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. 2020 Jul 9;10(19):8744-8756.
doi: 10.7150/thno.45845. eCollection 2020.

Erythropoietin receptor in B cells plays a role in bone remodeling in mice

Naamit Deshet-Unger  1 , Albert Kolomansky  1   2 , Nathalie Ben-Califa  1 , Sahar Hiram-Bab  3 , Dafna Gilboa  1 , Tamar Liron  3 , Maria Ibrahim  1 , Zamzam Awida  1 , Anton Gorodov  1 , Howard S Oster  2 , Moshe Mittelman  2 , Martina Rauner  4 , Ben Wielockx  5 , Yankel Gabet  3 , Drorit Neumann  1
Affiliations

Erythropoietin receptor in B cells plays a role in bone remodeling in mice

Naamit Deshet-Unger et al. Theranostics. .

Abstract

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.

Keywords: Pro-B cells; bone marrow; cFMS/CD115/CSF1R; erythropoietin; lymphocytes; osteoclastogenesis; transdifferentiation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
EPO may regulate bone metabolism via bone marrow (BM) B cells by a dual mechanism, involving both an effect on the expression of RANKL by these cells and B cell-derived osteoclastogenesis. (A) Representative histogram of flow cytometry analysis of RANKL-expressing BM B cells (B220+CD19+). (B) Surface expression of RANKL by BM B cells in EPO- versus diluent-treated female mice as measured by mean fluorescence intensity (MFI). Data are mean ± SEM, n = 8-9 mice in each group. (C) TRAP staining (indicating differentiated osteoclasts) of sorted B220+CD19+ (left) and B220-CD19- (right) cells (180,000 cells per well) cultured with M-CSF and RANKL. Representative images were acquired at ✕4 magnification. (D) Total area (left) and number (right) of TRAP-positive multinucleated (≥ 3 nuclei) cells. Data are mean ± SEM, n = 8-9 mice in each group. (E) B-cell-derived osteoclastogenesis in vitro as demonstrated by the differentiation of sorted B220+CD19+ BM cells derived from CD19-Cre;R26R-EYFP B cell-specific reporter mice (upper panels) and R26R-EYFP (Cre negative) mice (lower panels), which served as a negative control for the anti-GFP staining. Blue - nuclear stain (by DRAQ5©), green - EYFP (enhanced by Alexa Fluor 488-conjugated anti-GFP). Right - conventional TRAP staining performed on the following day (dark gray) (✕20 magnification). (F) Lineage tracing in vivo: B-cell-derived osteoclastogenesis as demonstrated by immunofluorescent staining of formalin fixed OCT-embedded (FFOE) bone sections derived from MB1-Cre;R26R-EYFP female mice. The EYFP+/TRAP+ (the former signifying B cell origin) osteoclasts (arrow) are shown in paired panels where the left side shows the fluorescent staining followed by a conventional TRAP staining (right panels) (both at x20 magnification). YFP- (conventional) osteoclast is marked by an arrowhead. The number of TRAP+ osteoclasts's per bone perimeter in the specimen (lumbar vertebra) was 7.2 per millimeter (consistent with previous reports for wild-type C57BL female mice , .
Figure 2
Figure 2
Lymphoid-osteoclastic differentiation is restricted to CD115+ Pro-B cells. (A) Definition of BM B cell precursor populations by flow cytometry using CD19, surface IgM (IgM) and CD43 antigens. Note, all CD19+ cells were also B220+ (B, C) TRAP staining of osteoclasts derived from the indicated sorted BM cells. Plots represent the percentage of osteoclast area (left) and osteoclast number (right); (B) Left panel - osteoclast differentiation from sorted Pro-B cells (B220+CD19+CD43HighIgM), middle - Pre-B cells (B220+CD19+CD43LowIgM-) and right - immature B cells (B220+CD19+CD43-IgM+)(180,000 cells/well); (C) TRAP staining of osteoclasts derived from the indicated BM sorted cells (10,000 cells per well). Left -positive control of monocyte lineage (CD19-CD115+). Middle - Pro-B cells expressing CD115. Right - Pro-B cells negative for CD115. n = 5-9 mice in each group; (D) Expression of β3 integrin (CD61) by CD115+ Pro-B cells (E) Pit resorption area from the indicated sorted cells (10,000 cells per well) cultured on calcium phosphate-coated 96-well plates with M-CSF and RANKL. Left - positive control of monocyte lineage (CD19-CD115+), stopped after 5 days in culture. Middle - Pro-B cells expressing CD115, and right - Pro-B cells not expressing CD115, stopped after 8 days in culture. Note that white area indicates bone resorption while the brown regions are negative for osteoclast activity. Representative images were acquired at x4 magnification. Values in the scatter plot represent the quantification of the pit resorption area (resorbed area is white and non-resorbed is brown). For (B), (C) and (E) the p values were calculated by 1-way ANOVA with Bonferroni post-hoc test. In the “Box and Whisker” plots error bars represent 5-95 percentile range.
Figure 3
Figure 3
EPO augments B cell-derived osteoclastogenesis in vitro and enriches the pool of B cell lineage-traced osteoclast precursors. (A) Osteoclast differentiation from sorted BM B cells (B220+CD19+) and Pro-B (B220+CD19+CD43HighIGM‑) cells treated with either diluent (DIL) (upper & lower left) or EPO (upper & lower right). Representative images were acquired at x4 magnification. (B) Dot plots indicating the osteoclast area (normalized to the mean of the diluent group) and osteoclast number of the corresponding experiments described in (A) (at least 7 mice in each group). (C) Flow cytometry analysis of the CD115 expression by Pro-B cells in DIL- versus EPO-injected (for one week) female mice. Upper panel - mean fluorescence intensity, lower panel - % of CD115 cells (n > 14 mice in each group). (D) Representative histograms (left panels) and density plots (right panels) of the bone marrow cells isolated from either diluent- (upper panels) or EPO-treated (lower panels) CD19-Cre;R26R-EYFP mice (180 U thrice weekly for 2 weeks). (E) Scatter plot indicates the percentage of EYFP+/β3+/CD115+ cells and represent a summary of 8-9 mice in each group. Data are mean ± SEM; p values calculated by Student's t-test.
Figure 4
Figure 4
B-cell-specific EPO-R knockdown is associated with increased bone mass phenotype. (A) The expression of the murine EPO-R, as measured by RT-qPCR, in the Pro-B cells of the control and MB1-Cre;EPO-Rfl/fl mice. The B220-/CD19- non-B cell fraction (containing erythroid precursors) and 5T33 myeloma cell line were used as positive and negative controls, respectively . Dashed lines and bars are mean ± SEM, n = 4-5 mice in each group. Mann-Whitney nonparametric test was used to calculate the p values shown in the figure. (B) Volumetric bone mineral density (vBMD) in the cortical and trabecular bone (upper left and right panels, respectively) as well as trabecular number (Tb.N) and thickness (Tb.Th) (lower left and right panels, respectively) as measured by μCT in the distal femoral metaphyses of transgenic mice carrying a conditional knockdown of EPO-R in the B cells lineage (MB1-Cre;EPO-Rfl/fl) as compared to MB1-Cre;EPO-Rwt/wt controls. n = 11 in each group. (C) Representative μCT images of the femoral cortex of the MB1-Cre;EPO-Rfl/fl (right) compared to control mice (left). Color spectrum reflects the vBMD gradient across the section of the cortical bone. Data are mean ± SEM, n = 11 in each group. P values were calculated by the Mann-Whitney test. In all “Box and Whisker” plots, error bars represent 5-95 percentile range.
Figure 5
Figure 5
B-cell-specific EPO-R knockdown attenuates EPO-induced bone loss. (A) Hemoglobin levels of EPO- versus diluent (DIL)-treated control mice or mice harboring a conditional knockdown of EPO-R in the B cell lineage (MB1-Cre;EPO-Rfl/fl). p value was calculated by 2-way ANOVA (B) trabecular bone volume (BV/TV) and (C) trabecular thickness (Tb.Th) in the proximal part of the distal femoral metaphysis of EPO- versus diluent (DIL)-treated control or MB1-Cre;EPO-Rfl/fl female mice. (D) Representative 3D µCT images of the distal femur of mice described in Figures (B) and (C). (E) Proportion of CD115+ Pro-B cells (measured by multi-color flow cytometry) in the EPO- versus diluent (DIL)-treated control or MB1-Cre;EPO-Rfl/fl mice. Values for each EPO group were normalized to the diluent controls of the same experiment. (F) Pro-B cell fraction (out of live cell gate) in the bone marrow of the corresponding experimental animal groups described in (D); (G) mRNA expression of RANKL (left) and OPG (right) in the whole bone (proximal tibia) of mice described in (A-F). p values were calculated by 2-way ANOVA. For all panels, n = 9-10 mice in each group. In all “Box and Whisker” plots, error bars represent 5-95 percentile range, except in (E) where error bars are represented by the Tukey method.
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