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Erythropoietin (EPO) is a key regulator of erythropoiesis. However, EPO receptors (EPO-Rs) are also expressed on non-erythroid cell types, including myeloid and bone cells. Immune cells also participate in bone homeostasis. B cells produce receptor activator of nuclear factor kappa-Β ligand (RANKL) and osteoprotegerin (OPG), two pivotal regulators of bone metabolism. Here we explored the ability of B cells to transdifferentiate into functional osteoclasts and examined the role of EPO in this process in a murine model. Methods: We have combined specifically-designed experimental mouse models and in vitro based osteoclastogenesis assays, as well as PCR analysis of gene expression. Results: (i) EPO treatment in vivo increased RANKL expression in bone marrow (BM) B cells, suggesting a paracrine effect on osteoclastogenesis; (ii) B cell-derived osteoclastogenesis occured in vivo and in vitro, as demonstrated by B cell lineage tracing in murine models; (iii) B-cell-derived osteoclastogenesis in vitro was restricted to Pro-B cells expressing CD115/CSF1-R and is enhanced by EPO; (iv) EPO treatment increased the number of B-cell-derived preosteoclasts (β3+CD115+), suggesting a physiological rationale for B cell derived osteoclastogenesis; (v) finally, mice with conditional EPO-R knockdown in the B cell lineage (cKD) displayed a higher cortical and trabecular bone mass. Moreover, cKD displayed attenuated EPO-driven trabecular bone loss, an effect that was observed despite the fact that cKD mice attained higher hemoglobin levels following EPO treatment. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling and suggests their involvement in the regulation of bone homeostasis and possibly in EPO-stimulated erythropoietic response. Importantly, we present here for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo.

In vitro osteoclastogenesis is a central assay in bone biology to study the effect of genetic and pharmacologic cues on the differentiation of bone resorbing osteoclasts. To date, identification of TRAP+ multinucleated cells and measurements of osteoclast number and surface rely on a manual tracing requiring specially trained lab personnel. This task is tedious, time-consuming, and prone to operator bias. Here, we propose to replace this laborious manual task with a completely automatic process using algorithms developed for computer vision. To this end, we manually annotated full cultures by contouring each cell, and trained a machine learning algorithm to detect and classify cells into preosteoclast (TRAP+ cells with 1–2 nuclei), osteoclast type I (cells with more than 3 nuclei and less than 15 nuclei), and osteoclast type II (cells with more than 15 nuclei). The training usually requires thousands of annotated samples and we developed an approach to minimize this requirement. Our novel strategy was to train the algorithm by working at “patch-level” instead of on the full culture, thus amplifying by >20-fold the number of patches to train on. To assess the accuracy of our algorithm, we asked whether our model measures osteoclast number and area at least as well as any two trained human annotators. The results indicated that for osteoclast type I cells, our new model achieves a Pearson correlation (r) of 0.916 to 0.951 with human annotators in the estimation of osteoclast number, and 0.773 to 0.879 for estimating the osteoclast area. Because the correlation between 3 different trained annotators ranged between 0.948 and 0.958 for the cell count and between 0.915 and 0.936 for the area, we can conclude that our trained model is in good agreement with trained lab personnel, with a correlation that is similar to inter-annotator correlation. Automation of osteoclast culture quantification is a useful labor-saving and unbiased technique, and we suggest that a similar machine-learning approach may prove beneficial for other morphometrical analyses.

The common use of dental and orthopedic implants calls for special attention to the immune response leading to peri-prosthetic bone loss and implant failure. In addition to the well-established microbial etiology for oral implant failure, wear debris and in particular titanium (Ti) particles (TiP) in the implant vicinity are an important trigger of inflammation and activation of bone resorption around oral and orthopedic implants, presenting an unmet medical need. Here, we employed bacterial-derived lipopolysaccharides (LPS) to model infection and TiP to model aseptic inflammation and osteolysis. We assessed inflammation in vitro by measuring IL1β, IL6 and TNFα mRNA expression in primary macrophages, osteoclastogenesis in RANKL-induced bone marrow derived pre-osteoclasts and osteolysis in vivo in a mouse calvarial model. We also assessed the trans-epithelial penetrability and safety of the tested compound in rats. Our results show that a lipophilic super-active derivative of vasoactive intestinal peptide (VIP), namely stearyl-norleucine-VIP (SNV) presented superior anti-inflammatory and anti-osteoclastogenic effects compared to VIP in vitro. In the bacterial infection model (LPS), SNV significantly reduced IL1β expression, while VIP increased IL6 expression. In the aseptic models of osteolysis, SNV showed greater suppression of in vitro osteoclastogenesis than VIP, and significantly inhibited inflammation-induced osteolysis in vivo. We also observed that expression levels of the VIP receptor VPAC-2, but not that of VPAC-1, dramatically decreased during osteoclast differentiation. Importantly, SNV previously shown to have an increased stability compared to VIP, showed here significant trans-epithelial penetration and a clean toxicological profile, presenting a novel drug candidate that could be applied topically to counter both aseptic and infection-related bone destruction.

Background and aims: Erythropoietin (EPO) is a pleiotropic cytokine, which besides its classical role in driving erythropoiesis, displays tissue protective and immunomodulatory activities. EPO also induces bone loss. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. Cibinetide (CIB), a non-erythropoietic analogue of EPO, specifically binds to the heteromeric receptor and confers tissue protection. Our published findings that EPO stimulates osteoclast precursors and entrains a decrease in bone density, raise questions regarding the underlying molecular mechanisms. Here, we evaluated the role of the heteromeric complex in bone metabolism using CIB alone and in combination with EPO in vivo and in vitro. Results: CIB injections to 12-week-old female mice (120 µg/kg thrice weekly for 4 weeks) resulted in a significant increase in tissue mineral density in cortical bone by 5.8% (1416.4±39.27 vs 1338.74±16.56 mgHA/cm 3) and in trabecular bone by 5.2% (1056.52±30.94 vs 1004.13±16.91 mg HA/cm 3) (n=10 in each group, p< 0.05 versus saline-injected controls), as measured by microCT (Figure 1A). To evaluate the capacity of CIB to attenuate EPO mediated bone loss, we administered CIB (300 µg/kg) for 5 consecutive days, to 13-week-old female mice that also received 2 injections of 120U EPO on days 1 and 4. Flow cytometry analysis revealed a 1.8-fold reduction in the number of osteoclast progenitors, defined as Lin -CD11b −CD115 +Ly6C hi, in the EPO + CIB injected mice, compared to the mice injected with EPO alone (n=7 in each group, p< 0.05). Hemoglobin levels and TER119 + bone marrow (BM) erythroid progenitors were similar in both groups. In vitro, EPO administration to BM-derived macrophages (BMDM) enhanced osteoclastogenesis, whereas CIB had an opposite, dose-dependent effect. Combining CIB with EPO inhibited osteoclastogenesis in BMDM, suggesting that CIB overrides the pro-osteoclastogenic effect of EPO (Figure 1B). Conclusions: Our findings highlight the increasing complexity of EPOR signaling in bone and pave the way for clinical translation through potential combination therapy of EPO and CIB in anemic and in cancer patients. Adjunctive administration of CIB may prevent or attenuate bone loss while preserving the erythropoietic actions of EPO. This study was supported by a grant from the Dotan Hemato-oncology Fund, the Cancer Biology Research Center, Tel Aviv University to DN and YG. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background and aims: Immunotherapy with anti-CD20-specific antibodies (e.g. rituximab), has become the standard of care for B cell lymphoproliferative disorders and many autoimmune diseases. Despite previously demonstrated role for B cells in bone metabolism, the effect of anti-CD20-mediated B cell depletion on bone mass in human patients has not been thoroughly studied. For example, in rheumatological patients the effect of rituximab on bone mass yielded conflicting results, while in lymphoma patients it has not yet been described. Here, we describe the effect of treatment with anti-CD20-specific antibodies on bone mass in a cohort of patients with follicular lymphoma and propose a plausible mechanism using murine model. Methods: To assess the effect of rituximab on bone mass in lymphoma patients, we retrospectively studied the bone mass in patients with follicular lymphoma (FL) during the maintenance phase of chemoimunotherapy, i.e. when rituximab is administered as monotherapy. FL patients on no maintenance (historical controls) or patients with marginal zone lymphoma were include as a control group. Cross-sectional X-ray imaging (CT/PET-CT), performed at the completion of the induction phase and 6-12 months thereafter, were used to serially assess bone density. Wild-type female C57BL/6J mice and mouse anti-mouse CD20 antibody (Genentech) were used for the animal experimental system. Murine bone structure was assessed by the microCT method. Immunophenotyping of the bone marrow (BM), spleen and peripheral blood cells was performed. ELISA and "real-time" quantitative PCR were used to measure the levels of key mediators of bone remodeling, e.g. RANKL, OPG and TNFα. Standard osteoclastogenic assay was used to assesses the osteoclastogenic potential of BM cells. Results: Rituximab treatment prevented the decline in bone mass observed in patients who did not receive active maintenance therapy, both in the lumbar spine (-2.6% vs -8%) and femoral head (-0.5% vs -5.1%) (n=12 patients in each group, p Conclusions: While many lymphoma patients may suffer from bone loss due to advanced age and glucocorticoid administration, our data suggest additional favorable effect of anti-CD20 treatment in bone preservation. Importantly, our murine studies indicate that B cell depletion has a direct effect on bone remodeling, primarily by reducing the osteoclastogenic signals, thus potentially diminishing bone resorption. This novel unrecognized effect should be taken into consideration when maintenance treatment is considered. MM and DN contributed equally to this work. Figure Disclosures No relevant conflicts of interest to declare.

Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production, commonly used in clinical practice to treat certain forms of anemia. Our studies and those of others have demonstrated that EPO administration induces substantial trabecular bone loss. We proposed that EPO-induced bone loss is partially mediated by subsets of bone marrow (BM) B cells that express EPO-R. Mechanistically, EPO upregulates the surface expression of RANKL by BM B cells and augments B cell-derived osteoclastogenesis in vitro. We showed that the latter is likely mediated by pro-B cells expressing the MCS-F receptor (CD115) and capable of transdifferentiation to osteoclasts (Abstract # 1007, EHA 2017). Here we address the role of B cell-specific EPO-R in EPO-induced bone loss (i.e. at supra-physiological EPO levels). Moreover, we demonstrate, for the first time, the occurrence of B cell-derived osteoclastogenesis in vivo, a finding of critical importance in the field of osteohematology. Methods: In order to trace the B cell lineage from its earliest precursors, we used the MB1-Cre mouse line combined with either the R26R-EYFP or the EPO-Rfl/fl mice for lineage tracing and B cell-specific EPO-R knockdown, respectively. Sequential fluorescence and light microscopy were used for the demonstration of B cell-derived osteoclastogenesis in vivo. Human recombinant EPO was administered in vivo at a dose of 180IU thrice weekly for two weeks. Immunophenotyping of BM B cell populations was assessed by multi-color flow cytometry. Results: Using female MB1-Cre; EPO-Rfl/fl (cKD) mice, we found that B cell-specific EPO-R knockdown attenuated the profound EPO-induced trabecular bone loss in the proximal part of the femoral distal metaphysis (proximal BV/TV 0.034±0.012% vs 0.007±0.003% in the cKD vs control mice, p Using the MB1-Cre;R26R-EYFP murine model for B cell lineage tracing, we could demonstrate that some of the TRAP+/ β3 integrin+ bone lining cells were also positive for EYFP (Figure 2). This demonstrates the B cell origin of some of the osteoclasts in vivo. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling that regulates bone homeostasis and might also indirectly affect EPO-stimulated erythropoietic response. The relevance and the mechanisms of the latter phenomenon merits further investigation. Importantly, we present here, for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo, thus opening novel research avenues. DN and YG Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and by the Israel Science Foundation (ISF) Grant No. 343/17 to DN. Disclosures Mittelman: Novartis: Honoraria, Research Funding, Speakers Bureau.