Effects of RANKL-Targeted Therapy in Immunity and Cancer.

The role of the receptor activator of nuclear factor-κB ligand (RANKL)/RANK system is well characterized within bone, where RANKL/RANK signaling mediates osteoclastogenesis and bone resorption. However, this system has also been shown to influence biologic processes beyond the skeletal system, including in the immune system and in cancer. RANKL/RANK signaling is important in lymph-node development, lymphocyte differentiation, dendritic cell survival, T-cell activation, and tolerance induction. The RANKL/RANK axis may also have direct, osteoclast-independent effects on tumor cells. Indeed, activity of the RANKL/RANK pathway in cancer cells has been correlated with tumor progression and advanced disease. Denosumab, a fully human monoclonal antibody against RANKL, inhibits osteoclastogenesis and is widely used not just for the treatment of osteoporosis, but for the prevention of skeletal-related events from bone metastases in solid malignancies such as breast and prostate cancer. The potential effects of denosumab on the immune system have been largely ignored. Nevertheless, with the emergence of immunotherapies for cancer, denosumab may impact the effectiveness of these therapies, especially if they are given in combination. In this article, we review the role of RANKL/RANK in bone, immunity, and cancer. Examining the potential effects of routine treatment with denosumab beyond the bone represents an important area of investigation

Histamine and Histamine H4 Receptor Promotes Osteoclastogenesis in Rheumatoid Arthritis.

Histamine H4 receptor (H4R) has immune-modulatory and chemotaxic effects in various immune cells. This study aimed to determine the osteoclastogenic role of H4R in rheumatoid arthritis (RA). The concentration of histamine in synovial fluid (SF) and sera in patients with RA was measured using ELISA. After RA SF and peripheral blood (PB) CD14+ monocytes were treated with histamine, IL-17, IL-21 and IL-22, and a H4R antagonist (JNJ7777120), the gene expression H4R and RANKL was determined by real-time PCR. Osteoclastogenesis was assessed by counting TRAP-positive multinucleated cells in PB CD14+ monocytes cultured with histamine, Th17 cytokines and JNJ7777120. SF and serum concentration of histamine was higher in RA, compared with osteoarthritis and healthy controls. The expression of H4R was increased in PB monocytes in RA patients. Histamine, IL-6, IL-17, IL-21 and IL-22 induced the expression of H4R in monocytes. Histamine, IL-17, and IL-22 stimulated RANKL expression in RA monocytes and JNJ7777120 reduced the RANKL expression. Histamine and Th17 cytokines induced the osteoclast differentiation from monocytes and JNJ7777120 decreased the osteoclastogenesis. H4R mediates RANKL expression and osteoclast differentiation induced by histamine and Th17 cytokines. The blockage of H4R could be a new therapeutic modality for prevention of bone destruction in RA

Osteoprotegerin reduces osteoclast resorption activity without affecting osteogenesis on nanoparticulate mineralized collagen scaffolds.

The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration

Development of Functional Microfold (M) Cells from Intestinal Stem Cells in Primary Human Enteroids.

Background & aimsIntestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting.MethodsHuman intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium.ResultsFunctional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells.ConclusionsHuman intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome

The adaptive nature of the bone-periodontal ligament-cementum complex in a ligature-induced periodontitis rat model.

The novel aspect of this study involves illustrating significant adaptation of a functionally loaded bone-PDL-cementum complex in a ligature-induced periodontitis rat model. Following 4, 8, and 15 days of ligation, proinflammatory cytokines (TNF- α and RANKL), a mineral resorption indicator (TRAP), and a cell migration and adhesion molecule for tissue regeneration (fibronectin) within the complex were localized and correlated with changes in PDL-space (functional space). At 4 days of ligation, the functional space of the distal complex was widened compared to controls and was positively correlated with an increased expression of TNF- α. At 8 and 15 days, the number of RANKL(+) cells decreased near the mesial alveolar bone crest (ABC) but increased at the distal ABC. TRAP(+) cells on both sides of the complex significantly increased at 8 days. A gradual change in fibronectin expression from the distal PDL-secondary cementum interfaces through precementum layers was observed when compared to increased and abrupt changes at the mesial PDL-cementum and PDL-bone interfaces in ligated and control groups. Based on our results, we hypothesize that compromised strain fields can be created in a diseased periodontium, which in response to prolonged function can significantly alter the original bone and apical cementum formations

The Direct Effect of Low-Magnitude High-Frequency Mechanical Vibration on Osteoclast Formation from RAW267.4 Monocytes

Low-magnitude high-frequency (LMHF) mechanical vibration has been demonstrated to enhance bone formation possibly through inhibition of osteoclastogenesis of bone. Earlier research has demonstrated osteoclast formation from RAW264.7 monocytes was inhibited by a chewing cycle mimicking vibration through inhibition of dendritic cell-specific transmembrane protein (DC-STAMP). We hypothesize that application of LMHF mechanical vibration directly inhibits osteoclast formation from RAW264.7 monocytes in a frequency dependent manner. RAW264.7 monocytes (ATCC) were cultured in alpha minimal essential medium (MEM) with 10% fetal bovine serum (FBS ) and 1% Pen/Strep at 37°C and 5% CO2. The cells were seeded at a density of 2000 cells/well in 96-well cell culture plates. After allowing growth overnight, the cells were treated with 20ng/ml receptor activator nuclear factor kappa-B ligand (RANKL) and refreshed every 2 days to induce osteoclast formation. In the meantime, the cells were subjected to a low-magnitude (0.3g acceleration) mechanical vibration at various frequencies (0, 30, 60 and 90 Hz) respectively. For each frequency group, the vibration was applied for 1 hour per day for 5 consecutive days. By the end of the 5th day, the cells were rinsed with 1X PBS and fixed in 4% formaldehyde for 5 minutes. Tartrate-resistant Acidic Phosphatase (TRAP, a marker enzyme of osteoclast) staining was performed. The TRAP+ multi nuclei (\u3e = 3) cells were counted and calculated. For statistical analysis, one-way ANOVA was used to test differences among the different frequency groups with Tukey post hoc comparison was used to compare between the groups, with p value being set at 0.05. Three days after RANKL stimulation, osteoclasts started to form from RAW264.7 monocytes, with a peak observed on the 5th day. After 5 days, the cells underwent apoptosis and death. Compared to the control group (0 Hz), the 30 Hz but not 60 Hz and 90 Hz frequencies of vibration group showed significant reduction of osteoclast formation by approximately 21% (p \u3c 0.05, n = 6). No significant difference was found among the three frequency groups. Low-magnitude high-frequency mechanical vibration directly inhibits osteoclast formation from RAW264.7 monocytes, and is frequency dependent

Interleukin-17A upregulates receptor activator of NF-kappaB on osteoclast precursors.

IntroductionThe interaction between the immune and skeletal systems is evidenced by the bone loss observed in autoimmune diseases such as rheumatoid arthritis. In this paper we describe a new mechanism by which the immune cytokine IL-17A directly affects osteoclastogenesis.MethodsHuman CD14+ cells were isolated from healthy donors, cultured on dentine slices and coverslips and stimulated with IL-17A and/or receptor activator of NF-kappaB ligand (RANKL). Osteoclast differentiation was evaluated by gene expression, flow cytometry, tartrate-resistant acid phosphatase staining, fluorescence and electron microscopy. Physiologic bone remodelling was studied in wild-type (Wt) and IL-17A-/- mice using micro-computer tomography and serum RANKL/osteoprotegerin concentration. Functional osteoclastogenesis assays were performed using bone marrow macrophages isolated from IL-17A-/- and Wt mice.ResultsIL-17A upregulates the receptor activator for NF-kappaB receptor on human osteoclast precursors in vitro, leading to increased sensitivity to RANKL signalling, osteoclast differentiation and bone loss. IL-17A-/- mice have physiological bone homeostasis indistinguishable from Wt mice, and bone marrow macrophages isolated from these mice develop fully functional normal osteoclasts.ConclusionsCollectively our data demonstrate anti-IL-17A treatment as a selective therapeutic target for bone loss associated with autoimmune diseases