Cell fusion-mediated formation of multinuclear osteoclasts (OCs) plays a key role in bone resorption. It is reported that 2 unique OC-specific fusogens [ i.e., OC-stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP)], and permissive fusogen CD9, are involved in OC fusion. In contrast to DC-STAMP-knockout (KO) mice, which show the osteopetrotic phenotype, OC-STAMP-KO mice show no difference in systemic bone mineral density. Nonetheless, according to the ligature-induced periodontitis model, significantly lower level of bone resorption was found in OC-STAMP-KO mice compared to WT mice. Anti-OC-STAMP-neutralizing mAb down-modulated in vitro: 1) the emergence of large multinuclear tartrate-resistant acid phosphatase-positive cells, 2) pit formation, and 3) mRNA and protein expression of CD9, but not DC-STAMP, in receptor activator of NF-κB ligand (RANKL)-stimulated OC precursor cells (OCps). While anti-DC-STAMP-mAb also down-regulated RANKL-induced osteoclastogenesis in vitro, it had no effect on CD9 expression. In our mouse model, systemic administration of anti-OC-STAMP-mAb suppressed the expression of CD9 mRNA, but not DC-STAMP mRNA, in periodontal tissue, along with diminished alveolar bone loss and reduced emergence of CD9 OCps and tartrate-resistant acid phosphatase-positive multinuclear OCs. The present study demonstrated that OC-STAMP partners CD9 to promote periodontal bone destruction by up-regulation of fusion during osteoclastogenesis, suggesting that anti-OC-STAMP-mAb may lead to the development of a novel therapeutic regimen for periodontitis.-Ishii, T., Ruiz-Torruella, M., Ikeda, A., Shindo, S., Movila, A., Mawardi, H., Albassam, A., Kayal, R. A., Al-Dharrab, A. A., Egashira, K., Wisitrasameewong, W., Yamamoto, K., Mira, A. I., Sueishi, K., Han, X., Taubman, M. A., Miyamoto, T., Kawai, T. OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

By binding to its chemokine receptor CXCR4 on osteoclast precursor cells (OCPs), it is well known that stromal cell-derived factor-1 (SDF-1) promotes the chemotactic recruitment of circulating OCPs to the homeostatic bone remodeling site. However, the engagement of circulating OCPs in pathogenic bone resorption remains to be elucidated. The present study investigated a possible chemoattractant role of macrophage migration inhibitory factor (MIF), another ligand for C-X-C chemokine receptor type 4 (CXCR4), in the recruitment of circulating OCPs to the bone lytic lesion. To accomplish this, we used Csf1r-eGFP-knock-in (KI) mice to establish an animal model of polymethylmethacrylate (PMMA) particle-induced calvarial osteolysis. In the circulating Csf1r-eGFPþ cells of healthy Csf1r-eGFP-KI mice, Csf1rþ/CD11bþ cells showed a greater degree of RANKL-induced osteoclastogenesis compared to a subset of Csf1rþ/RANKþ cells in vitro. Therefore, Csf1r-eGFPþ/CD11bþ cells were targeted as functionally relevant OCPs in the present study. Although expression of the two cognate receptors for MIF, CXCR2 and CXCR4, was elevated on Csf1rþ/CD11bþ cells, transmigration of OCPs toward recombinant MIF in vitro was facilitated by ligation with CXCR4, but not CXCR2. Meanwhile, the level of PMMA-induced bone resorption in calvaria was markedly greater in wild-type (WT) mice compared to that detected in MIF-knockout (KO) mice. Interestingly, in contrast to the elevated MIF, diminished SDF-1 was detected in a particle-induced bone lytic lesion of WT mice in conjunction with an increased number of infiltrating CXCR4þ OCPs. However, such diminished SDF-1 was not found in the PMMA-injected calvaria of MIF-KO mice. Furthermore, stimulation of osteoblasts with MIF in vitro suppressed their production of SDF-1, suggesting that MIF can downmodulate SDF-1 production in bone tissue. Systemically administered anti-MIF neutralizing monoclonal antibody (mAb) inhibited the homing of CXCR4þ OCPs, as well as bone resorption, in the PMMA-injected calvaria, while increasing locally produced SDF-1. Collectively, these data suggest that locally produced MIF in the inflammatory bone lytic site is engaged in the chemoattraction of circulating CXCR4þ OCPs.

SummaryOsteopontin (OPN) is a pro-inflammatory protein that paradoxically protects against inflammation and bone destruction in a mouse model of endodontic infection. Here we have tested the hypothesis that this effect of OPN is mediated by effects on migration of innate immune cells to the site of infection. Using the air pouch as a model of endodontic infection in mice, we showed that neutrophil accumulation at the site of infection with a mixture of endodontic pathogens is significantly reduced in OPNdeficient mice. Reduced neutrophil accumulation in the absence of OPN was accompanied by an increase in bacterial load. OPN-deficiency did not affect neutrophil survival, CXCR2 ligand expression, or the production of inflammatory cytokines in the air pouch. In vitro, OPN enhanced neutrophil migration to CXCL1, whereas in vivo, inhibition of CXCR2 suppressed cellular infiltration in air pouches of infected wild-type mice by > 50%, but had no effect in OPN-deficient mice. OPN increased cell surface expression of CXCR2 on bone marrow neutrophils in an integrina v -dependent manner, and suppressed the internalization of CXCR2 in the absence of ligand. Together, these results support a model where the protective effect of OPN results from enhanced initial neutrophil accumulation at sites of infection resulting in optimal bacterial killing. We describe a novel mechanism for this effect of OPN: integrin-a v -dependent suppression of CXCR2 internalization in neutrophils, which increases the ability of these cells to migrate to sites of infection in response to CXCR2 ligands.

It is well known that Semaphorin 4D (Sema4D) inhibits IGF-1-mediated osteogenesis by binding with PlexinB1 expressed on osteoblasts. However, its elevated level in the gingival crevice fluid of periodontitis patients and the broader scope of its activities in the context of potential upregulation of osteoclast-mediated periodontal bone-resorption suggest the need for further investigation of this multifaceted molecule. In short, the pathophysiological role of Sema4D in periodontitis requires further study. Accordingly, attachment of the ligature to the maxillary molar of mice for 7 days induced alveolar bone-resorption accompanied by locally elevated, soluble Sema4D (sSema4D), TNF-α and RANKL. Removal of the ligature induced spontaneous bone regeneration during the following 14 days, which was significantly promoted by anti-Sema4D-mAb administration. Anti-Sema4D-mAb was also suppressed in vitro osteoclastogenesis and pit formation by RANKL-stimulated BMMCs. While anti-Sema4D-mAb downmodulated the bone-resorption induced in mouse periodontitis, it neither affected local production of TNF-α and RANKL nor systemic skeletal bone remodeling. RANKL-induced osteoclastogenesis and resorptive activity were also suppressed by blocking of CD72, but not Plexin B2, suggesting that sSema4D released by osteoclasts promotes osteoclastogenesis via ligation to CD72 receptor. Overall, our data indicated that ssSema4D released by osteoclasts may play a dual function by decreasing bone formation, while upregulating bone-resorption.

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors.Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is