Human Osteoclasts Research Articles

Siglec-15 on Osteoclasts Is Crucial for Bone Erosion in Serum-Transfer Arthritis.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15-/- mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.

Novel 2,7-Diazaspiro[4,4]nonane Derivatives to Inhibit Mouse and Human Osteoclast Activities and Prevent Bone Loss in Ovariectomized Mice without Affecting Bone Formation.

Osteoporosis is currently treated with drugs targeting the differentiation or viability osteoclasts, the cells responsible for physiological and pathological bone resorption. Nevertheless, osteoporosis drugs that target only osteoclast activity are expected to preserve bone formation by osteoblasts in contrast to current treatments. We report here the design, synthesis, and biological characterization of a series of novel N-arylsufonamides featuring a diazaspiro[4,4]nonane nucleus to target the guanine nucleotide exchange activity of DOCK5, which is essential for bone resorption by osteoclasts. These compounds can inhibit both mouse and human osteoclast activity. In particular, 4-chlorobenzyl-4-hydroxy-2-phenyl-1-thia-2,7-diazaspiro[4,4]nonane 1,1-dioxide (compound E197) prevented pathological bone loss in mice. Most interestingly, treatment with E197 did not affect osteoclast and osteoblast numbers and hence did not impair bone formation. E197 could represent a lead molecule to develop new antiosteoporotic drugs targeting the mechanism of osteoclast adhesion onto the bone.

Osteoclastogenesis and sphingosine-1-phosphate secretion from human osteoclast precursor monocytes are modulated by the cystic fibrosis transmembrane conductance regulator

Osteopenia and increased fracture rates are well-recognized in patients with cystic fibrosis (CF) disease. In CF pathology, F508del is the most common CFTR mutation, with more than 85% of patients carrying it on at least one allele. The underlying molecular defect in CFTR caused by the F508del-CFTR mutation in osteoclastogenesis, i.e., on the generation and bone-resorption activity of osteoclasts (OCs) from peripheral blood-derived monocytes (PBMCs) remained unexplored. We therefore investigated whether the F508del mutation could affect the osteoclastogenic capacity of PBMCs collected from 15 adult patients bearing the F508del-CFTR mutation, to modulate their bone-resorptive abilities and the level of sphingosine-1-phosphate (S1P) produced by OCs, a key factor in the bone mineral density and formation. In the present study, a severe, defective differentiation of CF-F508del PBMCs to CF-F508del OCs without any significant difference in nuclei number per OC was found compared to non-CF healthy PBMCs from 13 subjects after 7–14-days culture periods. We observed a reduced number of formed non-CF healthy OCs in the presence of a selective inhibitor of CFTR chloride conductance (CFTR-Inh172). Our data regarding OCs resorptive capabilites revealed that a loss of CFTR chloride activity in OCs led to a marked reduction in their trench-resorption mode. A 7-fold increase of the S1P release by CF-F508del OCs was found compared to non-CF healthy OCs after a 21-days culture period. We hypothesize that defective maturation of F508del-OCs precursor monocytes associated with high S1P production in the bone environment might contribute to low bone mineral density observed in the CF population.

Establishment and validation of an in vitro co-culture model for oral cell lines using human PBMC-derived osteoclasts, osteoblasts, fibroblasts and keratinocytes

Indirect co-culture models with osteoclasts including oral cell lines may be influenced by M-CSF and RANKL in the common cell medium. Therefore, we investigated the viability and proliferation of osteoblasts (OB), fibroblasts (FB) and oral keratinocytes (OK) under stratified medium modification and assessed the differentiation of osteoclasts in each co-culture. The impact of M-CSF and RANKL in the common OC co-culture was assessed for OB, FB and OK via MTT assay via DAPI control. The multinuclearity and function of OC were evaluated by light microscopy, DAPI staining, resorption assay and FACS analysis. The PBMC showed the highest differentiation into OC after an incubation period of 7 days. Furthermore, co-culture with OB enhanced the number of differentiated multinucleated OC in comparison with monoculture, whereas co-culture with OK decreased PBMC multinuclearity and OC differentiation. FB did not influence the number of differentiated OC in a co-culture. RANKL and M-CSF reduction had no impact on OC differentiation in co-culture with FB or OB, whereas this medium modification for OK attenuated PBMC multinuclearity and OC differentiation in all approaches. Supplementation of RANKL and M-CSF can be modified for a co-culture of PBMC with FB or OB without disturbing OC differentiation. Thus, pathogenic processes of bone remodelling involving OB, OC, FB and OK in the oral cavity can be investigated thoroughly.

Fusion Potential of Human Osteoclasts In Vitro Reflects Age, Menopause, and In Vivo Bone Resorption Levels of Their Donors-A Possible Involvement of DC-STAMP.

It is well established that multinucleation is central for osteoclastic bone resorption. However, our knowledge on the mechanisms regulating how many nuclei an osteoclast will have is limited. The objective of this study was to investigate donor-related variations in the fusion potential of in vitro-generated osteoclasts. Therefore, CD14+ monocytes were isolated from 49 healthy female donors. Donor demographics were compared to the in vivo bone biomarker levels and their monocytes’ ability to differentiate into osteoclasts, showing that: (1) C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) levels increase with age, (2) the number of nuclei per osteoclast in vitro increases with age, and (3) there is a positive correlation between the number of nuclei per osteoclast in vitro and CTX levels in vivo. Furthermore, the expression levels of the gene encoding dendritic cell-specific transmembrane protein (DCSTAMP) of osteoclasts in vitro correlated positively with the number of nuclei per osteoclast, CTX levels in vivo, and donor age. Our results furthermore suggest that these changes in gene expression may be mediated through age-related changes in DNA methylation levels. We conclude that both intrinsic factors and age-induced increase in fusion potential of osteoclasts could be contributing factors for the enhanced bone resorption in vivo, possibly caused by increased expression levels of DCSTAMP.

Human dendritic cell-derived osteoclasts with high bone resorption capacity and T cell stimulation ability.

Osteoclasts are typically differentiated from monocytes (Mo-OC). A subset of osteoclasts (DC-OC) that are differentiated from dendritic cells (DC) has been reported in the arthritic mice model. However, little information is available on DC-OC in humans. The present study applied both in vitro and in vivo experiments to determine the function and pathological significance of DC-OC. DC-OC were differentiated from human monocyte-derived DC and their bone resorption and antigen-presenting functions were investigated. Synovial tissue samples from patients with rheumatoid arthritis were examined for the presence and characteristics of DC-OC. DC-OC differentiated from DC in the presence of M-CSF and RANKL in vitro were demonstrated to be cathepsin K-positive and TRAP-positive multinucleated giant cells. The DC-OC showed stronger bone resorption ability than monocyte-derived osteoclast (Mo-OC) as observed with the pit formation assay. The DC-OC retained CD11c positivity and expressed costimulatory molecules, unlike Mo-OC. T-cells proliferated when co-cultured with DC-OC, but not with Mo-OC. The addition of abatacept to the cocultures reduced T-cell stimulating activity of DC-OC. Abatacept inhibited the differentiation of monocytes into Mo-OC but did not suppress the differentiation of DC into DC-OC. TRAP-positive and CD86-positive DC-OC were detected in the synovial membranes of rheumatoid arthritis patients but not in patients with osteoarthritis. Human DC-OC demonstrated T-cell stimulating activity in addition to osteolytic activity. We further observed this subset of osteoclasts in the inflammatory synovial membrane of patients with rheumatoid arthritis. Such deviations from normal bone metabolism contribute to the inflammation and bone destruction in chronic inflammatory diseases such as rheumatoid arthritis.

Multiple Myeloma-Derived Extracellular Vesicles Induce Osteoclastogenesis through the Activation of the XBP1/IRE1α Axis.

Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression; therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1α/XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1α by phosphorylation in S724; accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1α (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption.

LncRNA‐mRNA expression profiles and functional networks in osteoclast differentiation

Human osteoclasts are differentiated from CD14+ monocytes and are responsible for bone resorption. Long non‐coding RNAs (lncRNAs) have been proved to be significantly involved in multiple biologic processes, especially in cell differentiation. However, the effect of lncRNAs in osteoclast differentiation is less appreciated. In our study, RNA sequencing (RNA‐seq) was used to identify the expression profiles of lncRNAs and mRNAs in osteoclast differentiation. The results demonstrated that expressions of 1117 lncRNAs and 296 mRNAs were significantly altered after osteoclast differentiation. qRT‐PCR assays were performed to confirm the expression profiles, and the results were almost consistent with the RNA‐seq data. GO and KEGG analyses were used to predict the functions of these differentially expressed mRNA and lncRNAs. The Path‐net analysis demonstrated that MAPK pathway, PI3K‐AKT pathway and NF‐kappa B pathway played important roles in osteoclast differentiation. Co‐expression networks and competing endogenous RNA networks indicated that ENSG00000257764.2‐miR‐106a‐5p‐TIMP2 may play a central role in osteoclast differentiation. Our study provides a foundation to further understand the role and underlying mechanism of lncRNAs in osteoclast differentiation, in which many of them could be potential targets for bone metabolic disease.

Osteoclast signaling-targeting miR-146a-3p and miR-155-5p are downregulated in Paget's disease of bone

MicroRNA (miRs) are small, non-coding RNA that post-transcriptionally regulate DNA expression. We hypothesized that specific miR profiles may be a feature of overactive osteoclasts in Paget's disease of bone (PDB), a disorder characterized by an increased and disorganized bone remodeling that typically begins with excessive bone resorption. We compared the expression profile of 13 miRs in human osteoclasts differentiated in vitro from peripheral blood mononuclear cells (PBMCs) of patients with PDB (n = 10) or age- and sex- matched healthy subjects (n = 10). We selected 13 miRs for testing, on the basis of their previously reported roles either in human osteoclast differentiation, in bone diseases, or in osteoclast important signaling pathways. From those expression results, 3 miRNAs were further selected for in-vitro studies aiming at modulating miR expression in human cord blood monocyte derived osteoclasts: 2 miRs (miR-146a-3p and miR-155-5p) whose expression was significantly reduced in pagetic osteoclasts, as well as miRNA-133a-3p, stable in PDB relative to controls, but with known regulatory importance within osteoclasts. We demonstrated a positive (miR-133a-3p) or negative (miR-155-5p, miR-146a-3p) impact of those miRs on the formation of osteoclasts and/or their bone resorption capacity in this human model. Signaling pathways were significantly affected, including p38 MAP-kinase (miR-133a-3p), RANKL-induced TRAF6/NFκB signaling (miR-146a-3p), and MITF expression (miR-155-5p). Osteoclast miRNA profiles might have an important value to yield significant new insights into the osteoclast phenotype in PDB and in other bone diseases with hyperactive osteoclasts.

Primary myeloid cell proteomics and transcriptomics: importance of β-tubulin isotypes for osteoclast function.

Among hematopoietic cells, osteoclasts (OCs) and immature dendritic cells (DCs) are closely related myeloid cells with distinct functions: OCs participate skeleton maintenance while DCs sample the environment for foreign antigens. Such specificities rely on profound modifications of gene and protein expression during OC and DC differentiation. We provide global proteomic and transcriptomic analyses of primary mouse OCs and DCs, based on original stable isotope labeling with amino acids in cell culture (SILAC) and RNAseq data. We established specific signatures for OCs and DCs, including genes and proteins of unknown functions. In particular, we showed that OCs and DCs have the same α- and β-tubulin isotype repertoire but that OCs express much more of the β tubulin isotype Tubb6 (also known as TBB6). In both mouse and human OCs, we demonstrate that elevated expression of Tubb6 in OCs is necessary for correct podosomes organization and thus for the structure of the sealing zone, which sustains the bone resorption apparatus. Hence, lowering Tubb6 expression hinders OC resorption activity. Overall, we highlight here potential new regulators of OC and DC biology, and illustrate the functional importance of the tubulin isotype repertoire in the biology of differentiated cells.

SUN-347 Glucagon-like Peptide 1 (GLP-1) Acts Directly On Human Osteoclasts To Increase Differentiation And Bone Resorptive Activity

Glucagon-like peptide 1 (GLP-1) is an intestinal hormone released in response to nutrient intake that promotes glucose-dependent insulin secretion by acting upon the pancreatic GLP-1 receptor (GLP-1R). GLP-1R agonists (GLP-1RAs) are widely used in treatment of type 2 diabetes. Preclinical data indicate that GLP-1RAs could be repurposed to treat low bone mass as GLP-1R-depleted mice have higher bone resorption and thinner cortical bones, while insulinopenic and insulin resistant rats have improved bone formation and reduced bone mass deterioration when treated with GLP-1 or GLP-1RAs. However, the effect of GLP-1 and GLP-1RAs on human bone cells remains undetermined. We aimed to elucidate the effect of GLP-1 on primary human osteoclast (OC) and osteoblast (OB) cultures. OCs were differentiated over 10 days from human blood-derived CD14+ monocytes and OBs over 4–6 weeks from human bone. Cells were seeded on bovine bone slices and studies performed using fetal bovine serum, MCSF and RANKL (OC monocultures) or MCSF only (OB monocultures and OB+OC co-cultures). We first investigated the effect of GLP-1 on bone resorptive activity of mature OCs on bovine bone slices. GLP-1 increased the eroded bone surface percentage compared to vehicle in both OC monocultures (1nM P=0.002; 10nM P=0.023; n=8 donors) and OC+OB co-cultures (1nM P=0.013; 10nM P=0.012; n=8 donors). We then tested the effects of GLP-1 on osteoblast activity in OC+OB co-cultures by measuring alkaline phosphatase (ALP). We found that GLP-1 increased ALP in OC+OB cultures (1nM, P=0.049; 10nM, P=0.019) and these effects were reversed by the GLP-1R antagonist exendin 9–39 (1nM, P=0.93, 10nM, P=0.64). However, in OB monocultures GLP-1 had no effects on ALP (1nM P=0.93, 10nM P=0.64) indicating a GLP-1-driven increase in osteoblast activity through osteoclast-osteoblast coupling. We then assessed the effect of GLP-1 on OC differentiation by assessing TRAcP activity. Although there was a trend towards increased TRAcP activity upon stimulation with GLP-1 on day 10 of osteoclastogenesis, this was not statistically significant (1nM P=0.12; n=8 donors; 10nM P=0.29, n=4 donors). Our studies indicated GLP-1 may have a direct effect on osteoclasts, and we therefore sought to characterise GLP-1-mediated signalling in these cells. We assessed the effect of GLP-1 on cAMP signalling using LANCE assays and assessed phosphorylation of ERK proteins by Western blot analysis in human OC cultures. OCs treated with 10nM GLP-1 for 30 minutes had increased cAMP signaling (P=0.004, n=12 bone slices from 2 donors) when compared to vehicle. Furthermore, 10nM GLP-1 induced rapid increases in phosphorylated ERK (P=0.03 following 2 minutes exposure, n=4 blots). In conclusion, our studies reveal that GLP-1 increases activity in primary mature human OCs, and OBs, via OCs. Our signaling studies in OCs indicate this is mediated by direct action of GLP-1 on human bone cells.

Characterisation of genetic regulatory effects for osteoporosis risk variants in human osteoclasts

BackgroundOsteoporosis is a complex disease with a strong genetic contribution. A recently published genome-wide association study (GWAS) for estimated bone mineral density (eBMD) identified 1103 independent genome-wide significant association signals. Most of these variants are non-coding, suggesting that regulatory effects may drive many of the associations. To identify genes with a role in osteoporosis, we integrate the eBMD GWAS association results with those from our previous osteoclast expression quantitative trait locus (eQTL) dataset.ResultsWe identify sixty-nine significant cis-eQTL effects for eBMD GWAS variants after correction for multiple testing. We detect co-localisation of eBMD GWAS and osteoclast eQTL association signals for 21 of the 69 loci, implicating a number of genes including CCR5, ZBTB38, CPE, GNA12, RIPK3, IQGAP1 and FLCN. Summary-data-based Mendelian Randomisation analysis of the eBMD GWAS and osteoclast eQTL datasets identifies significant associations for 53 genes, with TULP4 presenting as a strong candidate for pleiotropic effects on eBMD and gene expression in osteoclasts. By performing analysis using the GARFIELD software, we demonstrate significant enrichment of osteoporosis risk variants among high-confidence osteoclast eQTL across multiple GWAS P value thresholds. Mice lacking one of the genes of interest, the apoptosis/necroptosis gene RIPK3, show disturbed bone micro-architecture and increased osteoclast number, highlighting a new biological pathway relevant to osteoporosis.ConclusionWe utilise a unique osteoclast eQTL dataset to identify a number of potential effector genes for osteoporosis risk variants, which will help focus functional studies in this area.

Modulation of Transient Receptor Potential Channels 3 and 6 Regulates Osteoclast Function with Impact on Trabecular Bone Loss

Enhanced osteoclast formation and function is a fundamental cause of alterations to bone structure and plays an important role in several diseases impairing bone quality. Recent work revealed that TRP calcium channels 3 and 6 might play a special role in this context. By analyzing the bone phenotype of TRPC6-deficient mice we detected a regulatory effect of TRPC3 on osteoclast function. These mice exhibit a significant decrease in bone volume per tissue volume, trabecular thickness and -number together with an increased number of osteoclasts found on the surface of trabecular bone. Primary bone marrow mononuclear cells from TRPC6-deficient mice showed enhanced osteoclastic differentiation and resorptive activity. This was confirmed in vitro by using TRPC6-deficient RAW 264.7 cells. TRPC6 deficiency led to an increase of TRPC3 in osteoclasts, suggesting that TRPC3 overcompensates for the loss of TRPC6. Raised intracellular calcium levels led to enhanced NFAT-luciferase reporter gene activity in the absence of TRPC6. In line with these findings inhibition of TRPC3 using the specific inhibitor Pyr3 significantly reduced intracellular calcium concentrations and normalized osteoclastic differentiation and resorptive activity of TRPC6-deficient cells. Interestingly, an up-regulation of TRPC3 could be detected in a cohort of patients with low bone mineral density by comparing micro array data sets of circulating human osteoclast precursor cells to those from patients with high bone mineral density, suggesting a noticeable contribution of TRP calcium channels on bone quality. These observations demonstrate a novel regulatory function of TRPC channels in the process of osteoclastic differentiation and bone loss.

TGFβ1 Regulates Human RANKL-Induced Osteoclastogenesis via Suppression of NFATc1 Expression.

Osteoclasts are multinucleated giant cells responsible for bone resorption. Various mediators involved in osteoclast differentiation have been investigated as possible therapeutic targets for osteoporosis and rheumatoid arthritis (RA). Although transforming growth factor beta1 (TGFβ1) has been described as one such multifunctional cytokine essential for bone remodeling, its effect on osteoclastogenesis remains controversial. Therefore, we sought to examine the effect of TGFβ1 on osteoclast generation induced by receptor activator of nuclear factor (NF)-κB ligand (RANKL) in humans. Peripheral blood monocytes, isolated using magnetic bead sorting, were cultured with macrophage-colony stimulating factor (M-CSF) or RANKL with or without TGFβ1. Tartrate-resistant acid phosphatase (TRAP) staining, as well as bone resorption assays, revealed that TGFβ1 suppressed RANKL-mediated human osteoclast development. Real-time reverse transcription PCR and Western blotting revealed that TGFβ1 reduced the gene and protein expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), the master regulator of osteoclast differentiation, respectively. Luciferase assays indicated that TGFβ1 inhibited the NF-κB p65-stimulated promoter activity of NFATc1. Immunofluorescence analysis demonstrated that TGFβ1 abrogated RANKL-induced nuclear translocation of p65. Thus, TGFβ1 regulates human RANKL-induced osteoclastogenesis via downregulation of NFATc1 by blocking nuclear translocation of NF-κB, suggesting that TGFβ1 may be a potential therapeutic target for RA.

Effects of zoledronic acid and geranylgeraniol on angiogenic gene expression in primary human osteoclasts.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication associated with bisphosphonate treatment. Zoledronic acid (ZA) is a commonly used bisphosphonate due to its effectiveness in increasing bone density and reducing skeletal events, with evidence that it alters angiogenesis. Replacement of the mevalonate pathway using geranylgeraniol (GGOH) was studied to determine the effects of ZA on angiogenic gene expression in primary human osteoclasts. Osteoclast cultures were generated from peripheral blood mononuclear cells of three patients using the peripheral blood mononuclear cell isolation. These cells were phenotyped by phase-contrast microscopy, tartrate-resistant acid phosphatase staining, and pit assays. Primary osteoclasts were found to express a number of key angiogenic molecules at very high levels. Gene expression levels for 84 human angiogenic factors were determined using PCR arrays. Three genes with significant fold regulation (FR) in response to ZA were as follows: tumor necrosis factor (FR = +2.57, P = 0.050), CXCL9 (FR = +39.48, P = 0.028), and CXCL10 (FR = +18.52, P = 0.0009). The co-addition of geranylgeraniol with ZA resulted in the significant down-regulation of these three genes along with CCL2, TGFBR1, ENG, and CXCL1. GGOH reversed the gene changes induced by ZA and may offer a promising treatment for BRONJ.

Three-Dimensional Co-culture of Primary Human Osteocytes and Mature Human Osteoclasts in Collagen Gels.

Osteoclasts are pivotal cells for bone remodeling and their activity is coordinated by osteocytes that reside inside the bone matrix. In vitro co-cultures of osteocytes and osteoclasts are therefore advantageous to analyze the crosstalk between these cell species. In this study, primary osteocytes were isolated from human bone in a multistep isolation process and embedded into three-dimensional collagen gels. Mature human osteoclasts were generated by differentiation of human peripheral blood mononuclear cells (PBMCs). Different surfaces were tested for osteoclast formation: suspension dishes, collagen gels, and normal tissue culture polystyrene. After detachment from the surfaces, osteoclasts showed typical morphology and gene expression of osteoclast markers. Osteoclasts that were differentiated on collagen exhibited the highest osteoclast marker expression. Cocultivation of mature osteoclasts with osteocytes was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoclasts on the basal side of a porous polyethylen terephtalate membrane, which allowed the separate gene expression analysis for osteocytes and osteoclasts. After 7 days of co-culture both cell species showed their typical morphology, which is multinucleated giant cells for osteoclasts and star-shaped cells with dendritic extensions for osteocytes. Furthermore, osteoclast markers tartrate-resistant acid phosphatase, carbonic anhydrase II, and cathepsin K were detected both on gene expression and protein level in single and co-cultures. Osteocytes showed gene expression of typical osteocyte markers E11, sclerostin, dentin matrix protein 1, osteocalcin, and receptor activator of nuclear factor-κ ligand both in single and co-culture. Impact statement This study is the first to establish an in vitro bone model that contains both primary human osteocytes and primary human osteoclasts. Previous studies applied rodent osteocyte cell lines to examine the influence of osteocytes on osteoclast function. This model mimics the clinical situation better since osteocytes are postmitotic cells whose function might be different in primary state compared with a proliferating cell line. Furthermore, the co-culture model can be the basis for in vitro triple culture models involving osteoblasts as the third bone cell species.

Glycolaldehyde-modified advanced glycation end-products inhibit differentiation of human monocytes into osteoclasts via upregulation of IL-10.

Diabetes patients are at high risk of bone fracture due to accumulation of advanced glycation end products (AGEs) and low bone turnover. Although AGEs inhibit osteoblast functions, little is known about their roles in regulation of human osteoclast differentiation. The aim of this study was to determine the roles of AGEs in regulation of human osteoclast differentiation. Human CD14+ monocytes collected from healthy individuals were stimulated in vitro with conventional cytokines to induce osteoclast differentiation. Simultaneously, glucose-modified AGEs-BSA (Glu-AGEs-BSA) and glycolaldehyde-modified AGEs-BSA (Glyco-AGEs-BSA) were added to analyze their role in regulation of osteoclast differentiation. Human CD14+ cells expressed endogenous receptor for AGE (RAGE). Stimulation with Glyco-AGEs-BSA, but not Glu-AGEs-BSA, reduced the number of tartrate-resistant acid phosphatase-positive cells in a dose-dependent manner and suppressed mRNA expression of nuclear factor of activated T-cells 1 and cathepsin K. Glyco-AGEs-BSA up-regulated pro-inflammatory cytokines and anti-inflammatory cytokine IL-10. The addition of IL-10-neutralizing antibodies abrogated the suppressive effect of Glyco-AGEs-BSA on osteoclast differentiation. Stimulation of Glyco-AGE-BSA resulted in nuclear factor (NF)-κB phosphorylation, and addition of an inhibitor of κB kinase suppressed IL-10 production. We conclude that Glyco-AGEs-BSA inhibited human osteoclast differentiation through induction of IL-10 expression via NF-κB. It can be assumed that AGE bioaccumulation in diabetic patients increases the risk of bone fracture, through inhibition of osteoclast differentiation, reduction of bone turnover, and disruption of bone remodeling.

Human macrophages and osteoclasts resorb β-tricalcium phosphate in vitro but not mouse macrophages

β-TCP is a resorbable bony biomaterial but its biodegradation mechanisms in vivo remains unclear. Osteoclast can resorb β-TCP but a role for macrophages has also been suggested by in vivo studies. However no in vitro study has clearly evidenced the action of macrophages in the resorption process. We prepared flat β-TCP tablets with a smooth surface to investigate the in vitro capability of murine (RAW 264.7) and human macrophage cells (PBMCs) to resorb the biomaterial. In parallel, these cells were differentiated into multinucleated osteoclasts with M-CSF and RANK-L. The action of these cells was evaluated by scanning electron microscopy and Raman microspectroscopy after a 21 day culture on the tablets. Human macrophages and osteoclasts derived from PBMCs appeared able to resorb β-TCP by forming resorption pits at the surface of the flat tablets. RAW macrophages were unable to resorb β-TCP but they exhibited this possibility when they have been differentiated into osteoclasts. These cells can engulf β-TCP grains in their cytoplasm as evidenced by light and TEM microscopy with production of carbonic anhydrase (revealed by the immunogold technique in TEM). The resorbed areas were characterized by severe degradation of the grains showing speckled and stick-like aspects indicating a chemical corrosion. The effect was maximal at the grain boundaries which have a slightly different chemical composition. Changes in the Raman spectrum were observed between the resorbed and un-resorbed β-TCP suggesting crystal modifications. In contrast, un-differentiated murine macrophages were not able to chemically attack β-TCP and no resorption pit was observed. RAW cell is not a representative model of the macrophage-biomaterial interactions that occur in human. This in vitro study evidences that both human osteoclasts and macrophages represent active cell populations capable to resorb β-TCP.

Abstract 343: A potent and selective cfms inhibitor EI-1071 inhibits CSF1R signaling and regulates the tumor-associated macrophages

Abstract We have found that tumor-associated macrophages (TAMs) isolated from metastatic tumors displayed a predominant M2 phenotype. Preclinical studies show that M2-polarized TAMs forge an immunosuppressive microenvironment at metastatic niches to facilitate colonization. Colony-stimulating factor 1 receptor (CSF-1R) signaling is known to regulate the survival and maintenance of M2 TAMs, suggesting targeting CSF-1R might have therapeutic potential in halting cancer progression. To this end, we developed an orally active and selective small-molecule CSF-1R inhibitor EI-1071. Experimental procedures: EI-1071 was evaluated in kinase and cell-based bioassays, including macrophage proliferation and osteoclast differentiation. In vivo activity was verified using murine syngeneic models of colorectal cancer and breast cancer. EI-1071 was administered individually or in combination with an anti-PD1 antibody. Tumor infiltrating leucocytes were profiled using flow cytometric analysis. Results: EI-1071 exhibited nanomolar potency for inhibition of CSF-1R but not other 453 kinases tested. In cell-based bioassays, EI-1071 inhibited CSF-1R activity in human monocytes/macrophages, AML-5 leukemia, MNFS-60 leukemia, and human osteoclasts. In preclinical studies, EI-1071 displayed a favorable ADME properties, including hepatocyte stability, high Caco-2 permeability, and pharmacokinetic profiles. Oral administration of EI-1071 reduced TAM infiltration in the MC38 colorectal cancer and EMT6 breast cancer models. Moreover, EI-1071 dosed with an anti-PD-1 antibody showed further enhanced antitumor activity in vivo . Conclusions: Preclinical characterization of EI-1071 demonstrated that it is a potent and selective CSF-1R inhibitor and has a favorable drug like properties for further clinical evaluation. Therefore, EI-1071 is undergoing IND-enabling activities for the First-In-Human (FIH) Phase 1 study. Citation Format: Hung-Kai Chen, Huey-Wen Hsiao, Li-Ming Lu, Chih-Lun Hsiao, Yin-Ping Wang, Jing-Yi Huang, Chu-Bin Liao, Shao-Zheng Peng, Daw-Tsun Shih. A potent and selective cfms inhibitor EI-1071 inhibits CSF1R signaling and regulates the tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 343.

Histone deacetylases 1 and 2 inhibition suppresses cytokine production and osteoclast bone resorption in vitro.

The regulation of epigenetic factors is an emerging therapeutic target of immune function in a variety of osteolytic pathologies. Histone deacetylases (HDAC) modify core histone proteins and transcriptional processes, in addition to nonhistone protein activity. The activated immune response in rheumatoid arthritis, periodontitis, and prosthetic implant particle release stimulates the catabolic activity of osteoclasts. In this study, we investigated the effects of novel therapeutic agents targeting HDAC isozymes (HDAC 1, 2, and 5), previously shown to be upregulated in inflammatory bone disorders, in cytokine-stimulated human monocytes and osteoclasts in vitro. Inhibiting HDAC 1 and 2 significantly reduced gene expression of IL-1β, TNF, MCP-1, and MIP-1α in TNF-stimulated monocytes, while suppressing secretions of IL-1β, IL-10, INF-γ, and MCP-1 (P < .05). Osteoclast formation and bone resorption were also significantly diminished with HDAC 1 and 2 inhibition, through reduced NFATc1 expression and osteoclast specific target genes, TRAF6, CTR, TRAP, and Cathepsin K (P < .05). Similar trends were observed when inhibiting HDAC 1 and to a lesser extent, HDAC 2, in isolation. However, their combined inhibition had the greatest anti-inflammatory and antiosteoclastic effects. Targeting HDAC 5 had minimal effects on these processes investigated in this study, whereas a broad acting HDACi, 1179.4b, had widespread suppressive outcomes. This study demonstrates that targeting HDACs is a potent and effective way of regulating the inflammatory and catabolic processes in human monocytes and osteoclasts. It also demonstrates the importance of targeting individual HDACs with an overall aim to improve efficiency and reduce any potential off target effects.