Erythropoietin Administration Delays B Cell Maturation and Promotes B Cell to Erythroid Transdifferentiation in Mice

Abstract

Background: Erythropoietin (EPO) is a glycoprotein hormone which serves as a key regulator of erythropoiesis. Previous studies have shown that it also has a myriad of extraerythropoietic effects including on immune and skeletal systems. Specifically, it was shown that EPO reduces bone marrow (BM) B cell population, in part, by inducing an egress of IgM+ cells out of BM to the peripheral blood (PB). However, the effect of EPO on early B cells (IgM- Pro- and Pre-B cells) has not been studied. Moreover, a process of transdifferentiation of early B cells into erythroid cells under EPO stimulation has not been addressed. Methods: We used MB1-Cre;R26-eYFP mice for B-cell-specific lineage tracing as well as MB1-Cre; EPO-Rfl/fl and Osx-Cre; EPO-Rfl/fl mouse models for B-cell and osteoblast-specific EPO-R knockdown, respectively. Mice (10-12 week old females) were treated with either diluent or human recombinant EPO (hereafter, EPO) for one week (180IU x3/week). Immunophenotyping of BM cells was performed by multi-color flow cytometry (mFC) and immunofluorescence microscopy (IF) was used to demonstrate B cell-derived erythropoiesis in BM sections. Expression of PAX5 and IL-7 in the BM was determined by real-time quantitative PCR. Results: EPO treatment led to a significant 2.4-fold reduction in BM B cells (B220+/CD19+) manifested by a decrease in both IgM+ and IgM- cell fractions. While the IgM+ B cell fraction in PB after one week of EPO did not change, we did observe an almost 2-fold increase in IgM- B cells in EPO-treated as compared to diluent-treated mice (1.4±0.8% vs 0.7±0.5%, respectively, p<0.05). There was no change in IgM+ or IgM- B cells in the spleen, suggesting that IgM- B cells in PB originate from the BM. Concurring with these data, expression of CXCR4 adhesion molecule by BM Pro-B (IgM-) cells was significantly reduced (1.8-fold) upon EPO treatment. Since a decrease in BM B cells could not be solely explained by their egress from BM, we hypothesized that EPO induced a maturation delay at the level of Pre/Pro-B cell stages. Using both MB1-Cre; EPO-Rfl/fl and Osx-Cre; EPO-Rfl/fl mouse models we showed that this maturation delay is independent of EPO-EPO-R signaling in both early B cells and osteoblasts, i.e. following EPO treatment the amount and subpopulation distribution of BM B cells were similar in the control and corresponding EPO-R knockdown mice. However, we found significant perturbations in the key regulators of B cell lymphopoiesis in EPO-treated mice (EPO-R+/+): a 4-fold and 1.7-fold decrease in the expression of PAX5 and IL-7, respectively, while expression of IL-7 receptor by BM cells (both B220+ and B220-)was modestly but significantly reduced (as measured by mFC). Finally, applying IF on BM sections, we could detect eYFP+ BM cells (derived from MB1-Cre;R26-eYFP mice) that co-expressed the specific erythroid marker TER119 (Figure 1). These findings demonstrate that a fraction of BM B cells can transdifferentiate into erythroid cells upon EPO stimulation. Conclusions: Besides its effect on erythropoiesis, EPO compromises BM B cell compartment by a multifaceted mechanism, including BM egress, delay in maturation as well as transdifferentiation to the erythroid lineage. An understanding of the biological and clinical significance of this phenomenon requires further research. For example, the transdifferentiation might explain an improved erythropoiesis in patients with anemia who already have high EPO levels. This research was funded by Israel Science Foundation (ISF) grant number 1188/21 to DN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal