Erythropoietin May Affect B-Cell Maturation and Plasma Cell Antibody Production in Mice

Introduction: Recombinant human erythropoietin (EPO) treats anemia, but EPO also has non-erythroid effects. We have previously shown that EPO has anti-neoplastic immunomodulating effects in both patients and mice (Mittelman PNAS 2001; Mittelman Eur J Haematol 2004). EPO effects were demonstrated in both the cellular and humoral immune systems (Katz Acta Haematol 2005; Katz Eur J Immunol 2007; Prutchi-Sagiv Br J Haematol 2006; Prutchi-Sagiv Exp Hematol 2008; Lifshitz Mol Immunol 2009, Hassan Ren Fail 2003). In a previous study we found that EPO was associated with an improved antibody response to the seasonal influenza vaccine in patients (Oster Exp Hematol 2013). B-cell maturation begins in the bone marrow (BM), and continues primarily in the spleen. The cells mature either to marginal zone (MZ) or to Follicular B-cells, both of which can progress to antibody producing plasma cells (PC). This study evaluates EPO's effects on B-cell maturation and antibody production. Methods and Results: Two murine models: 1) Mice were injected (INJ) with either recombinant human EPO (rHuEPO 180units) or saline 3 times over one week (9+8 mice respectively). 2) Transgenic mice from the Tg6 line (TG), with constitutively increased levels of EPO from birth vs wild type (9+8) mice. The total B220+ (a pan B marker) cell number in EPO mice of both murine models was significantly reduced in the BM (similar to Singbrant Blood 2011; see Table). In the spleen, the total number of B220+ cells was similar, irrespective of EPO exposure. However, some B-cell populations were different (Table): splenic MZ precursor (MZP, B220+/CD21hi/CD24mid/CD23hi) as well as MZ B-cell (B220+/CD21hi/CD24mid/CD23lo) numbers were significantly smaller in EPO mice compared with controls. Splenic PC (B220-/CD138+) were tested in TG mice and their number was greater than in the WT controls (5+6 mice, respectively; see Table). Finally, serum antibodies and light chains were studied and found to be increased in TG compared with WT mice (3+4 mice). IgA: 140±14.1 vs 47±5.0 (x104 ng/ml), p<0.005; kappa TG/WT ratio: 1.6±0.08, p=0.005; and lambda TG/WT ratio: 2.0±0.18, p=0.03. Conclusions: Our findings demonstrate a multistep process, with reduced BM B-cells, reduced splenic MZP and MP cells, followed by increased splenic PC and increased antibody production. EPO may be involved in stimulating this dynamic process and as such may have the additional clinical application of augmenting the humoral immune response in patients.Table.Injected (EPO vs Saline) miceTransgenic vs Wild Type mice(mean%±SEM)EPOSalineTGWTBM B220+, total10.9 ±0.6**28.6 ±1.717.7 ±1.8**30.2 ±1.8Spleen MZP2.1 ±0.2**4.8 ±0.24. 9 ±0.6**9.4 ±1.2Spleen MZ2.2 ±0.4**4.4 ±0.43.8 ±0.5*6.4 ±0.9Spleen PCN/A2.5 ±0.4**0.5 ±0.1*depicts p<0.05; **depicts p<0.005; EPO - erythropoietin, TG - transgenic, WT - wild type, BM - bone marrow, MZ - marginal zone, MZP - marginal zone precursors, PC - plasma cells Disclosures Mittelman: XTL Biotech company, interested in EPO: Consultancy.