Osteocytes directly regulate osteolysis via MYD88 signaling in bacterial bone infection

AUTHORS

Tetsuya Yoshimoto, Mizuho Kittaka, Andrew Anh Phuong Doan, Rina Urata, Matthew Prideaux, Roxana E. Rojas, Clifford V. Harding, W. Henry Boom, Lynda F. Bonewald, Edward M. Greenfield & Yasuyoshi Ueki

ABSTRACT

The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.

Aberrant adenosine signaling in patients with focal cortical dysplasia

AUTHORS

Mengyi Guo, Jing Zhang, Jing Wang, Xiongfei Wang, Qing Gao, Chongyang Tang, Jiahui Deng, Zhonghua Xiong, Xiangru Kong, Yuguang Guan, Jian Zhou, Detlev Boison, Guoming Luan, Tianfu Li

ABSTRACT

Focal cortical dysplasia (FCD), a common malformation of cortical development, is frequently associated with pharmacoresistant epilepsy in both children and adults. Adenosine is an inhibitory modulator of brain activity and a prospective anti-seizure agent with potential for clinical translation. Our previous results demonstrated that the major adenosine-metabolizing enzyme adenosine kinase (ADK) was upregulated in balloon cells (BCs) within FCD type IIB lesions, suggesting that dysfunction of the adenosine system is implicated in the pathophysiology of FCD. In our current study, we therefore performed a comprehensive analysis of adenosine metabolism and signaling in surgically resected cortical specimens from patients with FCD type I and type II via immunohistochemistry and immunoblot analysis. Adenosine metabolism was assessed by quantifying the levels of the key enzymes of adenosine metabolism, i.e., ADK, adenosine deaminase (ADA), and 5’-ectonucleotidase (CD73). Adenosine signaling was assessed by quantifying the levels of adenosine A2A receptor (A2AR) and putative downstream mediators of adenosine, namely, glutamate transporter-1 (GLT-1) and mammalian target of rapamycin (mTOR). Within lesions in FCD specimens, we found that the adenosine-metabolizing enzymes ADK and ADA, as well as the adenosine-producing enzyme CD73, were upregulated. We also observed an increase in A2AR expression, as well as a decrease in GLT-1 levels and an increase in mTOR levels, in FCD specimens compared with control tissue. These results suggest that dysregulation of the adenosine system is a common pathologic feature of both FCD type I and type II. The adenosine system might therefore be a therapeutic target for the treatment of epilepsy associated with FCD.

3D-printed Strontium-Titanium Scaffolds Incorporated with Highly Bioactive Serum Exosomes Promotes Critical Bone Defect Repair by Enhancing Osteogenesis and Angiogenesis

AUTHORS

Hao Liu, Ranli Gu, Wei Li, Lijun Zeng, Yuan Zhu, Siyi Wang, Xuenan Liu, Boon Chin Heng, Yunsong Liu, Yongsheng Zhou

ABSTRACT

Background

Large bone defect healing faces significant challenges because of inadequate bone regeneration and revascularization. Serum exosomes (sEXO) during bone defect repair are rich in osteogenic factors. Titanium (Ti) scaffolds and low dose strontium (Sr) can promote bone regeneration. Here, a “cell-free scaffold engineering” strategy that incorporates strontium and highly bioactive sEXO within a 3D-printed Ti scaffold is developed.

Methods

Sr-Ti-sEXO composite was prepared by ion implantation and ultra-high-speed centrifugation. Alkaline phosphatase (ALP), Alizarin red (ARS), immunofluorescence (IF) staining, and polymerase chain reaction (PCR) were used to detect the osteogenic effect of Sr-Ti-sExo on bone marrow mesenchymal stem cells (BMSCs). Tartrate-resistant acid phosphatase (TRAP) staining, and PCR were used to detect the osteoclast effect of Sr-Ti-sEXO on RAW264.7. The vascularization effect of Sr-Ti-sEXO on human umbilical vein endothelial cells (HUVECs) was investigated by scratch and migration experiments. Micro-CT and histological staining were used to study the osteogenic and vasculogenic effects of Sr-Ti-sEXO implanted in rabbit large radius defect at 6 and 12 weeks in vivo. RNA-seq was used to explore the potential mechanism.

Results

Sr-Ti-sEXO composite promoted early osteogenesis and inhibited osteoclast formation through the combined release of Sr ions and sEXO, and sustained release of Sr ions enhanced bone conduction, bone induction and inhibited fibroblasts. sEXO can promote the vascular reconstruction of CBD in fracture stage, which has the dual effect of promoting bone and promoting angiogenesis in critical bone defect repair. These effects are regulated by multiple miRNAs that shuttle in sEXO.

Conclusions

Sr-Ti-sEXO has favourable sustained release performance, osteogenic and vasogenic effects, which is a biocompatible and clinically feasible critical bone defect repair strategy. This study also broadens the biomedical potential of exosomes with specific functions such as sEXO in fracture stage. Based on the relative abundance of sEXO, a sEXO library for clinical treatment can be established.

Bone-targeting delivery of platelet lysate exosomes ameliorates glucocorticoid-induced osteoporosis by enhancing bone-vessel coupling

AUTHORS

Gang Zheng, Hai-Wei Ma, Guang-Heng Xiang, Gao-Lu He, Han-Chen Cai, Zi-Han Dai, Yan-Lin Chen, Yan Lin, Hua-Zi Xu, Wen-Fei Ni, Cong Xu, Hai-Xiao Liu & Xiang-Yang Wang

ABSTRACT

Background

Glucocorticoids (GCs) overuse is associated with decreased bone mass and osseous vasculature destruction, leading to severe osteoporosis. Platelet lysates (PL) as a pool of growth factors (GFs) were widely used in local bone repair by its potent pro-regeneration and pro-angiogenesis. However, it is still seldom applied for treating systemic osteopathia due to the lack of a suitable delivery strategy. The non-targeted distribution of GFs might cause tumorigenesis in other organs.

Results

In this study, PL-derived exosomes (PL-exo) were isolated to enrich the platelet-derived GFs, followed by conjugating with alendronate (ALN) grafted PEGylated phospholipid (DSPE-PEG-ALN) to establish a bone-targeting PL-exo (PL-exo-ALN). The in vitro hydroxyapatite binding affinity and in vivo bone targeting aggregation of PL-exo were significantly enhanced after ALN modification. Besides directly modulating the osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs), respectively, PL-exo-ALN also facilitate their coupling under GCs’ stimulation. Additionally, intravenous injection of PL-exo-ALN could successfully rescue GCs induced osteoporosis (GIOP) in vivo.

Conclusions

PL-exo-ALN may be utilized as a novel nanoplatform for precise infusion of GFs to bone sites and exerts promising therapeutic potential for GIOP.

Sex-specific differences in Gsα-mediated signaling downstream of PTH1R activation by abaloparatide in bone

AUTHORS

Srilatha Swami, Joshua Johnson, Lawrence Vecchi, Matthew Kim, Beate Lanske, Rachelle Johnson, Joy Wu

ABSTRACT

Teriparatide, recombinant parathyroid hormone (PTH[1-34]), and abaloparatide, an analogue of PTH related-peptide (PTHrP[1-34]), are both anabolic medications for osteoporosis that target the PTH receptor PTH1R. PTH1R is a G protein-coupled receptor, and the stimulatory Gs protein is an important mediator of the anabolic actions of PTH1R activation in bone. We have published that mice lacking the α subunit of Gs in osteoprogenitors do not increase bone mass in response to PTH[1-34]. Unexpectedly, however, PTH[1-34] still increases osteoblast numbers and bone formation rate in male mice, suggesting that PTH1R may have both Gs-dependent and independent actions in bone. Here we examine the role of Gs signaling in the anabolic actions of abaloparatide. We find that abaloparatide increases bone formation in male mice with postnatal deletion of Gsα in Osx-expressing osteoprogenitors (P-GsαOsxKO mice), but not in female P-GsαOsxKO mice. Therefore, abaloparatide has anabolic effects on bone in male but not female mice that appear to be independent of Gs-mediated signaling.

Context-Dependent Roles for Toll-Like Receptors 2 and 9 in the Pathogenesis of Staphylococcus aureus Osteomyelitis

AUTHORS

Jenna R. Petronglo, Nicole E. Putnam, Caleb A. Ford, Virginia Cruz-Victorio, Jacob M. Curry, Casey E. Butrico, Laura E. Fulbright, Joshua R. Johnson, Sun H. Peck, Sana R. Fatah, James E. Cassat

ABSTRACT

Staphylococcus aureus is the major causative agent of bacterial osteomyelitis, an invasive infection of bone. Inflammation generated by the immune response to S. aureus contributes to bone damage by altering bone homeostasis. Increases in the differentiation of monocyte lineage cells into bone-resorbing osteoclasts (osteoclastogenesis) promote bone loss in the setting of osteomyelitis. In this study, we sought to define the role of Toll-like receptor (TLR) signaling in the pathogenesis of S. aureus osteomyelitis. We hypothesized that S. aureus-sensing TLRs 2 and 9, both of which are known to alter osteoclastogenesis in vitro, promote pathological changes to bone, including increased osteoclast abundance, bone loss, and altered callus formation during osteomyelitis. Stimulation of osteoclast precursors with S. aureus supernatant increased osteoclastogenesis in a TLR2-dependent, but not a TLR9-dependent, manner. However, in vivo studies using a posttraumatic murine model of osteomyelitis revealed that TLR2-null mice experienced similar bone damage and increased osteoclastogenesis compared to wild type (WT) mice. Therefore, we tested the hypothesis that compensation between TLR2 and TLR9 contributes to osteomyelitis pathogenesis. We found that mice deficient in both TLR2 and TLR9 (Tlr2/9−/−) have decreased trabecular bone loss in response to infection compared to WT mice. However, osteoclastogenesis is comparable between WT and Tlr2/9−/− mice, suggesting that alternative mechanisms enhance osteoclastogenesis in vivo during osteomyelitis. Indeed, we discovered that osteoclast precursors intracellularly infected with S. aureus undergo significantly increased osteoclast formation, even in the absence of TLR2 and TLR9. These results suggest that TLR2 and TLR9 have context-dependent roles in the alteration of bone homeostasis during osteomyelitis.