Liver Macrophages (Kupffer cells) As Novel Targets of Erythropoietin
Authors: Gilboa Dafna , Ohana Haim Yasmin , Deshet Naamit , Gross Mor , Ben-Califa Nathalie , Gassmann Max , Souroujon Miriam C. , Mittelman Moshe , Neumann Drorit 
Published: October 2019

Abstract
Erythropoietin (EPO) synthesized by the kidney and by the fetal liver is the major hormone that drives mammalian erythropoiesis. It is thus used in treatment of anemia, associated with chronic renal failure, as well as for alleviating chemotherapy or radiotherapy induced anemia in certain cancer patients. The physiological role of EPO is to regulate production of red blood cells via its receptor, EPO-R. However, EPO-R was found also in various other cell types, as shown also by us, suggesting that EPO may have pleiotropic activities. Originally, we have shown EPO effects on bone-marrow derived macrophages. We now report on EPO effects on liver macrophages. Using transgenic mice, overexpressing human EPO (tg6 mice), we found that they have a 2 fold increased level of F4/80 positive liver macrophages, compared to wt mice (p=0.01). In addition, C57bl/6 wt mice, injected 3 times a week with 180 U/ml of recombinant human EPO (rHuEPO) displayed a 2 fold increase in the percentage of F4/80 expressing cells (p=0.02), and in double positive F4/80 and CD11b expressing cells (p=0.03), compared to their untreated littermates. Similar results were observed in an experimental murine model of multiple myeloma (5T33). Hence, 5T33 MM mice that were treated with rHuEPO (10 consecutive days of 30 U/ml followed by 3 weekly injections of 30 U/ml for 2 weeks), presented a 2.5 fold increase (p=0.0004) and a 3 fold increase (p<0.0001) in the percentage of cells positive for F4/80 and in the percentage of cells double positive for F4/80 and CD11b, respectively. In addition, these mice displayed a 20% increase in the mean fluorescence intensity of CD11b, indicating that EPO causes not only an increase in the proportion of Kupffer cells, but it also induces the activation of CD11b positive cells, as reflected by the elevated expression of this marker per cell. We thus questioned whether Kupffer cells are direct targets of EPO. Our results show that the rat Kupffer cell line (KCL3-2) expresses EPO-R mRNA and protein on the cell surface, as detected by the novel EPO-R antibody, GM1012 generated by the FP7-Health European commission EpoCan grant (Maxwell et al., British Journal of Haematology, accepted for publication). Following treatment of the cells with EPO, the levels of EPO-R mRNA increased by 1.5 fold (p=0.02) and surface EPO-R levels decreased by 2 fold (p≤0.01). Furthermore, stimulation of the KCL3-2 cells with EPO led to activated MAPK signaling, and induced a 50% increase (p<0.005) in cell migration. A 3 fold increase (p=0.04) in the levels of the CCL-2 chemokine mRNA following treatment with EPO, points to a possible mechanism by which monocytes are recruited to the liver, where they differentiate into liver macrophages. Our findings thus introduce and elucidate a new function of EPO, focused on the liver, and indicate its effects in normal as well as in pathological conditions.
The first two authors contributed equally to this study.
This work was supported by the FP7-Health European commission EpoCan grant (282551).

Disclosures
No relevant conflicts of interest to declare.