw by rescuing osteoclast function.

Unlike other antiresorptive medications, bisphosphonate molecules accumulate in the bone matrix. Previous studies of side-effects of anti-resorptive treatment focused mainly on systemic effects. We hypothesize that matrix-bound bisphosphonate molecules contribute to the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). In this study, we examined the effect of matrix-bound bisphosphonates on osteoclast differentiation in vitro using TRAP staining and resorption assay, with and without pretreatment with EDTA. We also tested the effect of zoledronate chelation on the healing of post-extraction defect in rats. Our results confirmed that bisphosphonates bind to, and can be chelated from, mineralized matrix in vitro in a dose-dependent manner. Matrix-bound bisphosphonates impaired the differentiation of osteoclasts, evidenced by TRAP activity and resorption assay. Zoledronate-treated rats that underwent bilateral dental extraction with unilateral EDTA treatment showed significant improvement in mucosal healing and micro-CT analysis on the chelated sides. The results suggest that matrix-bound bisphosphonates are accessible to osteoclasts and chelating agents and contribute to the pathogenesis of BRONJ. The use of topical chelating agents is a promising strategy for the prevention of BRONJ following dental procedures in bisphosphonate-treated patients.

Deletion of protein kinase D1 in osteoprogenitor cells results in decreased osteogenesis in vitro and reduced bone mineral density in vivo.

Protein kinase D1 (PRKD1) is thought to play a role in a number of cellular functions, including proliferation and differentiation. We hypothesized that PRKD1 in bone marrow-derived mesenchymal stem cells (BMMSC) could modulate osteogenesis. In BMMSCs from floxed PRKD1 mice, PRKD1 ablation with adenovirus-mediated Cre-recombinase expression inhibited BMMSC differentiation in vitro. In 3- and 6-month-old conditional knockout mice (cKO), in which PRKD1 was ablated in osteoprogenitor cells by osterix promoter-driven Cre-recombinase, bone mineral density (BMD) was significantly reduced compared with floxed control littermates. Microcomputed tomography analysis also demonstrated a decrease in trabecular thickness and bone volume fraction in cKO mice at these ages. Dynamic bone histomorphometry suggested a mineralization defect in the cKO mice. However, by 9 months of age, the bone appeared to compensate for the lack of PRKD1, and BMD was not different. Taken together, these results suggest a potentially important role for PRKD1 in bone formation.

Protein kinase D1 conditional null mice show minimal bone loss following ovariectomy.

We previously found that 3- and 6-month-old male mice with conditional ablation of protein kinase D1 (PRKD1) in osteoprogenitor cells (expressing Osterix) exhibited reduced bone mass. Others have demonstrated similar effects in young female PRKD1-deficient mice. Here we examined the bone resorptive response of adult female floxed control and conditional knockout (cKO) mice undergoing sham surgery or ovariectomy (OVX). Femoral and tibial bone mineral density (BMD) values were significantly reduced upon OVX in control, but not cKO, females compared to the respective sham-operated mice. Micro-CT analysis showed that OVX significantly increased trabecular number and decreased trabecular spacing in cKO but not control mice. Finally, in control mice serum levels of a marker of bone resorption (pyridinoline crosslinks) and the osteoclast activator RANKL significantly increased upon OVX; however, no such OVX-induced increase was observed in cKO mice. Our results suggest the potential importance of PRKD1 in response to estrogen loss in bone.

Increased Innate Lymphoid Cells in Periodontal Tissue of the Murine Model of Periodontitis: The Role of AMP-Activated Protein Kinase and Relevance for the Human Condition.

Innate lymphoid cells (ILCs) are master regulators of immune and inflammatory responses, but their own regulatory mechanisms and functional roles of their subtypes (i.e., ILC1s-ILC3s) remain largely unresolved. Interestingly, AMP-activated protein kinase (AMPK), influences inflammatory responses, but its role in modulation of ILCs is not known. Periodontitis is a prevalent disorder with impairment of immune and inflammatory responses contributing importantly to its pathogenesis; however, neither the role of ILCs nor AMPK has been explored in this condition. We tested the hypotheses that (a) periodontitis increases ILCs and expression of relevant cytokines thereby contributing to inflammation and (b) knockdown of AMPK worsens indices of periodontitis in association with further increases in subtypes of ILCs and cytokine expression. The studies utilized wild-type (WT) and AMPK knockout (KO) mice, subjected to ligature-induced periodontitis or sham operation, in association with the use of micro-CT for assessment of bone loss, immunogold electron microscopy to show presence of ILCs in periodontal tissues, flow cytometry for quantitative assessment of subtypes of ILCs and RT-polymerase chain reaction analyses to measure mRNA expression of several relevant cytokines. The results for the first time show (a) presence of each subtype of ILCs in periodontal tissues of sham control and periodontitis animals, (b) that periodontitis is associated with increased frequencies of ILC1s-ILC3s with the effect more marked for ILC2s and differential phenotypic marker expression for ILC3s, (c) that AMPK KO mice display exacerbation of indices of periodontitis in association with further increases in the frequency of subtypes of ILCs with persistence of ILC2s effect, and (d) that periodontitis increased mRNA for interleukin (IL)-33, but not IL-5 or IL-13, in WT mice but expression of these cytokines was markedly increased in AMPK KO mice with periodontitis. Subsequently, we showed that human periodontitis is associated with increases in each ILCs subtype with the effect more marked for ILC2s and that mRNA expressions for IL-33 and IL-5 are markedly greater for sites affected by periodontitis than healthy sites. Collectively, these novel observations indicate a pivotal role for ILCs in pathogenesis of periodontitis and that AMPK is a regulator of their phenotype expression in this condition.