Erythropoietin as a Novel Pro-Inflammatory Mediator of Macrophages.

Abstract 3596

Poster Board III-533

The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system were originally described by our group in multiple myeloma patients and have been further elucidated in murine experimental models (Mittelman, 2001; Katz 2005; 2007; Prutchi-Sagiv, 2006). However, the mechanisms of action by which EPO affects lymphocyte number and function are still unknown, particularly since lymphocytes do not carry EPO receptors (EPO-R). We thus set to unravel mechanisms underlying the anti-neoplastic immunomodulatory action of EPO. These studies led us to the novel discovery that dendritic cells (DCs) express EPO-R, and that EPO enhances their survival and function (Prutchi-Sagiv, 2008; Lifshitz, 2009). Here we focus on macrophages as an additional EPO target, since in analogy to DCs, macrophages are also antigen presenting cells, and serve as key effectors of the innate immune response.

Using murine models, we first explored the in-vivo effects of EPO using recombinant human EPO (rHuEPO, EPREX^R, JC)-injected mice, as well as transgenic mice over-expressing human EPO (termed tg6). EPO treatment was associated with an increased splenic macrophage population, detected by F4/80 expression, and an increased number of macrophages expressing CD11b, CD80 and MHC class II. We further explored the effect of in-vivo EPO administration in an inflammatory model exploiting thioglygollate injection to induce recruitment of peritoneal inflammatory macrophages. The inflammatory macrophages obtained from both EPO injected and from tg6 mice displayed increased expression of F4/80, CD11b, CD80 and MHC class II and augmented phagocytic activity, as compared to the control counterparts.

These results are supported by in-vitro studies in bone marrow derived macrophages (BMDMs). We show that BMDMs express EPO-R mRNA, as detected by RT-PCR. In-vitro stimulation of the BMDMs with rHuEPO activated multiple signaling pathways including STAT1, STAT5, MAPK, AKT and NFkB indicating macrophage activation via surface EPO-R. EPO treatment of the BMDMs up-regulated their surface expression of CD11b, F4/80 and CD80, as well as enhanced their phagocytic activity. EPO treatment of LPS-stimulated BMDMs augmented IL-12 secretion, and decreased IL-10 secretion.

In conclusion our results show that macrophages are direct targets of EPO and that EPO treatment enhances their pro-inflammatory activity and function. These findings point to the multifunctional role of EPO and may advance its clinical applications as an anti-neoplastic immunomodulator.