Erythropoietin Potentiates The Immune System Yet Induces Bone Resorption In a Myeloma Mouse Model
Authors: Deshet-Unger Naamit , Hiram-Bab Sahar , Gross Mor , Haim-Ohana Yasmin , Souroujon Miriam C. , Oster Howard S. , Mittelman Moshe , Gabet Yankel , Neumann Drorit 
Published: October 2019

Abstract

Background
Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells and monoclonal gammopathy. It is associated with various clinical manifestations including bone disease and anemia. Clinical introduction of recombinant human erythropoietin (rHuEPO) and its derivatives has been a breakthrough in treating patients with anemia, mainly those with chronic kidney disease and cancer patients on chemotherapy. EPO was found to have additional biological effects, including potentiation of both humoral and cell-mediated immune responses. Our studies and those of others, have shown that EPO treatment for anemia in MM patients was associated with general improved immunological functions and immune-mediated modulation (suppression) of MM. A role for EPO in bone metabolism was previously suggested, but its effects were often controversial and the underlying mechanisms remain to be resolved.

Objectives
To determine the immunological and skeletal effects as well as the underlying mechanisms of EPO signaling in the 5T33 MM mouse model.

Results
EPO administration led to a 50% decrease in the levels of the MM pathological κ light chain while attenuating the decrease in normal IgA. These immunoglobulin changes were accompanied by a 70% elevation (p=0.015) of B220+IGM+IgD- immature B cells in the BM of EPO-treated MM mice, compared to diluent-injected MM mice (9.8%±0.91 versus 5.68%±1.19 of B220-gated cells, respectively). We also found a 45% increase (p=0.01) in the level of BM macrophages (F4/80+CD11b+) following EPO administration in both healthy and MM mice. Moreover, stimulation in vitro of purified BM-derived macrophages with EPO, led to a 33% increase (p=0.01) in phagocytosis of the 5T33 MM cells.
Th17 cells play an important role in MM pathobiology. Our data demonstrate that in MM mice, rHuEPO administration induces a 70% decrease (p=0.021) in the expression level of RORγt, a Th17 hallmark transcription factor. These findings were associated with a 4-fold increase (p<0.001) in the transcript levels of the Th1-associated cytokine IFN-γ, in the BM of EPO-treated MM mice versus their diluent-injected MM counterparts. As MM is typically associated with a Th2 response and since Th17-associated cytokines in MM patients are suggested to inhibit the Th1 response, EPO may thus have a beneficial role in MM, by shifting the balance towards a Th1 response. Collectively, these findings demonstrate that EPO treatment potentiates humoral immune responses, and limits MM progression.
Bone disease is a major component of MM and is associated with significant morbidity. High-resolution CT analysis of the distal femurs confirmed bone loss in 5T33 MM mice. The femur bone density of 5T33 MM mice was 30% lower (p=0.04) than that of their healthy counterparts (3.1%±0.003 versus 4.4%±0.008, respectively). Unexpectedly, EPO treatment of MM mice, in comparison to diluent-injected MM mice, induced a 35% reduction (p=0.004) in trabecular bone density (3.1%±0.003 and 2%±0.007, respectively) and 41% (p<0.001) reduction in trabecular number (2.62mm-1±0.23 and 1.54mm-1±0.266, respectively). This was only partly compensated by a 19% increase (p=0.006) in trabecular thickness (0.043mm ±0.005 versus 0.036mm ±0.003 in controls) in EPO-treated MM mice. Bone loss correlated with a 50% increase in transcript levels of the osteoclastogenic nuclear factor-kappa B ligand (RANKL) in BM of EPO-treated MM mice compared to diluent-injected MM mice. In line with the premise that EPO-induced bone loss is mediated by increased osteoclastogenesis, we found an EPO-associated elevation in BM osteoclast progenitors, as determined by RANK and cFms immunostaining (cFms+/RANK−, 1.7 fold, p=0.01; cFms+/RANK+, 2.7 fold, p=0.007).

Conclusions
We show that in MM, EPO acts as a double-edged sword, by stimulating the immune response against MM on one hand, but also accelerating bone resorption on the other, which further alters the already compromised bone tissue. A better understanding of the osteoimmunological roles of EPO may advocate for the use of rHuEPO along with targeted bone protective treatment in MM patients, to attenuate the anemia and MM progression, while also preventing bone damage.
ND-U and SH-B have contributed equally. Supported by the Multiple Myeloma Research Foundation (DN).

Disclosures:
No relevant conflicts of interest to declare.